Quantification of ethyl carbamate in cachaça produced in different agro-industrial production systems

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1 Research article Received: 26 November 2015 Revised: 12 February 2016 Accepted: 27 February 2016 Published online in Wiley Online Library: 15 April 2016 (wileyonlinelibrary.com) DOI /jib.322 Quantification of ethyl carbamate in cachaça produced in different agro-industrial production systems Gabriel Biscotto d Avila, 1 Maria das Graças Cardoso, 2 * Wilder Douglas Santiago, 2 Leonardo Milani Avelar Rodrigues, 1 Bruno Leuzinger da Silva, 2 Rodolfo Romanielo Cardoso, 3 Alex Rodrigues Silva Caetano, 2 Cleusa de Fátima e Silva Ribeiro 1 and David Lee Nelson 4 Popularly known by various names such as caninha, dangerous, drips, damned and many other denominations, cachaça is the genuine Brazilian drink, produced by the fermentation of sugarcane juice by yeast, followed by distillation in alambics. Considering that cachaça is the most widely consumed distilled beverage from Brazil, the knowledge of the chemical composition and the presence of potentially toxic compounds such as ethyl carbamate, considered a human carcinogen, is important. The aim of this study was to evaluate the influence of different conditions of the agro-industrial cachaça production systems, including the variety of sugarcane, on the quantification of ethyl carbamate through the chromatographic. Thirteen unaged beverage samples, produced from different varieties of sugarcane, were analysed. Using analysis of variance and comparison of average concentrations of ethyl carbamate (Scott Knott, α = 5%), all of the samples were found to contain contaminant levels below the ceiling established by the legislation, which is 210 μgl 1. Copyright 2016 The. Keywords: sugarcane; organic contaminant; production; beverage Introduction A genuinely Brazilian beverage produced by the fermentation of sugarcane juice by yeast, followed by distillation in alambics, cachaça is the most widely consumed distilled beverage produced by Brazil. Brazil produces about 800 ML of cachaça annually, so it is important to verify the chemical composition and presence of potentially toxic compounds, such as ethyl carbamate (EC), which is considered to be a human carcinogen. There are 12,000 establishments that produce cachaça in the country; 4000 brands are currently marketed, and 90% of the production is derived from legalized producers. However, it is estimated that 85% of the producers are micro and small industries that are informal producers. Approximately 10,180,000 L of cachaça was exported to 66 countries in 2014 by more than 60 companies, generating an income of US$18.33 million (1). The sugarcane juice is obtained by a grinding process and, after passing through the fermentation and distillation steps, yields cachaça, in which the major component is ethanol. In addition, several other compounds are formed during these steps and during aging, and these are known as secondary components. They are responsible for the characteristic flavour and aroma of the beverage. In parallel with the secondary components, some contaminants can also be found in the beverage, including EC, which is the ester of carbamic acid. It is formed naturally during the fermentation process and is present in various foods and fermented beverages. Despite its origin, its mechanism of formation has not yet been fully elucidated. Ethyl carbamate has been classified as a possible human carcinogen by the International Agency for Research on Cancer (2), and the maximum limit allowed in distilled sugarcane spirits and cachaça in Brazil is 210 μgl 1 (3). Therefore,theaimofthis study was to quantify EC in new cachaça produced in copper stills by different agro-industrial production systems in which different varieties of sugarcane were used and, thereby, verify the relationship between the presence of this contaminant and the production system. Materials and methods Samples Thirteen samples of unaged sugarcane spirits distilled in pot stills in Minas Gerais were collected. Each sample represented an * Correspondence to: M. das Graças Cardoso, Department of Chemistry, Federal University of Lavras, Minas Gerais, Brazil. mcardoso@dqi.ufla.br 1 Department of Food Science, Federal University of Lavras, CP 3037, Lavras, MG, Brazil 2 Department of Chemistry, Federal University of Lavras, Minas Gerais, Brazil 3 Department of Environmental Engineering, Federal University of Lavras, Minas Gerais, Brazil 4 Foods Department, Faculty of Pharmacy, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil 299 J. Inst. Brew. 2016; 122: Copyright 2016 The

2 agro-industrial production system. Of the 13 samples, 10 were produced from different varieties of sugarcane, and three were produced from the same variety of sugarcane, but they were produced by different companies. Two samples were produced using a selected yeast (LNF CA-11), and the others were produced using natural yeast. The choice of the samples was based on data supplied by producers that used only one variety at a time during the fermentation steps. Furthermore, the liquor did not undergo any treatment that might change its characteristics, such as the addition of a dye or carbon. All of the producers were small-scale industries that used stainless steel vats for fermentation, and distillation was accomplished in copper stills. They did not perform burning of the cane fields before harvest. Samples were collected in glass bottles directly at the production sites and identified with the brand of the product, city, sample collection date and variety of sugarcane used in production. Analyses were performed at the Laboratório de Análises de Qualidade de Aguardentes of the Departamento de Química of the Universidade Federal de Lavras. Table 1 shows the sugarcane varieties used to produce the beverages and the respective towns where the cachaça was produced. Cachaças were produced in 2014 and analysed four months after production. Reagents and samples The reagents used for analysis were the EC standard (Acros Organics), ethanol, propanol, hexane, hydrochloric acid, ethyl acetate, sodium acetate, HPLC-grade acetonitrile (Merck), water type I and 9-xanthydrol (Acros Organics). Determination of EC The identification and quantification of EC were performed according to the method described by Madrera and Valles (4) and modified by Anjos et al. (5), Machado et al. (6) and Santiago et al. (7). External standardization was used, and derivation of the samples was performed prior to analysis by HPLC. G. B. d Avila et al. To ensure the analytical quality of the results, procedures were conducted to validate the method using the following parameters: linearity, limit of detection, limit of quantification and accuracy (6,8). The mathematical relationship between the sign and the concentration of the species of interest was expressed by line equations (analytical curves) and their respective coefficient of determination (R 2 ). The limits of detection (LD) and quantification (LQ) of the methods were estimated using the parameters of the analytical curve according to the following mathematical equations: LD = 3 (s/s) and LQ = 10 (s/s), in which s is the estimate of the standard deviation of the equation for the regression line, and S is the angular coefficient of the analytical curve (9). All of the samples were analysed in triplicate. Preparation of the derivative of the EC standard A propanol solution of 9-xanthydrol at a concentration of 0.2 mol L 1 was prepared in an amber flask. To the flask was added 20 ml of a standard solution of EC (4.0 g L 1 ) in 40% ethanol, followed by 2 ml of HCl (5.5 mol L 1 ). The reaction mixture was stirred for about 60 s and then maintained at rest for an additional 60 min. The crystals that formed were filtered and recrystallized from hexane. The quantitative analysis was validated by diluting a stock solution of the EC derivative, at a concentration of 10 mg L 1 in ethyl acetate, to give working solutions containing μgl 1 in 50% ethanol. Preparation of the sample derivatives Four millilitres of cachaça was added to an amber vial, followed by 0.8 ml of a 0.02 mol L 1 xanthydrol solution (prepared in propanol). After stirring, 0.4 ml HCl (1.5 mol L 1 ) was added with stirring, and the stirring was continued for an additional minute. This mixture was allowed to stand for 60 min, filtered through 0.45 μm polyethylene membranes (Millipore) and injected into the chromatograph. 300 Table 1. Varieties of sugarcane used to produce cachaça, types of yeast and cities where the samples were collected. Sample Type of yeast Variety of sugarcane City of origin A Natural CO421 Cel. Xavier Chaves B Natural RB Divinésia C Natural CO413 Guiricema D Natural SP Ponte Nova E Natural SP Ponte Nova F Natural RB Santa Bárbara do Tugúrio G Natural Caiana Poço Fundo H LNF CA-11 RB Toledo I LNF CA-11 RB Lavras J Natural SP Perdões K Natural RB Perdões L Natural SP Ponte Nova M Natural Espalha-rama Presidente Bernardes Chromatographic conditions and quantification of EC Analysis was performed by HPLC on a Shimadzu liquid chromatograph equipped with two model SPD-M20A highpressure pumps, a DGU-20A3 degasser, a SIL-10AF automatic injector with a CBM-20A autosampler interface, and an RF-10AXL model fluorescence detector. Separations were performed using an Agilent-Zorbax Eclipse AAA column ( mm, 5 μm) connected to an Agilent-Zorbax Eclipse AAA 4-Pack guard column ( mm, 5 μm). The excitation and emission wavelengths used for the quantification of EC were 233 and 600 nm, respectively. The flow rate used throughout the analysis was 0.75 ml min 1, and the volume of injected sample and standard was 20 μl. The mobile phase consisted of 20 mmol L 1 sodium acetate (solvent A) and acetonitrile (solvent B). Elution was performed in a gradient system: 0 5 min, 60% solvent A and 40% solvent B; 5 10 min, 40% solvent A and 60% solvent B; min 30% solvent A and 70% solvent B; min, 20% solvent A and 80% solvent B; min, 10% solvent A and 90% solvent B; and min, 60% solvent A and 40% solvent B. All the analyses were performed in triplicate. wileyonlinelibrary.com/journal/jib Copyright 2016 The J. Inst. Brew. 2016; 122:

3 Ethyl carbamate in cachaça production Statistical analysis The analysis of variance was based on a scheme involving variation among varieties and within the varieties. The varieties were obtained from different locations and different agro-industrial production systems and, therefore, did not permit randomization. When the F-test for the analysis of variance was significant in the quantification of EC, the means were compared by the Scott Knott test (1974) at a 95% level of confidence with the aid of SISVAR (10) statistical software. Results and discussion The chromatogram obtained by injection of the EC standard is shown in Fig. 1. The average retention time obtained for the compound was ± 0.09 min. Santiago et al. (7) evaluated EC levels in the production and aging process in oak and amburana barrels and found an average retention time of min. Anjos et al. (5) studied the EC in cachaças stored in oak and glass containers and observed an average retention time of min. Mendonça et al. (11) observed an average retention time of min while studying cachaça produced with selected wild yeasts and baker s yeast. These results are consistent with those found in this work. The LD and LQ values were 1.86 and 6.23, respectively. These values were lower than those found in a recent work on the determination of EC in a cachaça matrix using HPLC equipped with a fluorescence detector (HPLC-FLD) with prior derivatization of EC. Anjos et al. (5) observed LD and LQ values of 3.93 and μgl 1, respectively; Machado et al. (6) obtained values of 6.39 μgl 1 for the LD and μgl 1 for the LQ. Mendonça et al. (11) found an LD of 2.48 μgl 1 and an LQ of 8.26 μgl 1, and Santiago et al. (7) found values of 3.24 μgl 1 for the LD and μgl 1 for the LQ. The EC concentrations found in all the cachaça samples were within the legally established limit in Brazil, that is, <210 μgl 1, with a mean of μgl 1. The values obtained for the EC concentrations in the samples are presented in ascending order in Table 2. Sample B, which corresponds to the RB variety, contained the highest concentration of EC (35.35 μgl 1 ). Its concentration was significantly different from those of the second placed samples A and F, there being no significant differences between them (30.91 and μgl 1, respectively). The lowest concentration (12.29 μgl 1 ) of EC was observed in sample K (variety RB867515). The samples H, M and L (14.21, and μgl 1, respectively) did not differ significantly from one another (α = 0.05). The chromatograms of the samples B and K are shown in Figs. 2 and 3, respectively. The three samples that were produced from the same RB variety (samples F, H and K) were significantly different from one another at 5% probability. Sample F contained the second highest concentration of EC observed among the 13 analysed, whereas Sample K contained the lowest concentration of all the samples, followed by sample M. Barcelos et al. (12) evaluated cachaça samples from three regions of Minas Gerais (South, Zona da Mata and Jequitinhonha Valley) and obtained values ranging from not detected to 700 μgl 1 of EC. Among the regions studied, only the samples from the Jequitinhonha Valley contained levels greater than the limits set by the Ministério da Agricultura, Pecuária e Abastecimento (3). Figure 1. Chromatogram of the ethyl carbamate standard with fluorescence detection. Concentration of the standard is 160 μgl 1. Table 2. Concentrations of ethyl carbamate in the beverage samples Sample Ethyl carbamate (μgl 1 ) a Variety A ± 0.10 g CO421 B ± 0.19 h RB C ± 0.35 f CO413 D ± 0.32 f SP E ± 0.70 c SP F ± 0.42 g RB G ± 0.51 d Caiana H ± 0.31 b RB I ± 0.57 d RB J ± 0.53 e SP K ± 0.20 a RB L ± 0.06 b SP M ± 0.10 b Espalha-rama a Mean ± standard deviation. Coefficient of variation = 1.59%. The means followed by the same lower case letter do not differ significantly by the Scott Knott test at the 5% level of probability. Figure 2. HPLC chromatogram of the cachaça produced from the RB variety (sample B) of sugarcane showing the detection of ethyl carbamate. 301 J. Inst. Brew. 2016; 122: Copyright 2016 The wileyonlinelibrary.com/journal/jib

4 G. B. d Avila et al. 302 Figure 3. HPLC chromatogram of the cachaça produced from the RB variety (sample K) of sugarcane showing the detection of ethyl carbamate. Masson et al. (13) studied 63 samples of cachaça produced in small- to medium-sized copper stills in the northern and southern regions of Minas Gerais and found values ranging from 22.6 to 980 μgl 1. The author reported that the EC content found in cachaça did not correlate with the alcohol content, acidity or copper concentration in the samples. Although the acidity of samples B, G and M exceeded the allowable limit in the present study, and copper concentrations of samples E, G and M also exceeded the limit, there was no correlation with the EC concentrations present in these samples. Zacaroni et al. (14) analysed some contaminants in samples of distilled sugarcane spirits and observed that all the samples contained EC concentrations below the limit established by Brazilian legislation. The highest concentration found was 119 μgl 1, far superior to the highest concentration found in this study, which was μgl 1 (sample B). Serafim et al. (15) determined the EC concentration in three fractions from a cachaça still (head, heart and tail) and found that the sum of the concentrations of this contaminant in all three fractions tended to be lower than the carbamate concentrations found in cachaça distilled through columns where there is no separation of fractions. Fernandes (16) studied cachaças obtained from five varieties of cane harvested in three periods of maturation, but did not detect the presence of EC in their samples. In this study, only new cachaças were evaluated, that is, cachaça that had been distilled less than 2 months previously and packed in glass bottles, well sealed and kept away from light in a cool place, without major changes in temperature, until the time of analysis. Zacaroni et al. (17) reviewed the evolution of the EC, at two-month intervals for a period of six months, in 10 cachaça samplesagedinwoodenbarrelsandthenstoredinglassbottles inthepresenceandabsenceoflight.theyconcludedthatthe luminosity and storage period are factors that influenced the presence of EC in cachaça. In addition, the authors reported that the presence of nitrogen compounds (urea, arginine, citrulline and ornithine) and the conditions (temperature and ph) used during fermentation of cachaça may also contribute to the formation of EC. In this context, we cite the studies of Anjos et al. (5), who confirmed that the storage of the beverage, both in oak barrels and in a glass container, influenced the formation of EC and caused a significant increase in the concentration of this contaminant. The presence of light also had an influence, μgl 1 of EC being detected in cachaça stored in a glass container at the second month of storage, whereas the same concentration was only observed in cachaça stored in an oak barrel in the ninth month. Machado et al. (6) analysed EC in alambic cachaça obtained from sugarcane fertilized with urea and ammonium nitrate and packed in high-density polyethylene drums and glass containers. They used a solid-phase microextraction method and GC-MS, in selective ion monitoring mode, derivatization of the EC and HPLC with fluorescence detection. They found that the two methods can be employed to quantify the carbamate and that none of the samples had concentrations above the limit established by law. Despite having only worked with one variety of cane, the SP791011, the results corroborate those obtained in this work, where no concentrations of EC above the limit allowed by law were found. Santiago et al. (7) periodically accompanied the chromatographic profile of EC in the production process and the aging of cachaça in amburana barrels. The authors observed that the concentration of EC presented a value lower than the maximum legal limit for this compound during the production and aging stages. Mendonça et al. (11) studied various types of yeast and different materials for storage and noted that the EC concentration was influenced by both the oak wood and the glass containers in which the samples were stored. However, the concentrations were well below the maximum limit established by law for this compound. The cachaça that used rice bran as a nutrient source in the fermentation contained the highest EC concentrations before and after storage. Conclusions All of the samples studied contained EC concentrations well below the limit established by Brazilian legislation; those from the same variety of sugarcane contained distinct concentrations, showing that the characteristics of the agro-industrial systems used to produce the beverage did not influence the concentration of EC in the new beverages analysed. Acknowledgements The authors acknowledge the support of the Universidade Federal de Lavras, the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, the Fundação de Amparo à Pesquisa do Estado de Minas Gerais and the Conselho Nacional de Desenvolvimento Científico e Tecnológico. References 1. Instituto Brasileiro da Cachaça (2015) Mercado externo, IBRAC, Brasília. Available from: mercado-externo (last accessed February 2015). 2. Lachenmeier, D. W., Nerlich, U., and Kuballa, T. (2006) Automated determination of ethyl carbamate in stone-fruit spirits using headspace solid-phase microextraction and gas chromatography tandem mass spectrometry, J. Chromatogr. A 1108(1), MAPA (Brasil Ministérioda Agricultura, Pecuária e Abastecimento) (2014) Leis, decretos, etc. Instrução Normativa no. 28, de agosto de 2014, DiárioOficial da União, Brasília, 7, August. 4. Madrera, R. R., and Valles, B. S. (2009) Determination of ethyl carbamate in cider spirits by HPLC-FLD, Food Control 20, Anjos, J. P., Cardoso, M. G., Saczk, A. A., Zacaroni, L. M., Santiago, W. D., Dórea, H. S., and Machado, A. M. R. (2011) Identificação do carbamato de etila durante o armazenamento da cachaça em tonel de carvalho (Quercus sp) e recipiente de vidro, Quim. Nova 34, Machado, A. M. R., Cardoso, M. G., Saczk, A. A., Anjos, J. P., Zacaroni, L. M., Dorea, H. S., and Nelson, D. L. (2013) Determination of ethyl wileyonlinelibrary.com/journal/jib Copyright 2016 The J. Inst. Brew. 2016; 122:

5 303 Ethyl carbamate in cachaça production carbamate in cachaça produced from copper stills by HPLC, Food Chem. 138, Santiago, W. D., Cardoso, M. G., Duarte, F. C., Saczk, A. A., and Nelson, D. L. (2014) Ethyl carbamate in the production and aging of cachaça in oak (Quercus sp.) and amburana (Amburana cearensis) barrels, J. Inst. Brew. 120, Ribani, M., Bottoli, C. B. G., Collins, C. H., Jardim, I. C. S. F., and Melo, L. F. C. (2004) Validação em métodos cromatográficos e eletroforéticos, Quim. Nova 27, Harris, D. C. (2008) Análise Química Quantitativa, 7thed.,, LTC, Riode Janeiro. 10. Ferreira, D. F. (2011) Sisvar: A computer statistical analysis system, Cienc. Agrotecnol. 35, Mendonça, J. G. P., Cardoso, M. G., Santiago, W. D., Rodrigues, L. M. A., Nelson, D. L., Brandão, R. M., and Silva, B. L. (2016) Determination of ethyl carbamate in cachaças produced by selected yeast and spontaneous fermentation, J. Inst. Brew. 122, Barcelos, L. V. F., Cardoso, M. G., Vilela, F. J., and Anjos, J. P. (2007) Teores de carbamato de etila e outros componentes secundários em diferentes cachaças produzidas em três regiões do estado de Minas Gerais: Zona da Mata, Sul de Minas e Vale do Jequitinhonha, Quim. Nova 30, Masson, M., Cardoso, M. G., Zacaroni, L. M., Anjos, J. P., Sackz, A. A., Machado, A. M. R., and Nelson, D. L. (2012) Determination of acrolein, ethanol, volatile acidity, and copper in different samples of sugarcane spirits, Cienc. Tecnol. Aliment. (Campinas, Brazil) 32, Zacaroni, L. M., Cardoso, M. G., Saczk, A. A., Santiago, W. D., Anjos, J. P., Masson, J., Duarte, F. C., and Nelson, D.L. (2011). Caracterização e quantificação de contaminantes em aguardentes de cana, Quim. Nova 34, Serafim, F. A. T., Silva, A. A., Galinaro, C. A., and Franco, W. D. (2012) Comparação do perfil químico entre cachaças de um mesmo vinho destiladas em alambiques e em colunas, Quim. Nova 35, Fernandes, O. W. B. (2013) Avaliação da composição físico-química de cachaça de alambique de cinco cultivares de cana-de-açúcar colhidas em três épocas de maturação, Tese de Doutorado, Universidade Federal de Ouro Preto, Brasil. 17. Zacaroni, L. M., Cardoso, M. G., Santiago, W. D., Gomes, M. S., Duarte, F. C., and Nelson, D. L. (2015) Effect of light on the concentration of ethyl carbamate in cachaça stored in glass bottles, J. Inst. Brew. 121, J. Inst. Brew. 2016; 122: Copyright 2016 The wileyonlinelibrary.com/journal/jib