CN111721794A - Method for quantitatively detecting 5-hydroxymethylfurfural in yellow wine by utilizing nuclear magnetic resonance hydrogen spectrum - Google Patents
Method for quantitatively detecting 5-hydroxymethylfurfural in yellow wine by utilizing nuclear magnetic resonance hydrogen spectrum Download PDFInfo
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- NOEGNKMFWQHSLB-UHFFFAOYSA-N 5-hydroxymethylfurfural Chemical compound OCC1=CC=C(C=O)O1 NOEGNKMFWQHSLB-UHFFFAOYSA-N 0.000 title claims abstract description 92
- RJGBSYZFOCAGQY-UHFFFAOYSA-N hydroxymethylfurfural Natural products COC1=CC=C(C=O)O1 RJGBSYZFOCAGQY-UHFFFAOYSA-N 0.000 title claims abstract description 83
- 235000014101 wine Nutrition 0.000 title claims abstract description 51
- 238000000034 method Methods 0.000 title claims abstract description 40
- 229910052739 hydrogen Inorganic materials 0.000 title claims abstract description 34
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 title claims abstract description 33
- 239000001257 hydrogen Substances 0.000 title claims abstract description 33
- 238000001228 spectrum Methods 0.000 title claims abstract description 25
- 238000005481 NMR spectroscopy Methods 0.000 title claims abstract description 21
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 62
- 238000012937 correction Methods 0.000 claims abstract description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- 238000001514 detection method Methods 0.000 claims abstract description 12
- 238000003825 pressing Methods 0.000 claims abstract description 11
- 238000004364 calculation method Methods 0.000 claims abstract description 7
- 238000012360 testing method Methods 0.000 claims description 27
- 239000000126 substance Substances 0.000 claims description 24
- 239000003153 chemical reaction reagent Substances 0.000 claims description 22
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 14
- 238000002360 preparation method Methods 0.000 claims description 13
- 238000010813 internal standard method Methods 0.000 claims description 8
- 238000005259 measurement Methods 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 8
- 238000001208 nuclear magnetic resonance pulse sequence Methods 0.000 claims description 8
- 238000010812 external standard method Methods 0.000 claims description 7
- 238000005070 sampling Methods 0.000 claims description 7
- 230000010354 integration Effects 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 239000004576 sand Substances 0.000 claims description 6
- 238000012417 linear regression Methods 0.000 claims description 4
- 238000000079 presaturation Methods 0.000 claims description 4
- 238000007796 conventional method Methods 0.000 abstract description 2
- 238000004611 spectroscopical analysis Methods 0.000 abstract 2
- 239000000523 sample Substances 0.000 description 46
- XLYOFNOQVPJJNP-ZSJDYOACSA-N Heavy water Chemical compound [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 14
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 239000007853 buffer solution Substances 0.000 description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 4
- 230000005284 excitation Effects 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 244000062793 Sorghum vulgare Species 0.000 description 2
- -1 aldehyde compound Chemical class 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 238000002290 gas chromatography-mass spectrometry Methods 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 2
- 235000019713 millet Nutrition 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- JXKPEJDQGNYQSM-UHFFFAOYSA-M sodium propionate Chemical class [Na+].CCC([O-])=O JXKPEJDQGNYQSM-UHFFFAOYSA-M 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 125000000026 trimethylsilyl group Chemical group [H]C([H])([H])[Si]([*])(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- YZCKVEUIGOORGS-OUBTZVSYSA-N Deuterium Chemical compound [2H] YZCKVEUIGOORGS-OUBTZVSYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 235000013334 alcoholic beverage Nutrition 0.000 description 1
- 235000013405 beer Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 235000016213 coffee Nutrition 0.000 description 1
- 235000013353 coffee beverage Nutrition 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 229910052805 deuterium Inorganic materials 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- 230000001738 genotoxic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 235000012907 honey Nutrition 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 235000013555 soy sauce Nutrition 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000012956 testing procedure Methods 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
- G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N24/00—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
- G01N24/08—Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects by using nuclear magnetic resonance
- G01N24/087—Structure determination of a chemical compound, e.g. of a biomolecule such as a protein
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- High Energy & Nuclear Physics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
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- Crystallography & Structural Chemistry (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a method for quantitatively detecting 5-hydroxymethylfurfural in yellow wine by utilizing nuclear magnetic resonance hydrogen spectrum, wherein in the determination process, the hydrogen spectrum is determined by pressing a water peak and an ethanol peak, and the determination result is substituted into a conventional quantitative calculation formula to obtain the quantitative concentration C of uncorrected 5-hydroxymethylfurfuralHMF‑0(ii) a C is to beHMF‑0Substituting the formula (I) to obtain the corrected quantitative concentration of the 5-hydroxymethylfurfural; cHMF＝CHMF‑0Alpha (I), wherein alpha is a correction factor. The method provided by the invention is mainly used for quantitatively detecting 5-hydroxymethylfurfural in yellow wine by utilizing nuclear magnetic resonance hydrogen spectrometry, and based on a conventional method for nuclear magnetic resonance hydrogen spectrometry, the influence of a pressing signal peak on the accurate quantitative determination is corrected by pressing a water peak and an ethanol signal and introducing a correction factor, so that the detection accuracy is improved. The method has no destructiveness to the sample, has simple pretreatment even does not need pretreatment operation, and can simply, quickly and accurately realize the quantitative detection of the 5-hydroxymethylfurfural in the yellow wine.
Description
Technical Field
The invention belongs to the technical field of food detection, and particularly relates to a method for quantitatively detecting 5-hydroxymethylfurfural in yellow wine by utilizing nuclear magnetic resonance hydrogen spectrum.
Background
Yellow wine is originated from the whole-year times of commerce three thousand years ago, only China has China, and is called world three ancient wines together with beer and wine, and the yellow wine is brewed wine which is prepared by taking rice, husked millet, corn, millet, wheat and the like as main raw materials through the steps of stewing, starter adding, saccharifying, fermenting, squeezing, filtering, wine decocting, storing and blending.
5-hydroxymethyl furfural (5-hydroxymethyl furfurfural, 5-HMF) has a chemical structure shown in formula I, is an aldehyde compound generated by catalytic dehydration of glucose or fructose and other monosaccharide compounds in a high-temperature or acidic environment, and is widely present in wine, soy sauce, coffee, raisin, dairy products, honey and certain natural products including yellow wine.
In recent years, the content of 5-hydroxymethylfurfural (5-HMF) has become a measure of whether many foods are excessively heated and stored for an excessively long time. Studies have also shown that 5-HMF may initiate and promote abnormal growth of the colon pouch with some degree of genotoxicity. After a certain dose of 5-HMF is absorbed by the body, the 5-HMF can irritate eyes, mucous membranes or skin, and the 5-HMF is poisoned by excessive intake. Therefore, the limitation of the 5-HMF content is a necessary trend.
The existing detection method for 5-HMF in yellow wine mainly comprises ultraviolet spectrophotometry (UV), High Performance Liquid Chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS) and the like. The ultraviolet spectrophotometry has higher sensitivity and longer sample measuring time. The liquid chromatography-mass spectrometry has the advantages of both high-efficiency separation of liquid chromatography and mass spectrum structure identification, but the liquid chromatography needs to use toxic solvents. Gas chromatography-mass spectrometry has certain limitations for the detection of high boiling point substances or substances with poor thermal stability.
However, the prior art never discloses the utilization of nuclear magnetic resonance to detect 5-HMF in yellow wine, and because the content of 5-HMF in yellow wine is low, the sensitivity for directly detecting 5-HMF is too low or cannot be detected, and the accurate quantification purpose cannot be achieved.
Disclosure of Invention
The invention provides a method for quantitatively detecting 5-HMF in yellow wine by utilizing nuclear magnetic resonance hydrogen spectrum, which is simple, rapid and accurate.
The invention provides a method for quantitatively detecting 5-hydroxymethylfurfural in yellow wine by utilizing nuclear magnetic resonance hydrogen spectrum,
in the measuring process, the hydrogen spectrum is measured by pressing a water peak and an ethanol peak, and the measuring result is substituted into a conventional quantitative calculation formula to obtain the quantitative concentration C of the uncorrected 5-hydroxymethylfurfuralHMF-0(ii) a C is to beHMF-0Substituting formula (I) to obtain the corrected quantitative concentration C of 5-hydroxymethylfurfuralHMF；
CHMF＝CHMF-0Alpha (I), wherein alpha is a correction factor.
Further, the method for determining the correction factor comprises the following steps:
taking the concentration of the 5-hydroxymethylfurfural standard product as an ordinate y and the quantitative concentration of uncorrected 5-hydroxymethylfurfural as an abscissa x, establishing a linear regression equation y alpha x + beta, and calculating alpha; wherein, alpha is a correction factor.
Further, the water peak and the ethanol peak are suppressed by adjusting the hydrogen measuring pulse sequence of the water peak and the ethanol peak by adopting a pre-saturation method.
Further, in the determination process, a lock field reagent is added into a sample to be determined; the field-locking reagent comprises a deuterated field-locking reagent or a fluorinated field-locking reagent.
Further, the method for the quantitative detection of the nuclear magnetic resonance hydrogen spectrum comprises an internal standard method and an external standard method.
Further, the internal standard method comprises the following steps:
a) sample preparation: adding an internal standard substance and a field locking reagent into a sample to be detected, mixing, adding into a nuclear magnetic tube, and detecting;
b) sampling the mixture to be tested obtained in the step a), calling a hydrogen measurement pulse sequence for suppressing a water peak and an ethanol peak, and then locking a field, shimming and tuning;
c) b) carrying out Fourier transform, phase correction, baseline correction, quantitative peak and internal standard peak integration on the original data obtained after sampling is finished, and then calculating according to a formula (I) and a conventional quantitative formula (I-0-1) to obtain the quantitative concentration of the 5-hydroxymethylfurfural in the corrected yellow wine;
CHMF＝CHMF-0ɑ (I)；
wherein M isRIs the mass of the internal standard, NRNumber of hydrogens represented by the quantitative peak of the internal standard, NHMFIs the number of hydrogens represented by the quantitative peak for 5-hydroxymethylfurfural, PRAs the purity of the internal standard substance, A is the integral area ratio of the quantitative peak of 5-hydroxymethylfurfural to the quantitative peak of the internal standard substance, WHMFIs the molecular weight of 5-hydroxymethylfurfural, WRMolecular weight of internal standard, VSAnd the volume of a sample to be detected is used for sample preparation, and alpha is a correction factor.
Further, the external standard method comprises the following steps:
A) sample preparation: respectively preparing an external standard and a sample to be detected, adding a field locking reagent into the sample to be detected and the external standard, transferring the sample to be detected and the external standard into a nuclear magnetic tube, and detecting the sample to be detected;
B) after the mixture obtained in the step A) is injected, measuring the 90-degree pulse width of an external standard and a sample to be measured, and testing a hydrogen spectrum by a method of suppressing a water peak and ethanol;
C) b) performing Fourier transform, phase adjustment, baseline correction, quantitative peak and internal standard peak integration on the original data obtained after the test is finished, and then calculating the quantitative concentration of the 5-hydroxymethylfurfural in the yellow wine according to the formula (I) and a conventional quantitative formula (I-0-2);
CHMF＝CHMF-0ɑ (I)；
wherein M isRIs the mass of the external standard, VSVolume of sample to be tested, PRA is the ratio of the integrated areas of the quantitative peak of 5-HMF and the quantitative peak of the external standard, W, for the purity of the external standardHMFIs the molecular weight of 5-hydroxymethylfurfural, WRIs the molecular weight of the external standard, NRNumber of hydrogens represented by the quantitative peak of the external standard, NHMFIs the number of hydrogens represented by the quantitative peak for 5-hydroxymethylfurfural, P1SFor a sample to be tested 90 DEG pulse width, P1RIs the 90 DEG pulse width, T, of the external standardRAs test temperature, T, of external standardSFor the test temperature, NS, of the sample to be testedRFor the number of scans of external targets, NSSAnd alpha is the scanning frequency of the sample to be detected, and alpha is a correction factor.
The invention has the following advantages:
the invention provides a method for quantitatively detecting 5-hydroxymethylfurfural in yellow wine by utilizing a nuclear magnetic resonance hydrogen spectrum, which is based on a conventional method for quantitatively detecting the nuclear magnetic resonance hydrogen spectrum, corrects the influence of a pressing signal peak on the precise quantitative determination by pressing a water peak and an ethanol peak and introducing a correction factor, and improves the detection accuracy. The method has no damage to the sample, the pretreatment is simple, even no pretreatment operation is needed, no toxic reagent is used, and the quantitative detection of the 5-hydroxymethylfurfural in the yellow wine can be simply, quickly and accurately realized.
Detailed Description
It should be noted that the embodiments and features of the embodiments may be combined with each other without conflict.
The invention provides a method for quantitatively detecting 5-hydroxymethylfurfural in yellow wine by utilizing nuclear magnetic resonance hydrogen spectrum, which comprises the following steps:
in the measuring process, the hydrogen spectrum is measured by pressing a water peak and an ethanol peak, and the measuring result is substituted into a conventional quantitative calculation formula to obtain the quantitative concentration C of the uncorrected 5-hydroxymethylfurfuralHMF-0(ii) a C is to beHMF-0Substituting formula (I) to obtain the corrected quantitative concentration C of 5-hydroxymethylfurfuralHMF；
CHMF＝CHMF-0Alpha (I), wherein alpha is a correction factor.
The inventor of the application finds that yellow wine mainly comprises water and ethanol, the sensitivity of 5-HMF quantitative detection directly by utilizing nuclear magnetic resonance hydrogen spectrum is too low or cannot be detected, the accurate quantitative purpose cannot be achieved, the influence of a pressing signal peak on accurate quantitative determination is corrected by pressing water peak and ethanol signals and introducing correction factors, and therefore the method for detecting 5-HMF in yellow wine quantitatively is simple, rapid and accurate.
In an embodiment of the present invention, the method for measuring the calibration factor includes:
and establishing a linear regression equation y alpha x + beta by taking the concentration of the 5-hydroxymethylfurfural standard product as an ordinate y and the quantitative concentration of the uncorrected 5-hydroxymethylfurfural as an abscissa x, so as to obtain alpha, wherein alpha is a correction factor.
Specifically, the concentration range of the 5-hydroxymethylfurfural standard is larger than the quantitative concentration of uncorrected 5-hydroxymethylfurfural obtained through testing.
In one embodiment of the invention, a pre-saturation method [ Monakhova Y B,
H,Humpfer E,etal.Application of automated eightfold suppression of water and ethanolsignals in1H NMR to provide sensitivity for analyzing alcoholic beverages[J]magnetic Resonance in Chemistry,2011,49(11): 734-.
Specifically, the test is carried out by using a Brookfield nuclear magnetic resonance instrument, wherein the peak-pressing pulse sequence of the Brookfield nuclear magnetic resonance instrument comprises zgpr, noesygpps1d, p3919gp, zggpwg, WET, zgesgp and the like.
In an embodiment of the present invention, a lock field reagent is added to a sample to be tested. Further, the lock field agent comprises a deuterated lock field agent or a fluorinated lock field agent. For example, the deuterated field-locking agent can be deuterium oxide, or the like.
In an embodiment of the present invention, a buffer solution is added to a sample to be tested. The buffer solution is phosphoric acid buffer solution with pH value of 4.0. To avoid drift in the chemical shift of the quantitative peak due to differences in pH, buffer solutions can be added to stabilize the pH.
In one embodiment of the invention, the method for the quantitative detection of the nuclear magnetic resonance hydrogen spectrum comprises an internal standard method and an external standard method.
In a preferred embodiment of the present invention, the internal standard method comprises the following steps:
a) sample preparation: adding an internal standard substance and a field locking reagent into a sample to be detected, mixing, adding into a nuclear magnetic tube, and detecting;
b) sampling the mixture to be tested obtained in the step a), calling a hydrogen measurement pulse sequence for suppressing a water peak and an ethanol peak, and then locking a field, shimming and tuning;
c) b) carrying out Fourier transform, phase correction, baseline correction, quantitative peak and internal standard peak integration on the original data obtained after sampling is finished, and then calculating according to a formula (I) and a conventional quantitative formula (I-0-1) to obtain the quantitative concentration of the 5-hydroxymethylfurfural in the corrected yellow wine;
CHMF＝CHMF-0ɑ (I)；
wherein M isRIs the mass of the internal standard, NRNumber of hydrogens represented by the quantitative peak of the internal standard, NHMFIs the number of hydrogens represented by the quantitative peak for 5-hydroxymethylfurfural, PRAs the purity of the internal standard substance, A is the integral area ratio of the quantitative peak of 5-hydroxymethylfurfural to the quantitative peak of the internal standard substance, WHMFIs the molecular weight of 5-hydroxymethylfurfural, WRMolecular weight of internal standard, VSAnd the volume of a sample to be detected is used for sample preparation, and alpha is a correction factor.
Further, in step a): the internal standard substance should not react with 5-HMF and yellow wine, and the quantitative signal does not overlap with the signals of yellow wine and 5-HMF, and can be dissolved in yellow wine. The quantitative peak of 5-HMF should be separated from the chemical baseline of the quantitative peak of the internal standard.
Furthermore, the lock field reagent is a lock field reagent which does not react with the sample to be detected and the internal standard substance and can be dissolved in the sample to be detected; the lock-field agent can be a deuterated or fluorinated agent. The amount of the lock field reagent to be added may be determined depending on the sensitivity of the apparatus, for example, the sensitivity of the apparatus is high, and it is sufficient that the volume of the lock field reagent is 5. mu.l for a 5ml nuclear magnetic tube.
Further, in step b): further comprising, after hydrogen measurement pulse sequences of water peak and ethanol peak are suppressed, adjusting other test parameters, such as longitudinal relaxation time with relaxation delay time greater than 5 times; the excitation center is located between the quantitative peak and the internal standard peak; the angle of excitation may be at a small angle, such as 30 °; the number of scans is such that the signal to noise ratio of the quantitative peak is at least greater than 10.
In a preferred embodiment of the present invention, the external standard method comprises the following steps:
A) sample preparation: respectively preparing an external standard and a sample to be detected, adding a field locking reagent into the sample to be detected and the external standard, transferring the sample to be detected and the external standard into a nuclear magnetic tube, and detecting the sample to be detected;
B) after the mixture obtained in the step A) is injected, measuring the 90-degree pulse width of an external standard and a sample to be measured, and testing a hydrogen spectrum by a method of suppressing a water peak and ethanol;
C) b) performing Fourier transform, phase adjustment, baseline correction, quantitative peak and internal standard peak integration on the original data obtained after the test is finished, and then calculating the quantitative concentration of the 5-hydroxymethylfurfural in the yellow wine according to the formula (I) and a conventional quantitative formula (I-0-2);
CHMF＝CHMF-0ɑ (I)；
MRis the mass of the external standard, VSVolume of sample to be tested, PRA is the ratio of the integrated areas of the quantitative peak of 5-HMF and the quantitative peak of the external standard, W, for the purity of the external standardHMFIs the molecular weight of 5-hydroxymethylfurfural, WRIs the molecular weight of the external standard, NRNumber of hydrogens represented by the quantitative peak of the external standard, NHMFIs the number of hydrogens represented by the quantitative peak for 5-hydroxymethylfurfural, P1SFor a sample to be tested 90 DEG pulse width, P1RIs the 90 DEG pulse width, T, of the external standardRAs test temperature, T, of external standardSFor the test temperature, NS, of the sample to be testedRFor the number of scans of external targets, NSSAnd alpha is the scanning frequency of the sample to be detected, and alpha is a correction factor.
Furthermore, in the step A), the external standard does not need to consider interaction and signal overlapping with the sample to be detected, and only needs to be stable in property, not easy to volatilize and soluble in yellow wine. The external standard can be citric acid, maleic acid, etc.
Further, in step B): further comprising, after hydrogen measurement pulse sequences of water peak and ethanol peak are suppressed, adjusting other test parameters, such as longitudinal relaxation time with relaxation delay time greater than 5 times; the excitation center is located between the quantitative peak and the internal standard peak; the angle of excitation may be at a small angle, such as 30 °; the number of scans is such that the signal to noise ratio of the quantitative peak is at least greater than 10.
In the embodiment of the present invention, when an external standard method is adopted, the conventional quantitative calculation formula is formula (I-0-2); when an internal standard method is adopted, the conventional quantitative calculation formula is the formula (I-0-1).
The present invention will be described in detail with reference to examples.
Example 1Method for quantitatively detecting 5-hydroxymethylfurfural in yellow wine by nuclear magnetic resonance hydrogen spectrum by internal standard method
1 reagent materials and instruments and equipment therefor
1.1 reagents used: the yellow wine sample is provided by a yellow wine manufacturer; 5-HMF (99%, sigma aldrich); sodium azide (high purity, NaN)3Biotopped, China); sodium 3- (trimethylsilyl) deuteropropionate (98%, CIL, USA); potassium dihydrogen phosphate (98%, CIL, USA); heavy water (99.9%, tenglong microwave technologies, inc., Qingdao); ethanol (analytical grade, Beijing chemical plant); potassium hydroxide (analytical grade, Beijing chemical plant); sodium hydroxide (analytical grade, Beijing chemical plant; phosphoric acid (analytical grade, Beijing chemical plant); hydrochloric acid (analytical grade, Beijing chemical plant).
1.2 apparatus used: bruker Avance III HD 400M spectrometer (Bruker Biospin, Rheinstetten, Germany); bruker autosampler (SampleJet); bruker SampleJet 5mm high throughput nuclear magnetic tubes (Bruker Biospin, Rheinstetten, Germany); vortex mixer (MX-S), Darongxing laboratory instruments (Beijing) Inc.; bruker Biospin, Rheinstetten, Germany.
2 sample preparation and testing procedure
2.1Preparing a buffer solution: 10.21 g of monopotassium phosphate and 9.75 mg of sodium azide were accurately weighed and dissolved in 50 ml of heavy water (D)2O), adjusting the pH value of the buffer solution to 4 by using phosphoric acid or potassium hydroxide for later use;
2.2 preparation of samples: transfer 800 microliter (V)S) The yellow wine sample to be tested and 20 microliter of internal standard containing 0.05 wt% (M can be obtained by calculation)R) And (2) uniformly mixing 3- (trimethylsilyl) deuterated sodium propionate heavy water, adding 100 microliters of buffer solution with the pH value of 4.0, adding 90 microliters of TSP-free heavy water, adding 1-2 microliters of 1M hydrochloric acid solution or sodium hydroxide solution, adjusting the pH value of the mixed solution to about 4.5, uniformly mixing, transferring 600 microliters of the mixed solution, and placing the mixed solution in a 5mm nuclear magnetic tube to be tested. 6 parts are prepared in parallel.
2.3 preparation of 5-HMF standard: 5 concentration gradients of 5-HMF solution were made up with 20% vol ethanol at concentrations ranging from 5.012mg/L to 80.198 mg/L. Respectively transferring 800 microliters of 5-HMF solution and 20 microliters of heavy water containing 0.05 wt% of internal standard 3- (trimethylsilyl) deuterated sodium propionate, uniformly mixing, adding 100 microliters of buffer solution with the pH value of 4.0, then adding 90 microliters of heavy water without TSP, then adding 1-2 microliters of hydrochloric acid solution or sodium hydroxide solution with the concentration of 1M, adjusting the pH value of the mixed solution to about 4.5, uniformly mixing, transferring 600 microliters of the mixed solution, and placing the mixed solution in a 5mm nuclear magnetic tube for testing.
2.4 adjusting test parameters and testing: the hydrogen spectra of the suppressed water and ethanol peaks were measured first by injection, then by deuterium water lock field and shimming, using a Bruker presaturation standard pulse NOESYPS 1D. The test temperature is 300K (± 0.1), the relaxation delay time D1 is 10s, the number of blank scans DS is 4, the number of scans NS is 128, the sampling time AQ is 4s, and the spectrum width SW is 20 ppm.
2.5 data processing: and (3) when the line width factor LB is 0.3, carrying out Fourier transform, phase correction and baseline correction, and integrating a signal of 9.45ppm of a quantitative peak of the sample and a signal of 0ppm of an internal standard peak to obtain a quantitative peak of the 5-hydroxymethylfurfural and a quantitative peak integral area ratio A of the internal standard substance. Calculating the concentration C of 5-HMF in the yellow wine before correction according to the formula (I-0-1)HMF-0See table 1 for details.
M in the formula (I-0-1)RIs the mass of the internal standard, NRNumber of hydrogens represented by the quantitative peak of the internal standard, NHMFIs the number of hydrogens represented by the quantitative peak for 5-hydroxymethylfurfural, PRAs the purity of the internal standard substance, A is the integral area ratio of the quantitative peak of 5-hydroxymethylfurfural to the quantitative peak of the internal standard substance, WHMFIs the molecular weight of 5-hydroxymethylfurfural, WRMolecular weight of internal standard, VSThe volume of the sample to be tested was used for the sample preparation.
Linear relationship investigation of 35-HMF and testing of correction factor
The actual concentration of the 5-HMF standard obtained at 2.3 is used as the ordinate, and the actual test concentration C obtained at 2.5 is used as the ordinateHMF-0Making linear regression for abscissa to obtain linear equation of y-1.0616 x +0.0003, R2It can be seen that the linear relationship is good at 0.9998, with a correction factor of 1.0616.
4 results of measurement
And (3) calculating the concentration quantitative result of the 5-hydroxymethylfurfural in the corrected yellow wine: according to formula (I) CHMF＝CHMF-0And alpha, calculating to obtain the concentration of the corrected 5-HMF, which is specifically shown in Table 1.
TABLE 1 results of sample testing
Analysis of 5-HMF assay results in yellow wine
5.1 precision and repeatability of 5-HMF determination in yellow wine: another yellow wine sample is taken, 6 sample solutions are prepared in parallel according to the method for testing, each sample is tested for 5 times, the average value is taken, the standard deviation is calculated, and the result is shown in table 2. As can be seen from Table 2, the precision and reproducibility were good.
TABLE 2 content determination precision and repeatability analysis of 5-HMF in yellow wine
5.2 assay of recovery from 5-HMF assay in yellow wine
20 yellow wine samples were taken in parallel, 5 of which were control samples, 15 of which were added with 3 different levels of 5-HMF standard solutions of 7.5mg/L, 15mg/L, 60mg/L, respectively, and the measurements were repeated 5 times per level, and the results were averaged to give an average recovery of 99.546% as shown in Table 3.
TABLE 35 HMF recovery normalized
5.3 assay of stability of 5-HMF in yellow wine
The same sample solution is taken, and the results are measured in 0 th, 2 th, 4 th, 6 th, 8 th, 10 th, 12 th, 14 th, 16 th, 18 th, 20 th and 24 th hours under the test conditions, the results are averaged and the relative standard deviation is calculated, the results are shown in table 4, the relative standard deviation is 1.717%, and the test stability is very good.
TABLE 4 stability analysis of 5-HMF assay in yellow wine
Conclusion 6
The invention establishes the method for quantitatively determining 5-HMF in yellow wine by nuclear magnetic resonance hydrogen spectrum, the yellow wine sample is simple and quick to test without pretreatment, the accuracy and the repeatability are good, the quantitative limit can reach 2.86mg/L (S/N is 10), and the detection limit can reach 0.86mg/L (S/N is 3).
Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (7)
1. A method for quantitatively detecting 5-hydroxymethylfurfural in yellow wine by utilizing nuclear magnetic resonance hydrogen spectrum is characterized in that,
in the measuring process, the hydrogen spectrum is measured by pressing a water peak and an ethanol peak, and the measuring result is substituted into a conventional quantitative calculation formula to obtain the quantitative concentration C of the uncorrected 5-hydroxymethylfurfuralHMF-0(ii) a C is to beHMF-0Substituting formula (I) to obtain the corrected quantitative concentration C of 5-hydroxymethylfurfuralHMF；
CHMF＝CHMF-0Alpha (I), wherein alpha is a correction factor.
2. The method of claim 1,
the method for determining the correction factor comprises the following steps:
establishing a linear regression equation y alpha x + beta by taking the concentration of the 5-hydroxymethylfurfural standard product as an ordinate y and the quantitative concentration of the uncorrected 5-hydroxymethylfurfural as an abscissa x to obtain alpha; wherein, alpha is a correction factor.
3. The method of claim 1,
and (3) adopting a pre-saturation method, and suppressing a water peak and an ethanol peak by adjusting hydrogen measurement pulse sequences of the water peak and the ethanol peak.
4. The method of claim 1,
in the determination process, a lock field reagent is added into a sample to be determined; the field-locking reagent comprises a deuterated field-locking reagent or a fluorinated field-locking reagent.
5. The method of claim 1,
the method for the quantitative detection of the nuclear magnetic resonance hydrogen spectrum comprises an internal standard method and an external standard method.
6. The method of claim 5,
the internal standard method comprises the following steps:
a) sample preparation: adding an internal standard substance and a field locking reagent into a sample to be detected, mixing, adding into a nuclear magnetic tube, and detecting;
b) sampling the mixture to be tested obtained in the step a), calling a hydrogen measurement pulse sequence for suppressing a water peak and an ethanol peak, and then locking a field, shimming and tuning;
c) b) carrying out Fourier transform, phase correction, baseline correction, quantitative peak and internal standard peak integration on the original data obtained after sampling is finished, and then calculating according to a formula (I) and a conventional quantitative formula (I-0-1) to obtain the quantitative concentration of the 5-hydroxymethylfurfural in the corrected yellow wine;
CHMF＝CHMF-0ɑ (I)；
wherein M isRIs the mass of the internal standard, NRNumber of hydrogens represented by the quantitative peak of the internal standard, NHMFIs the number of hydrogens represented by the quantitative peak for 5-hydroxymethylfurfural, PRAs the purity of the internal standard substance, A is the integral area ratio of the quantitative peak of 5-hydroxymethylfurfural to the quantitative peak of the internal standard substance, WHMFIs the molecular weight of 5-hydroxymethylfurfural, WRMolecular weight of internal standard, VSAnd the volume of a sample to be detected is used for sample preparation, and alpha is a correction factor.
7. The method of claim 5,
the external standard method comprises the following steps:
A) sample preparation: respectively preparing an external standard and a sample to be detected, adding a field locking reagent into the sample to be detected and the external standard, transferring the sample to be detected and the external standard into a nuclear magnetic tube, and detecting the sample to be detected;
B) after the mixture obtained in the step A) is injected, measuring the 90-degree pulse width of an external standard and a sample to be measured, and testing a hydrogen spectrum by a method of suppressing a water peak and ethanol;
C) b) performing Fourier transform, phase adjustment, baseline correction, quantitative peak and internal standard peak integration on the original data obtained after the test is finished, and then calculating the quantitative concentration of the 5-hydroxymethylfurfural in the yellow wine according to the formula (I) and a conventional quantitative formula (I-0-2);
CHMF＝CHMF-0ɑ (I)；
wherein M isRIs the mass of the external standard, VSVolume of sample to be tested, PRA is the ratio of the integrated areas of the quantitative peak of 5-HMF and the quantitative peak of the external standard, W, for the purity of the external standardHMFIs the molecular weight of 5-hydroxymethylfurfural, WRIs the molecular weight of the external standard, NRNumber of hydrogens represented by the quantitative peak of the external standard, NHMFIs the number of hydrogens represented by the quantitative peak for 5-hydroxymethylfurfural, P1SFor a sample to be tested 90 DEG pulse width, P1RIs the 90 DEG pulse width, T, of the external standardRAs test temperature, T, of external standardSFor the test temperature, NS, of the sample to be testedRFor the number of scans of external targets, NSSAnd alpha is the scanning frequency of the sample to be detected, and alpha is a correction factor.
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