CN116359389A - Pretreatment method for determining acetic acid non-exchangeable hydrogen stable isotope ratio in vinegar - Google Patents
Pretreatment method for determining acetic acid non-exchangeable hydrogen stable isotope ratio in vinegar Download PDFInfo
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- CN116359389A CN116359389A CN202310375206.1A CN202310375206A CN116359389A CN 116359389 A CN116359389 A CN 116359389A CN 202310375206 A CN202310375206 A CN 202310375206A CN 116359389 A CN116359389 A CN 116359389A
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 title claims abstract description 399
- 235000021419 vinegar Nutrition 0.000 title claims abstract description 99
- 239000000052 vinegar Substances 0.000 title claims abstract description 98
- 229910052739 hydrogen Inorganic materials 0.000 title claims abstract description 78
- 239000001257 hydrogen Substances 0.000 title claims abstract description 77
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 title claims abstract description 69
- 238000002203 pretreatment Methods 0.000 title claims abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 37
- 238000006243 chemical reaction Methods 0.000 claims abstract description 21
- 239000000523 sample Substances 0.000 claims description 75
- 238000000034 method Methods 0.000 claims description 60
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 28
- 239000000243 solution Substances 0.000 claims description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 25
- 239000007789 gas Substances 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 18
- 238000005259 measurement Methods 0.000 claims description 18
- 239000012488 sample solution Substances 0.000 claims description 17
- 239000012086 standard solution Substances 0.000 claims description 17
- 238000000605 extraction Methods 0.000 claims description 13
- 238000001546 stable isotope ratio mass spectrometry Methods 0.000 claims description 13
- 235000007164 Oryza sativa Nutrition 0.000 claims description 11
- 235000009566 rice Nutrition 0.000 claims description 11
- 239000002253 acid Substances 0.000 claims description 9
- 238000000622 liquid--liquid extraction Methods 0.000 claims description 8
- 238000000638 solvent extraction Methods 0.000 claims description 8
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 6
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 4
- VZGDMQKNWNREIO-UHFFFAOYSA-N tetrachloromethane Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims description 4
- 230000002378 acidificating effect Effects 0.000 claims description 3
- 235000013399 edible fruits Nutrition 0.000 claims description 3
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- 239000000376 reactant Substances 0.000 claims description 3
- 230000000155 isotopic effect Effects 0.000 claims description 2
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- 240000007594 Oryza sativa Species 0.000 claims 1
- 238000004587 chromatography analysis Methods 0.000 claims 1
- 238000011282 treatment Methods 0.000 claims 1
- 238000004458 analytical method Methods 0.000 abstract description 21
- 235000013305 food Nutrition 0.000 abstract description 9
- -1 carboxyl hydrogen Chemical compound 0.000 abstract description 8
- 238000011160 research Methods 0.000 abstract description 5
- 238000001514 detection method Methods 0.000 abstract description 3
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- 235000011054 acetic acid Nutrition 0.000 description 96
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- 238000012360 testing method Methods 0.000 description 21
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- 238000004817 gas chromatography Methods 0.000 description 8
- 238000001819 mass spectrum Methods 0.000 description 6
- 150000007524 organic acids Chemical class 0.000 description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 5
- 238000005336 cracking Methods 0.000 description 5
- 235000011194 food seasoning agent Nutrition 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- MYRTYDVEIRVNKP-UHFFFAOYSA-N 1,2-Divinylbenzene Chemical compound C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 3
- 150000001242 acetic acid derivatives Chemical class 0.000 description 3
- 235000009508 confectionery Nutrition 0.000 description 3
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- 229940093915 gynecological organic acid Drugs 0.000 description 3
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- DBCAQXHNJOFNGC-UHFFFAOYSA-N 4-bromo-1,1,1-trifluorobutane Chemical compound FC(F)(F)CCCBr DBCAQXHNJOFNGC-UHFFFAOYSA-N 0.000 description 2
- YZCKVEUIGOORGS-OUBTZVSYSA-N Deuterium Chemical compound [2H] YZCKVEUIGOORGS-OUBTZVSYSA-N 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 235000013361 beverage Nutrition 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
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- 229910052805 deuterium Inorganic materials 0.000 description 2
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Substances CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 description 2
- 238000001319 headspace solid-phase micro-extraction Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000002307 isotope ratio mass spectrometry Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- BJEPYKJPYRNKOW-REOHCLBHSA-N (S)-malic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O BJEPYKJPYRNKOW-REOHCLBHSA-N 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 235000009917 Crataegus X brevipes Nutrition 0.000 description 1
- 235000013204 Crataegus X haemacarpa Nutrition 0.000 description 1
- 235000009685 Crataegus X maligna Nutrition 0.000 description 1
- 235000009444 Crataegus X rubrocarnea Nutrition 0.000 description 1
- 235000009486 Crataegus bullatus Nutrition 0.000 description 1
- 235000017181 Crataegus chrysocarpa Nutrition 0.000 description 1
- 235000009682 Crataegus limnophila Nutrition 0.000 description 1
- 235000004423 Crataegus monogyna Nutrition 0.000 description 1
- 240000000171 Crataegus monogyna Species 0.000 description 1
- 235000002313 Crataegus paludosa Nutrition 0.000 description 1
- 235000009840 Crataegus x incaedua Nutrition 0.000 description 1
- 238000005361 D2 NMR spectroscopy Methods 0.000 description 1
- 239000004278 EU approved seasoning Substances 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 206010022998 Irritability Diseases 0.000 description 1
- 239000004698 Polyethylene Substances 0.000 description 1
- 238000006935 Simonis synthesis reaction Methods 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 125000000218 acetic acid group Chemical class C(C)(=O)* 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N alpha-hydroxysuccinic acid Natural products OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 235000013409 condiments Nutrition 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 235000021186 dishes Nutrition 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000010981 drying operation Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 150000002431 hydrogen Chemical class 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- ZDGGJQMSELMHLK-UHFFFAOYSA-N m-Trifluoromethylhippuric acid Chemical compound OC(=O)CNC(=O)C1=CC=CC(C(F)(F)F)=C1 ZDGGJQMSELMHLK-UHFFFAOYSA-N 0.000 description 1
- 239000001630 malic acid Substances 0.000 description 1
- 235000011090 malic acid Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 235000013379 molasses Nutrition 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 235000008373 pickled product Nutrition 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 210000000697 sensory organ Anatomy 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
- G01N1/4044—Concentrating samples by chemical techniques; Digestion; Chemical decomposition
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
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- G—PHYSICS
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7206—Mass spectrometers interfaced to gas chromatograph
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/02—Food
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention provides a pretreatment method for determining the hydrogen isotope ratio of acetic acid in vinegar, which is used for determining the hydrogen isotope ratio of acetic acid in vinegar, and belongs to the technical field of food stable isotope analysis. The pretreatment method comprises the following steps: adding a reaction reagent into the vinegar sample extract, and replacing original carboxyl hydrogen of acetic acid with the exchangeable hydrogen to reach isotope exchange balance. The invention can be used for simply and rapidly determining the isotope ratio of the acetic acid non-exchangeable hydrogen in the vinegar products, and can be used for the detection research and application of the authenticity or adulteration of the vinegar products.
Description
Technical Field
The invention belongs to the field of stable isotope analysis and test, and relates to a pretreatment method for determining the stable isotope ratio of acetic acid in vinegar.
Background
Vinegar is a traditional seasoning in various dishes in China, and is also a necessity in daily life of people. GB 2719-2018, "national standard for food safety vinegar", was formally implemented in 2019, 12, 21, and provides vinegar which is a liquid acidic condiment prepared by fermenting and brewing various materials containing starch and sugar, edible alcohol, singly or in combination, by microorganisms, and is required to be unavailable for vinegar. Whereas the prior formulated products are managed according to GB 31644-2018 national Standard composite seasonings for food safety.
In 2021, 6 months, the national market supervision and administration issues bulletin on strengthening soy and vinegar quality safety supervision and administration, and illegal activities such as "use of raw materials such as glacial acetic acid for preparing and producing vinegar and false labeling of product identifiers" are checked and treated in law. At present, food detection personnel often conduct vinegar authenticity identification research based on characteristic analysis of sense organs, organic acid volatile components and the like, fingerprint spectrum characteristics and the like, but the practical application effect of the existing method is not ideal due to similar physicochemical and sensory characteristics between vinegar and a compound seasoning (vinegar flavor seasoning juice) product.
Foreign researches show that whether industrial acetic acid [ Hermann, A. (2001) Determination of D/H isotope ratio in acetic acid from vinegars and pickled products by H-2-NMR spectrometer. Eur Food Res technical, 212 683-686 is mixed into vinegar beverage can be detected according to the difference of stable hydrogen isotope ratio of non-exchangeable hydrogen of acetic acid; fauhl, C., wittkowski, R. (1996) On-line 1H-NMR to facilitate tube preparation in SNIF-NMR analysis.Z Lebensm Unters Forsch (1996), 541-545; hsieh C W, li P H, cheng J Y, 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 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 which process the stable hydrogen isotope ratio of acetic acid non-exchangeable hydrogen is analyzed using point specific fractionation-nuclear magnetic resonance (SNIF-NMR). For this technology, the European Union related institutions organized inter-laboratory method alignments [ 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 ]. Analytica chimica acta,2009,649 (1): 98-105], validating the assay technology. However, this method has some problems in practical operation: since deuterium is low in abundance in nature (natural abundance is about 150 x 10-6), and the resonance frequency of deuterons is low and the spectral line dispersion degree is low, the sensitivity of the SNIF-NMR method is low, and a large amount of sample (more than 300 mL) and a long analysis time (more than 8 h) are required to obtain satisfactory signal-to-noise ratio signals and measurement accuracy, in addition to acetic acid, water, ethanol and other hydrogen-containing compounds are contained in vinegar, and a special distillation device (Cadiot Column) is required to be used for purifying ethanol before the measurement of the samples, and the distillation device is not only a patent product of european Eurofins company, but also consumes a long time of 6h in the whole sample purification process, and a user also acknowledges that the purification procedure of acetic acid is a bottleneck difficult to exceed in the application process of the technology [ Ko W C, cheng Y, chen Y, et al, optimal extraction method of acetic acid in vinegar and its effect on SNIF-NMRanalysis to control the authenticity of vinegar [ J ]. Food and bioprocess technology,2013,6 (8): 2206 ].
In order to reduce the analysis difficulty, the hydrogen isotope analysis of acetic acid is also researched by utilizing a stable isotope ratio mass spectrum, but the basic principle and the purity requirement of stable isotope technology analysis and test are considered, the influence of water on the conversion of acetic acid into hydrogen is required to be eliminated, and the influence of water in an original solution on the carboxyl hydrogen of acetic acid is required to be eliminated. In 2009 Milanka Glavanovic and Ivan samjloc, a us patent is filed, wherein it is mentioned that acetic acid in a solution can be converted into acetate, a solid sample containing acetate is obtained after drying to remove water and leaching with an organic reagent to remove soluble organic matters, and the solid sample is measured by a stable isotope ratio mass spectrometer after being converted into hydrogen by a high-temperature cracking/elemental analyzer, however, the technology has great defects in principle and technology: in addition to acetic acid, the organic acids of the food matrix, formic acid, oxalic acid, lactic acid, malic acid, etc., are also converted into organic acid salts at the same time when acetic acid is converted into acetic acid salts, and no matter the organic acids have the same physical characteristics as acetic acid salts, the acetic acid salts cannot be removed, so that the obtained "solid samples" are impure. The inventors believe that the method determines the stable hydrogen isotope ratio of acetate, which is indeed that of other organic acids; in addition, acetate is very hygroscopic, and because the process of packing and waiting for sample introduction are open-ended when using pyrolysis/elemental analyzer to convert, moisture is easily absorbed from the air and additional hydrogen atoms are introduced, and then the measurement is inaccurate, this patent is withdrawn/rejected in 2014. There is also a method of Ryota Hattori et al [ 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 japan, which established a headspace solid-phase microextraction method for extracting acetic acid in vinegar and measuring the hydrogen isotope ratio of acetic acid by gas chromatography-cleavage-stable isotope ratio mass spectrometry in 2010, but the method used a headspace solid-phase microextraction technique to achieve separation of acetic acid from other hydrogen-containing components, but the extraction head had a great influence on extraction efficiency and stability, and researchers have questioned the feasibility of the technique for stable isotope analysis, and the technique was measured for the hydrogen isotope ratio of acetic acid as a whole in a system, thus having no practical significance. As such, the feasibility of stable isotope ratio mass spectrometry in acetic acid non-exchangeable hydrogen-hydrogen isotope ratio analysis is not seen at home and abroad, and the technology has not been broken through for about 20 years, so that the 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) ] is formulated by the international grape and wine organization in 2015 by adopting SNIF-NMR technology.
The national patent of invention of the top of the same is "method for measuring stable hydrogen isotope ratio of acetic acid non-exchangeable hydrogen in 2020100815968 vinegar beverage", which adopts: adding an alkaline reagent into vinegar to form an organic acid salt system taking acetate as a main body, removing most of water and volatile alcohol aldehyde ester organic matters in a frozen or heated and dried mode to obtain solid residues, adding a strong acid solution into the solid residues to acidify each organic acid, wherein the solution contains water, acetic acid, organic acid and sulfuric acid, separating the water from the acetic acid by a proper chromatographic column, and determining the stable hydrogen isotope ratio of the acetic acid by using a gas chromatography-cracking-stable isotope ratio mass spectrum (average characteristic of 4 hydrogens), and finally determining the non-exchangeable hydrogen stable hydrogen isotope ratio of the acetic acid by using the stable isotope ratio mass spectrum through standard calibration. This technique is a qualitative breakthrough compared to previous studies, including the method standard relative to OIV: the sample can be pretreated in batches, the test time is reduced from 5 hours to within 20 minutes, the analysis and test cost is greatly reduced, the efficiency is greatly improved, and especially, the dependence on a micro-distillation device of a patent outside OIV China is avoided when the method is popularized. However, the pretreatment process takes a long time (more than 24 hours for freeze drying and more than 6 hours for drying operation) due to the three key steps of alkalization, water removal and acidification involved, especially the water removal process; in addition, since the sample to be measured contains a large amount of water, a special chromatographic column for separating water from acetic acid, which is a polymerizable filler, is required, and there is a risk that the conventional polar column may affect the measurement of hydrogen acetate isotopes due to incomplete separation of water. In order to further improve the analysis efficiency, the invention provides another key thought based on the basic principle and the requirement of stable isotope analysis, and a novel pretreatment method is developed accordingly.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention develops a pretreatment method of a vinegar sample to determine the isotope ratio of an acetic acid non-exchangeable hydrogen site in vinegar. The method is simpler to operate, has high safety coefficient, is suitable for analysis and test of a large number of samples, and is favorable for promoting further research and application of stable isotope technology in the vinegar field. The invention provides a method for measuring isotope ratio of acetic acid non-exchangeable hydrogen sites in vinegar, which can eliminate the influence of water in solution vinegar on the isotope characteristic measurement of acetic acid exchangeable hydrogen, and further can accurately and stably analyze the isotope ratio of acetic acid non-exchangeable hydrogen by utilizing a gas chromatography-cracking-stable isotope ratio mass spectrometry technology, and comprises the following steps:
extracting acetic acid from the vinegar sample by liquid-liquid extraction, and reacting exchangeable hydrogen ions in the reaction reagent with exchangeable hydrogen ions in the acetic acid to achieve the purpose of substitution, thereby obtaining a sample solution.
Further, in the present invention, the reagent is methanol or ethanol.
Further, in the present invention, the volume fraction of the reagent is 5 times or more, preferably 5 to 100 times, more preferably 30 times that of acetic acid in the extraction solution.
Further, the method of the present invention further comprises one, two, three, four or five of the following steps:
1) Determining the total acid content of the vinegar sample;
2) Extracting acetic acid by a liquid-liquid extraction mode to obtain an extract, and adding a reaction reagent into the extract to obtain a sample solution;
3) Determining the stable hydrogen isotope ratio of acetic acid in the sample solution by using a gas chromatography-cleavage-stable isotope ratio mass spectrometry;
4) The method comprises the steps of (1) taking an acetic acid standard solution, treating the acetic acid standard solution with an organic reagent and a reaction reagent which are the same as a sample in the same or similar proportion to enable the acetic acid standard solution to have the same or similar component characteristics as the sample solution, and measuring the stable hydrogen isotope ratio of acetic acid in the standard solution by using gas chromatography-cracking-stable isotope ratio mass spectrometry;
5) And constructing a standard curve according to the given value of the non-exchangeable hydrogen of the acetic acid standard substance and the isotope measurement value of the acetic acid hydrogen of the standard substance solution, and correcting to obtain the isotope ratio of the non-exchangeable hydrogen of the acetic acid in the acetic acid sample.
Further, in the present invention, acetic acid is extracted from the vinegar sample using an organic reagent such as ethyl acetate, n-hexane, petroleum ether, chloroform, carbon tetrachloride, diethyl ether and the like, preferably diethyl ether at the time of liquid-liquid extraction.
Further, in the present invention, in order to facilitate the batch sample operation, the vinegar sample is diluted with water to the same acidity after the total acid content is measured, and then the same extraction is performed, and the same reagent is added, with the acidity preferably being 3.5g/100mL.
Further, in the present invention, the vinegar-like solution and the standard solution are further diluted with an organic reagent to a proper sample concentration or directly injected when measured by gas chromatography-cleavage-stable isotope ratio mass spectrometry.
Further, in the present invention, the gas chromatograph-cracker-stable isotope ratio mass spectrometer determines the stable hydrogen isotope ratio of acetic acid in each solution, which is the isotope characteristic average value of 4 hydrogen atoms of acetic acid molecules, including non-exchangeable hydrogen and exchangeable hydrogen.
Further, in the present invention, a capillary column is provided in the gas chromatography-fragmentation-stable isotope ratio mass spectrometer.
Further, in the present invention, wherein the vinegar sample is mature vinegar, rice vinegar, fruit vinegar, white vinegar, or an acidic seasoning liquid having characteristics of a vinegar sample.
Illustratively, the present invention replaces the original exchangeable hydrogen in the vinegar-like acetic acid with a reactant to bring the exchangeable hydrogen to a hydrogen isotope exchange equilibrium, wherein the reactant, such as methanol, ethanol, etc., may be directly added to the vinegar-like; alternatively, acetic acid may be extracted by liquid-liquid extraction followed by isotope exchange with the reagent. Preferably, the reagent is added after extraction to achieve hydrogen isotope exchange equilibrium.
In the method of the present invention, the standard is treated with the same organic reagent and the same reaction reagent in the same or similar proportion as the sample so as to have the same or similar component characteristics as the sample solution, and the stable hydrogen isotope ratio of acetic acid in the standard solution is determined by gas chromatography-cleavage-stable isotope ratio mass spectrometry, characterized in that the isotope characteristics of exchangeable hydrogen in the sample solution and the standard solution are consistent. In the method of the invention, when gas chromatography-cleavage-stable isotope ratio mass spectrometry is used, the vinegar sample and the standard solution are diluted to proper sample injection concentration by using an organic reagent or water, or are directly injected.
The organic reagent used for extraction in the present invention is one or more of alkanes, esters, alcohols, ethers, etc. which are anhydrous and have miscibility with acetic acid, preferably diethyl ether.
Illustratively, the replacement of the original exchangeable hydrogen in acetic acid in the vinegar-like reaction with a reagent that is 5-100 times, preferably 5-50 times, more preferably 30 times diluted, e.g. 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 more than the molecule having exchangeable hydrogen in the sample. For example, acetic acid may be extracted with an organic reagent and then a reagent may be added, wherein the volume fraction of 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 to 50 times, most preferably 30 times.
Illustratively, the vinegar sample and acetic acid standard are diluted with water to the same acidity after the total acid content is determined, for example 0.2g/100mL, 0.5g/100mL, 1g/100mL, 1.2g/100mL, 1.4g/100mL, 1.6g/100mL, 1.8g/100mL, 2.0g/100mL, 2.5g/100mL, 3.0g/100mL, 3.5g/100mL, preferably 3.5g/100mL.
In the present invention, the gas chromatograph-cracker-stable isotope ratio mass spectrometer determines the stable hydrogen isotope ratio of acetic acid in each solution, which is the isotope characteristic average value of 4 hydrogen atoms of acetic acid molecules, including non-exchangeable hydrogen and exchangeable hydrogen.
Obviously, a capillary chromatographic column is provided in the gas chromatograph-split-stable isotope ratio mass spectrometer.
The vinegar in the invention is, for example, a product with a vinegar sample labeled as edible vinegar or fruit vinegar, such as mature vinegar, rice vinegar, apple vinegar, white vinegar, hawthorn vinegar, etc.
Further, in the method of the present invention, the condition parameters of the gas chromatograph-split-stable isotope ratio mass spectrometer are adjusted: the temperature of the sample inlet is 270 ℃, the sample inlet volume is 2uL, the gas chromatography flow rate is constant current 1.2mL/min, the split ratio of the gas chromatography sample inlet is 20:1, and the column temperature box program is constant temperature of 180 ℃; the cracking module ensures constant temperature of 1420 ℃.
Optionally, confirming stable parityThe working environment, air tightness and vacuum degree of an ion chamber of the element ratio mass spectrometer all meet the analysis requirements, and then an inspection instrument measures H 2 Precision and linearity of δd, and ion source parameter values are adjusted as necessary.
More specifically, the invention provides a method for determining the isotope ratio of acetic acid non-exchangeable hydrogen in vinegar, which comprises the following steps:
1) Determining the total acid content of the vinegar sample;
2) Separating and extracting acetic acid from vinegar by liquid-liquid extraction to obtain an extract, and adding a reaction reagent sample solution into the extract, wherein the addition of the reaction reagent can lead exchangeable hydrogen in the sample solution to reach hydrogen isotope exchange balance;
3) Determining the stable hydrogen isotope ratio of acetic acid in the sample solution by using a gas chromatography-cleavage-stable isotope ratio mass spectrometry;
4) The method comprises the steps of taking an acetic acid standard solution, treating the acetic acid standard solution with the same organic reagent and the same reaction reagent in the same or similar proportion as a sample to ensure that the acetic acid standard solution has the same or similar component characteristics as the sample solution, and measuring the stable hydrogen isotope ratio of acetic acid in the standard solution by using gas chromatography-cracking-stable isotope ratio mass spectrometry;
5) And constructing a standard curve according to the given value of the non-exchangeable hydrogen of the acetic acid standard substance and the isotope measurement value of the acetic acid hydrogen of the standard substance solution, and correcting to obtain the isotope ratio of the non-exchangeable hydrogen of the acetic acid in the acetic acid sample.
Wherein the reaction reagent is methanol, and the extraction solvent is diethyl ether.
The operation scheme provided by the invention provides a rapid and stable method for isotope analysis of acetic acid in vinegar and detection/authenticity guarantee of adulteration.
Detailed Description
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
Test example 1Method stability study
a) Respectively taking mature vinegar, rice vinegar, white vinegar, sweet vinegar and glacial acetic acid, wherein the glacial acetic acid simulates the acidity of common vinegar and is diluted by 20 times to obtain acetic acid solution, and determining the total acid content of the 5 samples according to a third method of GB 12456-2021, wherein the results are 6.71g/100mL, 5.37g/100mL, 4.52g/100mL,3.73g/100mL and 4.83g/100mL respectively;
b) The above 4 vinegar samples and acetic acid solutions were each taken at 30mL and diluted with water to 3.5g/100mL.
c) 10mL of each diluted solution is taken out, 10mL of diethyl ether is added respectively, after vortex mixing is carried out, the upper layer solution is taken out for standby, and each sample is treated for 3 times.
d) Taking 1mL of each sample solution after extraction, taking methanol as a reaction reagent, adding 0.3mL of each reaction reagent, and uniformly mixing by vortex to obtain a sample to be measured.
e) The method for determining the ethanol hydrogen isotope ratio by establishing a gas chromatography-cracking-stable isotope ratio mass spectrum comprises the following steps: the temperature of the sample inlet is 270 ℃, the flow rate of gas chromatography (a polar bonding divinylbenzene/ethylene glycol dimethacrylate column is 30m multiplied by 0.32mm multiplied by 10 mu m) is constant current 1.2mL/min, the split ratio of the gas chromatography sample inlet is 20:1, and the column temperature box program is constant temperature of 180 ℃; the temperature of the cracking reaction tube is 1420 ℃; the measurement results are shown in Table 1.
TABLE 1 determination of various samples acetic acid delta D (mill)
| Sample type | Repeat-1 | Repeat-2 | Repeat-3 | Average value of | Standard deviation of |
| Mature 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 |
As can be seen from the table, the method researches the repeatability of the pretreatment process, and the standard deviation of the measurement results after each sample is simultaneously treated for 3 times is less than 3 per mill, so that the method meets the requirement of measuring hydrogen isotopes by a stable isotope ratio mass spectrometer.
Test example 2 method accuracy verification
a) Two glacial acetic acid samples with isotopic gradients (labeled glacial acetic acid # 2 and glacial acetic acid # 3 respectively) were alternatively selected relative to glacial acetic acid in test example 1 (labeled glacial acetic acid # 1);
b) Determination of D/H values of 3 glacial acetic acids by nuclear magnetic resonance techniques according to the method standard of OIV RESOLUTION OIV-OENO 527-2015, and conversion of the acetic acid to δD without hydrogen exchange CH3 The values are-225.6 per mill, -41.7 per mill and +163.4 per mill respectively;
c) 2# glacial acetic acid and 3# glacial acetic acid are diluted by 20 times with water to obtain two acetic acid solutions (2 # and 3# respectively), and the total acid content of the acetic acid solutions is respectively 4.91g/100mL and 4.84g/100mL according to the third method of GB 12456-2021;
d) 30mL of each acetic acid solution was diluted with water to 3.5g/100mL.
e) 10mL of each diluted solution is taken out, 10mL of diethyl ether is added respectively, after vortex mixing is carried out, the upper layer solution is taken out for standby, and each sample is treated for 3 times. Taking 1mL of each sample solution after extraction, adding 0.3mL of reaction reagent methanol respectively, and mixing uniformly by vortex to obtain a sample solution to be measured.
f) The method for determining the ethanol hydrogen isotope ratio by establishing a gas chromatography-cracking-stable isotope ratio mass spectrum comprises the following steps: the temperature of the sample inlet is 270 ℃, the flow rate of gas chromatography (a polar bonding divinylbenzene/ethylene glycol dimethacrylate column is 30m multiplied by 0.32mm multiplied by 10 mu m) is constant current 1.2mL/min, the split ratio of the gas chromatography sample inlet is 20:1, and the column temperature box program is constant temperature of 180 ℃; the temperature of the cracking reaction tube is 1420 ℃; the measurement results are shown in Table 2.
TABLE 2 determination of acetic acid delta D in glacial acetic acid samples (mill)
| Sample type | Repeat-1 | Repeat-2 | Repeat-3 | Average value of | Standard deviation of |
| 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 |
As can be seen from the above table, the table 2 has similar repeatability characteristics as the table 1, the standard deviation is less than 3 per mill, and the stable isotope determination requirements are met. Delta D of acetic acid non-exchangeable hydrogen measured by the method of OIV and the average of 3 glacial acetic acid delta D CH3 The values were fitted linearly (δD CH3 Let R be defined as = 1.3176 ×δd-7.1564) 2 =0.9997, which can indicate that the method has the same accuracy characteristics as the OIV method. Thus, the acetic acid δD measured by the method can be used for the acetic acid non-exchangeable hydrogen isotope ratio δD CH3 Is a result of the analysis and calculation of (a).
Example 1
a) The data obtained in Table 1 of test example 1 were taken together with the standard curve obtained in Table 2 of test example 2 to obtain acetic acid δD of the vinegar sample of test example 1 CH3 Values, results are shown in Table 3.
TABLE 3 acetic acid delta D for each sample CH3 Analysis of test results (mill)
| Sample type | Mature vinegar sample | Rice vinegar sample | White vinegar sample | Sweet vinegar sample | Acetic acid solution |
| Delta D value | -254.2 | -272.0 | -221.5 | -290.3 | -168.6 |
| δD CH3 Value of | -342.1 | -365.5 | -298.9 | -389.7 | -227.6 |
As can be seen from Table 3, the difference between the result of calibration of acetic acid solution according to the standard curve of-227.6%o and the result of measurement of-225.6%o by OIV method is less than 3%o, therefore, the method can be used for measuring the delta D of various acetic acid samples of which acetic acid is not exchangeable hydrogen CH3 Values.
Example 2
a) Taking mature vinegar, rice vinegar and glacial acetic acid samples in test example 1 and 2 glacial acetic acid samples in test example 2 respectively;
b) Taking samples after extraction of each sample in test example 1 and test example 2, and testing the samples;
c) The method for determining the ethanol hydrogen isotope ratio by establishing a gas chromatography-cracking-stable isotope ratio mass spectrum comprises the following steps: the temperature of the sample inlet is 270 ℃, the flow rate of gas chromatography (a polar wax column of a bonding/crosslinking polyethylene alcohol stationary phase is 50m multiplied by 0.25mm multiplied by 0.20 mu m) is constant current 1.2mL/min, the split ratio of the gas chromatography sample inlet is 20:1, and the temperature of a column temperature box is programmed: maintaining at 120deg.C for 1min, heating to 200deg.C at 15deg.C/min, and maintaining for 2min; the temperature of the cracking reaction tube is 1420 ℃; the results of the acetic acid δd measurement are shown in table 4.
TABLE 4 measurement results of acetic acid delta D for each sample (mill)
| Sample type | Mature vinegar sample | Rice vinegar sample | Glacial acetic acid 1# | Glacial acetic acid 2# | Glacial acetic acid 3# |
| Delta D value | -266.1 | -280.3 | -177.6 | -36.9 | 115.1 |
As can be seen from Table 4, comparing the data of tables 1 and 2, the results of the same sample were different under different column conditions. The measurement value of glacial acetic acid and the measurement result of OIV method are subjected to linear fitting to obtain a fitting curve delta D CH3 The linear variance obtained in test example 2 is slightly different from = 1.3291 δd+9.4197, but the fitting coefficient R 2 =0.9999, in accordance with test example 2. The fitting curve is used for data calculation to obtain delta D of acetic acid of mature vinegar and rice vinegar samples CH3 Values, results are shown in Table 5.
TABLE 5 acetic acid delta D for each sample CH3 Analysis of test results (mill)
| Sample type | Mature vinegar sample | Rice vinegar sample |
| δD CH3 Value of | -344.3 | -363.1 |
As can be seen from a comparison of tables 5 and 3, the same sample was shown to have a delta D under both chromatographic conditions CH3 The absolute deviation of the results is less than 3 per mill, and meets the measurement requirements, which shows that different test conditions do not influence the final result.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (10)
1. A pretreatment method for determining the isotope ratio of acetic acid non-exchangeable hydrogen in vinegar, which is used for determining the isotope ratio of acetic acid non-exchangeable hydrogen in vinegar, comprising the following steps:
acetic acid is extracted from the vinegar sample, and a reaction reagent is added to obtain a sample solution.
2. The method according to claim 1, characterized in that the reactant is methanol or ethanol.
3. The method according to claim 1, wherein the volume fraction of the reagent is more than 5 times, preferably 5-100 times, more preferably 30 times the volume fraction of acetic acid in the extraction solution.
4. The method of claim 1, further comprising one, two, three, four, or five of the following steps:
1) Determining the total acid content of the vinegar sample;
2) Extracting acetic acid by a liquid-liquid extraction mode, and adding a reaction reagent into the extract to obtain a sample solution;
3) Determining the stable hydrogen isotope ratio of acetic acid in the sample solution by using a gas chromatography-cleavage-stable isotope ratio mass spectrometry;
4) The method comprises the steps of (1) taking an acetic acid standard solution, treating the acetic acid standard solution with an organic reagent and a reaction reagent which are the same as a sample in the same or similar proportion to enable the acetic acid standard solution to have the same or similar component characteristics as the sample solution, and measuring the stable hydrogen isotope ratio of acetic acid in the standard solution by using gas chromatography-cracking-stable isotope ratio mass spectrometry;
5) And constructing a standard curve according to the given value of the non-exchangeable hydrogen of the acetic acid standard substance and the isotope measurement value of the acetic acid hydrogen of the standard substance solution, and correcting to obtain the isotope ratio of the non-exchangeable hydrogen of the acetic acid in the acetic acid sample.
5. The method according to claim 1, characterized in that the acetic acid is extracted from the vinegar sample during liquid-liquid extraction using an organic reagent, which is ethyl acetate, n-hexane, petroleum ether, chloroform, carbon tetrachloride, diethyl ether, etc., preferably diethyl ether.
6. The method according to claim 4, wherein for facilitating the batch sample operation, the vinegar sample is diluted with water to the same acidity as the acetic acid standard after the vinegar sample is assayed for total acid content, and then subjected to the same extraction and isotope exchange treatments, the acidity is preferably 3.5g/100mL.
7. The method of claim 1, wherein the vinegar-like solution and the standard solution are further diluted with an organic reagent or injected directly when measured by gas chromatography-cleavage-stable isotope ratio mass spectrometry.
8. The method of any one of claims 1-7, wherein the gas chromatograph-cracker-stable isotope ratio mass spectrometer determines the stable hydrogen isotope ratio of acetic acid in each solution, which is the isotopic characteristic average of 4 hydrogen atoms of acetic acid molecules, including non-exchangeable hydrogen and exchangeable hydrogen.
9. The method according to any one of claims 1-7, wherein a capillary chromatography column is provided in the gas chromatography-fragmentation-stable isotope ratio mass spectrometer.
10. The method of any one of claims 1-7, wherein the vinegar sample is mature vinegar, rice vinegar, fruit vinegar, white vinegar, or an acidic flavoring liquid having characteristics of a vinegar sample, optionally extracting acetic acid from the vinegar sample by liquid-liquid extraction.
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