C: Food Chemistry
A Fluorescence-Based Method for Cyanate Analysis in Ethanol/Water Media: Correlation between Cyanate Presence and Ethyl Carbamate Formation in Sugar Cane Spirit Thiago Hideyuki Kobe Ohe, Alexandre Ataide da Silva, Tha´ıs da Silva Rocha, Fl´avio Schutzer de Godoy, and Douglas Wagner Franco
Abstract: Based on the fluorescence properties of 2,4-(1H,3H)-quinazolinedione, a product of the reaction between cyanate and 2-aminobenzoic acid, a simple, sensitive, selective, and reproducible method for the cyanate analysis in aqueous ethanolic media is proposed. In this method, λexc and λem are 310 and 410 nm, respectively, and the limits of detection and quantification are 2.2 × 10−7 and 6.7 × 10−7 mol/L, respectively. Under optimal conditions (pH = 4.5, 40% ethanol), a concentration of 5.0 × 10−6 mol/L cyanate can be determined in a single measurement, at a 95% level of confidence, with an uncertainty of ± 0.13 × 10−6 mol/L. Cyanide, thiocyanate, chloride, nitrate, and sulfate ions, as well as urea and urethane in concentrations 1 × 103 higher than that of cyanate do not interfere with the measurement. The methodology was applied to cyanate analyses in the different fractions of the sugarcane distillate and the data strongly suggest a correlation between the presence of urea in wine, and the cyanate and ethyl carbamate concentrations in the spirit. Keywords: ethyl carbamate, fluorimetric cyanate analysis, sugar cane spirit, urea in wine
Based on the fluorescence properties of the reaction product between cyanate and 2-aminobenzoic acid, a method for assaying cyanate was devised. This procedure applied to the sugarcane distillate showed for the first time a correlation between cyanate presence and ethyl carbamate (EC) formation in the different fractions of the product. Therefore, the proposed methodology can be used to predict in freshly distillate sugar cane spirits the potential total concentration of EC to be formed. Therefore, these data could be used to advise about the necessity of implementing a procedure to reduce spirit EC concentration before the product reaches the market.
Practical Application:
Introduction Due to its carcinogenic properties, the presence of ethyl carbamate (EC) in food and beverages is controlled worldwide. Currently, a great deal of effort is being put into improving the quality of Brazilian sugar cane spirit (cachac¸a), the production of which is approximately 1.2 billion liters per year (Granato and others 2014). Therefore, it is important to understand the genesis of EC to prevent or reduce its presence in this beverage and, by extension, in the ethanol itself. Brazil is the world’s largest producer of ethanol from sugar cane (>20 billion liters per year), which is used both as a “green fuel” and by the pharmaceutical industry (Raele and others 2014). Previous studies (Zimmerli and Schlatter 1991; Taki and others 1992; EFSA 2007; Da Silva and others 2013) have strongly suggested that OCN− and CN− , as well as their corresponding acids, are precursors of EC. In our laboratory,
MS 20140683 Submitted 4/22/2014, Accepted 7/12/2014. Authors are with Dept. de Qu´ımica e F´ısica Molecular, Inst. de Qu´ımica de S˜ao Carlos, Univ. de S˜ao Paulo (USP), Avenida Trabalhador S˜ao-carlense 400, CP 780, CEP 13560-970, S˜ao Carlos – SP, Brazil. Direct inquiries to author Franco (E-mail: [email protected]).
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Journal of Food Science r Vol. 79, Nr. 10, 2014
we have collected, for the first time, evidence that HOCN is the most important EC precursor for cachac¸as, mostly when dealing with fresh distilled sugar cane spirit (Galinaro and Franco 2011; Da Silva and others 2013). During our experiments, it was necessary to develop a reliable, selective, and sensitive method for OCN− analysis in the presence of a large excess of CN− . 2-Aminobenzoic acid (2ABA) has previously been described as a colorimetric reagent for OCN− analysis in mine effluents (Zvinowanda and others 2008). The fluorescence properties of the cyanate/2-ABA reaction have been explored for analytical purposes in estuarine, seawater, and plasma analysis (Lundquist and others 1993; Widner and others 2013). Therefore, based on the reaction (Guilloton and Karst 1985) as shown in Scheme 1, and taking advantage of the fluorescence properties of 2,4-(1H,3H)-quinazolinedione, a simple and rapid analytical method was developed for OCN− quantification in a wide range of ethanol/water mixtures, pH values, and in the presence of a large excess of foreign species (KCN, NaSCN, NaCl, NaNO3 , Na2 SO4 , urea, and EC) without any previous separation. In addition, it was also applied to cyanate analysis in the different fractions (head, heart, and tail) of sugar cane distillate. R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12587 Further reproduction without permission is prohibited
Material and Methods Reagents and solutions All reagents were of analytical reagent grade or higher purity. The water used was obtained from a Millipore Milli-Q water purification system. The aqueous ethanolic solutions were prepared using analytical grade ethanol (Panreac, Barcelona, Spain) and buffered using an acetic acid/sodium acetate buffer system (Mallinckrodt Baker, Phillipsburg, NJ, USA). The effect of hydrogen ion concentration on the emission measurement was evaluated at pH 3.5, 4.5, and 5.5. The influence of ethanol concentration on the cyanate determination was evaluated in water/ethanol mixtures ranging from 0% to 70% (v/v) ethanol. Aliquots of freshly prepared potassium cyanate (Fluka, Buchs, Switzerland) stock solution were added to these solutions. Calibration curves were obtained by plotting the cyanate concentration versus emission measurements as a function of pH value
Scheme 1–Cyanate derivatization reaction.
and ethanol concentration as shown in Figures 1 and 2, respectively. For the foreign species interference experiments, KCN, NaSCN, NaCl, NaNO3 , Na2 SO4 , urea, and EC (Sigma-Aldrich, Steinheim, Germany) solutions were prepared by dissolving the appropriate amount of the solid in the buffered water/ethanol solutions. A 0.01 mol/L 2-aminobenzoic acid (Sigma-Aldrich) solution, which was prepared daily and kept in the dark, and a 4.1 mol/L hydrochloric acid (Mallinckrodt Baker) solution were used in the cyanate ion derivatization reaction.
Equipment The fluorescence measurements were performed on a model F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). Emission spectra were obtained using 310 nm as the excitation wavelength, and the maximum emission was observed at 410 nm. 1 H NMR spectroscopy was employed for the identification of the product forming in the reaction showed in Scheme 1. The 1 H NMR spectra were recorded in an NMR spectrometer Agilent 500/54 Premium Shielded (500 MHz), at 26 ˚C using deuterated dimethyl sulfoxide (DMSO-d6, Sigma-Aldrich) solutions containing tetramethylsilane (TMS, Sigma-Aldrich) as a standard reference. The determination of EC concentration was performed through direct sample injection, without previous treatment, into a gas chromatograph model GC-2010 (Shimadzu, Tokyo, Japan) provided with an auto-injector AOC-20i (Shimadzu) interfaced to a mass selective detector model GCMS-QP2010 Plus
Figure 1–Excitation (A) and the emission (B) spectra for solutions containing the 2,4-(1H,3H)-quinazolinedione formed by reacting cyanate (1 × 10-3 mol/L) and 2-aminobenzoic acid (1 × 10−3 mol/L) in a water/ethanol (40%) mixture. Blank emission (C) spectra and inset (D) characteristics 1 H NMR signals of 2,4-(1H,3H)-quinazolinedione: δ = 11.26 ppm (s, 1H); 11.17 ppm (s, 1H); in DMSO-d6. Vol. 79, Nr. 10, 2014 r Journal of Food Science C1951
C: Food Chemistry
Ethyl carbamate formation from cyanate . . .
Ethyl carbamate formation from cyanate . . . (Shimadzu) using electron impact (70 eV) as the ionization by Guilloton and Karst (1985) for plasma analysis. This method involves the reaction between cyanate ion and 2-aminobenzoic acid source. in the presence of HCl to form the 2,4-(1H,3H)-quinazolinedione Cyanate determination (Scheme 1). The formation of this product was confirmed by charThe determination of cyanate ions in the aqueous ethanolic me- acteristic 1 H NMR signals (Figure 3, inset), namely δ = 11.26 ppm dia was carried out based on an adaptation of the method suggested (s, 1H) and 11.17 ppm (s, 1H), which were previously reported in
C: Food Chemistry Figure 2–Ethyl carbamate ( ) formation from cyanate () in buffered (CH3 COOH/CH3 COONa, pH 4.5) solution containing 1 × 10-3 mol/L potassium cyanate and 40% ethanol at 45 ࢪ C.
Figure 3–Plots of cyanate concentration versus fluorescence emission values for aqueous ethanolic (40%) solutions: () pH = 3.5; () pH = 4.5; (•) pH = 5.5.
C1952 Journal of Food Science r Vol. 79, Nr. 10, 2014
Ethyl carbamate formation from cyanate . . . Table 1–Cyanate analysisa in the presence of selected foreign propyl carbamate was used as an internal standard. An HP-FFAP ions, urea, and ethyl carbamate. (50 m, 0.2 mm, 0.3 μm, Agilent Technologies, Santa Clara, Calif., CN−
∗ SCN−
Cl− NO3 − SO4 2− Urea Ethyl carbamate
Mean 10−6
5.01 × 4.97 × 10−6 4.81 × 10−6 4.94 × 10−6 5.12 × 10−6 5.12 × 10−6 5.00 × 10−6
% RSD U.S.A.) capillary column was used in the EC separation. The inlet
SD 10−8
9.54 × 1.23 × 10−7 1.68 × 10−7 1.53 × 10−7 1.21 × 10−7 1.63 × 10−7 9.52 × 10−8
1.91 2.47 3.49 3.01 2.37 3.18 1.90
cKOCN = 5.0 × 10−6 mol/L, pH = 4.5. Sodium salts, except the ∗ thiocyanate which is a potassium salt, in concentrations 1000 times greater than that of KOCN.
a
b
Table 2–Cyanatea and ethyl carbamateb concentrations in the distilled “heart” fraction of cachac¸a prepared with urea addition. Urea (mol/L) 0 7.50 × 10−3 2.50 × 10−2 0.05 0.10 0.20 0.40
Cyanatea in cachac¸a “heart” fraction (mol/L)
Ethyl carbamateb (mol/L)
n.d. 2.70× 10−6 6.67× 10−6 1.99× 10−5 4.98 × 10−5 1.04 × 10−4 2.72 × 10−4
1.07 × 10−6 6.15× 10−6 1.45× 10−5 3.11 × 10−5 3.86 × 10−5 8.32 × 10−5 1.92 × 10−4
n.d. – not detected; a – freshly distilled; b – ethyl carbamate concentration from “heart” fraction 10 d after the distillation.
literature (Gao and others 2010). These signals are not observed in the 1 H NMR precursor (2-aminobenzoic acid) spectrum (Figure S1), confirming that the reaction shown in Scheme 1 yields 2,4-(1H,3H)-quinazolinedione. The entire 1 H NMR spectrum of this product is shown in Figure S2, which is in accordance with that reported for 2,4-(1H,3H)-quinazolinedione (Gao and others 2010). Experimental 1 H NMR (500 MHz, DMSO-d6): δ (ppm) = 11.26 (s, 1H); 11.17 (s, 1H); 7.89 (d, J = 7.85 Hz, 1H); 7.70 (dd, J = 7.64 e 1.57 Hz, 1H); 7.63 (t, J = 7.82 Hz, 1H); 7.18 (d, J = 5.41 Hz, 1H); 6.78 (d, J = 8.41 Hz, 1H), 6.54 (t, J = 7.49 Hz, 1H). To carry out this reaction first, 2.0 mL aliquots of aqueous ethanolic solutions containing KOCN were mixed with 2.0 mL of the 2-aminobenzoic acid solution. The resulting solutions were stored in glass vials, sealed with rubber septa (to minimize ethanol loss), and wrapped with aluminum foil before being heated at 40 ± 0.5 °C in a dry bath for 10 min, after which 4 mL of HCl (4 mol/L) was added. This resulting solution was then heated at 75 ± 0.5 °C for 10 min in a dry bath. The final solution containing the quinazolinedione was cooled to room temperature. After about 10 min, the solution was analyzed using a fluorescence spectrophotometer. The emission spectra were recorded over a range of 320 to 600 nm at a resolution of 240 nm min−1 using a 5-nm slit and a 1-cm quartz cell path. The reproducibility (Meier and Z¨und 1993) was evaluated by preparing 10 samples of the same concentration and following the proposed methodology.
and detector interface temperatures were 250 and 230 ˚C, respectively. The oven program temperature used was: 90 ˚C (2 min), followed by an increase to 150 ˚C at 10 ˚C min−1 (0 min), then up to 230 ˚C at 40 ˚C min−1 (10 min). The injected volume was 1.0 μL in the splitless mode.
Results and Discussion Effect of pH and ethanol Figure 3 shows the excitation and emission spectra for solutions containing the 2,4-(1H,3H)-quinazolinedione formed by reacting cyanate and 2-aminobenzoic acid in a water/ethanol (40%) mixture. The λexc and λem under these conditions were 310 and 410 nm, respectively. The emission wavelengths were only slightly influenced (less than 2 nm) by the ethanol concentration in the range of 0% to 70% (v/v). No noticeable changes in λexc and λem values were observed for pH (Table S1) variation in the range of 3.5 to 5.5. Also no substantial changes (less than 1%) were observed on the emission values when, at the same pH, the ethanol concentration was varied from 40 ± 2%, which is the experimental condition usually found in commercial sugar cane spirits. Nevertheless, for large variations of ethanol concentration (±10% ethanol) in the sample, noticeable changes in emission values were observed (Table S2). Therefore, for analytical purposes the calibration plots must reproduce the experimental conditions of samples. The plots of fluorescence intensity compared with cyanate concentration at 410 nm were linear in the range of 6.7 × 10−7 to 5.0 × 10−4 mol/L in the pH (Table S1) range of 3.5 to 5.5 and ethanol (Table S2) concentrations ranging from 0% to 70% (v/v). The limits of detection (LOD) and quantification (LOQ) were 2.2 × 10−7 and 6.6 × 10−7 mol/L, respectively. The LOD was calculated as 3.3 times the standard deviation of 3 blanks and the LOQ as 10 times the standard deviation of 3 blanks (Meier and Z¨und 1993).
Foreign ions, urea, and the EC Based on the reproducibility of the measurements, the best experimental conditions were an ethanol concentration of 40% and a pH of 4.5. Under these conditions, a solution containing 5 × 10−6 mol/L of cyanate can be analyzed and the cyanate concentration can be determined in a single measurement at a confidence level of 95% with an uncertainty of ± 0.13 × 10−6 mol/L (2.6%). The accuracy and precision of the method were checked using 3 concentrations of cyanate (1.0 × 10−6 , 2.0 × 10−6 , and 4.0 × 10−6 mol/L). The experimental data obtained showed a mean recovery of 100 ± 3.8% for the analyte and a maximum coefficient of variation of 1.8%. This analysis can be performed in the presence of cyanide, thiocyanate, chloride, nitrate, sulfate, sodium, and potassium ions, as well as the EC and urea without interference (Table 1), even when present at concentrations 1000 times greater than that of the analyte. The analysis of cyanate was also performed in distillates previEC determination ously fortified with KOCN. Samples of the Brazilian sugar cane The EC determination was carried out according to the method spirit, cachac¸a, which is 40% ethanol, were used for this purpose. described by Andrade-Sobrinho and others (2002). The mass spec- The results obtained were in very good agreement with those for trometer detector operated in the SIM mode (m/z = 62), and the water plus ethanol mixtures. Vol. 79, Nr. 10, 2014 r Journal of Food Science C1953
C: Food Chemistry
Foreign speciesb
Ethyl carbamate formation from cyanate . . . Table 3–Cyanatea and ethyl carbamateb concentrations in different fractions of the sugarcane spirit prepared with urea addition. Cyanatea in sugarcane spirit fractions (mol/L) Urea (mol/L)
C: Food Chemistry
0.00 7.50 × 10−3 2.50 × 10−2 0.05 0.10 0.20 0.40
Head n.d. 1.06 × 10−6 3.07 × 10−6 7.44 × 10−6 1.16 × 10−5 2.37 × 10−5 5.15 × 10−5
Heart n.d. 2.70 × 10−6 6.67 × 10−6 1.99 × 10−5 4.98 × 10−5 1.04 × 10−4 2.72 × 10−4
Tail n.d. 3.91 × 10−6 1.32 × 10−5 3.31 × 10−5 7.24 × 10−5 1.33 × 10−4 2.63 × 10−4
CEb in sugarcane spirit fractions (mol/L) Head 10−6
1.47 × 2.19 × 10−6 7.08 × 10−6 1.32 × 10−5 1.47 × 10−5 3.33 × 10−5 7.92 × 10−5
Heart 10−6
1.07 × 6.15 × 10−6 1.45 × 10−5 3.11 × 10−5 3.86 × 10−5 8.32 × 10−5 1.92 × 10−4
Tail 9.48 × 10−7 9.89 × 10−6 2.34 × 10−5 5.07 × 10−5 4.98 × 10−5 9.98 × 10−5 1.89 × 10−4
n.d. – not detected; a - freshly distilled; b - ethyl carbamate concentration from cachac¸a fractions 10 d after the distillation.
Usually, in small-scale production the distillation process occurs in alembics (pot stills) being the distillate separated into 3 different fractions, through operations named cuts. These fractions are called head (70% to 54% alcohol), heart (the noble distillate, 54% to 38% alcohol), and tail (below 38% alcohol). Therefore, cyanate determination was carried out in the “head,” “heart,” and “tail” fractions of just-distilled cachac¸a, in which urea was used in the fermentation as a nitrogen source. The “heart fraction” corresponds to the main part of the spirit, which is the commercialized one. The results included in Table 2 clearly show increases in cyanate concentration in the distillate when the urea concentration added to the wine increases. Furthermore, the data also pointed out that the cyanate concentration and, as a consequence, the EC presence increases as follows: head
A Fluorescence-Based Method for Cyanate Analysis in Ethanol/Water Media: Correlation between Cyanate Presence and Ethyl Carbamate Formation in Sugar Cane Spirit Thiago Hideyuki Kobe Ohe, Alexandre Ataide da Silva, Tha´ıs da Silva Rocha, Fl´avio Schutzer de Godoy, and Douglas Wagner Franco
Abstract: Based on the fluorescence properties of 2,4-(1H,3H)-quinazolinedione, a product of the reaction between cyanate and 2-aminobenzoic acid, a simple, sensitive, selective, and reproducible method for the cyanate analysis in aqueous ethanolic media is proposed. In this method, λexc and λem are 310 and 410 nm, respectively, and the limits of detection and quantification are 2.2 × 10−7 and 6.7 × 10−7 mol/L, respectively. Under optimal conditions (pH = 4.5, 40% ethanol), a concentration of 5.0 × 10−6 mol/L cyanate can be determined in a single measurement, at a 95% level of confidence, with an uncertainty of ± 0.13 × 10−6 mol/L. Cyanide, thiocyanate, chloride, nitrate, and sulfate ions, as well as urea and urethane in concentrations 1 × 103 higher than that of cyanate do not interfere with the measurement. The methodology was applied to cyanate analyses in the different fractions of the sugarcane distillate and the data strongly suggest a correlation between the presence of urea in wine, and the cyanate and ethyl carbamate concentrations in the spirit. Keywords: ethyl carbamate, fluorimetric cyanate analysis, sugar cane spirit, urea in wine
Based on the fluorescence properties of the reaction product between cyanate and 2-aminobenzoic acid, a method for assaying cyanate was devised. This procedure applied to the sugarcane distillate showed for the first time a correlation between cyanate presence and ethyl carbamate (EC) formation in the different fractions of the product. Therefore, the proposed methodology can be used to predict in freshly distillate sugar cane spirits the potential total concentration of EC to be formed. Therefore, these data could be used to advise about the necessity of implementing a procedure to reduce spirit EC concentration before the product reaches the market.
Practical Application:
Introduction Due to its carcinogenic properties, the presence of ethyl carbamate (EC) in food and beverages is controlled worldwide. Currently, a great deal of effort is being put into improving the quality of Brazilian sugar cane spirit (cachac¸a), the production of which is approximately 1.2 billion liters per year (Granato and others 2014). Therefore, it is important to understand the genesis of EC to prevent or reduce its presence in this beverage and, by extension, in the ethanol itself. Brazil is the world’s largest producer of ethanol from sugar cane (>20 billion liters per year), which is used both as a “green fuel” and by the pharmaceutical industry (Raele and others 2014). Previous studies (Zimmerli and Schlatter 1991; Taki and others 1992; EFSA 2007; Da Silva and others 2013) have strongly suggested that OCN− and CN− , as well as their corresponding acids, are precursors of EC. In our laboratory,
MS 20140683 Submitted 4/22/2014, Accepted 7/12/2014. Authors are with Dept. de Qu´ımica e F´ısica Molecular, Inst. de Qu´ımica de S˜ao Carlos, Univ. de S˜ao Paulo (USP), Avenida Trabalhador S˜ao-carlense 400, CP 780, CEP 13560-970, S˜ao Carlos – SP, Brazil. Direct inquiries to author Franco (E-mail: [email protected]).
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Journal of Food Science r Vol. 79, Nr. 10, 2014
we have collected, for the first time, evidence that HOCN is the most important EC precursor for cachac¸as, mostly when dealing with fresh distilled sugar cane spirit (Galinaro and Franco 2011; Da Silva and others 2013). During our experiments, it was necessary to develop a reliable, selective, and sensitive method for OCN− analysis in the presence of a large excess of CN− . 2-Aminobenzoic acid (2ABA) has previously been described as a colorimetric reagent for OCN− analysis in mine effluents (Zvinowanda and others 2008). The fluorescence properties of the cyanate/2-ABA reaction have been explored for analytical purposes in estuarine, seawater, and plasma analysis (Lundquist and others 1993; Widner and others 2013). Therefore, based on the reaction (Guilloton and Karst 1985) as shown in Scheme 1, and taking advantage of the fluorescence properties of 2,4-(1H,3H)-quinazolinedione, a simple and rapid analytical method was developed for OCN− quantification in a wide range of ethanol/water mixtures, pH values, and in the presence of a large excess of foreign species (KCN, NaSCN, NaCl, NaNO3 , Na2 SO4 , urea, and EC) without any previous separation. In addition, it was also applied to cyanate analysis in the different fractions (head, heart, and tail) of sugar cane distillate. R C 2014 Institute of Food Technologists
doi: 10.1111/1750-3841.12587 Further reproduction without permission is prohibited
Material and Methods Reagents and solutions All reagents were of analytical reagent grade or higher purity. The water used was obtained from a Millipore Milli-Q water purification system. The aqueous ethanolic solutions were prepared using analytical grade ethanol (Panreac, Barcelona, Spain) and buffered using an acetic acid/sodium acetate buffer system (Mallinckrodt Baker, Phillipsburg, NJ, USA). The effect of hydrogen ion concentration on the emission measurement was evaluated at pH 3.5, 4.5, and 5.5. The influence of ethanol concentration on the cyanate determination was evaluated in water/ethanol mixtures ranging from 0% to 70% (v/v) ethanol. Aliquots of freshly prepared potassium cyanate (Fluka, Buchs, Switzerland) stock solution were added to these solutions. Calibration curves were obtained by plotting the cyanate concentration versus emission measurements as a function of pH value
Scheme 1–Cyanate derivatization reaction.
and ethanol concentration as shown in Figures 1 and 2, respectively. For the foreign species interference experiments, KCN, NaSCN, NaCl, NaNO3 , Na2 SO4 , urea, and EC (Sigma-Aldrich, Steinheim, Germany) solutions were prepared by dissolving the appropriate amount of the solid in the buffered water/ethanol solutions. A 0.01 mol/L 2-aminobenzoic acid (Sigma-Aldrich) solution, which was prepared daily and kept in the dark, and a 4.1 mol/L hydrochloric acid (Mallinckrodt Baker) solution were used in the cyanate ion derivatization reaction.
Equipment The fluorescence measurements were performed on a model F-4500 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). Emission spectra were obtained using 310 nm as the excitation wavelength, and the maximum emission was observed at 410 nm. 1 H NMR spectroscopy was employed for the identification of the product forming in the reaction showed in Scheme 1. The 1 H NMR spectra were recorded in an NMR spectrometer Agilent 500/54 Premium Shielded (500 MHz), at 26 ˚C using deuterated dimethyl sulfoxide (DMSO-d6, Sigma-Aldrich) solutions containing tetramethylsilane (TMS, Sigma-Aldrich) as a standard reference. The determination of EC concentration was performed through direct sample injection, without previous treatment, into a gas chromatograph model GC-2010 (Shimadzu, Tokyo, Japan) provided with an auto-injector AOC-20i (Shimadzu) interfaced to a mass selective detector model GCMS-QP2010 Plus
Figure 1–Excitation (A) and the emission (B) spectra for solutions containing the 2,4-(1H,3H)-quinazolinedione formed by reacting cyanate (1 × 10-3 mol/L) and 2-aminobenzoic acid (1 × 10−3 mol/L) in a water/ethanol (40%) mixture. Blank emission (C) spectra and inset (D) characteristics 1 H NMR signals of 2,4-(1H,3H)-quinazolinedione: δ = 11.26 ppm (s, 1H); 11.17 ppm (s, 1H); in DMSO-d6. Vol. 79, Nr. 10, 2014 r Journal of Food Science C1951
C: Food Chemistry
Ethyl carbamate formation from cyanate . . .
Ethyl carbamate formation from cyanate . . . (Shimadzu) using electron impact (70 eV) as the ionization by Guilloton and Karst (1985) for plasma analysis. This method involves the reaction between cyanate ion and 2-aminobenzoic acid source. in the presence of HCl to form the 2,4-(1H,3H)-quinazolinedione Cyanate determination (Scheme 1). The formation of this product was confirmed by charThe determination of cyanate ions in the aqueous ethanolic me- acteristic 1 H NMR signals (Figure 3, inset), namely δ = 11.26 ppm dia was carried out based on an adaptation of the method suggested (s, 1H) and 11.17 ppm (s, 1H), which were previously reported in
C: Food Chemistry Figure 2–Ethyl carbamate ( ) formation from cyanate () in buffered (CH3 COOH/CH3 COONa, pH 4.5) solution containing 1 × 10-3 mol/L potassium cyanate and 40% ethanol at 45 ࢪ C.
Figure 3–Plots of cyanate concentration versus fluorescence emission values for aqueous ethanolic (40%) solutions: () pH = 3.5; () pH = 4.5; (•) pH = 5.5.
C1952 Journal of Food Science r Vol. 79, Nr. 10, 2014
Ethyl carbamate formation from cyanate . . . Table 1–Cyanate analysisa in the presence of selected foreign propyl carbamate was used as an internal standard. An HP-FFAP ions, urea, and ethyl carbamate. (50 m, 0.2 mm, 0.3 μm, Agilent Technologies, Santa Clara, Calif., CN−
∗ SCN−
Cl− NO3 − SO4 2− Urea Ethyl carbamate
Mean 10−6
5.01 × 4.97 × 10−6 4.81 × 10−6 4.94 × 10−6 5.12 × 10−6 5.12 × 10−6 5.00 × 10−6
% RSD U.S.A.) capillary column was used in the EC separation. The inlet
SD 10−8
9.54 × 1.23 × 10−7 1.68 × 10−7 1.53 × 10−7 1.21 × 10−7 1.63 × 10−7 9.52 × 10−8
1.91 2.47 3.49 3.01 2.37 3.18 1.90
cKOCN = 5.0 × 10−6 mol/L, pH = 4.5. Sodium salts, except the ∗ thiocyanate which is a potassium salt, in concentrations 1000 times greater than that of KOCN.
a
b
Table 2–Cyanatea and ethyl carbamateb concentrations in the distilled “heart” fraction of cachac¸a prepared with urea addition. Urea (mol/L) 0 7.50 × 10−3 2.50 × 10−2 0.05 0.10 0.20 0.40
Cyanatea in cachac¸a “heart” fraction (mol/L)
Ethyl carbamateb (mol/L)
n.d. 2.70× 10−6 6.67× 10−6 1.99× 10−5 4.98 × 10−5 1.04 × 10−4 2.72 × 10−4
1.07 × 10−6 6.15× 10−6 1.45× 10−5 3.11 × 10−5 3.86 × 10−5 8.32 × 10−5 1.92 × 10−4
n.d. – not detected; a – freshly distilled; b – ethyl carbamate concentration from “heart” fraction 10 d after the distillation.
literature (Gao and others 2010). These signals are not observed in the 1 H NMR precursor (2-aminobenzoic acid) spectrum (Figure S1), confirming that the reaction shown in Scheme 1 yields 2,4-(1H,3H)-quinazolinedione. The entire 1 H NMR spectrum of this product is shown in Figure S2, which is in accordance with that reported for 2,4-(1H,3H)-quinazolinedione (Gao and others 2010). Experimental 1 H NMR (500 MHz, DMSO-d6): δ (ppm) = 11.26 (s, 1H); 11.17 (s, 1H); 7.89 (d, J = 7.85 Hz, 1H); 7.70 (dd, J = 7.64 e 1.57 Hz, 1H); 7.63 (t, J = 7.82 Hz, 1H); 7.18 (d, J = 5.41 Hz, 1H); 6.78 (d, J = 8.41 Hz, 1H), 6.54 (t, J = 7.49 Hz, 1H). To carry out this reaction first, 2.0 mL aliquots of aqueous ethanolic solutions containing KOCN were mixed with 2.0 mL of the 2-aminobenzoic acid solution. The resulting solutions were stored in glass vials, sealed with rubber septa (to minimize ethanol loss), and wrapped with aluminum foil before being heated at 40 ± 0.5 °C in a dry bath for 10 min, after which 4 mL of HCl (4 mol/L) was added. This resulting solution was then heated at 75 ± 0.5 °C for 10 min in a dry bath. The final solution containing the quinazolinedione was cooled to room temperature. After about 10 min, the solution was analyzed using a fluorescence spectrophotometer. The emission spectra were recorded over a range of 320 to 600 nm at a resolution of 240 nm min−1 using a 5-nm slit and a 1-cm quartz cell path. The reproducibility (Meier and Z¨und 1993) was evaluated by preparing 10 samples of the same concentration and following the proposed methodology.
and detector interface temperatures were 250 and 230 ˚C, respectively. The oven program temperature used was: 90 ˚C (2 min), followed by an increase to 150 ˚C at 10 ˚C min−1 (0 min), then up to 230 ˚C at 40 ˚C min−1 (10 min). The injected volume was 1.0 μL in the splitless mode.
Results and Discussion Effect of pH and ethanol Figure 3 shows the excitation and emission spectra for solutions containing the 2,4-(1H,3H)-quinazolinedione formed by reacting cyanate and 2-aminobenzoic acid in a water/ethanol (40%) mixture. The λexc and λem under these conditions were 310 and 410 nm, respectively. The emission wavelengths were only slightly influenced (less than 2 nm) by the ethanol concentration in the range of 0% to 70% (v/v). No noticeable changes in λexc and λem values were observed for pH (Table S1) variation in the range of 3.5 to 5.5. Also no substantial changes (less than 1%) were observed on the emission values when, at the same pH, the ethanol concentration was varied from 40 ± 2%, which is the experimental condition usually found in commercial sugar cane spirits. Nevertheless, for large variations of ethanol concentration (±10% ethanol) in the sample, noticeable changes in emission values were observed (Table S2). Therefore, for analytical purposes the calibration plots must reproduce the experimental conditions of samples. The plots of fluorescence intensity compared with cyanate concentration at 410 nm were linear in the range of 6.7 × 10−7 to 5.0 × 10−4 mol/L in the pH (Table S1) range of 3.5 to 5.5 and ethanol (Table S2) concentrations ranging from 0% to 70% (v/v). The limits of detection (LOD) and quantification (LOQ) were 2.2 × 10−7 and 6.6 × 10−7 mol/L, respectively. The LOD was calculated as 3.3 times the standard deviation of 3 blanks and the LOQ as 10 times the standard deviation of 3 blanks (Meier and Z¨und 1993).
Foreign ions, urea, and the EC Based on the reproducibility of the measurements, the best experimental conditions were an ethanol concentration of 40% and a pH of 4.5. Under these conditions, a solution containing 5 × 10−6 mol/L of cyanate can be analyzed and the cyanate concentration can be determined in a single measurement at a confidence level of 95% with an uncertainty of ± 0.13 × 10−6 mol/L (2.6%). The accuracy and precision of the method were checked using 3 concentrations of cyanate (1.0 × 10−6 , 2.0 × 10−6 , and 4.0 × 10−6 mol/L). The experimental data obtained showed a mean recovery of 100 ± 3.8% for the analyte and a maximum coefficient of variation of 1.8%. This analysis can be performed in the presence of cyanide, thiocyanate, chloride, nitrate, sulfate, sodium, and potassium ions, as well as the EC and urea without interference (Table 1), even when present at concentrations 1000 times greater than that of the analyte. The analysis of cyanate was also performed in distillates previEC determination ously fortified with KOCN. Samples of the Brazilian sugar cane The EC determination was carried out according to the method spirit, cachac¸a, which is 40% ethanol, were used for this purpose. described by Andrade-Sobrinho and others (2002). The mass spec- The results obtained were in very good agreement with those for trometer detector operated in the SIM mode (m/z = 62), and the water plus ethanol mixtures. Vol. 79, Nr. 10, 2014 r Journal of Food Science C1953
C: Food Chemistry
Foreign speciesb
Ethyl carbamate formation from cyanate . . . Table 3–Cyanatea and ethyl carbamateb concentrations in different fractions of the sugarcane spirit prepared with urea addition. Cyanatea in sugarcane spirit fractions (mol/L) Urea (mol/L)
C: Food Chemistry
0.00 7.50 × 10−3 2.50 × 10−2 0.05 0.10 0.20 0.40
Head n.d. 1.06 × 10−6 3.07 × 10−6 7.44 × 10−6 1.16 × 10−5 2.37 × 10−5 5.15 × 10−5
Heart n.d. 2.70 × 10−6 6.67 × 10−6 1.99 × 10−5 4.98 × 10−5 1.04 × 10−4 2.72 × 10−4
Tail n.d. 3.91 × 10−6 1.32 × 10−5 3.31 × 10−5 7.24 × 10−5 1.33 × 10−4 2.63 × 10−4
CEb in sugarcane spirit fractions (mol/L) Head 10−6
1.47 × 2.19 × 10−6 7.08 × 10−6 1.32 × 10−5 1.47 × 10−5 3.33 × 10−5 7.92 × 10−5
Heart 10−6
1.07 × 6.15 × 10−6 1.45 × 10−5 3.11 × 10−5 3.86 × 10−5 8.32 × 10−5 1.92 × 10−4
Tail 9.48 × 10−7 9.89 × 10−6 2.34 × 10−5 5.07 × 10−5 4.98 × 10−5 9.98 × 10−5 1.89 × 10−4
n.d. – not detected; a - freshly distilled; b - ethyl carbamate concentration from cachac¸a fractions 10 d after the distillation.
Usually, in small-scale production the distillation process occurs in alembics (pot stills) being the distillate separated into 3 different fractions, through operations named cuts. These fractions are called head (70% to 54% alcohol), heart (the noble distillate, 54% to 38% alcohol), and tail (below 38% alcohol). Therefore, cyanate determination was carried out in the “head,” “heart,” and “tail” fractions of just-distilled cachac¸a, in which urea was used in the fermentation as a nitrogen source. The “heart fraction” corresponds to the main part of the spirit, which is the commercialized one. The results included in Table 2 clearly show increases in cyanate concentration in the distillate when the urea concentration added to the wine increases. Furthermore, the data also pointed out that the cyanate concentration and, as a consequence, the EC presence increases as follows: head
