| Ethyl carbamate, a pluripotent carcinogen, has resulted in an increased incidence of lung, lymphocytic leukemia, liver, and melanotic tumors of the skin, which was first demonstrated in 1943. The International Agency for Research on Cancer (IARC) has classifed ethyl carbamate as a Group 2B, possibly carcinogenic to humans. Most fermented foods and beverages, including wine, contain trace amounts of ethyl carbamate. Health Protection Branch of Canada set the upper limit of 30μg/L ethyl carbamate in table wine; and the U.S. Food and Drug Administration has established a voluntary target for ethyl carbamate of 15μg/L for table wine≤14% alcohol by volume starting with the 1998 harvest.The purpose of the paper was to develop a simple, accurate and rapid ethyl carbamat analysis standard method for measuring ethyl carbamate in table wine. The developed method could have the application in routine quantification of ethyl carbamate for Chinese table wine. Furthermore, preliminary measures to reduce ethyl carbamate levels in table wine were investigated.The paper mainly describes the sample-pretreated methods for analyzing ethyl carbamate in table wines in combination with a gas chromatographic/mass spectrometric (GC/MS) system, and n-propyl carbamate used as the internal standard. Parameters were optimized of the sample-pretreated methods including liquid/liquid extraction, solid phase extraction, solid phase microextraction and solid/liquid extraction. Furthermore, the method for determining arginine in grape juice with Sakaguchi reaction following separating arginine with strong cation - exchange resins was developed. The results were as follows:The parameters of the liquid/liquid extraction method were optimized: the pH of the samples was natural pH, KCl 16 g (40%), dichloromethane 300mL. The limits of detection were 10μg/mL. The recoveries and the precision were 76.56%-80.39% and 4.42-9.82%, respectively. Liquid/liquid extraction method used for determination of ethyl carbamate in table wines dispenses with expensive instruments, however it has drawbacks: severe emulsification; the large amounts of organic solvents used, resulting in a large volume of waste; the low precision and accuracy due to manual concentration steps.The optimized parameters of the solid phase microextraction of table wine were: sample pH was 10.2, and 9.0 mL sample and 2.0-2.5 g (20-25%) NaCl were added to the vial; the sample vial was shaken for 30min at 40℃at 800 rpm in the agitator for equilibration; A 70μm CW/DVB fiber was inserted into the headspace for 35min at 40℃, then the fiber removed from the sample vial and inserted into the injection port of the GC for 10min. Under this extraction conditions, the recoveries and the presision were 76.00-84.80%, and 6.62-10.95%, respectively. The limits of detection were 8.0μg/mL. The solid phase microextraction technique was successfully applied to determine the ethyl carbamate levels in table wines, and it allows the extraction and concentration to be focused in a single step. The extraction method was rapid and simple, and without the need for any organic solvent, and it was an environmental protecting extraction method. But it has low precision and accuracy due to unstable fibers.The parameters of the solid phase extraction method were: the pH of the samples was natural pH, dichloromethane 175 mL. The recoveries and the precision were 85.76%-102.82% and 4.22-8.20%, respectivly. The limit of detection was 3.0μg/mL.And the optimized parameters of the solid/liquid extraction method were: the pH of the samples was natural pH, dichloromethane 70 mL. The recoveries and the precision were 82.67-93.39% and 2.91-6.67%, respectivly. The limits of detection were 2.0μg/mL.Both solid/liquid extraction and solid phase extraction methods provided good recovery and reproducibility. There was no significant difference between the two methods (p<0.05). Solid phase extraction method was recommended by OIV, but in the OIV method, anhydrous ethanol, one of the reagents, contained ethyl carbamate in itself. Therefore, there was drawback in this method. Furthermore, comparison with the solid phase extraction method, solid/liquid extraction method was less cost, environmental protecting, fast and simple. In the study, the solid/liquid extraction method was selected as the standard method to extract the ethyl carbamate in table wines.Solid/liquid extraction method in combination with GC/MS was applied to analysis for ethyl carbamate in 61Chinese table wine samples. The results showed that the real contents of ethyl carbamate in 81.82% samples were lower than 15μg/L. According to the U.S. Food and Drug Administration and the U.S. Bureau of Alcohol, Tobacco, and Firearms established voluntary programs, that is, 15μg/L weighted average for table wines≤14% alcohol by volume starting with the 1998 harvest, so 15μg/L was recommended as the maximum limits for ethyl carbamate in Chinese table wine.According to analysis of investigation of ethyl carbamate levels, the minimum was not detection, whatever in red or white table wine, and the maximum 28.6μg/L in red table wine and 13.9μg/L in white table wine. Obviously, ethyl carbamate levels in red table wines were higher than those in white table wines. There were no significant difference between wine region (p<0.05), and there were significant difference at storage time (p<0.05), namely, the longer storage time, the higher ethyl carbamate levels.Arginine, one of the most abundant amino acids in grape juice, is closely related to the levels of ethyl carbamate in wines. Arginine is degraded to urea and citrulline, precursors of ethyl carbamate in wine, by the yeast and malolactic bacteria, respectively. Therefore, control of arginine levels in grape juice is one of the most important steps for winemakers to reduce ethyl carbamate in wine. A simple, accurate and rapid arginine separation method by use of 732# ion exchange resins in combination with Sakaguchi reaction for measuring arginine in grape juice was developed. The results were as follows:Sixteen grams of 732#pretreated ion exchange resins was packed in chromatographic column. Natural grape juice was centrifuged and the upper (10.0 mL, pH 2.0) was loaded to the column, then rinsed with the distilled water, finally, it was eluted with 1mol/L NaOH solution. To a tube containing 5 mL eluate sample (1.5 -12μg/mL arginine), 1.0 mL 0.02% 8-hydroxyquinoline and 1.0 mL 10% NaOH were added sequentially, the solution was mixed thoroughly in the ice bath, after 10min, 0.5 mL 0.4% sodium hypobromite was added rapidly to develop the color. After mixing, 1.0 mL 40% urea was added within 30 seconds, and then its absorbance was read at 500 nm. According to the standard curve and dilution rate, arginine concentration was determined.The Lambert-Beer law was followed up to an arginine concentration from1.5 to 12μg/mL, and apparent molar absorptivity of the method was 1.45×104 L·mol-1·cm-1, its recoveries and precison of grape juice samples were 87.0-100.7% and 1.13-2.42% respectivly. The method presented in this paper is accurate and reliable, and it is suitable for determination of L-arginine in grape juice.The developed standard method of determination of ethyl carbamate in table wines was simple, fast, reproducible and accurate, and the method could be applicable for routine determination of ethyl carbamate in table wines. The method of determination of arginine in grape juice could be applied for grape growers and winemakers to take necessary measure for controlling the level of ethyl carbamate in wine.
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