CN113149893A - Acrolein-serine adduct and preparation method and application thereof - Google Patents
Acrolein-serine adduct and preparation method and application thereof Download PDFInfo
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- SZAIYEQSUPKOEL-DKWTVANSSA-N (2s)-2-amino-3-hydroxypropanoic acid;prop-2-enal Chemical compound C=CC=O.OC[C@H](N)C(O)=O SZAIYEQSUPKOEL-DKWTVANSSA-N 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title claims abstract description 10
- HGINCPLSRVDWNT-UHFFFAOYSA-N Acrolein Chemical compound C=CC=O HGINCPLSRVDWNT-UHFFFAOYSA-N 0.000 claims abstract description 62
- 238000000034 method Methods 0.000 claims abstract description 25
- 238000006243 chemical reaction Methods 0.000 claims abstract description 22
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- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 claims abstract description 18
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- HORQAOAYAYGIBM-UHFFFAOYSA-N 2,4-dinitrophenylhydrazine Chemical compound NNC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O HORQAOAYAYGIBM-UHFFFAOYSA-N 0.000 claims description 3
- 238000011161 development Methods 0.000 claims description 3
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- 244000061456 Solanum tuberosum Species 0.000 description 6
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- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 4
- 235000012015 potatoes Nutrition 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 3
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- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 150000001299 aldehydes Chemical class 0.000 description 2
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- 238000006845 Michael addition reaction Methods 0.000 description 1
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- 125000003172 aldehyde group Chemical group 0.000 description 1
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- 229910052757 nitrogen Inorganic materials 0.000 description 1
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- 230000000269 nucleophilic effect Effects 0.000 description 1
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- 150000002894 organic compounds Chemical class 0.000 description 1
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- 230000008569 process Effects 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000005173 quadrupole mass spectroscopy Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
- C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D211/68—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member
- C07D211/70—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having one double bond between ring members or between a ring member and a non-ring member with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/02—Food
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
Abstract
本发明属于食品检测的技术领域，公开了一种丙烯醛‑丝氨酸加合物及其制备方法与应用。所述丙烯醛‑丝氨酸加合物的制备方法，包括以下步骤：在水中，将丙烯醛与丝氨酸进行恒温加热反应，后续处理，获得丙烯醛‑丝氨酸加合物；所述丙烯醛与丝氨酸的摩尔比为1∶(1～3)。本发明的方法简单，所获得产物产率高，纯度高。所述丙烯醛‑丝氨酸加合物在食品检测领域的应用。The invention belongs to the technical field of food detection, and discloses an acrolein-serine adduct and a preparation method and application thereof. The preparation method of the acrolein-serine adduct comprises the following steps: performing a constant temperature heating reaction with acrolein and serine in water, and subsequent processing to obtain the acrolein-serine adduct; the molar ratio of the acrolein and serine The ratio is 1:(1～3). The method of the invention is simple, and the obtained product has high yield and high purity. Application of the acrolein-serine adduct in the field of food detection.
Description
Technical Field
The invention belongs to the technical field of organic compound synthesis, and particularly relates to an acrolein-serine adduct and a preparation method and application thereof.
Background
Acrolein is the most reactive aldehyde of the α, β -unsaturated carbonyl structure, widely found in ambient and thermally processed foods, and can also be produced by endogenous metabolism. Studies have shown that acrolein reacts very readily with nucleophilic macromolecules in organisms, interfering with DNA replication and transcription, leading to protein cross-linking and ultimately to various diseases (30 and 50mg/kg body weight for mouse and rat LD 50). In addition, it can also lower the level of intracellular Glutathione (GSH) and interfere with cellular signaling pathways, making the safety problem of acrolein a major concern.
It has been found that after frying food products such as potatoes, the acrolein content in the fried potato chips is much lower (2-3 orders of magnitude) than the sum of the acrolein content in the frying oil and the acrolein content in the potatoes, while after digestion, the acrolein content in the human body is much higher (more than 200 times) than that in the potato chips, which is very likely to cause food safety hazards because the acrolein reacts with food ingredients during frying, so that the actual exposure level of the acrolein in the food is covered.
Serine, also known as beta-hydroxyalanine, plays an important role in the metabolism of fats and fatty acids and in muscle growth, and is also essential for maintaining a healthy immune system because it contributes to the production of immunocytokines and antibodies. The human body can obtain serine from a large amount of plant and animal proteins, and if necessary, can synthesize serine from glycine. Studies have shown that acrolein is rapidly reduced simultaneously with a decrease in serine content of several 10-fold before and after frying of potatoes. Therefore, we speculate that serine performs a great deal of elimination reaction on acrolein in the frying process, but the nature and safety of elimination products are in need of research. Because the food contains various compounds and is difficult to separate, and the food contains various compounds and different contents, if a certain organic substance is singly researched, the time consumption and the low efficiency are realized by directly extracting and separating the food without a standard substance.
There is a need for further studies on how to obtain adducts in high yield, high purity and as standards for the detection of acrolein reaction products in foods.
Disclosure of Invention
In order to overcome the disadvantages and drawbacks of the prior art, it is an object of the present invention to provide an acrolein-serine adduct and a method for preparing the same. The method is simple, and the obtained product has high yield and high purity.
It is another object of the present invention to provide the use of the above acrolein-serine adduct. The application of the acrolein-serine adduct in the field of food detection is used as a standard substance for detecting acrolein reaction products in food so as to analyze the performance and the content of the acrolein adduct in the food more quickly.
The purpose of the invention is realized by the following technical scheme:
a method for preparing an acrolein-serine adduct comprising the steps of: heating acrolein and serine in water at constant temperature for reaction, and performing subsequent treatment to obtain acrolein-serine adduct.
The molar ratio of the acrolein to the serine is 1: 1-3.
The temperature of the heating reaction is 20-60 ℃, and preferably 45-55 ℃; the heating reaction time is 3-12 h, preferably 4-6 h. The heating reaction is carried out in a dark place.
The reaction is carried out with stirring. The stirring speed is 100-200 r/min.
The dosage ratio of the acrolein to the water is (1-3) mmol to (1-50) mL.
And the subsequent treatment refers to concentrating the reacted system, filtering with a filter membrane, and purifying by a reverse phase silica gel chromatography to obtain a purified product. The filter membrane is a 0.45 mu m filter membrane; the concentration is a reduced pressure concentration.
The reverse phase silica gel chromatography specifically comprises the following steps: selecting reverse phase silica gel ODS resin, loading, eluting with 5% methanol water solution, collecting target adduct by 2, 4-dinitrophenylhydrazine solution color development method, detecting purity by high performance liquid detection method, collecting eluent, and freeze drying to obtain purified sample. The freeze drying condition is preferably-70 to-40 ℃ and 1 to 100 Pa.
According to the high performance liquid chromatography detection result and the nuclear magnetic resonance spectrum result, the purity of the adduct prepared by the method is up to 99%, wherein the peak purity of a target single peak under each display wavelength (200-400 nm) of the high performance liquid chromatography is up to 99%.
The conditions of HPLC determination are as follows: column Zorbax SB-Aq C18(4.6 mm. times.250 mm, 5 μm), mobile phase A being pure water and mobile phase B being methanol; the chromatographic conditions are as follows: the detection wavelength is 220nm, the flow rate is 0.6mL/min, the column temperature is 40 ℃, and the sample injection amount is 2 mu L; the volume fraction of the mobile phase A is 95%, and the volume fraction of the mobile phase B is 5%, and the elution is carried out for 25min in an equal gradient manner.
Under the detection method, the retention time of the target is 5.635min, and the target presents a good peak pattern which is repeatable, high in separation degree and sharp without tailing.
The acrolein-serine adduct has the formula: c9H13O4N, molecular weight: 199, structural formula:
the reaction mechanism is as follows:
the olefinic bond of the acrolein is firstly connected to the nitrogen atom of an amino acid through Michael addition reaction, then two free aldehyde groups are subjected to aldol condensation reaction to generate the aldol, and under the heated condition, the aldol is further dehydrated to generate alpha, beta-unsaturated aldehyde.
The application of the acrolein-serine adduct in the field of food detection is used as a standard substance for detecting acrolein reaction products in food so as to analyze the performance and the content of the acrolein adduct in the food more quickly.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention provides a preparation method of an acrolein-serine adduct, which has high yield, the purity of the prepared adduct is up to 99%, wherein the peak purity of a target single peak under each display wavelength (200-400 nm) of a high performance liquid chromatogram is up to 99%, and the adduct in food can be detected by using the adduct as a standard substance.
Drawings
FIG. 1 is a first order mass spectrum (positive ion mode) of an acrolein-serine adduct prepared in example 1;
FIG. 2 is a first order mass spectrum (negative ion mode) of an acrolein-serine adduct prepared in example 1;
FIG. 3 is a secondary mass spectrum (positive ion mode) of the acrolein-serine adduct prepared in example 1;
FIG. 4 is a secondary mass spectrum (negative ion mode) of the acrolein-serine adduct prepared in example 1;
FIG. 5 is a high resolution mass spectrum (positive ion mode) of the acrolein-serine adduct prepared in example 1;
FIG. 6 is a schematic representation of the acrolein-serine adduct prepared in example 113C NMR nuclear magnetic spectrum;
FIG. 7 is a Dept-135 NMR spectrum of an acrolein-serine adduct prepared in example 1;
FIG. 8 is a schematic representation of the acrolein-serine adduct prepared in example 11H NMR nuclear magnetic spectrum;
FIG. 9 is a high performance liquid chromatogram of the acrolein-serine adduct prepared in example 1;
FIG. 10 is a UV spectrum of an acrolein-serine adduct prepared in example 1.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1
A method for preparing an acrolein-serine adduct comprising the steps of: in water, heating acrolein and serine at a molar ratio of 1: 2 in the dark (reaction at 50 ℃ for 5h and rotation speed of 130r/min), concentrating under reduced pressure for 1-2 mL, filtering with a 0.45-micrometer filter membrane to obtain a filtrate, and purifying by reverse phase silica gel chromatography to obtain an acrolein-serine adduct with a yield of 83%.
Reverse phase silica gel chromatography (C18 reverse phase silica gel resin chromatography): selecting 250mL of reverse phase silica gel ODS resin, enabling the specification of a chromatographic column to be 30 x 500mm, enabling an evaporated and concentrated sample to pass through a 0.45-micrometer filter membrane, then loading the sample, taking a 5% methanol aqueous solution as an eluent, carrying out full-flow-rate isocratic elution, determining the existence of an adduct by a 2, 4-dinitrophenylhydrazine solution color development method, detecting the purity by using a high performance liquid chromatography (HPLC method), collecting a target adduct, and carrying out freeze drying to obtain the high-purity target adduct of about 50 mg; the freeze drying condition is-70 to-40 ℃, 1 to 100Pa and 48 hours.
The characterization patterns of the acrolein-serine adduct prepared in this example are shown in FIGS. 1 to 10 and the analysis results Table 1. FIG. 1 is a primary mass spectrum (positive ion mode); FIG. 2 is a primary mass spectrum (negative ion mode); FIG. 3 is a secondary mass spectrum (positive ion mode); FIG. 4 is a secondary mass spectrum (negative ion mode); FIG. 5 is a high resolution mass spectrum (positive ion mode); FIG. 6 is a drawing showing13C NMR nuclear magnetic spectrum; FIG. 7 is a Dept-135 NMR spectrum; FIG. 8 is a drawing showing1H NMR nuclear magnetic spectrum; FIG. 9 is a high performance liquid chromatogram; FIG. 10 is a UV spectrum; table 1 shows the nuclear magnetic data table and the analysis of the secondary mass spectrum. According to the high performance liquid chromatography results and the nuclear magnetic resonance spectrum results shown in fig. 1 to fig. 10, the purity of the adduct prepared by the method of the present invention is as high as 99%, wherein the peak purity of the target single peak under each display wavelength (200 to 400nm) of the high performance liquid chromatogram is as high as 99%. Under the detection method, the retention time of the target is 5.635min, and the target presents a good peak shape which is repeatable, high in separation degree and sharp without tailing.
TABLE 1 Nuclear magnetic data and Secondary Mass Spectroscopy Structure of the adduct of example 1
Example 2
A method for preparing an acrolein-serine adduct comprising the steps of: in water, heating acrolein and serine in a molar ratio of 1: 1 in a dark place for reaction (reaction at 45 ℃ for 6 hours, rotation speed of 130r/min), concentrating under reduced pressure for 1-2 mL, filtering with a 0.45-micrometer filter membrane to obtain a filtrate, and purifying by using a reverse phase silica gel chromatography to obtain an acrolein-serine adduct; the yield was 84% and the purity of the adduct was 95%.
Example 3
A method for preparing an acrolein-serine adduct comprising the steps of: in water, heating acrolein and serine in a molar ratio of 1: 3 in a dark place for reaction (reaction at 60 ℃ for 5 hours, rotation speed of 130r/min), concentrating under reduced pressure for 1-2 mL, filtering with a 0.45-micrometer filter membrane to obtain a filtrate, and purifying by using a reverse phase silica gel chromatography to obtain an acrolein-serine adduct; yield 88% and purity of adduct 90%.
Comparative example 1 (too long or too short reaction time)
A method for preparing an acrolein-serine adduct comprising the steps of: in water, heating acrolein and serine in a molar ratio of 1: 2 in a dark place for reaction (reaction is carried out for 1h or 8h at 50 ℃ and at a rotation speed of 130r/min), concentrating under reduced pressure for 1-2 mL, filtering with a 0.45-micrometer filter membrane to obtain filtrate, and purifying by using a reverse phase silica gel chromatography to obtain the acrolein-serine adduct. The yields were 27% and 45%, respectively, and the purity of the adduct was less than 45%.
Comparative example 2 (serine excess)
A method for preparing an acrolein-serine adduct comprising the steps of: reacting acrolein and serine at 60 ℃ for 5 hours in water according to a molar ratio of 1: 5, rotating at 130r/min, concentrating under reduced pressure for 1-2 mL, filtering with a 0.45-micron filter membrane to obtain a filtrate, and purifying by using a reverse phase silica gel chromatography to obtain an acrolein-serine adduct, wherein the yield is 85% and the purity of the adduct is 60%.
Comparative example 3 (reaction temperature too high)
A method for preparing an acrolein-serine adduct comprising the steps of: in water, heating acrolein and serine in a molar ratio of 1: 3 in a dark place for reaction (reaction at 70 ℃ for 8 hours, rotation speed of 130r/min), concentrating under reduced pressure for 1-2 mL, filtering with a 0.45-micrometer filter membrane to obtain a filtrate, and purifying by using a reverse phase silica gel chromatography to obtain an acrolein-serine adduct, wherein the yield is 69%, and the purity of the adduct is 73%.
Application examples
Acrolein reaction product in fried potatoes was analyzed by detection using the acrolein-serine adduct prepared in example 1 as a standard.
Application example 1
S0. obtaining raw materials:
preparing potato chips: fresh potato → cleaning, peeling → cutting into 1.5mm potato pieces → washing with deionized water three times, cleaning surface starch → frying peanut oil (165 deg.C, 5min) → draining → storing under dry condition
S1, adduct extraction:
taking a proper amount of n-hexane for deoiling, continuously adding n-hexane into the potato chips for three times (placing the potato chips in a fume hood for 30 min/time and 3 times), stirring at intervals, weighing 1g of sample after the n-hexane is volatilized, sequentially adding 10mL of 50% methanol/water for extraction (three times and 10 min/time), centrifuging at 10000r/min for 30min, taking supernatant for spin drying, adding 6mL of 50% methanol/water for redissolving, and filtering with a 0.22 mu m organic filter membrane.
S2.HPLC _ MS/MS detection of adduct content
Adopting MRM positive ion mode, and selecting ion pairs as follows: 200/118 (quantitative ion pairs, collision voltage 20 eV); 200/154, respectively; 200/182 (collision voltage 22eV), mobile phase: phase A/phase B: water/acetonitrile (0.1% formic acid), elution procedure: the flow rate is 0.5 mL/min; 0-2min, 2% B-100% B; 2-8min, 100% B-100% B; 8-8.01min, 100% B-2% B; 8.01-18min, 2% B. Taking the concentration of adduct as x-axis and the peak area of adduct ion as y-axis, a standard curve was prepared with y being 7.04e +003x +859 (R)20.9960). Finally, the content of the additive in the potato chips is 23.72 +/-0.40 mu g/kg.
Application example 2
S0. obtaining raw materials:
potato chips purchased from local supermarket (Bengao sand house)
S1, adduct extraction:
taking a proper amount of n-hexane for deoiling, continuously adding n-hexane into the potato chips for three times (placing the potato chips in a fume hood for 30 min/time and 3 times), stirring at intervals, weighing 1g of sample after the n-hexane is volatilized, sequentially adding 10mL of 50% methanol/water for extraction (three times and 10 min/time), centrifuging at 10000r/min for 30min, taking supernatant for spin drying, adding 6mL of 50% methanol/water for redissolving, and filtering with a 0.22 mu m organic filter membrane.
S2.HPLC _ MS/MS detection of adduct content
Adopting MRM positive ion mode, and selecting ion pairs as follows: 200/118 (quantitative ion pairs, collision voltage 20 eV); 200/154, respectively; 200/182 (collision voltage 22eV), mobile phase: phase A/phase B: water/acetonitrile (0.1% formic acid), elution procedure: the flow rate is 0.5 mL/min; 0-2min, 2% B-100% B; 2-8min, 100% B-100% B; 8-8.01min, 100% B-2% B; 8.01-18min, 2% B. Taking the concentration of adduct as x-axis and the peak area of adduct ion as y-axis, a standard curve was prepared with y being 7.04e +003x +859 (R)20.9960). Finally, the content of the adduct in the potato chips is measured to be 13.79 +/-0.01 mu g/kg.
The MRM mode is a highly sensitive quantitative analysis method that can exclude a large number of interfering ions by two-stage ion selection. According to the triple quadrupole mass spectrometry principle, a double quadrupole detects specific parent ions, the double quadrupole selects the specific parent ions for collision, and if specific ion pairs can be obtained in a standard substance and a sample, quantitative analysis can be performed on substances through the signal intensity of the specific ion pairs.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
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