Pretreatment method for determining the stable isotope ratio of non-exchangeable hydrogen in acetic acid

Abstract

The present invention provides a pretreatment method for determining the stable isotope ratio of non-exchangeable hydrogen in acetic acid, belonging to the field of stable isotope analysis of foods. The pretreatment method comprises the following steps: adding a reaction reagent to a vinegar sample extract to replace the carboxyl hydrogen in the acetic acid with the exchangeable hydrogen to achieve isotope exchange equilibrium. The present invention can be used to simply and rapidly determine the isotope ratio of non-exchangeable hydrogen in acetic acid in vinegar products and can be used in research and applications for detecting the authenticity or adulteration of vinegar products.

Description

Technical Field

[0001] The invention belongs to the field of stable isotope analysis and testing, and relates to a pretreatment method for determining the stable isotope ratio of non-exchangeable hydrogen of acetic acid in vinegar, so as to be used for determining the isotope ratio of non-exchangeable hydrogen of acetic acid in a vinegar sample. Background Art

[0002] Vinegar is a traditional condiment used in all major Chinese cuisines and a daily necessity. GB 2719-2018, "National Food Safety Standard for Edible Vinegar," officially came into effect on December 21, 2019. It defines vinegar as "a liquid acidic condiment produced through microbial fermentation using various materials containing starch, sugar, and edible alcohol, either alone or in combination." It also mandates that "glacial acetic acid (also known as acetic acid) and glacial acetic acid (low-pressure hydroxylation method) are not to be used in vinegar." Previously formulated products will now be managed under GB 31644-2018, "National Food Safety Standard for Compound Condiments."

[0003] In June 2021, the State Administration for Market Regulation issued the "Announcement on Strengthening the Supervision and Administration of Soy Sauce and Vinegar Quality Safety," which stipulates that violations such as the use of glacial acetic acid and other raw materials in the production of vinegar and false product labeling will be investigated and punished in accordance with the law. Currently, food inspectors often conduct vinegar authenticity verification based on sensory analysis, analysis of organic acid volatile components, and fingerprint analysis. However, due to the similar physical, chemical, and sensory characteristics between vinegar and compound seasonings (vinegar-flavored sauces), the practical application of existing methods is not ideal.

[0004] Foreign studies have shown that the presence of industrial acetic acid in vinegar drinks can be detected based on the difference in the stable hydrogen isotope ratio of non-exchangeable hydrogen in acetic acid [Hermann, A. (2001) Determination of D / H isotope ratio in acetic acid from vinegars and pickled products by H-2-NMR spectroscopy. Eur Food Res Techn, 212 683-686; Fauhl, C., Wittkowski, R. (1996) Online 1H-NMR to facilitate tube preparation in SNIF-NMR analysis. Z Lebensm Unters Forsch (1996), 541-545; Hsieh CW, Li PH, Cheng JY, et al. Using SNIF-NMR method to identify the adulteration of molasses spirit vinegar by synthetic acetic acid in rice vinegar [J]. Industrial crops and products, 2013, 50: 904-908; Perini et al. M, Paolini M, Simoni M, et al. Stable isotope ratio analysis for verifying the authenticity of balsamic and wine vinegar[J]. Journal of agricultural and food chemistry, 2014, 62(32):8197-8203]. In this method, the stable hydrogen isotope ratio of non-exchangeable hydrogen in acetic acid was analyzed using site-specific fractionation-nuclear magnetic resonance technology (SNIF-NMR).Regarding this technology, relevant EU agencies organized an inter-laboratory method comparison [Thomas F, Jamin E. 2H NMR and 13C-IRMS analyses of acetic acid from vinegar, 18O-IRMS analysis of water in vinegar: International collaborative study report [J]. Analytical chimica acta, 2009, 649(1): 98-105] to verify the effectiveness of the determination technology. However, this method has some practical problems. Due to the low abundance of deuterium in nature (approximately 150 x 10⁻⁶), the low resonance frequency and spectral dispersion of deuterium nuclei, the sensitivity of the SNIF-NMR method is very low, requiring large sample volumes (>300 mL) and long analysis times (>8 hours) to achieve a satisfactory signal-to-noise ratio and measurement accuracy. Furthermore, since vinegar contains hydrogen-containing compounds such as water and ethanol in addition to acetic acid, the ethanol must be purified using a special distillation device (Cadiot Column) before sample analysis. This distillation device is not only a patented product of Eurofins, but the entire sample purification process takes up to 6 hours. Some users have also acknowledged that the purification of acetic acid is an insurmountable bottleneck in the application of this technology [Ko WC, Cheng JY, Chen PY, et al. Optimized extraction method of acetic acid in vinegar and its effect on SNIF-NMR analysis to control the authenticity of vinegar [J]. Food and bioprocesstechnology,2013,6(8):2202-2206.].

[0005] To reduce the analytical complexity, some have investigated the use of stable isotope ratio mass spectrometry for hydrogen isotope analysis of acetic acid. However, this approach must consider the fundamental principles of stable isotope analysis and purity requirements. It is necessary to eliminate the effects of water on the conversion of acetic acid to hydrogen, and even more so, to eliminate the effects of water in the original solution on the carboxyl hydrogen groups of acetic acid. In 2009, Milanka Glavanovic and Ivan Samjlovic applied for a US patent, which proposed converting acetic acid in solution into acetate. After drying to remove water and extracting with an organic reagent to remove soluble organic matter, a solid sample containing acetate was obtained. This acetate was then converted into hydrogen using a pyrolysis / elemental analyzer and analyzed using a stable isotope ratio mass spectrometer. However, this technique has significant limitations, both in principle and technically. In addition to acetic acid, organic acids in food matrices also include formic acid, oxalic acid, lactic acid, and malic acid. When acetic acid is converted to acetate, these organic acids are also converted into organic acid salts. These organic acids, regardless of their physical characteristics, cannot be removed, resulting in an impure acetate in the resulting "solid sample." The inventors believed that this method measured the stable hydrogen isotope ratio of acetate, which actually contains other organic acids. In addition, acetate is very hygroscopic. Since the sample packaging process and the waiting time for injection are open when using a high-temperature pyrolysis / elemental analyzer for conversion, it is easy to absorb moisture from the air and introduce additional hydrogen atoms, which in turn leads to inaccurate measurements. Therefore, this patent was withdrawn / rejected in 2014. In addition, Ryota Hattori et al. of Japan [Hattori, R., Yamada, K., Shibata, H., Hirano, S., Tajima, O., & Yoshida, N. (2010). Measurement of the Isotope Ratio of Acetic Acid in Vinegar by HS-SPME-GC-TC / C-IRMS. Journal of Agricultural and Food Chemistry, 58(12), 7115–7118.doi:10.1021 / jf100406y] In 2010, a method for extracting acetic acid from vinegar by headspace solid-phase microextraction was established and the hydrogen isotope ratio of acetic acid was determined by gas chromatography-pyrolysis-stable isotope ratio mass spectrometry. However, this method uses headspace solid-phase microextraction technology to separate acetic acid from other hydrogen-containing components. However, the extraction head has a great influence on the extraction efficiency and stability. Some researchers have questioned the feasibility of applying this technology to stable isotope analysis. In addition, the hydrogen isotope ratio of acetic acid as a whole is measured under this technical system, so it has no practical application significance.For this reason, both domestic and international researchers were not optimistic about the feasibility of using stable isotope ratio mass spectrometry to analyze the isotope ratios of non-exchangeable hydrogens in acetic acid. This technology has not made any breakthroughs in the past 20 years. Therefore, in 2015, the International Organization of Vine and Wine chose to use SNIF-NMR technology to develop a method standard [RESOLUTION OIV-OENO 527-2015 Determination of the distribution of deuterium in acetic acid extracted from wine vinegar using nuclear magnetic resonance (NMR)].

[0006] The national invention patent "2020100815968 Method for Determining the Stable Hydrogen Isotope Ratio of Non-exchangeable Hydrogen of Acetic Acid in Vinegar Beverages" applied for by Zhong Qiting et al. adopts: adding an alkaline reagent to vinegar to form an organic acid salt system with acetate as the main body, removing most of the water and volatile alcohol, aldehyde and ester organic matter by freezing or heating and drying to obtain a solid residue, and finally adding a strong acid solution to the solid residue to acidify the organic acids. At this time, the solution contains water, acetic acid, organic acid and sulfuric acid. By screening a suitable chromatographic column, water, acetic acid, etc. are separated and the stable hydrogen isotope ratio of acetic acid is determined by gas chromatography-pyrolysis-stable isotope ratio mass spectrometry (the average characteristics of the four hydrogens). Finally, the stable hydrogen isotope ratio mass spectrometry is used to determine the stable hydrogen isotope ratio of non-exchangeable hydrogen in acetic acid through standard calibration. Compared with previous research, this technology is a qualitative breakthrough, including compared with the OIV method standards: samples can be pre-treated in batches, and the test time is reduced from 5 hours to less than 20 minutes. The cost of analysis and testing is greatly reduced, and the efficiency is greatly improved. In particular, it avoids the dependence on the OIV's overseas patented micro-distillation device when promoting the method. However, since this pre-treatment process involves three key steps: alkalization-dehydration-acidification, the dehydration process in particular takes a long time (freeze drying requires more than 24 hours, and the drying operation requires more than 6 hours); and because the sample to be tested contains a large amount of water, a special chromatographic column with a polymeric filler that can separate water and acetic acid is required. Ordinary polar columns have the risk of affecting the determination of hydrogen acetate isotopes due to incomplete separation of water. To further improve the efficiency of analysis, the present invention proposes another key idea based on the basic principles and requirements of stable isotope analysis, and has developed a new pretreatment method accordingly. Summary of the Invention

[0007] To overcome the shortcomings of the prior art, the present invention develops a vinegar sample pretreatment method to determine the isotope ratio of the non-exchangeable hydrogen sites of acetic acid in vinegar. This invention is simpler to operate, has a high safety factor, is suitable for the analysis and testing of large quantities of samples, and is conducive to promoting the further research and application of stable isotope technology in the vinegar field. The present invention provides a method for determining the isotope ratio of the non-exchangeable hydrogen sites of acetic acid in vinegar. The method of the present invention can eliminate the influence of moisture in the vinegar solution on the determination of the isotope characteristics of the exchangeable hydrogen of acetic acid, and thus can accurately and stably analyze the isotope ratio of the non-exchangeable hydrogen of acetic acid using gas chromatography-pyrolysis-stable isotope ratio mass spectrometry technology. The method comprises the following steps:

[0008] Acetic acid is extracted from a vinegar sample by liquid-liquid extraction, and exchangeable hydrogen ions in a reaction reagent are reacted with exchangeable hydrogen ions in acetic acid to achieve the purpose of replacement, thereby obtaining a sample solution.

[0009] Furthermore, in the present invention, the reagent is methanol or ethanol.

[0010] Furthermore, in the present invention, the volume fraction of the reagent is more than 5 times that of acetic acid in the extraction solution, preferably 5-100 times, more preferably 30 times.

[0011] Furthermore, the method of the present invention further comprises one, two, three, four or five of the following steps:

[0012] 1) Determine the total acid content of the vinegar sample;

[0013] 2) extracting acetic acid by liquid-liquid extraction to obtain an extract, and adding a reaction reagent to the extract to obtain a sample solution;

[0014] 3) determining the stable hydrogen isotope ratio of acetic acid in the sample solution using gas chromatography-pyrolysis-stable isotope ratio mass spectrometry;

[0015] 4) treating a standard acetic acid solution with the same organic reagents and reaction reagents as the sample in the same or similar proportions to obtain a compositional profile identical or similar to that of the sample solution, and determining the stable hydrogen isotope ratio of the acetic acid in the standard solution by gas chromatography-pyrolysis-stable isotope ratio mass spectrometry;

[0016] 5) A standard curve is constructed based on the given value of the non-exchangeable hydrogen in the acetic acid standard and the measured isotope value of the acetic acid hydrogen in the standard solution, and the isotope ratio of the non-exchangeable hydrogen in the acetic acid in the vinegar sample is obtained by calibration.

[0017] Furthermore, in the present invention, an organic reagent is used to extract acetic acid from the vinegar sample during liquid-liquid extraction. The organic reagent is ethyl acetate, n-hexane, petroleum ether, chloroform, carbon tetrachloride and diethyl ether, etc., preferably diethyl ether.

[0018] Furthermore, in the present invention, in order to facilitate batch sample operation, after the total acid content of the vinegar sample is determined, the vinegar sample and the acetic acid standard are diluted with water to the same acidity, and then the same extraction is performed and the same reaction reagent is added. The acidity is preferably 3.5g / 100mL.

[0019] Furthermore, in the present invention, when gas chromatography-pyrolysis-stable isotope ratio mass spectrometry is used for determination, the vinegar sample solution and the standard solution are further diluted with an organic reagent to a suitable injection concentration, or are directly injected.

[0020] Furthermore, in the present invention, the gas chromatography-pyrolysis-stable isotope ratio mass spectrometer measures the stable hydrogen isotope ratio of acetic acid in each solution, which is the average value of the isotopic characteristics of the four hydrogen atoms in the acetic acid molecule, including non-exchangeable hydrogen and exchangeable hydrogen.

[0021] Furthermore, in the present invention, the gas chromatography-pyrolysis-stable isotope ratio mass spectrometer is equipped with a capillary chromatographic column.

[0022] Furthermore, in the present invention, the vinegar sample is aged vinegar, rice vinegar, fruit vinegar, white vinegar, or an acidic seasoning liquid having characteristics of a vinegar sample.

[0023] Illustratively, the present invention uses a reagent to replace the exchangeable hydrogen in the acetic acid of the vinegar sample, allowing the exchangeable hydrogen to reach hydrogen isotope exchange equilibrium. The reagent, such as methanol or ethanol, can be added directly to the vinegar sample. Alternatively, the acetic acid can be extracted by liquid-liquid extraction and then isotope exchange is performed with the reagent. Preferably, the reagent is added after extraction to achieve hydrogen isotope exchange equilibrium.

[0024] In the method of the present invention, the standard is treated with the same organic reagents and reaction reagents as the sample in the same or similar ratios to obtain the same or similar compositional characteristics as the sample solution. The stable hydrogen isotope ratio of acetic acid in the standard solution is determined by gas chromatography-pyrolysis-stable isotope ratio mass spectrometry, wherein the isotopic characteristics of exchangeable hydrogen in the sample solution and the standard solution are consistent. In the method of the present invention, when using gas chromatography-pyrolysis-stable isotope ratio mass spectrometry for determination, the vinegar sample and the standard solution are diluted with organic reagents or water to a suitable injection concentration, or are directly injected.

[0025] In the present invention, the organic reagent used for extraction is one or more of anhydrous alkanes, esters, alcohols, ethers and the like that are miscible with acetic acid, preferably diethyl ether.

[0026] Illustratively, the reaction reagent is used to replace the original exchangeable hydrogen in the acetic acid of the vinegar sample, and the multiple of the reagent having exchangeable hydrogen molecules in the original sample is 5-100 times, preferably 5-50 times, more preferably diluted 30 times, 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. For example, acetic acid can be extracted with an organic reagent and then the reaction reagent is added, wherein the volume fraction ratio of the reaction reagent to acetic acid is 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, preferably 5-50 times, and most preferably 30 times.

[0027] Illustratively, after the total acid content of the vinegar sample is determined, the vinegar sample and the acetic acid standard are diluted with water to the same acidity, for example, 0.2 g / 100 mL, 0.5 g / 100 mL, 1 g / 100 mL, 1.2 g / 100 mL, 1.4 g / 100 mL, 1.6 g / 100 mL, 1.8 g / 100 mL, 2.0 g / 100 mL, 2.5 g / 100 mL, 3.0 g / 100 mL, 3.5 g / 100 mL, preferably 3.5 g / 100 mL.

[0028] In the present invention, the gas chromatography-pyrolysis-stable isotope ratio mass spectrometer measures the stable hydrogen isotope ratio of acetic acid in each solution, which is the average value of the isotopic characteristics of the four hydrogen atoms in the acetic acid molecule, including non-exchangeable hydrogen and exchangeable hydrogen.

[0029] Obviously, the gas chromatography-pyrolysis-stable isotope ratio mass spectrometer is equipped with a capillary chromatography column.

[0030] In the present invention, the vinegar is, for example, a product whose vinegar sample is labeled as edible vinegar or fruit vinegar, such as aged vinegar, rice vinegar, apple vinegar, white vinegar, hawthorn vinegar, etc.

[0031] Furthermore, in the method of the present invention, the conditions of the gas chromatography-pyrolysis-stable isotope ratio mass spectrometer are adjusted as follows: the injection port temperature is 270°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, and the column oven program is a constant temperature of 180°C; the pyrolysis module ensures a constant temperature of 1420°C.

[0032] 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.

[0033] More specifically, the present invention provides a method for determining the isotope ratio of non-exchangeable hydrogen in acetic acid in vinegar, comprising the following steps:

[0034] 1) Determine the total acid content of the vinegar sample;

[0035] 2) separating and extracting acetic acid from the vinegar by liquid-liquid extraction to obtain an extract, and adding a reaction reagent sample solution to the extract, wherein the addition of the reaction reagent can allow exchangeable hydrogen in the sample solution to reach hydrogen isotope exchange equilibrium;

[0036] 3) determining the stable hydrogen isotope ratio of acetic acid in the sample solution using gas chromatography-pyrolysis-stable isotope ratio mass spectrometry;

[0037] 4) treating a standard acetic acid solution with the same organic reagents and reaction reagents as the sample solution in the same or similar proportions to obtain a compositional profile identical or similar to that of the sample solution, and determining the stable hydrogen isotope ratio of the acetic acid in the standard solution by gas chromatography-pyrolysis-stable isotope ratio mass spectrometry;

[0038] 5) A standard curve is constructed based on the given value of the non-exchangeable hydrogen in the acetic acid standard and the measured isotope value of the acetic acid hydrogen in the standard solution, and the isotope ratio of the non-exchangeable hydrogen in the acetic acid in the vinegar sample is obtained by calibration.

[0039] The reaction reagent is methanol and the extraction solvent is ether.

[0040] The operating scheme provided by the present invention provides a rapid and stable method for isotopic analysis of non-exchangeable hydrogen of acetic acid in vinegar and its application in adulteration detection / authenticity assurance. DETAILED DESCRIPTION

[0041] 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.

[0042] Test Example 1 Method stability study

[0043] a) take respectively aged vinegar, rice vinegar, white vinegar, sweet vinegar and glacial acetic acid each one, wherein the acidity of glacial acetic acid simulation common vinegar is diluted 20 times to obtain acetic acid solution, and the total acid content of above-mentioned 5 samples is measured according to the third method of GB 12456-2021, and the result is 6.71g / 100mL, 5.37g / 100mL, 4.52g / 100mL, 3.73g / 100mL and 4.83g / 100mL respectively;

[0044] b) Take 30 mL of each of the four vinegar samples and acetic acid solution and dilute them with water to 3.5 g / 100 mL.

[0045] c) Take 10 mL of each diluted solution, add 10 mL of ether, vortex mix, remove the upper layer solution and set aside. Treat each sample 3 times.

[0046] d) Take 1 mL of each sample solution after extraction, add 0.3 mL of methanol as the reaction reagent, vortex mix, and then test.

[0047] e) A method for determining the hydrogen isotope ratio of ethanol by gas chromatography-pyrolysis-stable isotope ratio mass spectrometry was established: the inlet temperature was 270°C, the gas chromatography (polar bonded divinylbenzene / ethylene glycol dimethacrylate column, 30 m × 0.32 mm × 10 μm) flow rate was a constant 1.2 mL / min, the gas chromatography inlet split ratio was 20:1, and the column oven was programmed for a constant temperature of 180°C; the pyrolysis reaction tube temperature was 1420°C; the determination results are shown in Table 1.

[0048] Table 1 Determination results of δD of acetic acid for various samples (‰)

[0049] Sample type Repeat -1 Repeat -2 Repeat -3 average value Standard Deviation Aged vinegar sample -252.3 -254.4 -256.0 -254.2 1.8 Rice vinegar sample -273.1 -271.5 -271.3 -272.0 1.0 White vinegar sample -221.1 -220.5 -222.7 -221.5 1.1 Sweet vinegar sample -288.7 -290.2 -292.0 -290.3 1.6 Acetic acid solution -167.3 -170.2 -168.4 -168.6 1.5

[0050] As can be seen from the table above, this method studies the repeatability of the pretreatment process. After each sample was processed three times simultaneously, the standard deviation of the measurement results was less than 3‰, which meets the requirements for the determination of hydrogen isotopes by stable isotope ratio mass spectrometry.

[0051] Test Example 2: Verification of Method Accuracy

[0052] a) With respect to the glacial acetic acid in Experimental Example 1 (referred to as glacial acetic acid 1#), two glacial acetic acid samples having an isotope gradient were selected (referred to as glacial acetic acid 2# and glacial acetic acid 3#, respectively);

[0053] b) Determine the D / H values ​​of three glacial acetic acids using nuclear magnetic resonance technology according to the method standard of OIV RESOLUTION OIV-OENO 527-2015, and calculate the δD of the non-exchangeable hydrogen of acetic acid after conversion. CH3 The values ​​were -225.6‰, -41.7‰, and +163.4‰;

[0054] c) Glacial acetic acid 2# and glacial acetic acid 3# were each diluted 20-fold with water to obtain two acetic acid solutions (2# and 3#). The total acid content of the acetic acid solutions was determined according to the third method of GB12456-2021, and the results were 4.91 g / 100 mL and 4.84 g / 100 mL, respectively;

[0055] d) Take 30 mL of each acetic acid solution and dilute with water to 3.5 g / 100 mL.

[0056] e) Take 10 mL of each diluted solution and add 10 mL of ether. Vortex mix thoroughly, remove the upper layer and set aside. Treat each sample three times. Take 1 mL of each extracted sample solution and add 0.3 mL of methanol, the reaction reagent. Vortex mix thoroughly, and then test.

[0057] f) A method for determining the hydrogen isotope ratio of ethanol by gas chromatography-pyrolysis-stable isotope ratio mass spectrometry was established: the inlet temperature was 270°C, the gas chromatography (polar bonded divinylbenzene / ethylene glycol dimethacrylate column, 30 m × 0.32 mm × 10 μm) flow rate was a constant 1.2 mL / min, the gas chromatography inlet split ratio was 20:1, and the column oven was programmed for a constant temperature of 180°C; the pyrolysis reaction tube temperature was 1420°C; the determination results are shown in Table 2.

[0058] Table 2 Determination results of acetic acid δD in glacial acetic acid samples (‰)

[0059] Sample type Repeat -1 Repeat -2 Repeat -3 average value Standard Deviation Glacial acetic acid 1# -167.3 -170.2 -168.4 -168.6 1.5 Glacial acetic acid 2# -23.7 -21.5 -24.5 -23.2 1.6 Glacial acetic acid 3# 128.9 129.1 126.1 128.0 1.7

[0060] As can be seen from the above table, Table 2 has similar repeatability characteristics to Table 1, and the standard deviation is less than 3‰, which meets the requirements for the determination of stable isotopes. The average value of the δD of 3 glacial acetic acid is compared with the δD of the non-exchangeable hydrogen of acetic acid determined by the OIV method. CH3 Linear fitting (δD CH3 =1.3176*δD-7.1564), we can know that R 2 =0.9997, which shows that this method has the same accuracy characteristics as the OIV method. Therefore, the acetic acid δD measured by this method can be used to calculate the acetic acid non-exchangeable hydrogen isotope ratio δD CH3 Analytical calculation.

[0061] Example 1

[0062] a) Taking the data obtained from Table 1 in Test Example 1 and combining it with the standard curve obtained from Table 2 in Test Example 2, the acetic acid δD of the vinegar sample in Test Example 1 can be obtained: CH3 The results are shown in Table 3.

[0063] Table 3 Acetic acid δD of each sample CH3 Analysis test results (‰)

[0064] Sample type Aged vinegar sample Rice vinegar sample White vinegar sample Sweet vinegar sample Acetic acid solution δD value -254.2 -272.0 -221.5 -290.3 -168.6 <![CDATA[δD CH3 Value]]> -342.1 -365.5 -298.9 -389.7 -227.6

[0065] As shown in Table 3, the result of acetic acid solution calibrated according to the standard curve is -227.6‰, which is less than 3‰ different from the result of -225.6‰ determined by the OIV method. Therefore, this method can be used to determine the δD of non-exchangeable hydrogen in acetic acid of various vinegar samples. CH3 value.

[0066] Example 2

[0067] a) take the aged vinegar, rice vinegar and glacial acetic acid sample in test example 1, and 2 glacial acetic acid samples in test example 2 respectively;

[0068] b) taking the extracted samples from Test Example 1 and Test Example 2 for testing;

[0069] c) A method for determining ethanol hydrogen isotope ratios using gas chromatography-pyrolysis-stable isotope ratio mass spectrometry was established: the inlet temperature was 270°C, the gas chromatography (polar wax column with bonded / cross-linked polyethylene glycol stationary phase, 50 m × 0.25 mm × 0.20 μm) flow rate was constant at 1.2 mL / min, the gas chromatography inlet split ratio was 20:1, and the column oven temperature was programmed: starting at 120°C, holding for 1 min, then increasing at 15°C / min to 200°C, and holding for 2 min; the pyrolysis reaction tube temperature was 1420°C; the results of the acetic acid δD determination are shown in Table 4.

[0070] Table 4 Determination results of acetic acid δD of each sample (‰)

[0071] Sample type Aged vinegar sample Rice vinegar sample Glacial acetic acid 1# Glacial acetic acid 2# Glacial acetic acid 3# δD value -266.1 -280.3 -177.6 -36.9 115.1

[0072] As shown in Table 4, the results of the same sample measured under different chromatographic column conditions are different when comparing the data in Table 1 and Table 2. The linear fitting of the measured values ​​of glacial acetic acid and the results of the OIV method can be obtained to obtain the fitting curve δD CH3 =1.3291*δD+9.4197 is slightly different from the linear variance obtained in Experiment 2, but the fitting coefficient R 2 =0.9999, which is consistent with Experiment 2. The fitting curve was used for data calculation to obtain the δD of acetic acid in the aged vinegar and rice vinegar samples. CH3 The results are shown in Table 5.

[0073] Table 5 Acetic acid δD of each sample CH3 Analysis test results (‰)

[0074] Sample type Aged vinegar sample Rice vinegar sample <![CDATA[δD CH3 Value]]> -344.3 -363.1

[0075] Comparing Table 5 and Table 3, we can see that under the two chromatographic conditions, the δD CH3The absolute deviations of the results were all less than 3‰, which met the measurement requirements, indicating that different test conditions did not affect the final results.

[0076] 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 stable isotope ratio of non-exchangeable hydrogen in acetic acid, characterized in that: The steps include: 1) Determine the total acid content of the vinegar sample; after determining the total acid content of the vinegar sample, dilute the vinegar sample and acetic acid standard with water to the same acidity; 2) extracting acetic acid from the vinegar sample by liquid-liquid extraction, adding a reaction reagent to the upper solution after extraction and separation, and vortex mixing to obtain a sample solution; the extraction reagent used in the liquid-liquid extraction is diethyl ether; the reaction reagent is methanol; 3) determining the stable hydrogen isotope ratio of acetic acid in the sample solution using gas chromatography-pyrolysis-stable isotope ratio mass spectrometry; 4) taking a standard acetic acid solution, performing the same extraction and isotope exchange treatment using the same extraction reagents and reaction reagents as the sample in the same proportions, and determining the stable hydrogen isotope ratio of the acetic acid in the standard solution by gas chromatography-pyrolysis-stable isotope ratio mass spectrometry; the stable hydrogen isotope ratio of acetic acid is the characteristic average of the isotopes of the four hydrogen atoms in the acetic acid molecule, including non-exchangeable hydrogen and exchangeable hydrogen; 5) A standard curve is constructed based on the given value of the non-exchangeable hydrogen in the acetic acid standard and the measured isotope value of the acetic acid hydrogen in the standard solution, and the isotope ratio of the non-exchangeable hydrogen in the acetic acid in the vinegar sample is obtained by calibration.

2. The method according to claim 1, characterized in that The volume fraction of the reaction reagent is more than 5 times that of acetic acid in the extraction solution.

3. The method according to claim 1, wherein the volume fraction of the reaction reagent is 5-100 times that of the acetic acid in the extraction solution.

4. The method according to claim 1, wherein The same acidity is 3.5g / 100mL.

5. The method according to claim 1, wherein When using gas chromatography-pyrolysis-stable isotope ratio mass spectrometry for determination, the vinegar sample solution and the standard solution are further diluted with an organic reagent or injected directly.

6. The method according to any one of claims 1 to 5, characterized in that The gas chromatography-pyrolysis-stable isotope ratio mass spectrometer is equipped with a capillary chromatographic column.

7. The method according to any one of claims 1 to 5, wherein the vinegar sample is aged vinegar, rice vinegar, fruit vinegar or white vinegar.