CN113325113A - Method for detecting content of acetaldehyde and furfural in wine - Google Patents
Method for detecting content of acetaldehyde and furfural in wine Download PDFInfo
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- IKHGUXGNUITLKF-UHFFFAOYSA-N Acetaldehyde Chemical compound CC=O IKHGUXGNUITLKF-UHFFFAOYSA-N 0.000 title claims abstract description 178
- HYBBIBNJHNGZAN-UHFFFAOYSA-N furfural Chemical compound O=CC1=CC=CO1 HYBBIBNJHNGZAN-UHFFFAOYSA-N 0.000 title claims abstract description 158
- 235000014101 wine Nutrition 0.000 title claims abstract description 90
- 238000000034 method Methods 0.000 title claims abstract description 56
- 238000001514 detection method Methods 0.000 claims abstract description 32
- 239000000126 substance Substances 0.000 claims abstract description 24
- 239000012086 standard solution Substances 0.000 claims abstract description 13
- 238000010813 internal standard method Methods 0.000 claims abstract description 10
- 239000000243 solution Substances 0.000 claims description 46
- 239000000523 sample Substances 0.000 claims description 41
- PGMYKACGEOXYJE-UHFFFAOYSA-N pentyl acetate Chemical compound CCCCCOC(C)=O PGMYKACGEOXYJE-UHFFFAOYSA-N 0.000 claims description 26
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- WVYWICLMDOOCFB-UHFFFAOYSA-N 4-methyl-2-pentanol Chemical compound CC(C)CC(C)O WVYWICLMDOOCFB-UHFFFAOYSA-N 0.000 claims description 13
- KYQCOXFCLRTKLS-UHFFFAOYSA-N Pyrazine Chemical compound C1=CN=CC=N1 KYQCOXFCLRTKLS-UHFFFAOYSA-N 0.000 claims description 12
- 238000004821 distillation Methods 0.000 claims description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 239000007789 gas Substances 0.000 claims description 9
- 239000011550 stock solution Substances 0.000 claims description 9
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 7
- 239000001257 hydrogen Substances 0.000 claims description 7
- 229910052739 hydrogen Inorganic materials 0.000 claims description 7
- PCNDJXKNXGMECE-UHFFFAOYSA-N Phenazine Natural products C1=CC=CC2=NC3=CC=CC=C3N=C21 PCNDJXKNXGMECE-UHFFFAOYSA-N 0.000 claims description 6
- 235000009754 Vitis X bourquina Nutrition 0.000 claims description 6
- 235000012333 Vitis X labruscana Nutrition 0.000 claims description 6
- 235000014787 Vitis vinifera Nutrition 0.000 claims description 6
- 238000004458 analytical method Methods 0.000 claims description 6
- 239000000796 flavoring agent Substances 0.000 claims description 6
- 235000019634 flavors Nutrition 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 6
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 6
- 239000012498 ultrapure water Substances 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 238000007664 blowing Methods 0.000 claims description 5
- 239000012159 carrier gas Substances 0.000 claims description 5
- 238000004817 gas chromatography Methods 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 235000019441 ethanol Nutrition 0.000 claims description 4
- 239000012496 blank sample Substances 0.000 claims description 3
- 239000000498 cooling water Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 239000012528 membrane Substances 0.000 claims description 3
- 239000012488 sample solution Substances 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 238000005303 weighing Methods 0.000 claims description 3
- 239000010457 zeolite Substances 0.000 claims description 3
- 229910021536 Zeolite Inorganic materials 0.000 claims description 2
- HNPSIPDUKPIQMN-UHFFFAOYSA-N dioxosilane;oxo(oxoalumanyloxy)alumane Chemical compound O=[Si]=O.O=[Al]O[Al]=O HNPSIPDUKPIQMN-UHFFFAOYSA-N 0.000 claims description 2
- -1 furfural compound Chemical class 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
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- 241000219095 Vitis Species 0.000 claims 3
- 238000001212 derivatisation Methods 0.000 abstract description 7
- 238000010998 test method Methods 0.000 abstract description 7
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- 238000011084 recovery Methods 0.000 description 14
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- 238000004587 chromatography analysis Methods 0.000 description 5
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 4
- HORQAOAYAYGIBM-UHFFFAOYSA-N 2,4-dinitrophenylhydrazine Chemical compound NNC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O HORQAOAYAYGIBM-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 240000006365 Vitis vinifera Species 0.000 description 3
- 150000001299 aldehydes Chemical class 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000002203 pretreatment Methods 0.000 description 3
- 238000002470 solid-phase micro-extraction Methods 0.000 description 3
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- 235000003095 Vaccinium corymbosum Nutrition 0.000 description 1
- 235000017537 Vaccinium myrtillus Nutrition 0.000 description 1
- 239000008168 almond oil Substances 0.000 description 1
- 235000021014 blueberries Nutrition 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 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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- 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
- G01N30/14—Preparation by elimination of some components
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/64—Electrical detectors
- G01N30/68—Flame ionisation detectors
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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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Abstract
The invention relates to food safety detection, and discloses a method for detecting acetaldehyde and furfural content in wine in order to solve the problems of complex pretreatment and high detection cost, which comprises the following steps: distilling the wine sample; adding an internal standard solution; analytical detection using a gas chromatography-flame ionization detector (GC-FID); and quantitatively calculating the content of acetaldehyde and furfural in the wine by an internal standard method. According to the invention, the distilling method is used for removing the saccharides and pigment interfering substances in the wine, the gas chromatograph is used for accurately detecting and analyzing the acetaldehyde and the furfural in the wine, the pretreatment of the test method is free from derivatization, the test method is quick and simple, and the detection result is reliable and stable.
Description
Technical Field
The invention relates to the technical field of food safety detection, in particular to a method for detecting acetaldehyde and furfural content in wine.
Background
Aldehydes are important components of flavor substances in wine, wherein acetaldehyde occupies an important position, low-concentration acetaldehyde has the pleasant flavor of fruits, and pungent odor is generated once the concentration is increased, however, in 2009, acetaldehyde is considered to be a strong carcinogen, furfural, namely 2-furfural, is an important aroma-generating substance of wine, is a colorless liquid with the flavor of almond oil, can highlight the characteristic of the wine that the wine is fragrant in an empty cup, and can be said that the aldehydes are important components of the flavor components of the wine, so that the characteristic of harmonious, soft and endless aftertaste of the wine is formed.
At present, the detection aiming at the acetaldehyde content in the wine mainly comprises the following steps: dawn et al use 2, 4-Dinitrophenylhydrazine (DNPH) pre-column derivatization gas chromatography mass spectrometry (GC/MS-SIM) to detect acetaldehyde in wine, Liaose et al use headspace gas chromatography (GC/FID) to detect trace methanol and acetaldehyde in wine, and Wanglechong uses 2, 4-Dinitrophenylhydrazine (DNPH) pre-column derivatization combined with HPLC to detect total acetaldehyde and free acetaldehyde in wine. Few main documents for detecting the furfural content in wine at home and abroad are as follows: plum blossom and the like are used for measuring furfural in wine by adopting a stirring rod adsorption extraction-gas chromatography-mass spectrometry combined method, and plum blossom and the like are used for measuring furfural in wine by adopting a solid phase microextraction internal standard method combined with gas chromatography-mass spectrometry. The method mainly adopts a pre-column derivatization method for detecting acetaldehyde in the wine, namely a method for detecting acetaldehyde in the wine by using 2,4 Dinitrophenylhydrazine (DNPH) pre-column derivatization and then determining the acetaldehyde by using a gas chromatography-mass spectrometry method or a method for detecting the acetaldehyde by using an HPLC (high performance liquid chromatography), has the problems of complicated pre-treatment, expensive instrument and equipment and overhigh test cost, has the problem of overhigh commercial extraction cost in a pre-treatment method of a solid phase micro-extraction technology, and has few documents for simultaneously detecting acetaldehyde and furfural in the wine.
Therefore, the method for determining acetaldehyde and furfural in wine by using distillation method-gas chromatography is established, is simple, stable and efficient, can remove interference factors such as sugar and pigment in wine on a gas chromatograph, greatly reduces the detection cost, and better solves the problems of complex pretreatment and overhigh test cost for determination of the aldehyde substances in wine.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a method for detecting the content of acetaldehyde and furfural in wine.
In order to achieve the purpose, the invention adopts the following technical scheme:
a method for detecting the content of acetaldehyde and furfural in wine comprises the following steps:
step 1: distilling the wine sample;
step 2: adding an internal standard solution;
and step 3: analytical detection using a gas chromatography-flame ionization detector (GC-FID);
and 4, step 4: and quantitatively calculating the content of acetaldehyde and furfural in the wine by an internal standard method.
In the step 1, the method for distilling the wine sample comprises the following steps:
accurately measuring 100mL of wine with a liquid temperature of 20 ℃ by using a volumetric flask, pouring the wine into a 500mL distillation flask, washing the volumetric flask with 50mL of ultrapure water for 3 times, adding the washing liquid into the distillation flask together, adding zeolite, connecting a condenser pipe, taking the volumetric flask for sampling as a receiver, carrying out ice bath, starting cooling water with the temperature lower than 15 ℃, slowly heating and distilling, collecting distillate, taking down the volumetric flask when the liquid in the volumetric flask is close to a scale, covering a stopper, carrying out heat preservation in a water bath at 20 ℃ for 30min, adding water to the scale, and mixing uniformly for later use;
the alcohol concentration of the wine is 8-15 degrees;
the grape wine is prepared by fermenting grape peel, pulp and kernel which are used as raw materials, and is a natural grape wine without adding foreign flavor and aroma substances and edible alcohol.
In the step 2, the addition of the internal standard solution comprises the following steps:
taking 10mL of wine distillate, putting the wine distillate into a 10mL volumetric flask for constant volume, respectively adding 0.1mL of 2% n-amyl acetate solution and 2% 4-methyl-2-pentanol solution, uniformly mixing, and filtering with a 0.22 mu m filter membrane to be tested;
the preparation method of the n-amyl acetate solution with the concentration of 2 percent comprises the following steps: absorbing 2mL of an n-amyl acetate standard substance, and using 12% absolute ethyl alcohol solution to fix the volume to 100 mL;
the preparation method of the 4-methyl-2-pentanol solution with the concentration of 2 percent comprises the following steps: 2mL of 4-methyl-2-pentanol standard is sucked, and the volume is increased to 100mL by using 12% absolute ethyl alcohol solution.
The step 3 is an analysis detection by using a gas chromatography-flame ionization detector (GC-FID), and comprises the following steps:
the gas chromatography conditions were as follows: a chromatographic column: ZKAT-LZP930.2 capillary column (30m × 0.32mm, 0.25um, China, Zhongke Antai Co.); column temperature control procedure: the initial temperature is 50 ℃, the temperature is raised to 90 ℃ at the speed of 5 ℃/min after the initial temperature is kept for 5min, and then the temperature is raised to 200 ℃ at the speed of 15 ℃/min; keeping the temperature of a sample inlet at 200 ℃ for 2 min; the temperature of the detector is 200 ℃; the carrier gas is high-purity nitrogen (the purity is more than 99.999 percent) and the flow rate is 1 mL/min;
flame ionization detector conditions: hydrogen flow rate is 30 mL/min; the air flow is 400 mL/min; the tail blowing flow is 25 mL/min; the split ratio is 5: 1; the sample size was 1.0 uL.
The method for quantitatively calculating the content of acetaldehyde and furfural in the wine by the internal standard method in the step 4 comprises the following steps:
the acetaldehyde and furfural standard stock solution is prepared by the following method: accurately weighing 10mg of acetaldehyde and furfural standard, and using 12% absolute ethyl alcohol solution to fix the volume to 100mL to respectively obtain 1000ug/mL standard stock solutions;
the acetaldehyde and furfural standard use solution is prepared by the following method: respectively sucking a proper amount of acetaldehyde and a proper amount of furfural standard stock solution into a 10mL volumetric flask, using 12% absolute ethanol solution to perform constant volume to 10mL, respectively adding 2% n-amyl acetate solution and 2% 4-methyl-2-pentanol solution to each 0.1mL to respectively obtain a series of acetaldehyde standard use solutions with the concentrations of 5.0, 15.0, 25.0, 50.0, 70.0 and 100.0ug/mL and a series of furfural standard use solutions with the concentrations of 0.5, 1.0, 5.0, 10.0, 20.0 and 50.0 ug/mL; measuring peak area, and drawing a working curve of the standard solution by taking the mass concentration as a horizontal coordinate and the peak area as a vertical coordinate;
acetaldehyde and furfural are respectively in the mass concentration ranges of 5.0-70.0ug/mL and 0.5-50.0ug/mL, the detection limit is determined by taking S/N as 3, and the detection limit of the acetaldehyde and the detection limit of the furfural are both less than 5.0 ug/mL;
and (3) carrying out sample injection on the distilled wine sample to obtain the peak areas of acetaldehyde and furfural, and quantitatively calculating the concentrations of corresponding acetaldehyde and furfural compounds by using an internal standard method according to the following formula.
In the formula:
x-the content of acetaldehyde and furfural in the sample, wherein the unit is mg/L;
c1, obtaining the concentration of pyrazine in the sample solution by standard curve, wherein the unit is ug/mL;
c2, checking the concentration of pyrazine in the blank sample injection solution by a standard curve, wherein the unit ug/mL;
v1-volume constant of sample, unit is mL;
v2-sample size in mL.
According to the invention, the distillation method is used for removing the saccharides and pigment interfering substances in the wine, the gas chromatograph is used for accurately detecting and analyzing acetaldehyde and furfural in the wine, the pre-treatment of the test method is free from derivatization, the test method is rapid and simple, and the detection result is reliable and stable.
Drawings
FIG. 1 is a bar graph of the effect of pure water addition on acetaldehyde and furfural recovery;
FIG. 2 is a chromatogram of two standard substances and two internal standards in a standard solution;
FIG. 3 is a chromatogram of two standard substances and two internal standards in a wine sample.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1
Step 1: instruments and reagents
An acetaldehyde standard; a furfural standard; n-pentyl acetate standard (for internal standard); 4-methyl-2-pentanol solution (as internal standard); ethanol (chromatographically pure); commercial wine; ultrapure water, primary water Milli-Q academy ultrapure water system specified in GB/T6682-2008.
The high performance gas chromatograph was equipped with a hydrogen flame ionization detector (1260II, agilent, usa); detector (hydrogen flame ionization FID); an all glass distiller (500 mL); constant temperature water bath (temperature control + -0.1 deg.C).
Step 2: conditions of instrumental analysis
Chromatography column, ZKAT-LZP930.2 capillary column (30m × 0.32mm, 0.25 μm, China, Zhongke Antai Co.); column temperature control procedure: the initial temperature is 50 ℃, the temperature is raised to 90 ℃ at the speed of 5 ℃/min after the initial temperature is kept for 5min, and then the temperature is raised to 200 ℃ at the speed of 15 ℃/min; keeping the temperature of a sample inlet at 200 ℃ for 2 min; the temperature of the detector is 200 ℃; the carrier gas is high-purity nitrogen (the purity is more than 99.999 percent) and the flow rate is 1 mL/min; hydrogen flow rate is 30 mL/min; the air flow is 400 mL/min; the tail blowing flow is 25 mL/min; the split ratio is 5: 1; the amount of the sample was 1.0. mu.L.
And step 3: solution preparation
1) Simulating a wine sample: simulating the alcohol content of 12% of the wine, taking 12mL of absolute ethyl alcohol, and fixing the volume of 100mL of the absolute ethyl alcohol by using ultrapure water to ensure that the volume fraction of the absolute ethyl alcohol is 12%;
2) internal standard solution: n-amyl acetate solution at 2% concentration: the standard substance is used as acetaldehyde internal standard, 2mL of n-amyl acetate standard substance is absorbed, and the volume is determined to be 100mL by using 12 percent absolute ethyl alcohol solution;
2% strength 4-methyl-2-pentanol solution: the standard furfural sample is used as an internal standard furfural sample, 2mL of 4-methyl-2-pentanol standard sample is sucked, and the volume is determined to be 100mL by using 12% absolute ethyl alcohol solution;
3) standard stock solutions: accurately weighing 10mg of acetaldehyde and furfural standard, and using 12% absolute ethyl alcohol solution to fix the volume to 100mL to respectively obtain 1000 mug/mL standard stock solution;
4) standard use solutions: respectively sucking a proper amount of acetaldehyde and a proper amount of furfural standard stock solution into a 10mL volumetric flask, metering the volume to 10mL by using a 12% absolute ethanol solution, and respectively adding an n-amyl acetate solution with the concentration of 2% and a 4-methyl-2-pentanol solution with the concentration of 2%; 0.1mL of each of the two internal standard substances respectively obtain a series of acetaldehyde standard use solutions with the concentrations of 5.0, 15.0, 25.0, 50.0, 70.0 and 100.0 mu g/mL and a series of furfural standard use solutions with the concentrations of 0.5, 1.0, 5.0, 10.0, 20.0 and 50.0 mu g/mL;
and 4, step 4: sample pretreatment and GC-FID analysis detection
1) Wine sample distillation
Using a dry and clean 100mL volumetric flask, accurately measuring 100mL wine with the liquid temperature of 20 ℃, pouring the wine into a 500mL distillation flask, washing the volumetric flask with 50mL ultrapure water for 3 times, adding washing liquid into the distillation flask, adding a plurality of zeolites, connecting a condenser pipe, taking an original volumetric flask for sampling as a receiver and carrying out ice bath, starting cooling water with the temperature lower than 15 ℃, slowly heating and distilling, collecting distillate, taking down the volumetric flask and covering a stopper when the liquid in the volumetric flask is close to a scale, preserving the heat in a water bath at 20 ℃ for 30min, replenishing water to the scale, and uniformly mixing for later use;
2) addition of the internal Standard solution
Taking 10mL of liquor sample distillate, fixing the volume in a 10mL volumetric flask, respectively adding 0.1mL of an n-amyl acetate solution with the concentration of 2% and an internal standard substance of a 4-methyl-2-pentanol solution with the concentration of 2%, uniformly mixing, and filtering through a 0.22-micrometer filter membrane to be detected;
3) gas chromatography-flame ionization detector (GC-FID) analysis detection;
and 5: internal standard method for quantitatively calculating content of acetaldehyde and furfural in wine
And (3) quantitatively calculating the corresponding acetaldehyde and furfural compound concentrations by an internal standard method according to the peak areas of acetaldehyde and furfural measured by sample injection of the distilled wine sample:
in the formula:
the content of acetaldehyde and furfural in the x-sample is mg/L
C1, obtaining the concentration of pyrazine in the sample solution by standard curve, wherein the unit is ug/mL;
c2, checking the concentration of pyrazine in the blank sample injection solution by a standard curve, wherein the unit is ug/mL;
v1-volume constant of sample, unit is mL;
v2-sample size in mL;
the result retains 2 as a significant number.
Further, the invention also makes systematic research on the process conditions in the method for detecting the content of acetaldehyde and furfural in wine, and only the following test scheme for obviously influencing the detection effect of the change of the process conditions on the content of acetaldehyde and furfural in wine is explained, and the specific results are shown in comparative examples 1-6:
comparative example 1: optimization of pure Water addition
In the process of distilling the wine, too little pure water is added to cause the short time of distilling the wine to cause the insufficient volatilization of the standard substances, while too much pure water is added to cause the long time of distilling the wine to cause the long test time, the test examines the pure water addition amount (20, 30, 40, 50, 60 and 70mL) in the standard sample, each addition amount is parallelly measured for 3 times, the average recovery rate is obtained, and the influence of the average recovery rate of the acetaldehyde and furfural standard sample is examined, and the result is shown in figure 1. Test results show that when the pure water addition amount is 50mL, the recovery rates of acetaldehyde and furfural reach the highest, and then the recovery rates are basically kept unchanged along with the increase of the pure water addition amount, so that the optimal pure water addition amount is finally determined to be 50mL for the purposes of ensuring better recovery rates of acetaldehyde and furfural and saving test time.
Comparative example 2: optimization of chromatography columns
Through the review of a large body of literature data, the choice of chromatography columns is mainly: DB-5MS capillary column (30.0m × 0.25mm × 0.25 μm), DB-WAX capillary column (30.0m × 0.25mm × 0.25 μm), CP WAX 57CB capillary column (50.0m × 0.25mm × 0.25 μm). Tests show that although a target substance can be separated when a DB-5MS capillary column (30.0m multiplied by 0.25mm multiplied by 0.25 mu m) is selected as a chromatographic column, a target peak of an acetaldehyde substance is wider and has a certain tailing to reduce the detection sensitivity, the DB-WAX capillary column (30.0m multiplied by 0.25mm multiplied by 0.25 mu m) is selected as the chromatographic column, the chromatogram peak is better, but a target peak of a furfural substance partially coincides with an interfering substance when a sample is measured, the purchase cost of an imported chromatographic column is too high, the test is carried out to determine that a ZKAT-LZP930.2 capillary column (30m multiplied by 0.32mm, 0.25 mu m, China, Coutai) is used as a domestic chromatographic column, the conditions are optimized continuously, and the optimal chromatographic conditions are determined: the column temperature control procedure is that the initial temperature is 50 ℃, the temperature is raised to 90 ℃ at the speed of 5 ℃/min after being kept for 5min, and the temperature is raised to 200 ℃ at the speed of 15 ℃/min; keeping the temperature of a sample inlet at 200 ℃ for 2 min; the temperature of the detector is 200 ℃; the carrier gas is high-purity nitrogen (the purity is more than 99.999 percent) and the flow rate is 1 mL/min; hydrogen flow rate is 30 mL/min; the air flow is 400 mL/min; the tail blowing flow is 25 mL/min; the split ratio is 5: 1; the sample size was 1.0 uL. Under the optimal chromatographic conditions, each standard substance has better separation degree and better peak type. The results are shown in figures 2 and 3, wherein figure 2 is a chromatogram of two standard substances and two internal standards in the standard solution, and figure 3 is a chromatogram of two standard substances and two internal standards in the sample.
Comparative example 3: determination of precision
The standard solution with the concentration of 30ug/ml is repeatedly injected and measured for 6 times, and the result is as follows as the accuracy and repeatability evaluation of the method:
TABLE 1 results of precision determination of acetaldehyde and furfural standard solutions in wine
From table 1, it can be seen that RSD of both acetaldehyde and furfural compounds are less than 10%, indicating good process precision.
Comparative example 4: detection limit, quantitation limit, and linear range:
the linear ranges of acetaldehyde and furfural are 5.0-100.0ug/mL and 0.5-50.0ug/mL respectively, the correlation coefficients are 0.99933 and 0.99975, the correlation coefficient R2 is greater than 0.999, the detection limit of the method is determined by the signal-to-noise ratio 3(S/N is 3), and the detection limit is 3 times as the method quantification limit, and the results are as follows:
TABLE 2 Linear regression equation, correlation coefficient and detection limits for acetaldehyde and furfural in wine
As can be seen from Table 2, the detection limits of acetaldehyde and furfural in wine by the method were 4.48ug/ml and 0.54ug/ml, respectively, and the quantification limits were 13.44ug/ml and 1.62ug/ml, respectively.
Comparative example 5: recovery rate of added standard
The test was carried out according to the method of GB/T27404-2008, with 3 level tests (n-5) at the lower limit of the measurement, 2 times the lower limit of the measurement and 10 times the lower limit of the detection, and the average recovery was calculated for each level, respectively, and the results are shown in table 3. The method has the advantages that the lower limit added standard Relative Standard Deviation (RSD) for detecting acetaldehyde and furfural in the wine is 1.47-4.23%, the average recovery rate is 82.47-89.66%, the lower limit added standard Relative Standard Deviation (RSD) measured by a 2-time method is 1.66-5.33%, the average recovery rate is 89.44-92.15%, the lower limit detected standard deviation (RSD) detected by a 10-time method is 1.54-4.52%, and the average recovery rate is 93.14% -95.26%.
Table 3 relative standard deviation and normalized recovery of acetaldehyde and furfural in wine (n ═ 5)
As can be seen from Table 3, the standard recovery rates of acetaldehyde and furfural are 82.47% -95.26%, indicating that the method and the apparatus have good accuracy and can meet the test requirements.
Comparative example 6: content determination of acetaldehyde and furfural compounds in part of wine
The wine was analyzed using established analytical methods and the content of some representative wine-like acetaldehyde and furfural are shown in table 4.
TABLE 4 acetaldehyde and Furfural content (ug/ml) in part of the wines
As can be seen from Table 4, acetaldehyde was detected in all 5 kinds of commercially available wines, but the content difference was changed, wherein the acetaldehyde and furfural content in Hebei dry red wine was the highest at 65.24ug/mL and 5.78ug/mL, respectively, while the acetaldehyde and furfural content in Guizhou blueberry wine was the lowest at 10.22ug/mL and was not detected.
According to the method for detecting the content of acetaldehyde and furfural in the wine, the influence of interfering substances such as sugar and pigment in the wine on gas chromatography is removed by adopting a distillation method, the test steps and the test cost of the conventional derivatization method and solid-phase microextraction method are simplified, the addition amount of pure water in the distillation method is optimized, the addition amount of pure water is determined to be 50mL, the test time is saved, the gas chromatography column and the chromatography conditions are optimized, and the chromatography conditions are determined to be: ZKAT-LZP930.2 capillary column (30m × 0.32mm, 0.25um, China, Zhongke Antai Co., Ltd.), column temperature control program: the initial temperature is 50 ℃, the temperature is raised to 90 ℃ at the speed of 5 ℃/min after the initial temperature is kept for 5min, and then the temperature is raised to 200 ℃ at the speed of 15 ℃/min; keeping the temperature of a sample inlet at 200 ℃ for 2 min; the temperature of the detector is 200 ℃; the carrier gas is high-purity nitrogen (purity is more than 99.999%) and the flow rate is 1 mL/min; hydrogen flow rate is 30 mL/min; the air flow is 400 mL/min; the tail blowing flow is 25 mL/min; the split ratio is 5: 1; the sample size was 1.0 uL.
As can be seen from the above example 1 and comparative examples 1 to 6, acetaldehyde and furfural have good linear relations of 5.0-100.0ug/mL and 0.5-50.0ug/mL respectively, the correlation coefficients R2 are 0.99933 and 0.99975 respectively, the acetaldehyde and furfural repeatability detection values RSD are 1.55-1.80%, the standard addition average recovery rates of 3 horizontal samples are 82.47-89.66%, 89.44-92.15% and 93.14-95.26%, the method precisions (RSD) are 1.47-4.23%, 1.66-5.33% and 1.54-4.52% (n ═ 5), the lowest detection limits of acetaldehyde and furfural are 4.48ug/mL and 0.54ug/mL respectively, the reproducibility and accuracy of the test method are good, the acetaldehyde content range of 5 commercially available wines is 10.22-65.24ug/mL, the furfural content range is 1.51-5.78ug/mL, the method is simple, convenient and accurate, reduces pretreatment steps, uses cheaper detection equipment, reduces test cost, and provides a new method for detecting acetaldehyde and furfural in wine.
According to the invention, the interfering substances of saccharides and pigments in the wine are removed by a distillation method, and the acetaldehyde and the furfural in the wine are accurately detected and analyzed by using a gas chromatograph, the pretreatment of the test method is not required to be derivatized, the test method is rapid and simple, and the detection result is reliable and stable.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (5)
1. A method for detecting the content of acetaldehyde and furfural in wine is characterized by comprising the following steps:
step 1: distilling the wine sample;
step 2: adding an internal standard solution;
and step 3: analytical detection using a gas chromatography-flame ionization detector (GC-FID);
and 4, step 4: and quantitatively calculating the content of acetaldehyde and furfural in the wine by an internal standard method.
2. The method for detecting the acetaldehyde and furfural content in wine according to claim 1, wherein in the step 1, the method for distilling the wine sample comprises the following steps:
accurately measuring 100mL of wine with a liquid temperature of 20 ℃ by using a volumetric flask, pouring the wine into a 500mL distillation flask, washing the volumetric flask with 50mL of ultrapure water for 3 times, adding the washing liquid into the distillation flask together, adding zeolite, connecting a condenser pipe, taking the volumetric flask for sampling as a receiver, carrying out ice bath, starting cooling water with the temperature lower than 15 ℃, slowly heating and distilling, collecting distillate, taking down the volumetric flask when the liquid in the volumetric flask is close to a scale, covering a stopper, carrying out heat preservation in a water bath at 20 ℃ for 30min, adding water to the scale, and mixing uniformly for later use;
the alcohol concentration of the wine is 8-15 degrees;
the grape wine is prepared by fermenting grape peel, pulp and kernel which are used as raw materials, and is a natural grape wine without adding foreign flavor and aroma substances and edible alcohol.
3. The method for detecting the content of acetaldehyde and furfural in wine according to claim 1, wherein the step 2 of adding the internal standard solution comprises the following steps:
taking 10mL of wine distillate, putting the wine distillate into a 10mL volumetric flask for constant volume, respectively adding 0.1mL of 2% n-amyl acetate solution and 2% 4-methyl-2-pentanol solution, uniformly mixing, and filtering with a 0.22 mu m filter membrane to be tested;
the preparation method of the n-amyl acetate solution with the concentration of 2 percent comprises the following steps: absorbing 2mL of an n-amyl acetate standard substance, and using 12% absolute ethyl alcohol solution to fix the volume to 100 mL;
the preparation method of the 4-methyl-2-pentanol solution with the concentration of 2 percent comprises the following steps: 2mL of 4-methyl-2-pentanol standard is sucked, and the volume is increased to 100mL by using 12% absolute ethyl alcohol solution.
4. The method for detecting the content of acetaldehyde and furfural in wine according to claim 1, wherein the analysis and detection in step 3 is performed by using a gas chromatography-flame ionization detector (GC-FID), and the method comprises the following steps:
the gas chromatography conditions were as follows: a chromatographic column: ZKAT-LZP930.2 capillary column (30m × 0.32mm, 0.25um, China, Zhongke Antai Co.); column temperature control procedure: the initial temperature is 50 ℃, the temperature is raised to 90 ℃ at the speed of 5 ℃/min after the initial temperature is kept for 5min, and then the temperature is raised to 200 ℃ at the speed of 15 ℃/min; keeping the temperature of a sample inlet at 200 ℃ for 2 min; the temperature of the detector is 200 ℃; the carrier gas is high-purity nitrogen (the purity is more than 99.999 percent) and the flow rate is 1 mL/min;
flame ionization detector conditions: hydrogen flow rate is 30 mL/min; the air flow is 400 mL/min; the tail blowing flow is 25 mL/min; the split ratio is 5: 1; the sample size was 1.0 uL.
5. The method for detecting the content of acetaldehyde and furfural in wine according to claim 1, wherein the method for quantitatively calculating the content of acetaldehyde and furfural in wine by using the internal standard method in the step 4 comprises the following steps:
the acetaldehyde and furfural standard stock solution is prepared by the following method: accurately weighing 10mg of acetaldehyde and furfural standard, and using 12% absolute ethyl alcohol solution to fix the volume to 100mL to respectively obtain 1000ug/mL standard stock solutions;
the acetaldehyde and furfural standard use solution is prepared by the following method: respectively sucking a proper amount of acetaldehyde and a proper amount of furfural standard stock solution into a 10mL volumetric flask, using 12% absolute ethanol solution to perform constant volume to 10mL, respectively adding 2% n-amyl acetate solution and 2% 4-methyl-2-pentanol solution to each 0.1mL to respectively obtain a series of acetaldehyde standard use solutions with the concentrations of 5.0, 15.0, 25.0, 50.0, 70.0 and 100.0ug/mL and a series of furfural standard use solutions with the concentrations of 0.5, 1.0, 5.0, 10.0, 20.0 and 50.0 ug/mL; measuring peak area, and drawing a working curve of the standard solution by taking the mass concentration as a horizontal coordinate and the peak area as a vertical coordinate;
acetaldehyde and furfural are respectively in the mass concentration ranges of 5.0-70.0ug/mL and 0.5-50.0ug/mL, the detection limit is determined by taking S/N as 3, and the detection limit of the acetaldehyde and the detection limit of the furfural are both less than 5.0 ug/mL;
and (3) quantitatively calculating the corresponding acetaldehyde and furfural compound concentrations by an internal standard method according to the peak areas of acetaldehyde and furfural measured by sample injection of the distilled wine sample:
in the formula:
x-the content of acetaldehyde and furfural in the sample, wherein the unit is mg/L;
c1, obtaining the concentration of pyrazine in the sample solution by standard curve, wherein the unit is ug/mL;
c2, checking the concentration of pyrazine in the blank sample injection solution by a standard curve, wherein the unit ug/mL;
v1-volume constant of sample, unit is mL;
v2-sample size in mL.
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