阅读说明：本技术 一种丙烯醛-甲醛-γ-氨基丁酸加合物及其制备方法与应用 (Acrolein-formaldehyde-gamma-aminobutyric acid adduct and preparation method and application thereof ) 是由 宋媛 于 2021-09-16 设计创作，主要内容包括：本发明公开了一种丙烯醛-甲醛-γ-氨基丁酸加合物及其制备方法与应用,属于有机化合物合成和食品检测技术领域,所述丙烯醛、甲醛和γ-氨基丁酸的摩尔比为(1～2)：1：(1～10),以水作为溶剂,对丙烯醛、甲醛和γ-氨基丁酸持续加热进行反应,获得丙烯醛-甲醛-γ-氨基丁酸加合物,分子式为C-(11)H-(15)NO-(3),本发明所述方法操作简便,对丙烯醛和甲醛的消除效果显著,纯化产物的纯度和产率高。(The invention discloses an acrolein-formaldehyde-gamma-aminobutyric acid adduct and a preparation method and application thereof, belonging to the technical field of organic compound synthesis and food detection, wherein the molar ratio of the acrolein to the formaldehyde to the gamma-aminobutyric acid is (1-2): 1: (1-10), continuously heating acrolein, formaldehyde and gamma-aminobutyric acid to react by using water as a solvent to obtain an acrolein-formaldehyde-gamma-aminobutyric acid adduct with a molecular formula of C 11 H 15 NO 3 The method of the invention has simple operation, obvious elimination effect on acrolein and formaldehyde and high purity and yield of the purified product.)

1. An acrolein-formaldehyde-gamma-aminobutyric acid adduct characterized by the molecular formula: c₁₁H₁₅NO₃Molecular weight: 209, structural formula as follows:

2. a method for preparing an acrolein-formaldehyde- γ -aminobutyric acid adduct according to claim 1, comprising the steps of: adding acrolein, formaldehyde and gamma-aminobutyric acid into water as a solvent, and heating for reaction.

3. The preparation method according to claim 2, wherein the molar ratio of the acrolein to the formaldehyde to the gamma-aminobutyric acid is (1-2): 1: (1-10).

4. The preparation method according to claim 2, wherein the heating temperature is 30 to 120 ℃ and the reaction time is 30min to 12 h.

5. The preparation method according to claim 2, wherein the heating reaction further comprises concentrating the reacted system under reduced pressure, filtering, and separating and purifying by reverse phase silica gel column chromatography to obtain pure acrolein-formaldehyde-gamma-aminobutyric acid adduct.

6. The method according to claim 5, wherein the pure acrolein-formaldehyde- γ -aminobutyric acid adduct is obtained by a method comprising the following steps: performing primary identification on the pure adduct by adopting a high performance liquid chromatography-tandem mass spectrometry method to obtain primary and secondary mass spectra; and analyzing the adduct sample by using a high-resolution mass spectrometer and a nuclear magnetic resonance spectrometer to obtain accurate relative molecular mass and structural formula.

7. The method according to claim 6, wherein the high resolution mass spectrometer ion source is an electrospray ion source and the scanning range is m/z 50-1500.

8. The method of claim 6, wherein in the HPLC tandem mass spectrometry:

high performance liquid chromatography conditions: the chromatographic column is ZORBAX SB-Aq, 4.6mm × 250mm, 5 μm; the mobile phase is water-acetonitrile, 5:95, V/V, the sample injection volume is 5 mu L, the flow rate is 0.6mL/min, and the column temperature is 40 ℃; the detection wavelength is 267 nm;

mass spectrum conditions: the mobile phase is 0.1% of acetic acid water and methanol, 95:5, V/V, the sample injection volume is 0.1 mu L, the flow rate is 0.4mL/min, the column temperature is 40 ℃, the ion source is an electrospray ion source, and the scanning range is m/z 50-800; the source temperature was 300 deg.C, the desolvation temperature was 250 deg.C, the capillary voltage was 4000V, the scanning rate was 1000Da/sec, and the collision energy was 15 eV.

9. Use of the acrolein-formaldehyde-gamma-aminobutyric acid adduct of claim 1 for simultaneous elimination of acrolein and formaldehyde.

10. Use of the acrolein-formaldehyde-gamma-aminobutyric acid adduct of claim 1 in food detection field.

Technical Field

The invention relates to the technical field of organic compound synthesis and food detection, in particular to an acrolein-formaldehyde-gamma-aminobutyric acid adduct and a preparation method and application thereof.

Background

Acrolein (Acr) is an alpha, beta-unsaturated aldehyde with highly toxic properties, its LD in mice and rats₅₀30mg/kg and 50mg/kg, respectively. The exogenous acrolein is mainly generated in the natural environment and in food processing processes, such as forest fire, dye combustion, Maillard reaction of baked food and brewing process of alcoholic beverage; endogenous acrolein is mainly spontaneously generated through various ways such as lipid peroxidation, amino acid oxidation and polyamine oxidation, and intestinal microorganisms can be endogenousOne of the pathways for the formation of the derived acrolein. Acrolein is used as a strong electrophilic reagent, has high water solubility, can rapidly penetrate cell membranes to form adducts with glutathione, DNA, proteins and the like, and oxygen radicals are accumulated, so that the cell membranes are cracked, mitochondrial dysfunction is caused, even gene mutation is induced, protein cross-linking, cell necrosis and apoptosis are caused, and different degrees of harm are caused to respiratory systems, cardiovascular systems, reproductive systems, nervous systems and the like. Numerous studies have shown that acrolein is associated with a variety of pathological conditions including lung cancer, alzheimer's disease, cardiovascular disease, atherosclerosis and spinal cord injury. The world health organization sets the tolerable maximum intake of acrolein in human oral administration at 7.5. mu.g/kg-bw/day.

Formaldehyde (FA) is the most active fatty aldehyde, and can be produced endogenously in food, or can be present in food through exogenous addition or environmental pollution. Formaldehyde is a metabolic product of cells, so that formaldehyde is widely present in the human body and in various animal and plant tissues. The food can generate formaldehyde by Maillard reaction and thermal degradation reaction during processing or storage, especially for fermented food such as wine, bread and the like. Formaldehyde has been classified as a first carcinogen by the international agency for research on cancer (IARC), one of the most dangerous chemicals affecting human health. Formaldehyde can react not only with amine functions of lysine in rhodopsin proteins, but also with amino groups in other proteins (including many enzymes), affecting the function and biochemical metabolism of the enzyme.

Free amino acids are commonly found in various foods, and some amino acids are widely used in food ingredients, such as gamma-aminobutyric acid. Gamma-aminobutyric acid (GABA) is a non-protein amino acid and belongs to a new resource food. It is widely found in mammals, plants and microorganisms, while cereals, tea, vegetables and other food products are present in higher amounts, for example up to 660mg/kg in potatoes, next to asparagine. GABA has a sweet taste similar to glutamic acid and can enhance food flavor. GABA plays an important role in the regulation of the nervous system, for example, in promoting neuronal growth and preventing insomnia, anxiety and depression.

Currently, formaldehyde scavengers mainly comprise amino groups, polyphenols, natural extracts and strong oxidants. Wherein, the amino has good cleaning effect and is not easy to generate secondary pollution. In addition, studies have shown that some amino acids form adducts with acrolein to effectively remove acrolein and reduce the cytotoxicity of acrolein, such as cysteine. However, it has not been investigated whether amino acids can form adducts with acrolein and formaldehyde simultaneously to simultaneously remove these two aldehydes.

Disclosure of Invention

Not only has no study on whether the amino acid can form an adduct with acrolein and formaldehyde so as to simultaneously remove the two aldehydes, but also the adduct in food cannot be detected without a standard substance, the toxicity of the formed adduct is still to be studied, and the obtaining of the adduct standard substance is an important prerequisite for further development of a detection method and toxicological study. Therefore, the invention mainly solves the problem of how to obtain the standard substance of the acrolein-formaldehyde-gamma-aminobutyric acid adduct with high yield and high purity, and provides the acrolein-formaldehyde-gamma-aminobutyric acid adduct as well as a preparation method and application thereof.

In order to achieve the purpose, the invention provides the following scheme:

the invention provides an acrolein-formaldehyde-gamma-aminobutyric acid adduct, which has a molecular formula: c₁₁H₁₅NO₃Molecular weight: 209, structural formula as follows:

the invention provides a preparation method of an acrolein-formaldehyde-gamma-aminobutyric acid adduct, which comprises the following steps: adding acrolein, formaldehyde and gamma-aminobutyric acid into water as a solvent, and heating for reaction.

Further, the molar ratio of the acrolein to the formaldehyde to the gamma-aminobutyric acid is (1-2): 1: (1-10).

Further, the heating temperature is 30-120 ℃, and the reaction time is 30 min-12 h. The heating temperature is preferably 80 ℃ and the reaction time is preferably 4 h.

Further, after the heating reaction, the method also comprises the steps of decompressing and concentrating the system after the reaction, filtering, and separating and purifying by a reverse phase silica gel column chromatography (ODS filler) to obtain the pure product of the acrolein-formaldehyde-gamma-aminobutyric acid adduct.

In reverse phase silica gel chromatography: selecting reverse phase silica gel powder (ODS filler), eluting with 1-3 mL of 5% methanol water at equal intervals, collecting an adduct by an iodine fumigation chromogenic method, detecting the purity by a high performance liquid detection method, and collecting eluent to obtain a purified solid sample.

Further, the structure identification method of the pure product of the acrolein-formaldehyde-gamma-aminobutyric acid adduct comprises the following steps: performing primary identification on the pure adduct by using a high performance liquid chromatography-tandem mass spectrometry (HPLC) method on the fraction with the purity of more than 99% to obtain a primary mass spectrum and a secondary mass spectrum; and analyzing the adduct sample by using a high-resolution mass spectrometer and a nuclear magnetic resonance spectrometer to obtain accurate relative molecular mass and structural formula.

Dissolving the solid pure product in 600 μ L of heavy water, and detecting by using Avance III spectrometer (600MHz Bruker BioSpin, Switzerland) to obtain NMR of the adduct pure product¹H、NMR ¹³C. Dept-135, HSQC, H COSY and HMBC nuclear magnetic spectra.

Furthermore, the ion source of the high-resolution mass spectrometer is an electrospray ionization source (ESI), and the scanning range is m/z 50-1500.

Further, in the high performance liquid chromatography tandem mass spectrometry:

high performance liquid chromatography conditions: the chromatographic column is ZORBAX SB-Aq, 4.6mm × 250mm, 5 μm; the mobile phase is water-acetonitrile, 5:95, V/V, the sample injection volume is 5 mu L, the flow rate is 0.6mL/min, and the column temperature is 40 ℃; the detection wavelength is 267 nm;

mass spectrum conditions: the mobile phase is 0.1% of acetic acid water and methanol, 95:5, V/V, the sample injection volume is 0.1 mu L, the flow rate is 0.4mL/min, the column temperature is 40 ℃, the ion source is an electrospray ion source (ESI), and the scanning range is m/z 50-800; the source temperature was 300 deg.C, the desolvation temperature was 250 deg.C, the capillary voltage was 4000V, the scanning rate was 1000Da/sec, and the collision energy was 15 eV.

The invention provides the use of the acrolein-formaldehyde-gamma-aminobutyric acid adduct in the simultaneous elimination of acrolein and formaldehyde.

The invention provides application of the acrolein-formaldehyde-gamma-aminobutyric acid adduct in the field of food detection, so that the joint product of acrolein and formaldehyde can be analyzed and identified more quickly and the content can be detected more quickly. The invention discloses the following technical effects:

the acrolein-formaldehyde-gamma-aminobutyric acid adduct prepared by the method has high yield and high purity, the purity reaches more than 99%, and the acrolein-formaldehyde-gamma-aminobutyric acid adduct can be used for simultaneously eliminating acrolein and formaldehyde and detecting the contents of the acrolein and the formaldehyde in food. In the process of detecting acrolein and formaldehyde in food, the exposure level of the acrolein-formaldehyde-gamma-aminobutyric acid adduct in the food is evaluated by rapidly and quantitatively detecting the content of the acrolein-formaldehyde-gamma-aminobutyric acid adduct in the food, so that the safety of the food is comprehensively evaluated.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without inventive exercise.

FIG. 1 is a high performance liquid chromatogram (HPLC chart) of the adduct of gamma-aminobutyric acid with acrolein and formaldehyde of example 1;

FIG. 2 is a high performance liquid chromatogram of the purified product of the adduct of gamma-aminobutyric acid with acrolein and formaldehyde of example 1;

FIG. 3 is a graph of the UV absorption spectrum of the purified product of the adduct of gamma-aminobutyric acid with acrolein and formaldehyde of example 1;

FIG. 4 high resolution mass spectrum of purified product of adduct of gamma aminobutyric acid with acrolein and formaldehyde of example 1;

FIG. 5 is a first order mass spectrum (positive ion mode) of the purified product of the adduct of gamma-aminobutyric acid with acrolein and formaldehyde of example 1;

FIG. 6 is a secondary mass spectrum (positive ion mode) and major fragment ion structure of the purified product of the adduct of gamma-aminobutyric acid with acrolein and formaldehyde of example 1;

FIG. 7 is a graph of the purified product of the adduct of gamma-aminobutyric acid with acrolein and formaldehyde of example 1¹³C NMR nuclear magnetic spectrum;

FIG. 8 is a nuclear magnetic spectrum of a Dept-135 of the purified product of the adduct of gamma-aminobutyric acid with acrolein and formaldehyde of example 1;

FIG. 9 is a graph of the purified product of the adduct of gamma-aminobutyric acid with acrolein and formaldehyde of example 1¹H NMR nuclear magnetic spectrum;

FIG. 10 is an HSQC-NMR spectrum of the purified product of the adduct of gamma-aminobutyric acid with acrolein and formaldehyde of example 1;

FIG. 11 is a H, H COSY-NMR spectrum of an adduct of gamma-aminobutyric acid with acrolein and formaldehyde purified product of example 1;

FIG. 12 is an HMBC-NMR spectrum of the purified product of the adduct of gamma-aminobutyric acid with acrolein and formaldehyde of example 1.

Detailed Description

Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.

As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.

Example 1

Preparing Formaldehyde (FA), acrolein (Acr) and gamma-aminobutyric acid (GABA) solutions: respectively taking 10mL of acrolein, formaldehyde and gamma-aminobutyric acid into a 100mL Erlenmeyer flask, wherein the reaction molar ratio is 1:2:1, reacting for 4 hours at the temperature of 80 ℃ to obtain the acrolein-formaldehyde-gamma-aminobutyric acid adduct with the purity of 62%. Filtering the reaction solution after decompression concentration by a filter membrane, separating and purifying by using an ODS silica gel chromatographic column, and freeze-drying to obtain the purified product of the acrolein-formaldehyde-gamma-aminobutyric acid adduct, wherein the yield is 86%, and the purity is over 99%.

The specific implementation method of the ODS silica gel chromatography column comprises the following steps:

soaking 350mL of ODS silica gel powder in methanol, standing for 24h, loading a silica gel methanol solution into a chromatographic column (30X 600mm), performing rotary evaporation concentration on a reaction solution, filtering by using a membrane, loading the reaction solution into a sample, performing pressure elution by using a mobile phase of 5% methanol and water, detecting a fraction containing an adduct by using an iodine fumigation method, verifying the purity by using an HPLC method, concentrating the fraction with the purity of more than 99%, and performing freeze drying (-40 ℃, 90Pa and 24h) to obtain 0.1g of a fixed sample.

The characterization results of the acrolein-formaldehyde-gamma-aminobutyric acid adduct prepared in this example are shown in fig. 1 to 12.

As can be seen from the HPLC chart, the maximum absorption wavelength of the product was 267nm, and the retention time was 6.6 min. From [ M + H ] in the primary mass spectrogram]⁺m/z 210 can determine the relative molecular weight of the product to be 209, and a high resolution mass spectrometer further obtains the accurate molecular weight of the product to be 209.1125, wherein the molecular formula is C₁₁H₁₅NO₃. From the secondary mass spectrum, the target product is removed with one carboxyl group to obtain a fragment peak of m/z ═ 164, which indicates that the adduct contains one carboxyl group of gamma-aminobutyric acid. The peak at m/z of 107 is the 1-methyl-3-methylene-1, 2,3, 6-tetrahydropyridine ring structure. From the purified target product¹³A C spectrum,¹The H spectrum and the Dept-135 spectrum show that the product contains 6 methylene groups. The peak signal with chemical shift delta 9.41 in the hydrogen spectrum corresponds to the hydrogen atom on the aldehyde group, the peak signal delta 5.83 corresponds to the hydrogen atom on the carbon-carbon double bond in the ring, and the two groups of peaks delta 5.83 and delta 5.71 correspond to the hydrogen atom on the carbon-carbon double bond outside the ring. The correlation of the two-dimensional NMR spectrum signals of the adduct is shown in Table 1, and it can be further confirmed that the adduct of the present invention is obtained by reacting one molecule of gamma-aminobutyric acid, one molecule of formaldehyde and two molecules of acrolein and removing two molecules of water, and the structure is as follows

The adduct prepared by the method has high purity, good repeatability and high separation degree, can be applied to the field of food detection, and can be used for rapidly analyzing the performance and the content of the acrolein-formaldehyde-gamma-aminobutyric acid adduct in food.

TABLE 1 assignment of nuclear magnetic signals of acrolein-formaldehyde-gamma-aminobutyric acid adduct

Example 2

Preparing GABA, Acr and FA solutions: 10mL of each was placed in a 100mL Erlenmeyer flask and reacted at 37 ℃ for 12 hours in a molar ratio of 1:2: 1. The reaction solution was concentrated under reduced pressure, filtered through a membrane, and separated, purified and freeze-dried by means of an ODS silica gel column to obtain an acrolein-formaldehyde-gamma-aminobutyric acid adduct, which was purified and freeze-dried in the same manner as in example 1. The product generated in the system is single and easy to separate, and the purity of the acrolein-formaldehyde-gamma-aminobutyric acid adduct reaches more than 98%.

Example 3

Preparing GABA, Acr and FA solutions: respectively putting 2mL of the mixture into a 10mL steel tube, reacting for 30min at 120 ℃ according to the mol ratio of 1:2:1, concentrating the reaction solution under reduced pressure, filtering the reaction solution by using a filter membrane, separating, purifying and freeze-drying the reaction solution by using an ODS silica gel chromatographic column to obtain an acrolein-formaldehyde-gamma-aminobutyric acid adduct, wherein the purification and freeze-drying processes are the same as those in example 1. The purity of the acrolein-formaldehyde-gamma-aminobutyric acid target adduct generated by the system reaches more than 98 percent, and the reaction time is greatly shortened.

Comparative example 1

The preparation method was the same as example 1 except that the reaction time was adjusted to 30 min.

The yield of the acrolein-formaldehyde-gamma-aminobutyric acid adduct was 43%, and the purity after purification was 98%. After the reaction time is shortened, although the purity of the adduct does not change much, the amount of adduct formed is small and the yield is extremely low.

Comparative example 2

The preparation process was the same as in example 1, except that the reaction time was adjusted to 10 hours.

The yield of the acrolein-formaldehyde-gamma-aminobutyric acid adduct was 79%, and the purity after purification was 87%. The reaction time is too long, other impurities are generated, the purity of the purified adduct is reduced, and the yield is also reduced; and the reaction solution is too thick and is not easy to carry out the pretreatment of column chromatography.

Comparative example 3

The preparation process was the same as in example 2, except that the reaction time was adjusted to 0.2 h.

The yield of the acrolein-formaldehyde-gamma-aminobutyric acid adduct was 36%, and the purity after purification was 90%. The principle is the same as that of comparative example 1.

Comparative example 4

The preparation method is the same as example 1 except that the reaction molar ratio is adjusted to GABA: acr: FA is 5: 5: 1.

the yield of the acrolein-formaldehyde-gamma-aminobutyric acid adduct was 80%, and the purity after purification was 67%. The molar ratio is increased, GABA is excessive, the purification and separation of column chromatography are not facilitated, and the purity of the obtained adduct is low.

Comparative example 5

The preparation method is the same as example 1 except that the reaction molar ratio is adjusted to GABA: acr: FA is 10: 1: 2.

the yield of the acrolein-formaldehyde-gamma-aminobutyric acid adduct was 59%, and the purity after purification was 45%. The GABA ratio is too high to facilitate column chromatography separation, resulting in a substantial reduction in purity without ensuring an excess of acrolein and a reduction in adduct yield.

Application examples

The acrolein-formaldehyde-gamma-aminobutyric acid adduct prepared in example 1 was used as a standard to measure the content of the adduct in food.

Adduct detection application example 1

S0. raw material acquisition

The potato chips are purchased from local supermarkets, namely the happy-thing original-taste potato chips and the Liriomo potato chips which are wanted to be baked with the original taste.

S1. preparation of standard curve

Preparing standard series solutions of the acrolein-formaldehyde-gamma-aminobutyric acid adduct, wherein the mass concentrations are respectively 1, 5, 10, 50, 100 and 500 mu g/kg, a standard curve is drawn by taking the peak area of a product as a vertical coordinate and the concentration as a horizontal coordinate, a linear equation is that y is 6348910x +6327.09, and the correlation coefficient is 0.9995. Thus, the content of the acrolein-formaldehyde-gamma-aminobutyric acid adduct in the experiment was measured.

S2. extraction of adduct

Grinding 16g of potato chips in a mortar, respectively putting 5g of potato chips in three 50mL centrifuge tubes, respectively adding 20mL of methanol, centrifuging at 10000rpm/min for 10min after 5min of vortex, repeating the operation for three times, combining about 60mL of supernate, performing reduced pressure spin-drying on the solvent, then performing constant volume with 5mL of methanol, and performing HPLC-MS/MS detection on the content of the adduct after a filter membrane is used.

S3. content of HPLC-MS/MS additive in food

Adopting an MRM positive ion mode in HPLC-MS/MS, and selecting ion pairs respectively as follows: 210/124 (quantitative ion pairs, collision energy 20 eV); 210/164 (qualitative ion pair, collision energy 30 eV); 210/192 (qualitative ion pair, collision energy 38eV), mobile phase: 0.1% acetic acid water/methanol (70:30, V/V), isocratic elution; the flow rate is 0.4 mL/min; the sample size was 1. mu.L.

Finally, the content of the acrolein-formaldehyde-gamma-aminobutyric acid adduct in the leishi original-taste potato chips is measured to be 6 mu g/kg, and the content of the adduct in the Liriomo potato wishland roasted original-taste potato chips is measured to be 25 mu g/kg.

Adduct detection application example 2

S0. raw material acquisition

Coarse grain rice rolls were purchased from local supermarket, 21-grain coarse-grain-flavored brown rice cake and 101-grain vitality bar, respectively.

S1. preparation of standard curve

Preparing standard series solutions of the acrolein-formaldehyde-gamma-aminobutyric acid adduct, wherein the mass concentrations are respectively 1, 5, 10, 50, 100 and 500 mu g/kg, a standard curve is drawn by taking the peak area of a product as a vertical coordinate and the concentration as a horizontal coordinate, a linear equation is that y is 6348910x +6327.09, and the correlation coefficient is 0.9995. Thus, the content of the acrolein-formaldehyde-gamma-aminobutyric acid adduct in the experiment was measured.

S2. extraction of adduct

Taking 16g of coarse grain rice rolls, grinding the coarse grain rice rolls in a mortar, respectively taking 5g of coarse grain rice rolls in three 50mL centrifuge tubes, respectively adding 20mL of methanol, whirling for 5min, then centrifuging at 10000rpm/min for 10min, repeating the operation for three times, combining about 60mL of supernate, carrying out reduced pressure whirling to dry the solvent, then carrying out constant volume with 5mL of methanol, and carrying out filtration membrane until the content of the adduct is detected by HPLC-MS/MS.

S3. content of HPLC-MS/MS additive in food

Adopting an MRM positive ion mode in HPLC-MS/MS, and selecting ion pairs respectively as follows: 210/124 (quantitative ion pairs, collision energy 20 eV); 210/164 (qualitative ion pair, collision energy 30 eV); 210/192 (qualitative ion pair, collision energy 38eV), mobile phase: 0.1% acetic acid water/methanol (70:30, V/V), isocratic elution; the flow rate is 0.4 mL/min; the sample size was 1. mu.L.

Finally, the content of the acrolein-formaldehyde-gamma-aminobutyric acid adduct in the 21-cereal grain-flavored brown rice cake was measured to be 21 μ g/kg, and the content of the adduct in the 101-cereal grain vitality bar was measured to be 23 μ g/kg.

Adduct detection application example 3

S0. raw material acquisition

The biscuits are purchased from local supermarkets, namely letter milk biscuits and yeast soda biscuits.

S1. preparation of standard curve

Preparing standard series solutions of the acrolein-formaldehyde-gamma-aminobutyric acid adduct, wherein the mass concentrations are respectively 1, 5, 10, 50, 100 and 500 mu g/kg, a standard curve is drawn by taking the peak area of a product as a vertical coordinate and the concentration as a horizontal coordinate, a linear equation is that y is 6348910x +6327.09, and the correlation coefficient is 0.9995. The content of the acrolein-formaldehyde- γ -aminobutyric acid adduct in the present application example was thus measured.

S2. extraction of adduct

Grinding 16g of biscuits in a mortar, respectively putting 5g of biscuits in three 50mL centrifuge tubes, respectively adding 20mL of methanol, whirling for 5min, centrifuging at 10000rpm/min for 10min, repeating the operation for three times, combining about 60mL of supernate, carrying out reduced pressure spin-drying on a solvent, then carrying out constant volume with 5mL of methanol, and carrying out filtration membrane until the content of the adduct is detected by HPLC-MS/MS.

S3. content of HPLC-MS/MS additive in food

Adopting an MRM positive ion mode in HPLC-MS/MS, and selecting ion pairs respectively as follows: 210/124 (quantitative ion pairs, collision energy 20 eV); 210/164 (qualitative ion pair, collision energy 30 eV); 210/192 (qualitative ion pair, collision energy 38eV), mobile phase: 0.1% acetic acid water/methanol (70:30, V/V), isocratic elution; the flow rate is 0.4 mL/min; the sample size was 1. mu.L.

Finally, the acrolein-formaldehyde-gamma-aminobutyric acid adduct content in the alphabetical milk biscuits was measured to be 48 μ g/kg, and the adduct content in the yeast crackers was measured to be 62 μ g/kg.

The application examples show that the method can be used for quickly and quantitatively detecting the content of the acrolein-formaldehyde-gamma-aminobutyric acid adduct in food.

The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.