CN118090992A - Method for detecting content of gamma-aminobutyric acid in wine - Google Patents
Method for detecting content of gamma-aminobutyric acid in wine Download PDFInfo
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- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 title claims abstract description 110
- 235000014101 wine Nutrition 0.000 title claims abstract description 70
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 title claims abstract description 55
- 229960003692 gamma aminobutyric acid Drugs 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 46
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- 238000001514 detection method Methods 0.000 claims abstract description 8
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 14
- 238000012360 testing method Methods 0.000 claims description 14
- 238000001914 filtration Methods 0.000 claims description 13
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- 229910021641 deionized water Inorganic materials 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000012085 test solution Substances 0.000 claims description 10
- VTVWTPGLLAELLI-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzenesulfonyl chloride Chemical class C1=CC(N(C)C)=CC=C1N=NC1=CC=C(S(Cl)(=O)=O)C=C1 VTVWTPGLLAELLI-UHFFFAOYSA-N 0.000 claims description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims description 7
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- 229910000030 sodium bicarbonate Inorganic materials 0.000 claims description 7
- 238000010812 external standard method Methods 0.000 claims description 5
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 5
- 238000002347 injection Methods 0.000 claims description 5
- 239000007924 injection Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- BDKLKNJTMLIAFE-UHFFFAOYSA-N 2-(3-fluorophenyl)-1,3-oxazole-4-carbaldehyde Chemical compound FC1=CC=CC(C=2OC=C(C=O)N=2)=C1 BDKLKNJTMLIAFE-UHFFFAOYSA-N 0.000 claims description 4
- 235000013532 brandy Nutrition 0.000 claims description 4
- 238000001212 derivatisation Methods 0.000 claims description 4
- 235000013533 rum Nutrition 0.000 claims description 4
- 235000017281 sodium acetate Nutrition 0.000 claims description 4
- 229940087562 sodium acetate trihydrate Drugs 0.000 claims description 4
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- FINHMKGKINIASC-UHFFFAOYSA-N Tetramethylpyrazine Chemical compound CC1=NC(C)=C(C)N=C1C FINHMKGKINIASC-UHFFFAOYSA-N 0.000 description 2
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Classifications
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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
-
- 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
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
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- Physics & Mathematics (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
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Abstract
The invention discloses a method for detecting the content of gamma-aminobutyric acid in wine, and belongs to the technical field of detection. The method for detecting the content of the gamma-aminobutyric acid in the wine comprises the steps of concentrating the gamma-aminobutyric acid in the wine through rotary evaporation, and carrying out qualitative and quantitative determination by adopting a high performance liquid chromatograph. The method can accurately measure the content of the gamma-aminobutyric acid in the wine sample, and can effectively solve the problems that the existing detection method has high requirements on instruments and equipment and limits the popularity of the method.
Description
Technical Field
The invention belongs to the technical field of detection, and particularly relates to a method for detecting the content of gamma-aminobutyric acid in wine.
Background
Chinese white spirit has a long history and is mainly brewed by taking grain crops such as wheat, sorghum, corn, rice, peas and the like as main raw materials through main production processes such as starter making, beam steaming, material mixing, stacking fermentation, pit fermentation, wine steaming and the like. The white spirit has various types, and can be classified into a faint scent type, a strong scent type, a sauce scent type, a rice scent type and other various scent types due to the difference of the scent types. The distilled spirit is a unique distilled spirit in China, has unique brewing style and taste, is known worldwide, and is one of six distilled spirits which are similar to other famous distilled spirits in the world (such as brandy, whiskey, rum and the like).
The production of white spirit is subjected to a mixed fermentation process of various microorganisms, and in the process, the microorganisms can metabolize to generate various functional components such as tetramethylpyrazine, gamma-aminobutyric acid, nucleoside substances and the like. Gamma-aminobutyric acid is a natural amino acid, exists in natural foods and fermentation processes of microorganisms such as saccharomycetes and lactobacillus, and mainly plays a role in inhibiting neurotransmitters in human bodies. Studies have shown that proper intake of gamma-aminobutyric acid can improve sleep, alleviate anxiety, and gamma-aminobutyric acid can regulate body immunity by affecting macrophage activation reaction.
The high performance liquid chromatography has the advantages of high separation efficiency, high analysis speed, small sample consumption and the like, and the method is widely used for measuring the content of various components in biological fermentation, and the used mobile phase is simple and has good reproducibility. The method for detecting gamma-aminobutyric acid in the wine by high performance liquid chromatography method is established, lays a foundation for further standardized quality evaluation of Chinese white spirit and development of related research, and has positive effects and significance for guiding production improvement, marketing and deep scientific research of Chinese white spirit. Chinese patent CN106442766A discloses a method for rapidly detecting gamma-aminobutyric acid in white spirit, which is to accurately measure 50mL of white spirit sample to be detected in a 100mL rotary evaporation bottle, rotary evaporate the white spirit sample to 3-5mL, remove alcohol substances in the white spirit to be detected, then use ultrapure water to fix volume to 10mL, finally filter the white spirit sample by a 0.22 mu m filter membrane needle filter to obtain a sample to be detected, and detect the sample by an ultra-high performance liquid chromatograph tandem triple quadrupole mass spectrometer. Chinese patent CN106970165A discloses a method for measuring gamma-aminobutyric acid in wine, which directly takes 5mL of sample from wine in test tube, and places the test tube in boiling water bath, removes ethanol, cools, then fixes volume to 5mL, filters with 0.45 μm filter membrane as test solution, and uses liquid chromatography quadrupole tandem mass spectrometry to measure. The existing patent method uses a liquid chromatography mass spectrometer, has high requirements on the instrument, and limits the popularity of the method.
Disclosure of Invention
The invention aims to solve the technical problems that the existing method for detecting the content of gamma-aminobutyric acid in wine has high requirements on instruments and limits the popularity of the method.
The technical scheme adopted for solving the technical problems is as follows: the method for detecting the content of gamma-aminobutyric acid in the wine comprises the following steps:
(1) Preparing a sample to be tested: after alcohol is removed by rotary evaporation of the wine sample, dissolving the sample by deionized water, and filtering to obtain a sample test solution to be tested;
(2) And (3) derivatization treatment: accurately sucking a sample test solution and a standard working solution into a test tube with a plug, adding sodium bicarbonate solution and a 4-dimethylaminoazobenzene-4' -sulfonyl chloride derivative reagent, uniformly mixing, performing a derivative reaction, filtering by a microporous filter membrane, and then testing;
(3) Preparing a gamma-aminobutyric acid standard working solution: preparing a standard working solution of gamma-aminobutyric acid in a gradient way;
(4) And (3) measuring the standard working solution and the sample testing solution to be tested by adopting a high performance liquid chromatograph, and quantifying by an external standard method to obtain the gamma-aminobutyric acid content in the wine.
Wherein in the step (1), the wine sample is at least one of distilled wine, fermented wine, liqueur and compound wine.
Further, the distilled liquor is at least one of white spirit, brandy, whiskey, vodka, rum, juniper berry wine, agave or fruit distilled liquor.
Further, the fermented wine is at least one of yellow wine, beer, wine, fruit wine, sake or milk wine.
Wherein, in the step (1), the volume ratio of the initial wine sample to the deionized water is 100:1-2.
Wherein, in the steps (1) and (2), a filter membrane with the diameter of 0.10-0.45 μm is adopted for filtration.
Wherein, in the step (2), the concentration of the sodium bicarbonate solution is 0.04g/mL, and the concentration of the 4-dimethylaminoazobenzene-4' -sulfonyl chloride derivative reagent is 2.0mg/mL.
Wherein, in the step (3), the concentration gradient of the prepared standard working solution is 5.0mg/L, 10.0mg/L, 20.0mg/L, 30.0mg/L, 40.0mg/L and 60.0mg/L respectively.
Wherein, in the step (4), the high performance liquid chromatography conditions include:
Chromatographic column: a C 18 column, 150mm by 4.6mm,5 μm, or a chromatographic column with equivalent performance;
column temperature: 25-30 ℃;
Mobile phase: acetonitrile+6.8 g/L sodium acetate trihydrate (35+65);
flow rate: 0.8-1.0mL/min;
Detection wavelength: 436nm;
sample injection amount: 10-20 mu L.
The beneficial effects of the invention are as follows: the invention weighs gamma-aminobutyric acid standard substance, uses absolute ethyl alcohol to dissolve and constant volume as standard stock solution. Different amounts of standard stock solutions were drawn up for dilution to prepare a series of standard working solutions of concentration. And (3) measuring the standard working solution and the sample testing solution by adopting a high performance liquid chromatograph, and quantifying by an external standard method. Thereby establishing a method for measuring the content of the gamma-aminobutyric acid in the wine and providing reliable technical support for analysis and research of the gamma-aminobutyric acid in the wine. The invention is applied to the determination of the gamma-aminobutyric acid content in the wine, and has important significance for researching the health promotion effect of the wine.
Drawings
FIG. 1 is a high performance liquid chromatogram of a gamma-aminobutyric acid standard solution;
FIG. 2 is a high performance liquid chromatogram of yellow wine samples.
Detailed Description
The technical scheme of the invention can be implemented in the following way.
The method for detecting the content of gamma-aminobutyric acid in the wine comprises the following steps:
(1) Preparing a sample to be tested: after alcohol is removed by rotary evaporation of the wine sample, dissolving the sample by deionized water, and filtering to obtain a sample test solution to be tested;
(2) And (3) derivatization treatment: accurately sucking a sample test solution and a standard working solution into a test tube with a plug, adding sodium bicarbonate solution and a 4-dimethylaminoazobenzene-4' -sulfonyl chloride derivative reagent, uniformly mixing, performing a derivative reaction, filtering by a microporous filter membrane, and then testing;
(3) Preparing a gamma-aminobutyric acid standard working solution: preparing a standard working solution of gamma-aminobutyric acid in a gradient way;
(4) And (3) measuring the standard working solution and the sample testing solution to be tested by adopting a high performance liquid chromatograph, and quantifying by an external standard method to obtain the gamma-aminobutyric acid content in the wine.
Wherein in the step (1), the wine source is at least one of distilled wine, fermented wine, liqueur and compound wine. Preferably, the distilled liquor is at least one of white spirit, brandy, whiskey, vodka, rum, juniper berry wine, agave or fruit distilled liquor; the fermented wine is at least one of yellow wine, beer, wine, fruit wine, sake or milk wine.
In the step (1), taking a wine sample, performing rotary evaporation to obtain a near dry state, then, using deionized water to fix the volume to a scale, and filtering by using a filter membrane with the diameter of 0.10-0.45 mu m to obtain a sample test solution to be tested; the volume ratio of the initial wine sample to the deionized water is 100:1-2.
Wherein, in the step (2), the concentration of the sodium bicarbonate solution is 0.04g/mL, and the concentration of the 4-dimethylaminoazobenzene-4' -sulfonyl chloride derivative reagent is 2.0mg/mL.
Wherein, in the step (3), the concentration gradient of the mixed standard working solution is 5.0mg/L, 10.0mg/L, 20.0mg/L, 30.0mg/L, 40.0mg/L and 60.0mg/L respectively.
Wherein, in the step (4), the high performance liquid chromatography conditions include:
Chromatographic column: c18 column, 150mm 4.6mm,5 μm, or equivalent performance chromatographic column;
column temperature: 25-30 ℃;
Mobile phase: acetonitrile+6.8 g/L sodium acetate trihydrate (35+65);
flow rate: 0.8-1.0mL/min;
Detection wavelength: 436nm;
sample injection amount: 10-20 mu L.
The technical scheme and effect of the present invention will be further described by practical examples.
Examples
Example 1 determination of the content of gamma-aminobutyric acid in wine
1. Treatment of wine samples
Taking 100mL of white spirit sample, rotary evaporating to dryness, dissolving with 1mL of deionized water, and filtering with a 0.45 μm filter membrane to obtain a sample test solution. Taking 50mL of absolute ethyl alcohol and 50mL of water, preparing a blank sample, taking 100mL of blank sample, performing rotary evaporation at 55 ℃ for 130r/min until the blank sample is dry, dissolving the blank sample with 1mL of deionized water, and filtering the solution with a 0.45 mu m filter membrane to obtain a blank sample test solution. The rotary evaporation is to completely dry, and aims to remove volatile components in a sample, retain non-volatile components, and fix the volume with deionized water after evaporation.
2. Derivatization treatment
Accurately sucking 1mL of sample solution and standard working solution into a test tube with a plug, adding 0.04g/mL of sodium bicarbonate solution and 2.0mg/mL of 4-dimethylaminoazobenzene-4' -sulfonyl chloride derivative reagent into the test tube, uniformly mixing, carrying out derivative reaction for 20min in a water bath at 70 ℃, and filtering with a microporous filter membrane of 0.45 mu m, and measuring.
3. Instrument measurement
The sample and standard working solution (standard working solution preparation is shown as 4) are measured according to the conditions of the high performance liquid chromatograph, the concentration of the sample solution is corrected by a standard curve, and the sample is quantified by an external standard method.
Chromatographic conditions:
Chromatographic column: a C 18 column, 150mm by 4.6mm,5 μm, or a chromatographic column with equivalent performance;
column temperature: 25-30 ℃;
Mobile phase: acetonitrile+6.8 g/L sodium acetate trihydrate (35+65);
flow rate: 0.8-1.0mL/min;
Detection wavelength: 436nm;
sample injection amount: 10-20 mu L.
4. Linear range
Preparation of standard stock solution of gamma-aminobutyric acid: accurately weighing 10.0mg of gamma-aminobutyric acid reference substance into a 10mL volumetric flask, preparing standard stock solution with concentration of about 1000mg/L by deionized water, standing at-18deg.C, sealing, and storing for 3 months.
Preparing a gamma-aminobutyric acid standard working solution: accurately sucking 0.25mL, 0.5mL, 1.0mL, 1.5mL, 2.0mL and 3.0mL of gamma-aminobutyric acid standard stock solution into a 50mL volumetric flask, and fixing the volume to the scale by absolute ethyl alcohol + water=6+4 (V+V). The standard series working solutions with the concentration of 5.0mg/L, 10.0mg/L, 20.0mg/L, 30.0mg/L, 40.0mg/L and 60.0mg/L are obtained, and the standard series working solutions are prepared on site.
And drawing a standard curve by taking the mass concentration of gamma-aminobutyric acid as an abscissa and the peak area as an ordinate to obtain a regression equation and a correlation coefficient. The result shows that the linear relation of the gamma-aminobutyric acid is good within the range of 5.0mg/L-60.0mg/L, the regression equation is y=1.458 x, and the correlation coefficient is 0.9996.
5. Quantitative limits of the method
When the sample injection amount is 10 mu L, comparing the chromatographic signal peak of the gamma-aminobutyric acid with a baseline signal, and calculating the quantitative limit by using the concentration with the signal to noise ratio of about 10 to obtain the quantitative limit of the gamma-aminobutyric acid of 0.01mg/L.
6. Precision of the method
And (3) adding a mark into the blank sample for precision experiments, measuring 6 parts of the blank sample in parallel, extracting according to a pretreatment method, and performing on-machine measurement, wherein the mark adding concentration of gamma-aminobutyric acid is 2.0 mg/L. The results of 6 times of measurement of gamma-aminobutyric acid are 2.12mg/L, 2.03mg/L, 2.08mg/L, 2.16mg/L, 1.97mg/L and 1.99mg/L respectively, the average value of 6 times of measurement is 2.06mg/L, and the Relative Standard Deviation (RSD) is 3.63% and less than 5%, which indicates that the method precision is good.
7. Accuracy of the method
The three-level six-parallel labeling recovery experiment is carried out in a blank sample, the labeling concentration is respectively 1.0mg/L, 2.0mg/L and 3.0mg/L, the extraction is carried out according to the sample pretreatment method, and the measurement is carried out on the sample, and the result is shown in the table 1 below. The recovery rate of gamma-aminobutyric acid is 94.21-103.20%, and the relative standard deviation is 1.37-4.05%, which shows that the recovery rate of the method is good and the accuracy is high.
Table 1 gamma-aminobutyric acid recovery experimental results (n=6)
Through the method verification, the gamma-aminobutyric acid content in different types of wines can be accurately and precisely detected by adopting the detection method, and the method has good application value in the field of wine brewing.
Claims (10)
1. The method for detecting the content of gamma-aminobutyric acid in the wine is characterized by comprising the following steps of:
(1) Preparing a sample to be tested: after alcohol is removed by rotary evaporation of the wine sample, dissolving the sample by deionized water, and filtering to obtain a sample test solution to be tested;
(2) And (3) derivatization treatment: accurately sucking a sample test solution and a standard working solution into a test tube with a plug, adding sodium bicarbonate solution and a 4-dimethylaminoazobenzene-4' -sulfonyl chloride derivative reagent, uniformly mixing, performing a derivative reaction, filtering by a microporous filter membrane, and then testing;
(3) Preparing a gamma-aminobutyric acid standard working solution: preparing a standard working solution of gamma-aminobutyric acid in a gradient way;
(4) And (3) measuring the standard working solution and the sample testing solution to be tested by adopting a high performance liquid chromatograph, and quantifying by an external standard method to obtain the gamma-aminobutyric acid content in the wine.
2. The method for detecting the content of gamma-aminobutyric acid in wine according to claim 1, wherein the method comprises the following steps: in the step (1), the wine sample is at least one of distilled wine, fermented wine, liqueur and compound wine.
3. The method for detecting the content of gamma-aminobutyric acid in wine according to claim 2, wherein the method comprises the following steps: the distilled liquor is at least one of Chinese liquor, brandy, whiskey, vodka, rum, juniper berry wine, agave or fruit distilled liquor.
4. The method for detecting the content of gamma-aminobutyric acid in wine according to claim 2, wherein the method comprises the following steps: the fermented wine is at least one of yellow wine, beer, wine, fruit wine, sake or milk wine.
5. The method for detecting the content of gamma-aminobutyric acid in wine according to claim 1, wherein the method comprises the following steps: in the step (1), the volume ratio of the initial wine sample to the deionized water is 100:1-2.
6. The method for detecting the content of gamma-aminobutyric acid in wine according to claim 1, wherein the method comprises the following steps: in the step (1), a filter membrane of 0.10-0.45 μm is used for filtration.
7. The method for detecting the content of gamma-aminobutyric acid in wine according to claim 1, wherein the method comprises the following steps: in the step (2), the concentration of the sodium bicarbonate solution is 0.04g/mL, and the concentration of the 4-dimethylaminoazobenzene-4' -sulfonyl chloride derivative reagent is 2.0mg/mL.
8. The method for detecting the content of gamma-aminobutyric acid in wine according to claim 1, wherein the method comprises the following steps: in the step (2), a filter membrane of 0.10-0.45 μm is used for filtration.
9. The method for detecting the content of gamma-aminobutyric acid in wine according to claim 1, wherein the method comprises the following steps: in the step (3), the concentration gradient of the standard working solution is 5.0mg/L, 10.0mg/L, 20.0mg/L, 30.0mg/L, 40.0mg/L and 60.0mg/L respectively.
10. The method for detecting the content of gamma-aminobutyric acid in wine according to claim 1, wherein the method comprises the following steps: in the step (4), the high performance liquid chromatography conditions include:
Chromatographic column: a C 18 column, 150mm by 4.6mm,5 μm, or a chromatographic column with equivalent performance;
column temperature: 25-30 ℃;
Mobile phase: acetonitrile+6.8 g/L sodium acetate trihydrate (35+65);
flow rate: 0.8-1.0mL/min;
Detection wavelength: 436nm;
sample injection amount: 10-20 mu L.
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| CN202410220606.XA CN118090992A (en) | 2024-02-28 | 2024-02-28 | Method for detecting content of gamma-aminobutyric acid in wine |
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