Article pubs.acs.org/JAFC
Cyanate as an Active Precursor of Ethyl Carbamate Formation in Sugar Cane Spirit Carlos A. Galinaro,† Thiago H. K. Ohe,† Augusto C. H. da Silva,† Sebastiaõ C. da Silva,§ and Douglas W. Franco*,† †
Departamento de Quı ́mica e Fı ́sica Molecular, Instituto de Quı ́mica de São Carlos, Universidade de São Paulo, Avenida do Trabalhador São Carlense 400, CP 780, CEP 13560-970 São Carlos, SP, Brazil § Departamento de Quı ́mica, Universidade Federal de Mato Grosso, Instituto de Ciências Exatas e da Terra, Avenida Fernando Correia da Costa S/N, Boa Esperança, CEP 78060-900 Cuiabá, MT, Brazil J. Agric. Food Chem. 2015.63:7415-7420. Downloaded from pubs.acs.org by UNIV OF SUSSEX on 08/31/15. For personal use only.
S Supporting Information *
ABSTRACT: The thermodynamic and kinetic aspects of ethyl carbamate (EC) formation through the reaction between cyanate and ethanol were investigated. The rate constant values for cyanate ion decay and EC formation are (8.0 ± 0.4) × 10−5 and (8.9 ± 0.4) × 10−5 s−1, respectively, at 25 °C in 48% aqueous ethanolic solution at pH 4.5. Under the investigated experimental conditions, the rate constants are independent of the ethanol and cyanate concentrations but increase as the temperature increases (ΔH⧧1 = 19.4 ± 1 kcal/mol, ΔS⧧1 = −12.1 ± 1 cal/K, and ΔG⧧1 = 23.0 ± 1 kcal/mol) and decrease as the solution pH increases. According to molecular modeling (DFT) that was performed to analyze the reaction mechanism, the isocyanic acid (HNCO) is the active EC precursor. The calculated ΔG⧧1 , ΔH⧧1 , and ΔS⧧1 values are in very good agreement with the experimental ones. KEYWORDS: ethyl carbamate, cyanate, sugar cane spirit, cachaça, distillate
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INTRODUCTION
Currently, the available studies on EC in whiskey and cachaça are mostly descriptive and concerned with the occurrence of EC and with correlations between still material composition, still design, distillation procedure, and EC yield.6,9−16 To the best of our knowledge, few studies have been dedicated to explaining the chemical reactions that account for EC formation in distillate.2,8,17 Because of their ability to react with ethanol, cyanide-type compounds have been previously recognized as possible precursors of EC.1,2,8 These compounds could originate from the urea and amino acids that are added during fermentation or from the hydrolysis of glycoside compounds.1 In grains, the presence of these glycosides has been reported,18 but in sugar cane this subject remains a matter of discussion.1,18 During experiments performed in our laboratory with only distilled sugar cane spirit, it was observed that ethyl carbamate content increases as time passes, reaching a final and stable concentration after a week.5 This finding was taken as an indication of the strong kinetic dependence of EC formation in the distillate. Furthermore, we recently detected the presence of cyanate ions in freshly distilled sugar cane spirit and found that the decay of these species correlated with the formation of EC.19 Aiming to better understand the kinetic aspects of the reactions between cyanide-type compounds (cyanate, cyanide, arginine, citrulline, carbamoyl phosphate, and urea) and
Ethyl carbamate (EC) is well-known as a potentially carcinogenic compound.1,2 Therefore, its presence in foods and beverages is controlled in many countries.1,2 In wine and beer, EC is a fermentation product, and substantial effort has been dedicated toward understanding its genesis and developing control practices to reduce its formation.1,2 Although the fermentation step is also involved in the production of spirits, the presence of EC in these beverages is the result of a more complicated pathway. Because EC has a high boiling point (≈182 °C), a distillation operation should provide an efficient means to reduce or even avoid the presence of this compound in the distillate. Indeed, when commercial samples of whiskey3,4 or cachaça (sugar cane spirit)5 were redistilled, a substantial reduction in beverage EC concentration was observed. In fact, in the latter case, this reduction could be as high as 92% and was independent5 of the spirit’s alcohol contents (10−80% v/v or alcohol by volume (ABV)) prior to the redistillation. Furthermore, the type of distillation apparatus (columns or pot stills) and the materials used in its construction also exhibited a marked effect on the EC content of the distillate.6,7 Therefore, to explain the presence of EC in spirits, in addition to the reactions that occur during fermentation, which although relevant are not considered in the present study, those that take place during fermented sugar cane juice (also called “wine” or “must”) heating via the contact between the vapors and the walls of the distillation apparatus and in the freshly distilled spirit between the compounds formed during the distillation and the others already present in the fermented sugar cane juice must be accounted for.1,8 © 2015 American Chemical Society
Received: Revised: Accepted: Published: 7415
June 25, 2015 August 3, 2015 August 7, 2015 August 7, 2015 DOI: 10.1021/acs.jafc.5b03146 J. Agric. Food Chem. 2015, 63, 7415−7420
Article
Journal of Agricultural and Food Chemistry
28 mL glass bottles (vials), sealed with rubber septa and aluminum, and kept in the dark (wrapped in aluminum foil). During the kinetic experiments, the pH, the contents of copper(II) and iron(II), the alcoholic concentration of the cachaça, and the temperature (25 °C) were kept constant, except when the influences of these parameters were evaluated separately. During the experiments, aliquots of the cachaça model solution were collected with the aid of a glass syringe. Decay of the Cyanate Ion. The cyanate concentration decay was monitored using two different procedures that have been described in the literature.19,23 Both methods involve the reaction between cyanate ion and 2-aminobenzoic acid in the presence of HCl to form 2,4(1H,3H)-quinazolinedione. The choice of method was based on the sensitivity required. One method uses the light absorption,23 whereas the other uses the fluorescence19 of the derivatization reaction product. The absorbance measurements were performed at 310 nm (ε = 412 L/mol cm) on a UV−visible spectrophotometer (model U3501, Hitachi, Tokyo, Japan) with a 10 cm path length cylindrical quartz cell. The fluorescence measurements were carried out on a model F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan); 310 nm was used as the excitation wavelength, and the maximum emission was observed at 410 nm. The emission spectra were recorded over a range of 320−600 nm at a resolution of 240 nm/ min using a 5 nm slit and a 1 cm quartz cell. Cyanate Influence in the Formation of Ethyl Carbamate. Model solutions of cachaça (50 mL) were prepared at final cyanate ion concentrations of (0.5−4.5) × 10−5 mol/L. Subsequently, the influence of the OCN− ion on the formation and evolution of EC levels was evaluated at 25 °C. On the basis of the analytical results (linearity, reproducibility, and repeatability)24 obtained for the EC levels in these experiments, a concentration of approximately 2.1 × 10−5 mol/L of KOCN was selected for further experiments. Influence of pH on the Ethyl Carbamate Formation. The pH measurements were carried out in an Ion Analyzer Radiometer Analytical model PHM250 (MeterLab, Villeurbanne, France). Model solutions of spirits (50 mL) were prepared with the following pH values: 3.0 ± 0.1, 3.9 ± 0.1, 5.3 ± 0.1, and 6.0 ± 0.1. The pH was decreased and increased with hydrochloric acid (1.0 × 10−2 mol/L) and sodium hydroxide (1.0 × 10−2 mol/L) solutions, respectively. These solutions were fortified with approximately 2.1 × 10−5 mol/L of KOCN, and their EC contents were subsequently analyzed at 25 °C. Influence of Light in the Formation of Ethyl Carbamate. Cachaça model solutions (50 mL) were prepared with approximately 2.1 × 10−5 mol/L KOCN. One of these solutions was protected from light (wrapped in aluminum foil and kept in the dark), whereas the others were exposed to radiation in a photochemical bench with appropriate filters (250−500 nm). Subsequently, these solutions were analyzed for their EC contents at 25 °C. Influence of Alcohol Content in the Formation of Ethyl Carbamate. Two new standard solutions with alcohol levels of 13.3% v/v (2.3 mol/L ethanol) and 24.4% v/v (4.2 mol/L) were prepared by diluting the reference cachaça, which had an alcohol content of 48% v/ v (8.2 mol/L ethanol), with distilled water. These solutions were fortified with approximately 2.1 × 10−5 mol/L KOCN and subsequently analyzed for EC content at 25 °C. Then, aqueous ethanolic solutions were prepared with different alcohol concentrations (1.70−91.5% v/v), fortified with 2.1 × 10−5 mol/L KOCN, and analyzed for their EC contents at 35 °C. The alcoholic strength was decreased and increased using deionized water and ethanol (99.5% v/v), respectively. Molecular Energies and Structures Calculations. The reaction between cyanate and ethanol was modeled using ab initio DFT25,26 calculations with a B3LYP27 exchange correlation function. The allelectron 6-31+G(d,p)28−30 basis set function was used to describe the electronic structures of the studied systems. An isomeric equilibrium reaction (Scheme 1, path A) was considered for the corresponding acid species cyanic acid (HOCN) and isocyanic acid (HNCO). Both of these species can react with ethanol (EtOH) according to the mechanism (B, C) in Scheme 1, forming ethyl carbamate (EC). A hydrolysis reaction (D) was also considered.
ethanol, the reaction between cyanate and ethanol was studied as a model. Therefore, herein we describe the kinetic aspects of this reaction and use density functional theory (DFT) calculations to discuss potential mechanistic pathways.
J. Agric. Food Chem. 2015.63:7415-7420. Downloaded from pubs.acs.org by UNIV OF SUSSEX on 08/31/15. For personal use only.
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MATERIALS AND METHODS
Reagents. The reagents and solvents used were of analytical or chromatographic grade and were purchased from Sigma-Aldrich (Steinheim, Germany), Mallinckrodt Baker (Paris, KY, USA), and Carlo Erba (Milano, Italy). The water used was previously distilled and then deionized using a Milli-Q system (Millipore, Bedford, MA, USA). Reference Cachaça. One commercial, nonaged cachaça sample (200 L), which was provided and certified by a major producer, was used as a model (reference) solution during the experiments. Selected constituents were evaluated in terms of their initial concentrations in this matrix (Table S1, Supporting Information). Ethyl Carbamate Analysis. The samples were analyzed using a gas chromatograph system equipped with a mass selective detector (GC-MS) using electron impact (70 eV) as an ionization source. The mass spectrometer was operated in selected ion monitoring (SIM) mode (m/z 62), and propyl carbamate was added as an internal standard.10 The oven temperature program was as follows: 90 °C (2 min), followed by an increase to 150 °C at 10 °C/min (0 min) and then an increase to 230 °C at 40 °C/min (10 min). A HP-FFAP (50 m, 0.2 mm, 0.3 μm, Agilent Technologies, Santa Clara, CA, USA) capillary column was used in the EC separation. The inlet and detector interface temperatures were 250 and 230 °C, respectively. Sample aliquots of 1.0 μL were injected into the gas chromatograph system in the splitless mode. Copper and Iron Ion Analysis. Copper and iron ions were determined using methodology described in the literature.9 The analysis was performed using inductively coupled plasma atomic emission spectroscopy (Optima 3000 model dual view, PerkinElmer, Santa Clara, CA, USA). The calibration curves were constructed using an external standard method. Photochemical Experiments. The irradiation of commercial cachaças and the aqueous ethanolic model solutions was performed in a photochemical bench (model 68805 Universal) equipped with a 200 W xenon lamp with interference filters (Oriel Instruments, Irvine, CA, USA). The intensity of the incident radiation was 1.3 × 10−7 einstein/ min, which was calculated using a chemical actinometer (K3[Fe(C2O4)3], tris(oxalato)ferrate(III) of potassium, 0.2 mol/L), as previously described.20 Kinetic Experiments. Unless otherwise indicated, the kinetic experiments were carried out using a previously described cachaça model solution. During the kinetic studies, potassium cyanate was added to this solution, and the formation of EC was monitored. These analyses were performed as a function of the following parameters: alcoholic degree; pH; addition of salts of copper, iron, and EDTA; influence of light (photochemical irradiation bench); and temperature. In the experiments, the solution pH was controlled using an acetic acid/sodium acetate buffer system. Half-life (t1/2) data were calculated from plots of ethyl carbamate concentration ([EC]) as a function of reaction time. t1/2 was defined as the time corresponding to the formation of half of the maximum urethane concentration ([EC]∞).21,22 The experiments were carried out under pseudo-first-order conditions (excess of ethanol), and the rate constant kobs (observed rate constant) was calculated from plots of ln([EC]∞ − [EC]t) versus time.21,22 These plots were linear for at least 3 half-lives. To calculate the kinetic parameters, the temperature was controlled at ±0.2 °C using a thermostat, and the experiments were run at 15, 25, and 35 °C. The activation parameters (ΔG⧧1 , ΔH⧧1 , and ΔS⧧1 ) were determined graphically.21,22 Preparation, Storage, and Analysis of Model Solutions Fortified with Potassium Cyanate. All cachaça model solutions were obtained using the reference cachaça (Table S1) and buffered using an acetic acid/sodium acetate buffer system. These solutions were spiked with a stock solution of potassium cyanate and stored in 7416
DOI: 10.1021/acs.jafc.5b03146 J. Agric. Food Chem. 2015, 63, 7415−7420
Article
Journal of Agricultural and Food Chemistry
as a consequence of the addition of urea or cyanide. This residual EC formation is
