Method for rapidly detecting ethyl carbamate in wine
By using the combination of CoFe2O4@CSC carbonized material and specific solvents, the pre-detection of urethane in wine is simplified, and the problems of time and large solvent usage in the prior art are solved, achieving a fast and accurate detection effect.
Patent Information
- Application Number
- CN202510427227.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-07
- Publication Date
- 2025-06-27
AI Technical Summary
In the prior art, the pre-treatment steps for the detection method of urethane in wine are cumbersome, time-consuming, the amount of organic solvent used is large, and the amount of impurities remains is large, which affects the accuracy and efficiency of the detection.
The wine was pH adjusted by using CoFe2O4@CSC carbonized material to adsorb ethyl carbamate, combined with magnetic separation and specific solvent elution, simplifying the sample pretreatment process, and GC-MS detection was performed using a small amount of organic solvent.
Fast and accurate quantitative analysis of ethyl urethane is achieved, which reduces detection costs, improves the stability and recovery of detection results, and reduces the use of organic solvents and environmental pollution.
Smart Images
Figure CN120214176A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of food analysis, and more specifically, relates to a method for rapid detection of ethyl carbamate in wine. Background Art
[0002] Since ethyl carbamate was discovered in fermented foods and beverages in the 1970s, extensive research has been conducted on the detection methods of ethyl carbamate. Many literatures have reported the instrumental detection methods of ethyl carbamate in alcoholic beverages, and the sample pretreatment step has become an important link in instrumental analysis.
[0003] Common detection methods for ethyl carbamate include gas chromatography - mass spectrometry, high - performance liquid chromatography - mass spectrometry, and high - performance liquid chromatography - fluorescence method after fluorescence derivatization, etc. These analytical methods require relatively cumbersome pretreatment of samples before detection to extract and concentrate ethyl carbamate in the samples to achieve the goal of improving detection sensitivity. Currently, common pretreatment methods include solid - phase extraction, column chromatography, and multi - stage liquid - liquid extraction, etc. These sample pretreatment methods often take a long time, use a large amount of organic solvents, and have a large amount of impurity residues, thus affecting the accurate and efficient determination of ethyl carbamate in fermented wine beverages. Summary of the Invention
[0004] In view of the above - mentioned technical problems, the present invention provides a method for rapid detection of ethyl carbamate in wine.
[0005] The present invention specifically adopts the following technical solutions:
[0006] The present invention provides a method for rapid detection of ethyl carbamate in wine, and the method comprises the following steps: adjusting the pH value of the wine liquid to acidic, adding CoFe2O4@CSC carbonized material and oscillating and mixing, standing to adsorb ethyl carbamate, then separating the CoFe2O4@CSC carbonized material through a magnetic rack, rinsing the CoFe2O4@CSC carbonized material with n - hexane, and then eluting with an ethyl acetate - ether mixed solution to obtain an eluate containing ethyl carbamate. The eluate is dried by nitrogen blowing to obtain a preliminary sample loading solution, and the preliminary sample loading solution is fixed - volume to 1 mL with methanol and then subjected to GC - MS detection;
[0007] The volume - mass ratio of the n - hexane to the wine liquid is 1 - 2 mL: 1 - 3 g;
[0008] The volume - mass ratio of the ethyl acetate - ether mixed solution to the wine liquid is 1 - 2 mL: 1 - 3 g;
[0009] The volume - mass ratio of the preliminary sample loading solution to the wine liquid is 0.1 - 0.5 mL: 1 - 3 g.
[0010] The present invention can quickly and accurately perform quantitative analysis of trace ethyl carbamate in wine by simplifying the sample pretreatment process, reducing the detection cost, saving the detection time, and improving the recovery rate of the detection method and the stability of the detection results.
[0011] Further, the pH value is adjusted to 4 - 6.
[0012] Further, the mass ratio of the CoFe2O4@CSC carbonized material to the wine liquid is 5 - 7 mg: 1 - 3 g.
[0013] Further, the static adsorption condition is static adsorption at 30 - 33 °C for 1 - 2 min.
[0014] Further, the n - hexane elution time is 10 - 30 s.
[0015] Further, the volume ratio of ethyl acetate to diethyl ether in the ethyl acetate - diethyl ether mixed solution is 24 - 26:74 - 76.
[0016] Further, the GC - MS detection conditions include: the injection temperature is 200 - 220 °C, gradient heating to 200 - 240 °C, the solvent delay is 5 - 10 min, and the injection volume is 1 - 2 μL.
[0017] Further, the gradient heating steps are as follows: the initial temperature is 100 - 150 °C, maintained for 1 - 2 min; rising at 15 - 20 °C / min to 180 - 200 °C, holding for 1 - 6 min; rising at 30 - 35 °C / min to 240 - 260 °C, holding for 1 - 5 min.
[0018] The present invention has the following beneficial effects:
[0019] Fast extraction speed: This method uses magnetic materials. Due to the large specific surface area and short diffusion distance of magnetic nanoparticles, they can fully contact with the target analyte and complete adsorption in a short time. The adsorption time is about 2 min, greatly improving the experimental efficiency.
[0020] Less use of organic solvents: Compared with the traditional solid - phase extraction method, the use amount of organic solvents is reduced, the experimental cost is lowered, and at the same time, the environmental pollution caused by organic solvents is also reduced.
[0021] It can effectively remove the impurity interference in the sample and improve the accuracy and reliability of the analysis results. In the traditional solid - phase extraction method, the packing material of the solid - phase extraction column itself may contain impurities, and these impurities may be eluted during the extraction process, thus forming impurity peaks. The number of impurity peaks generated by the traditional experimental method is between 15 - 17 and some of the impurity peak areas are relatively large. After being treated by this method, the number of impurity peaks is between 9 - 12, and the generated impurity peak areas are relatively small. Brief Description of the Drawings
[0022] Figure 1 is an operating flow chart.
[0023] Figure 2 is a standard curve graph.
[0024] Figure 3 is an adsorption efficiency graph.
[0025] Figure 4 is a chromatographic control graph, where A is the chromatogram after sample loading treated by the present invention, and B is the chromatogram after sample loading treated by the traditional method. Detailed Description of the Invention
[0026] The present invention will be described in detail below in conjunction with the drawings and specific embodiments, but it should not be construed as a limitation of the present invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.
[0027] Example 1
[0028] I. Preparation of Standard Solutions
[0029] Ethyl carbamate stock solution (1 g / L): Accurately weigh 10 mg of ethyl carbamate standard, dissolve it with methanol and make up the volume to 10 mL.
[0030] Propyl carbamate stock solution (1 g / L): Accurately weigh 10 mg of propyl carbamate standard, dissolve it with methanol and make up the volume to 10 mL.
[0031] Ethyl carbamate intermediate solution (1 mg / L): Accurately pipette 1 mL of ethyl carbamate stock solution with a concentration of 100 μg / mL, and make up the volume to 100 mL with a 10% methanol solution by mass.
[0032] Propyl carbamate intermediate solution (1 mg / L): Accurately pipette 1 mL of propyl carbamate stock solution with a concentration of 100 μg / mL, and make up the volume to 100 mL with a 10% methanol solution by mass.
[0033] Standard curve working solution: Respectively pipette 10 μL, 20 μL, 30 μL, 40 μL, 50 μL, 60 μL of ethyl carbamate intermediate solution (1 mg / L) into 6 10-mL volumetric flasks, then add 30 μL of propyl carbamate intermediate solution (1 mg / L) respectively, and make up the volume to the mark with distilled water to obtain standard curve working solutions with concentrations of 40 μg / L, 50 μg / L, 60 μg / L, 70 μg / L, 80 μg / L, 90 μg / L and an internal standard of 10 μg / L of propyl carbamate.
[0034] II. Pretreatment before extraction
[0035] Take 2 g of wine liquid, adjust the pH to 5.0, add 6 mg of CoFe2O4@CSC carbonized material, mix by shaking, let stand and adsorb at 30 °C for 2 min, separate the CoFe2O4@CSC carbonized material with a magnetic rack, and discard the supernatant; rinse the CoFe2O4@CSC carbonized material with 2 mL of n-hexane for 30 s, and separate the CoFe2O4@CSC carbonized material with a magnetic rack; elute ethyl carbamate with 2 mL of 25% (mass fraction) ethyl acetate-ether solution to obtain an eluate; after the eluate is blown to about 0.5 mL with nitrogen, make up the volume to 1 mL with methanol for GC / MS analysis. The overall operation process is shown in Figure 1 .
[0036] Optimization of pretreatment conditions:
[0037] When extracting wine samples, study the recovery rate of ethyl carbamate at different adsorption times. The test results are shown in the following table.
[0038] Table 1: Optimization of adsorption time
[0039]
[0040] As can be seen from Table 1, at different adsorption times, the recovery rate and relative standard deviation vary greatly. When the adsorption time is 1 min, the average recovery rate of ethyl carbamate is the lowest, and the relative standard deviation is relatively large. As the extraction adsorption time of grape samples increases, the average recovery rate first increases and then decreases, while the relative standard deviation shows the opposite trend. When the adsorption time is 2 min, the average recovery rate is 93.9%, and the relative standard deviation is 2.16%. The effect of extracting ethyl carbamate at this adsorption time is the best.
[0041] When extracting wine samples, study the effect of different eluents on the recovery rate of ethyl carbamate. The test results are shown in the following table.
[0042] Table 2: Optimization of elution solvent
[0043]
[0044] As can be seen from Table 2, when toluene is used for elution, ethyl carbamate cannot be well retained on the solid-phase extraction column, so the average recovery rate is the lowest, and the relative standard deviation is 5.67%; when ethylenediaminetetraacetic acid is used for elution, the average recovery rate is relatively high, at 86.2%, but the relative standard deviation value is too large, at 8.94%; when n-hexane is used for elution, it is found that ethyl carbamate can be well retained on the solid-phase extraction column. At this time, the average recovery rate is 89.7%, and the relative standard deviation is 3.79. In comparison, the effect of extracting ethyl carbamate with n-hexane is the best.
[0045] When extracting the wine sample, the influence of different eluent concentrations on the recovery rate of ethyl carbamate was studied. The test results are shown in the following table
[0046] Table 3: Optimization of the volume ratio of ethyl acetate and diethyl ether
[0047]
[0048] As can be seen from Table 3, with the gradual increase of the volume ratio of ethyl acetate to diethyl ether, the average recovery rate and relative standard deviation show large fluctuations, indicating that the proportion of the elution solvent has a significant impact on the extraction of ethyl carbamate. Specifically, when the volume ratio of ethyl acetate to diethyl ether is 30:70, the average recovery rate drops to the lowest, which is 79.8%, and the relative standard deviation is 2.75%; while when the volume ratio is 25:75, the average recovery rate reaches 91.7%, and the relative standard deviation is 2.46%, showing an ideal extraction effect at this time.
[0049] From the above tests, it can be determined that when extracting ethyl carbamate from wine using an extraction column, the optimal pretreatment for extraction is: the adsorption time is 2 min, the elution solvent is n-hexane, and the elution solvent is ethyl acetate:diethyl ether with a volume ratio of 25:75.
[0050] III. GC-MS analysis
[0051] Chromatographic conditions for wine samples
[0052] Open capillary column: DB-WAX, with a specification of 60 m × 0.25 mm × 0.25 μm;
[0053] Injection temperature: 220 °C;
[0054] Column temperature: The initial temperature is 100 °C, maintained for 2 min, then increased to 180 °C at a rate of 15 °C / min. Kept for 6 min, and then increased to 240 °C at a rate of 30 °C / min, and kept for 5 min;
[0055] Carrier gas: Helium with a purity greater than 99.99%, and the flow rate is 1.0 mL / min;
[0056] Ionization method: Electron bombardment source with an energy of 70 eV;
[0057] Quadrupole temperature: 150 °C;
[0058] Ion source temperature: 230 °C; Transfer line temperature: 250 °C;
[0059] Solvent delay: 10 min;
[0060] Injection method: Split injection with a volume ratio of 10:1; Injection volume 1 μL;
[0061] Detection method: Select SIM for ion monitoring;
[0062] Selective monitoring ions (m / z) of ethyl carbamate: 44.0, 62.0, 74.0, 89.1, and the quantitative ion is 62.0;
[0063] Selective monitoring ions (m / z) of propyl carbamate: 44.0, 62.0, 74.0, and the quantitative ion is 88.1.
[0064] Sample injection operation: Accurately aspirate 1 μL of wine sample using a microsyringe.
[0065] Insert the needle of the microsyringe into the injection port and quickly inject the sample into the injection port. The sample is split at a volume ratio of 10:1 at the injection port.
[0066] After splitting, it enters the DB-WAX capillary column under the push of the carrier gas. Under the control of the column temperature program, different components in the sample are gradually separated in the capillary column according to their interaction with the stationary phase. After a series of temperature change processes, each component flows out of the capillary column successively and enters the detection system for detection.
[0067] IV. Quantitative analysis
[0068] After performing GC-MS analysis on the standard curve working solution, plot the standard curve with the concentration of ethyl carbamate as the abscissa and the ratio of the peak area of ethyl carbamate to the peak area of the internal standard propyl carbamate as the ordinate. Calculate according to the formula:
[0069]
[0070] In the formula: X is the content of ethyl carbamate in the sample (μg / L); c is the content of ethyl carbamate in the determination solution (μg / L); V1 is the constant volume of the determination solution (mL); V2 is the sample volume (mL); 1000 is the conversion coefficient.
[0071] Calibration standard curve drawing and recovery rate determination: The pH of the calibration standard solution is 5.0, the dosage of magnetic nanomaterials is 6 mg, and the desorption time is 6 min. Extract the series of calibration standard solutions, and obtain the calibration standard curve through GC-MS quantitative analysis as Figure 2 , in the range of ethyl carbamate content from 0 to 200 μg / L, the regression equation is Y = 0.2311x - 0.1806R 2 = 0.99941. Through the regression equation and the linear correlation coefficient, it can be determined that the accuracy of the calibration standard curve can be applied to the determination of the content of ethyl carbamate and shows a linear relationship.
[0072] When analyzing the ethyl carbamate content in wine using this method, the recovery rate is between 93% and 103%, which is relatively high. The results of the routine analysis of the ethyl carbamate content in wine are reliable.
[0073] V. Experimental Results
[0074] As Figure 2 shown: In the range of ethyl carbamate content from 0 to 200 μg / L, the regression equation is Y = 0.2311x - 0.1806, and the correlation coefficient R 2 = 0.99941. It shows that the curve fitting has good linearity and can be used for the detection of ethyl carbamate. The recovery rate is between 93% and 103%.
[0075] As Figure 3 shown: This method uses magnetic materials. Due to the large specific surface area and short diffusion distance of magnetic nanoparticles, they can fully contact and complete adsorption with the target analyte in a short time. The adsorption time is about 2 min, which greatly improves the experimental efficiency.
[0076] Table 4 results show that: Compared with the traditional solid-phase extraction method, the usage amount of organic solvents is reduced, the experimental cost is lowered, and at the same time, the environmental pollution caused by organic solvents is also reduced.
[0077] In this method, such as: the eluent n-hexane and the eluent ethyl acetate - diethyl ether, the usage amount of reagents is less, about 2 ml, while the above usage amount of the traditional solid-phase extraction method is about 10 ml. And this method does not require a vacuum environment during adsorption, and the key magnetic materials can be washed and reused after the experiment.
[0078] Table 4: Reagent Usage Amount
[0079] Pharmaceutical Dosage of pharmaceutical used in traditional method Dosage of pharmaceutical used in this method n-Hexane 10ml 2ml 10% Ethyl acetate - Ether solution 10ml / 25% Ethyl acetate - Ether solution / 2ml
[0080] Note: / indicates no addition amount.
[0081] As Figure 4As shown: The present invention can effectively remove impurity interference in the sample, improving the accuracy and reliability of the analysis results. In the traditional solid-phase extraction method, the packing material of the solid-phase extraction column itself may contain impurities, and these impurities may be eluted during the extraction process, thus forming impurity peaks. In addition, if the solid-phase extraction column is not sufficiently activated or cleaned before use, it may also lead to impurity residues, thereby affecting the extraction results. Secondly, some components in the sample may interact with the packing material of the solid-phase extraction column, resulting in these components being non-specifically adsorbed or retained on the column. During the subsequent elution process, these components may be eluted, forming impurity peaks; The magnetic solid-phase extraction method does not require the use of the above-mentioned materials and columns, etc. And in a complex sample matrix, the magnetic adsorbent can selectively adsorb the target analyte, while having a weak adsorption capacity for other impurities, and most of the impurities can be removed through a simple cleaning step; The number of impurity peaks generated by the traditional experimental method is between 15 and 17, and the area of some impurity peaks is relatively large. After being processed by this method, the number of impurity peaks is between 9 and 12, and the area of the generated impurity peaks is relatively small.
[0082] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid repetition, the present invention describes preferred embodiments.
[0083] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted to include the preferred embodiments and all changes and modifications falling within the scope of the present invention.
[0084] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these changes and modifications.
Claims
1. A method for rapid detection of ethyl carbamate in wine, characterized in that: The method comprises the following steps: adjusting the pH value of wine to acidic, adding CoFe2O4@CSC carbonized material and oscillating and mixing, separating the CoFe2O4@CSC carbonized material by a magnetic rack after standing to adsorb ethyl carbamate, eluting the CoFe2O4@CSC carbonized material with n-hexane and then adding an ethyl acetate-ether mixed solution to elute, obtaining an eluent containing ethyl carbamate, drying the eluent with nitrogen to obtain a pre-loaded solution, and diluting the pre-loaded solution to 1 mL with methanol and then performing GC-MS detection; The volume mass ratio of n-hexane to wine liquid is 1-2 mL: 1-3 g; The volume mass ratio of the ethyl acetate-ether mixed solution to the wine liquid is 1-2 mL: 1-3 g; The volume mass ratio of the prepared sample solution to the wine liquid is 0.1-0.5 mL: 1-3 g.
2. The method for rapid detection of ethyl carbamate in wine according to claim 1, characterized in that: The pH value is adjusted to 4-6.
3. The method for rapid detection of ethyl carbamate in wine according to claim 1, characterized in that: The mass ratio of the CoFe2O4@CSC carbonized material to the wine liquid is 5-7 mg:1-3 g.
4. The method for rapid detection of ethyl carbamate in wine according to claim 1, characterized in that: The static adsorption condition is 30-33° C. for 1-2 min.
5. The method for rapid detection of ethyl carbamate in wine according to claim 1, characterized in that: The n-hexane elution time is 10 to 30 seconds.
6. The method for rapid detection of ethyl carbamate in wine according to claim 1, characterized in that: The volume ratio of ethyl acetate to ether in the ethyl acetate-ether mixed solution is 24-26:74-76.
7. The method for rapid detection of ethyl carbamate in wine according to claim 1, characterized in that: The GC-MS detection conditions include: injection temperature of 200-220° C., gradient temperature increase to 200-240° C., solvent delay of 5-10 min, and injection volume of 1-2 μL.
8. The method for rapid detection of ethyl carbamate in wine according to claim 7, characterized in that: The gradient heating step comprises the following steps: the initial temperature is 100-150°C, maintained for 1-2 minutes; the temperature is increased to 180-200°C at 15-20°C / min, maintained for 1-6 minutes; the temperature is increased to 240-260°C at 30-35°C / min, maintained for 1-5 minutes.