A method for determining the isotope ratio of non-exchangeable hydrogen in ethanol in wine

Abstract

The present invention provides a method for determining the isotope ratio of non-exchangeable hydrogen in ethanol in wine. The method comprises the following steps: 1) determining the δD value of water in the wine; 2) adding water with a lower δD value than the water in the wine to prepare a wine solution; 3) taking an ethanol standard and diluting the ethanol standard with water having the same or similar δD value as the wine solution to the same alcohol content as the wine solution; and 4) determining the δD ratio of stable hydrogen ethanol in the wine sample solution and the standard solution using gas chromatography-pyrolysis-stable isotope ratio mass spectrometry. 测 5) A standard curve is constructed based on the given value of non-exchangeable hydrogen in the ethanol standard and the measured hydrogen isotope values, and the isotope ratio of the non-exchangeable hydrogen in the ethanol in the wine sample is calibrated and obtained. This method can be used to simply and rapidly determine the isotope ratio of the non-exchangeable hydrogen in the ethanol in wine and can be used in research and applications for wine authenticity and adulteration detection.

Description

Technical Field

[0001] The invention belongs to the field of stable isotope analysis and testing, and relates to a method for determining the isotope ratio of non-exchangeable hydrogen in ethanol in wine. Background Art

[0002] Stable isotope technology is an important means to detect wine adulteration. For example, based on carbon isotope characteristics, it can detect whether carbon-4 plant sugars (including cane sugar and high fructose corn syrup) and the alcohol converted from them are adulterated in wine. The International Organization of Vine and Wine (OIV) and my country have both established standard analytical methods for ethanol carbon isotopes. However, since carbon isotope technology can only detect sugars and their metabolites of different photosynthetic types, and beets and grapes have the same photosynthetic pathway, there is almost no difference in their carbon isotope characteristics. Such standards have no practical effect on the detection of beet sugar and its metabolites in wine.

[0003] Studies have shown that there are significant differences in the hydrogen isotope distribution characteristics between ethanol in wine and ethanol converted from beet sugar. However, determining the hydrogen isotope ratio of ethanol is a major technical challenge currently faced. This is because: ① The determination of ethanol hydrogen isotopes requires cracking and conversion into hydrogen. However, any hydrogen-containing substance can be converted into hydrogen in this process. The ethanol content in wine is approximately 12%, and the rest is water, organic acids and other substances. Therefore, high-purity ethanol needs to be separated in advance; ② Ethanol has three hydrogen sites: methyl hydrogen, methylene hydrogen, and hydroxyl hydrogen. According to the principle of isotope exchange reaction, the hydrogen atoms on the hydroxyl group are easily exchanged with water. However, since the hydrogen isotope ratio of water in wine varies, it is unscientific to directly use the hydrogen isotope ratio of ethanol. Only the isotope characteristics of the non-exchangeable hydrogen at the methyl and methine sites of ethanol are of practical value. Therefore, although Blanca O. Aguilar-Cisneros, Keita Yamada and Ryota Hattori established methods for extracting wine ethanol using headspace solid-phase microextraction in 2002, 2007 and 2008 respectively, and determined the hydrogen isotope ratio of ethanol using gas chromatography-pyrolysis-stable isotope ratio mass spectrometry, including the national invention patent "Method for Determining the Hydrogen Isotope Ratio of Ethanol in Wine 201610353866.X" applied for by Zhong Qiding et al., the methods only measured the overall hydrogen isotope ratio of ethanol and therefore have no practical application significance and cannot be included as wine industry regulatory methods.

[0004] The currently established method for determining hydrogen isotope ratios at non-exchangeable hydrogen sites in ethanol is the site-specific fractionation-nuclear magnetic resonance (SNIF-NMR) method, first developed in the 1980s. This method requires ① extracting ethanol from fermentation broth using a specialized microdistillation system without isotopic fractionation; ② accurately determining the ethanol concentration in the extract and precisely weighing the injected ethanol volume; and ③ determining the hydrogen isotope ratio of the ethanol methyl group using a high-powered nuclear magnetic resonance spectrometer. However, a literature review revealed two major drawbacks to this technique: ① The micro-distillation system, patented by Eurofins in France, requires specialized customization to achieve a recovery greater than 96.5% and an ethanol concentration greater than 95% v / v in the distillate. This system, a patented product of Eurofins in France, costs 1.6 million RMB per unit and requires high operator proficiency. Despite this, a single distillation cycle can take over four hours. ② Nuclear magnetic resonance (NMR) analysis of the hydrogen isotope ratio of ethanol methyl groups relies on determining the absolute deuterium content of methyl sites in a quantitative sample. Because deuterium naturally accounts for only 0.02% of hydrogen atoms, achieving sufficient signal-to-noise ratio and stability requires measurement times exceeding five hours per sample. These two drawbacks result in low analytical efficiency. Furthermore, few laboratories worldwide are proficient in using this method and generating accurate data, and none in my country have yet employed this technology, severely hindering research and application in the wine industry.

[0005] The national invention patent "2020106569064 A method for determining the stable hydrogen isotope ratio of ethanol methyl sites in beverage wine" applied for by Zhong Qiting et al. uses a series of transformation methods to achieve the determination of the hydrogen isotope ratio of ethanol methyl sites, but the operation steps are numerous and the pre-processing time is long.

[0006] Against this backdrop, the present invention has developed a simple method for determining the isotope ratio of non-exchangeable hydrogen sites in ethanol from wine. This method offers simplified operation, a high safety factor, and is suitable for analyzing and testing large quantities of samples. Summary of the Invention

[0007] The present invention provides a method for eliminating the influence of solution moisture on the isotopic characteristics of exchangeable hydrogen in ethanol, and then analyzing the isotopic ratio characteristics of non-exchangeable hydrogen in ethanol, comprising the following steps:

[0008] 1) Determination of the δD value of water in wine;

[0009] 2) If the δD value of the water in the wine is greater than or equal to -40‰, add reagent water A having a lower δD value to prepare a wine solution;

[0010] 3) Prepare an ethanol aqueous solution by taking an ethanol standard;

[0011] 4) Determination of the stable hydrogen isotope ratio δD of ethanol in wine solution and standard solution by gas chromatography-pyrolysis-stable isotope ratio mass spectrometry 测 ;

[0012] 5) Construct a standard curve based on the given value of non-exchangeable hydrogen in the ethanol standard and the measured value of hydrogen isotope, and calibrate the isotope ratio of non-exchangeable hydrogen in the ethanol in the wine sample.

[0013] The δD value of the reagent water A is between -90‰ and -260‰, such as -90‰-150‰, and illustratively, -97.1‰-144.7‰.

[0014] Furthermore, in step 1), the δD value of water in the wine is determined using the GasBench-IRMS method.

[0015] Furthermore, in step 2), the reagent water A having a lower δD value added to the wine can have a δD value of water in the prepared wine solution in the range of -40 to -150‰.

[0016] Illustratively, the ethanol-water solution has the same or similar δD value characteristics in water as the wine solution, and has the same alcohol content as the wine solution.

[0017] Further illustratively, when using gas chromatography-pyrolysis-stable isotope ratio mass spectrometry for determination, the wine solution and the standard solution can be diluted with an organic reagent or water to a suitable injection concentration, or can be directly injected.

[0018] Illustratively, the organic reagent is one or more of anhydrous esters, alcohols, nitriles, ethers, ketones, etc., which are miscible with water, and preferably acetone.

[0019] As an illustration, the stable hydrogen isotope ratio of ethanol in each solution was measured by gas chromatography-pyrolysis-stable isotope ratio mass spectrometry, which is the average of the isotopic characteristics of the six hydrogen atoms in the ethanol molecule, including non-exchangeable hydrogen and exchangeable hydrogen.

[0020] In the method of the present invention, a gas chromatography-pyrolysis-stable isotope ratio mass spectrometer is equipped with a capillary chromatographic column to effectively separate water and ethanol.

[0021] Furthermore, in step 2), the wine is diluted 1-50 times with reagent A.

[0022] Exemplarily, in step 2), the wine is diluted with reagent A, preferably 4, 5, 6, 7, 8, 9 or 10 times.

[0023] Further, in step 4), the condition parameters of the gas chromatography-pyrolysis-stable isotope ratio mass spectrometer were adjusted: the injection port temperature was 200°C, the injection volume was 2uL, the gas chromatography flow rate was a constant flow of 1.2mL / min, the gas chromatography injection port split ratio was 20:1, the column oven program was 80°C for 5min, increased to 180 at 20°C / min, and maintained for 10min; the temperature of the pyrolysis module was maintained at a constant temperature of 1420°C;

[0024] Optionally, confirm that the working environment, airtightness, and vacuum degree of the ion chamber of the stable isotope ratio mass spectrometer meet the analysis requirements, then check the precision and linearity of the instrument in measuring δD in H2, and adjust the ion source parameter values ​​if necessary.

[0025] To explain in more detail, the following examples are given.

[0026] 47, 48, 49, 50, 60, 70, 80, 90, 100, 100-fold.

[0027] In the method of the present invention, when the ethanol standard is diluted to the same ethanol concentration, the same diluent as that used for diluting wine is used, for example, the δD value of the hydroxyl site is lower than -90‰, -100‰, -105‰, -110‰, -115‰, -120‰, -125‰, -130‰, -135‰, -140‰, -145‰, -150‰, -155‰, -160‰, -165‰, -170‰, -175‰, -180‰, -190‰, -200‰, -210‰, -220‰, -230‰, The dilution of reagent water of -240‰, -250‰, -260‰, -270‰, etc. is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 times.

[0028] In the method of the present invention, when gas chromatography-pyrolysis-stable isotope ratio mass spectrometry is used for determination, the wine solution and the standard solution are diluted with an organic reagent or water to a suitable injection concentration, or are directly injected.

[0029] In the present invention, the organic reagent used for dilution before injection is anhydrous and one or more of esters, alcohols, nitriles, ethers, ketones and other reagents that are miscible with water, preferably acetone.

[0030] Illustratively, before injection, samples and standards are diluted 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100 times with esters, alcohols, nitriles, ethers, and ketones.

[0031] For example, samples and standards can be diluted 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, or 100-fold with acetone.

[0032] Illustratively, hydroxyl site δD values ​​below -90‰, -100‰, -105‰, -110‰, -115‰, -120‰, -125‰, -130‰, -135‰, -140‰, -145‰, -150‰, -155‰, -160‰, -165‰, -170‰, -175‰, -180‰, -190‰, -200‰, -210‰, -220‰, -230‰, -240‰, -250‰, -260‰, -270‰ 47,48,49,50,60,70,80,90,100 times.

[0033] Furthermore, in the method of the present invention, the condition parameters of the gas chromatography-pyrolysis-stable isotope ratio mass spectrometer are adjusted: the injection port temperature is 200°C, the injection volume is 2uL, the gas chromatography flow rate is a constant flow of 1.2mL / min, the gas chromatography injection port split ratio is 20:1, the column oven program is 80°C for 5min, increased to 180°C at 20°C / min, and maintained for 10min; the temperature of the pyrolysis module is maintained at a constant temperature of 1420°C.

[0034] Alternatively, further, in the method of the present invention, the condition parameters of the gas chromatography-pyrolysis-stable isotope ratio mass spectrometer are adjusted: the injection port temperature is 210°C, the injection volume is 2uL, the gas chromatography flow rate is a constant flow of 1.0mL / min, the gas chromatography injection port split ratio is 20:1, the column oven program is 80°C for 5min, increased to 180°C at 20°C / min, and maintained for 10min; the temperature of the pyrolysis module is maintained at a constant temperature of 1420°C.

[0035] Optionally, confirm that the working environment, airtightness, and vacuum degree of the ion chamber of the stable isotope ratio mass spectrometer meet the analysis requirements, then check the precision and linearity of the instrument in measuring δD in H2, and adjust the ion source parameter values ​​if necessary.

[0036] Specifically, the present invention provides a method for determining the isotope ratio of non-exchangeable hydrogen in ethanol in wine, characterized by comprising the following steps:

[0037] 1) Dilute the wine sample with diluent and dilute the ethanol standard to the same ethanol concentration;

[0038] 2) Determination of the stable hydrogen isotope ratio δD of ethanol in wine sample solution and standard solution by gas chromatography-pyrolysis-stable isotope ratio mass spectrometry 测 ;

[0039] 3) A standard curve is constructed based on the given value of non-exchangeable hydrogen in the ethanol standard and the measured hydrogen isotope value, and the isotope ratio of non-exchangeable hydrogen in the ethanol in the wine sample is obtained by calibration.

[0040] The diluent reagent water A with a lower δD value is as described above.

[0041] The operating scheme provided by the present invention provides a stable method for isotopic analysis of non-exchangeable hydrogen in ethanol in wine and its application in adulteration detection / authenticity assurance.

[0042] The present invention has the following advantages:

[0043] This invention, for the first time, rapidly measures the isotope ratio of non-exchangeable hydrogen in ethanol. This method, based on extensive experimental experience, eliminates the effect of the sample's inherent moisture on the ethanol's hydroxyl hydrogen by using water with a different δD value from the sample. The operational protocol provided by this invention offers a method for isotope analysis of non-exchangeable hydrogen in ethanol in wine and its application in adulteration detection and authenticity assurance. DETAILED DESCRIPTION

[0044] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0045] Example 1 Determination of Non-exchangeable Hydrogen in Wine Samples by δD

[0046] a) GasBench-I RMS method for determination of δD values ​​of water in wine and reagent water: 0.5 mL of wine sample #1, reagent water A1, and reagent water B were each pipetted into a reaction flask. A platinum rod was added, the cap was tightened, and a hydrogen-helium mixture with a H2 ratio of 2.1% was filled in. The air in the flask was evacuated and placed in a sample tray at 27°C for 1 hour. The δD value of the water in the wine sample was +28.9‰, the δD value of reagent water A1 was -144.7‰, and the δD value of reagent water B was -67.3‰.

[0047] b) The alcohol content of the wine sample was determined to be 12.9% vol in accordance with Method 2 of GB 5009.225-2016, “National Food Safety Standard - Determination of Ethanol Concentration in Wines”;

[0048] c) Dilute the wine sample with reagent water with δD = -144.7‰: Pipette 1 mL of the wine sample and add 4 mL of reagent water A1. Calculate that the wine sample solution has δD ≈ -111.8‰, and the alcohol content is approximately 2.6% vol.

[0049] d) Prepare reagent water C1 with δD≈-111.8‰ using reagent water A1 and reagent water B;

[0050] e) Take two ethanol laboratory working standards a and b with different non-exchangeable hydrogen isotope ratios (determine the deuterium-hydrogen ratio of non-exchangeable hydrogen using SNIF-NMR technology and convert it into δD Non-EX After that, the concentrations of ethanol and water were -270.5‰ and -121.8‰, respectively. 0.10 mL of each was taken and added to 3.74 mL of reagent water C1 to prepare ethanol aqueous solutions a1 and b1 with an alcohol content of about 2.6% vol.

[0051] f) Extract water from 100 mL of orange juice by vacuum distillation to obtain reagent water E1. Measure δD = +37.5‰ using the method described in a). Prepare reagent water F1 with δD ≈ +28.9‰ using reagent water E1 and reagent water B.

[0052] g) Take 0.2 mL each of ethanol working standards a and b, mix them evenly to obtain ethanol working standard solution c (δD Non-EX After about -196.2‰), diluted with reagent water F1 to an ethanol concentration of 12.9% vol, and then diluted according to the method in step c) to obtain an ethanol aqueous solution c1;

[0053] h) Take chromatographically pure acetone and treat it with anhydrous water (add 100g of acetone to molecular sieves), adding 50% anhydrous acetone by volume to the wine sample solution in step c) and the ethanol aqueous solutions in step e) and g);

[0054] i) Adjust the gas chromatography-pyrolysis-stable isotope ratio mass spectrometer parameters and operating conditions: inlet temperature 200°C, GC flow rate constant 1.2 mL / min, GC inlet split ratio 20:1, column oven program: 80°C for 5 min, increase to 180°C at 20°C / min, hold for 10 min; pyrolysis reaction tube temperature 1420°C;

[0055] j) Each sample was measured three times. The results are shown in Table 1.

[0056] Table 1 Determination results of ethanol δD in samples (‰)

[0057] sample 1st time 2nd time 3rd time average value Standard deviation SD 1# wine sample -172.8 -172.0 -172.1 -172.3 0.5 Ethanol standard solution a1 -186.9 -186.8 -198.4 -187.4 0.9 Ethanol standard solution b1 -70.6 -69.2 -70.0 -69.9 0.7 Ethanol standard solution c1 -127.4 -127.3 -126.5 -127.1 0.7

[0058] It can be seen from the above table that the standard deviations of the three determination results of wine samples and ethanol standard solutions are all less than 1.0‰.

[0059] The measured values ​​of δD of a1 and b1 are compared with the known non-exchangeable hydrogen δD Non-EX By establishing a standard curve, we can know the δD of ethanol in standard solution c1. Non-EX The value is -194.1‰, which is only 2.1‰ different from the predicted value of -196.2‰, which meets the standard deviation requirement of 3‰ for the determination of stable hydrogen isotope ratios of organic matter. Therefore, it can be shown that this method can accurately determine the stable hydrogen isotope ratio of non-exchangeable hydrogen sites in ethanol.

[0060] Using this method, we can know that the isotope ratio of non-exchangeable hydrogen in ethanol in wine sample 1 is -251.4‰.

[0061] Example 2 Determination of Non-exchangeable Hydrogen in Wine Samples by δD

[0062] a) GasBench-IRMS method for determination of δD values ​​of water in wine and reagent water: 0.5 mL of wine sample #2, reagent water A2, and reagent water B were each transferred to a reaction flask. A platinum rod was added, the cap was tightened, and a hydrogen-helium mixture with a H2 ratio of 2.1% was filled in. The air in the reaction flask was evacuated and placed in a sample tray at 27°C for 1 hour. The δD value of the water in the wine sample was measured to be +15.5‰, the δD value of reagent water A2 was -96.1‰, and the δD value of reagent water B was -67.3‰.

[0063] b) The alcohol content of the wine sample was determined to be 12.2% vol according to Method 2 of GB 5009.225-2016, “National Food Safety Standard - Determination of Ethanol Concentration in Wine”;

[0064] c) Dilute the wine sample with reagent water with δD = -97.1‰: Pipette 1 mL of the wine sample and add 4 mL of reagent water A2. Calculate that the wine sample solution has δD ≈ -74.9‰, and the alcohol content is approximately 2.4% vol.

[0065] d) Prepare reagent water C2 with a δD of -74.9‰ using reagent water A2 and reagent water B;

[0066] e) Take two ethanol laboratory working standards a and b with different non-exchangeable hydrogen isotope ratios (determine the deuterium-hydrogen ratio of non-exchangeable hydrogen using SNIF-NMR technology and convert it into δD Non-EX After that, 0.10 mL of each was taken and added to 4 mL of reagent water C2 to prepare ethanol aqueous solutions a2 and b2 with an alcohol content of about 2.4% vol;

[0067] f) Prepare reagent water F2 with δD≈+15.5‰ using reagent water E (δD=+37.5‰) and reagent water B;

[0068] g) Take 0.2 mL each of ethanol working standards a and b, mix them evenly to obtain ethanol working standard solution c (δD Non-EX After about -196.2‰), diluted with reagent water F to an ethanol concentration of 12.2% vol, and then diluted according to the method in step c) to obtain an ethanol aqueous solution c2;

[0069] h) Take chromatographically pure acetone and treat it with anhydrous water (add 100g of acetone to molecular sieves), adding 50% anhydrous acetone by volume to the wine sample solution in step c) and the ethanol aqueous solutions in step e) and g);

[0070] i) Adjust the gas chromatography-pyrolysis-stable isotope ratio mass spectrometer parameters and operating conditions: inlet temperature 200°C, GC flow rate constant 1.2 mL / min, GC inlet split ratio 20:1, column oven program: 80°C for 5 min, increase to 180°C at 20°C / min, hold for 10 min; pyrolysis reaction tube temperature 1420°C;

[0071] j) Each sample was measured three times. The results are shown in Table 2.

[0072] Table 2 Determination results of ethanol δD in samples (‰)

[0073] sample 1st time 2nd time 3rd time average value Standard deviation SD 2# wine sample -163.3 -163.9 -165.4 -164.2 1.1 Ethanol standard solution a2 -184.4 -186.2 -185.2 -185.3 0.9 Ethanol standard solution b2 -64.5 -66.1 -63.8 -64.8 1.2 Ethanol standard solution c2 -123.2 -124.2 -122.3 -123.2 0.9

[0074] As can be seen from the table above, the standard deviations of the three determination results for the wine sample and the ethanol standard solution are all less than 3‰, which meets the stability requirements for the determination of stable hydrogen isotope ratios in organic matter.

[0075] The measured values ​​of δD of a2 and b2 are compared with the known non-exchangeable hydrogen δD Non-EX By establishing a standard curve, we can know the δD of ethanol in standard solution c2. Non-EX The value is -193.9‰, which is only 2.3‰ different from the predicted value of -196.2‰, meeting the error requirement. Therefore, it can be shown that this method can accurately determine the stable hydrogen isotope ratio of the non-exchangeable hydrogen sites in ethanol.

[0076] Using this method, we can know that the isotope ratio of non-exchangeable hydrogen in ethanol in 2# wine is -244.5‰.

[0077] Example 3 Determination of Non-exchangeable Hydrogen in Wine Samples by δD

[0078] a) GasBench-IRMS method for determination of δD values ​​of water in wine and reagent water: 0.5 mL of wine sample #3, reagent water A3, and reagent water B were each transferred to a reaction flask. A platinum rod was added, the cap was tightened, and a hydrogen-helium mixture with a H2 ratio of 2.1% was filled in. The air in the flask was evacuated and placed in a sample tray at 27°C for 1 hour. The δD value of the water in the wine sample was +21.1‰, the δD value of reagent water A3 was -432.1‰, and the δD value of reagent water B was -67.3‰.

[0079] b) The alcohol content of the wine sample was 11.6% vol, as determined by Method 2 of GB 5009.225-2016, “National Food Safety Standard - Determination of Ethanol Concentration in Wines”;

[0080] c) Dilute the wine sample with reagent water with δD = -144.7‰: Pipette 1 mL of the wine sample and add 1 mL of reagent water A3. Calculate that the wine sample solution has δD ≈ -345.9‰, and the alcohol content is approximately 2.3% vol.

[0081] d) Prepare reagent water C3 with δD≈-345.9‰ using reagent water A3 and reagent water B;

[0082] e) Take two ethanol laboratory working standards a and b with different non-exchangeable hydrogen isotope ratios (determine the deuterium-hydrogen ratio of non-exchangeable hydrogen using SNIF-NMR technology and convert it into δD Non-EX After that, the concentrations of the two ethanol solutions a3 and b3 were respectively prepared. 0.10 mL of each solution was added to 3.74 mL of reagent water C3 to prepare ethanol aqueous solutions a3 and b3 with an alcohol content of about 2.6% vol.

[0083] f) Prepare reagent water F3 with δD≈+21.1‰ using reagent water E (+37.5‰) and reagent water B;

[0084] g) Take 0.2 mL each of ethanol working standards a and b, mix them evenly to obtain ethanol working standard solution c (δD Non-EX After about -196.2‰), diluted with reagent water F3 to an ethanol concentration of 11.6% vol, and then diluted according to the method in c) to obtain an ethanol aqueous solution c3;

[0085] h) Take chromatographically pure acetone and treat it with anhydrous water (add 100g of acetone to molecular sieves), adding 50% anhydrous acetone by volume to the wine sample solution in step c) and the ethanol aqueous solutions in step e) and g);

[0086] i) Adjust the gas chromatography-pyrolysis-stable isotope ratio mass spectrometer parameters and operating conditions: inlet temperature 200°C, GC flow rate constant 1.2 mL / min, GC inlet split ratio 20:1, column oven program: 80°C for 5 min, increase to 180°C at 20°C / min, hold for 10 min; pyrolysis reaction tube temperature 1420°C;

[0087] j) Each sample was measured three times. The results are shown in Table 3.

[0088] Table 3 Determination results of ethanol δD in samples (‰)

[0089] sample 1st time 2nd time 3rd time average value Standard deviation SD 3# wine sample -200.3 -202.7 -200.1 -201.0 1.4 Ethanol standard solution a3 -212.7 -212.1 -209.7 -211.5 1.5 Ethanol standard solution b3 -85.2 -86.5 -88.0 -86.6 1.4 Ethanol standard solution c3 -149.0 -151.9 -150.5 -150.5 1.5

[0090] As can be seen from the table above, the standard deviations of the three determination results for the wine sample and the ethanol standard solution are all less than 3.0‰, which meets the stability requirements for the determination of stable hydrogen isotope ratios in organic matter.

[0091] The measured values ​​of δD of a3 and b3 are compared with the known non-exchangeable hydrogen δD Non-EX By establishing a standard curve, we can know the δD of ethanol in standard solution c3. Non-EX The value is -197.9‰, which is only 1.7‰ different from the predicted value of -196.2‰, meeting the error requirement. Therefore, it can be shown that this method can accurately determine the stable hydrogen isotope ratio of the non-exchangeable hydrogen sites in ethanol.

[0092] Using this method, we can know that the isotope ratio of non-exchangeable hydrogen in ethanol in wine sample 3 is -258.0‰.

[0093] Example 4

[0094] a) Take wine sample #1, reagent water A3, and reagent water B as research objects;

[0095] b) Pipette 1 mL of wine sample and add 4 mL of reagent water A3. Calculate the δD of this wine sample solution to be -344.7‰, and the alcohol content is approximately 2.6% vol.

[0096] c) Prepare reagent water C4 with a δD of -344.7‰ using reagent water A3 and reagent water B;

[0097] d) Take two ethanol laboratory working standards a and b with different non-exchangeable hydrogen isotope ratios (determine the deuterium-hydrogen ratio of non-exchangeable hydrogen using SNIF-NMR technology and convert it into δD Non-EX After that, the concentrations of the two ethanol solutions a4 and b4 were respectively -270.5‰ and -121.8‰), 0.10 mL of each solution was added to 3.74 mL of reagent water C4 to prepare ethanol aqueous solutions a4 and b4 with an alcohol content of about 2.6% vol;

[0098] e) Prepare reagent water F1 with δD≈+28.9‰ using reagent water E (δD=+37.5‰) and reagent water B;

[0099] f) Take 0.2 mL each of ethanol working standards a and b, mix them evenly to obtain ethanol working standard solution c (δD Non-EX After about -196.2‰), diluted with reagent water F1 to an ethanol concentration of 12.9% vol, and then diluted according to the method in c) to obtain an ethanol aqueous solution c4;

[0100] g) Take chromatographically pure acetone and treat it with anhydrous water (add 100g of acetone to molecular sieves), adding 50% anhydrous acetone by volume to the wine sample solution in step c) and the ethanol aqueous solutions in step e) and g);

[0101] h) Adjust the gas chromatography-pyrolysis-stable isotope ratio mass spectrometer parameters and operating conditions: inlet temperature 200°C, GC flow rate constant 1.2 mL / min, GC inlet split ratio 20:1, column oven program: 80°C for 5 min, increase to 180°C at 20°C / min, hold for 10 min; pyrolysis reaction tube temperature 1420°C;

[0102] i) Each sample was measured 3 times. The results are shown in Table 4.

[0103] Table 4 Determination results of ethanol δD in samples (‰)

[0104]

[0105]

[0106] As can be seen from the above table, the standard deviations of the three determination results of wine samples and ethanol standard solutions are all less than 3.0‰, which meets the requirements for the determination of stable hydrogen isotope ratios of organic matter.

[0107] The measured values ​​of δD of a4 and b4 are compared with the known non-exchangeable hydrogen δD Non-EX By establishing a standard curve, we can know the δD of ethanol in standard solution c4. Non-EX The value is -198.2‰, which is only 2.0‰ different from the predicted value of -196.2‰. This shows that this method can accurately determine the stable hydrogen isotope ratio of the non-exchangeable hydrogen sites in ethanol.

[0108] Using this method, it can be seen that the isotope ratio of non-exchangeable hydrogen in ethanol in wine sample 1 is -254.4‰, which is 3.0‰ different from the result obtained in Example 1 (-251.4‰), meeting the accuracy requirement.

[0109] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for determining the isotope ratio of non-exchangeable hydrogen in ethanol in wine, characterized in that: The steps include: 1) Determination of the δD value of water in wine; 2) If the δD value of the water in the wine is greater than or equal to -40‰, add reagent water A having a lower δD value to prepare a wine solution; 3) preparing an ethanol aqueous solution from an ethanol standard; the ethanol aqueous solution has the same δD value characteristics as the wine solution and has the same alcohol content as the wine solution; 4) Determination of the stable hydrogen isotope ratio δD of ethanol in wine solution and standard solution by gas chromatography-pyrolysis-stable isotope ratio mass spectrometry 测 ;δD 测 It is the average isotopic characteristic of the six hydrogen atoms in the ethanol molecule, including non-exchangeable and exchangeable hydrogen atoms. When measured by gas chromatography-pyrolysis-stable isotope ratio mass spectrometry, the wine solution and the standard solution are directly injected. 5) Construct a standard curve based on the given value of non-exchangeable hydrogen in the ethanol standard and the measured value of hydrogen isotope, and calibrate the isotope ratio of non-exchangeable hydrogen in the ethanol in the wine sample. The δD value of the reagent water A is between -90‰ and -260‰. And in step 2), the wine is diluted 3-50 times with reagent water A.

2. The method according to claim 1, characterized in that Step 1) Determine the δD value of water in wine using the GasBench-IRMS method.

3. The method according to claim 1, characterized in that The reagent water A added to the wine in step 2) has a lower δD value, and the δD value of the water in the prepared wine solution is in the range of -40 to -150‰.

4. The method according to claim 1, 2 or 3, characterized in that The gas chromatography-pyrolysis-stable isotope ratio mass spectrometer is equipped with a capillary column to effectively separate water and ethanol.

5. The method according to claim 1, wherein in step 2), the wine is diluted with reagent water A by a dilution factor of 4, 5, 6, 7, 8, 9 or 10 times.

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