CN114019043B - Method for simultaneously identifying double bond positions and cis-trans isomerism of carbon-carbon double bond isomers - Google Patents
Method for simultaneously identifying double bond positions and cis-trans isomerism of carbon-carbon double bond isomers Download PDFInfo
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Abstract
The invention provides a method for simultaneously identifying double bond positions and cis-trans isomerism of carbon-carbon double bond isomers, which comprises the following steps: 1) dissolving a substrate containing unsaturated double bonds and cis-trans isomeric structures, a reaction reagent containing carbonyl and a photocatalyst in a solvent, and uniformly stirring to obtain a solution to be detected; 2) carrying out illumination reaction on the solution to be detected obtained in the step 1) under the excitation of visible light to obtain a reaction product; 3) injecting the reaction product obtained in the step 2) into a mass spectrum, and further performing CID fragmentation to obtain characteristic fragment ions after chromatographic analysis and ionization of an ion source; 4) analyzing the characteristic fragment ions obtained in the step 3), and reversely deducing the position of the unsaturated double bond; 5) and analyzing and comparing the chromatographic behaviors of the substrate and the reaction product to deduce the cis-trans isomeric structure of the unsaturated double bond. The method can simultaneously identify the double bond position and the cis-trans isomerism of the carbon-carbon double bond isomer of the lipid compound containing the unsaturated double bond and the cis-trans isomerism structure.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a method for simultaneously identifying double bond positions and cis-trans isomerism of carbon-carbon double bond isomers.
Background
Lipids are one of the important constituents of organisms, and a large number of isomers exist in organisms, and the diversity of lipid structures endows the organisms with a variety of important biological and physical functions. Abnormal metabolism of lipid compounds can cause various physiological diseases of the body. Therefore, the development of a new method for lipid isomer analysis, the definition of the change of the lipid isomer in the occurrence and development processes of diseases and the network mechanism of metabolic regulation of the lipid isomer, and important biological significance and clinical value.
With the development of modern mass spectrometry techniques and their data processing tools, systematic identification and analysis of the structures of these lipid compounds is possible. However, the current lipidomics analysis based on mass spectrum is greatly limited in the identification of the position of carbon-carbon double bonds. This is mainly because in the traditional lipidomics analysis workflow, CID technology in tandem mass spectrometry is generally adopted, and the target compound cannot be cleaved to generate characteristic ions related to the carbon-carbon double bond structure.
In view of the above technical problems, a new method for the identification of location isomers of unsaturated lipid bonds in biological samples by means of an on-line Patern oa-buchi (pb) photochemical reaction was developed by the concession yoga et al, the university of qinghua, 2015. The method comprises the steps of reacting with acetonide under ultraviolet irradiation to generate an oxygen-containing four-membered ring structure, and further fragmenting by CID to obtain characteristic fragment ions for judging the position of carbon-carbon double bonds. However, this method using uv excitation results in some side reactions and the low molecular weight of acetone, which results in some PB product overlapping with the mass of the substrate, increasing the complexity of data analysis.
The subject group in 2020 proposes a new method for identifying unsaturated double bond position isomers in a biological sample by [2+2] cycloaddition reaction carried out under visible light, and overcomes the problems of reaction defect caused by ultraviolet light irradiation, small molecular weight of carbonyl reagent and the like, but the yield of the reaction is low, and the solubility of the used anthraquinone reagent is low, so that the yield of analyzing unsaturated double bonds of a complex biological system is low, the product signal is weak, the difficulty of data analysis is increased, and the large-scale identification of the double bond position of unsaturated lipid in the biological sample is limited. In addition, no method for simultaneously identifying double bond position isomerism and cis-trans isomerism in unsaturated lipid based on mass spectrometry technology exists at present.
Therefore, it is necessary to provide a method for simultaneously identifying the position of the double bond and cis-trans isomerism of a carbon-carbon double bond isomer.
Disclosure of Invention
The present invention has been made to solve at least some of the above problems occurring in the prior art, and in a first aspect of the present invention, the present invention provides a method for simultaneously identifying the position of the double bond and cis-trans isomerism of a carbon-carbon double bond isomer, comprising the steps of:
1) dissolving a substrate containing unsaturated double bonds and cis-trans isomeric structures, a reaction reagent containing carbonyl and a photocatalyst in a solvent, and uniformly stirring to obtain a solution to be detected;
2) carrying out illumination reaction on the solution to be detected obtained in the step 1) under the excitation of visible light to obtain a reaction product;
3) injecting the reaction product obtained in the step 2) into a mass spectrum, and further performing CID fragmentation to obtain characteristic fragment ions after chromatographic analysis and ionization of an ion source;
4) analyzing the characteristic fragment ions obtained in the step 3), and reversely deducing the position of the unsaturated double bond;
5) analyzing and comparing the chromatographic behaviors of the substrate containing the unsaturated double bond and the cis-trans isomeric structure in the step 1) and the reaction product obtained in the step 2) to deduce the cis-trans isomeric structure of the unsaturated double bond.
In one or more embodiments of the present invention, in the step 1), the substrate having an unsaturated double bond and a cis-trans isomeric structure is a lipid compound having an unsaturated double bond and a cis-trans isomeric structure. Specifically, the substrate containing unsaturated double bond and cis-trans isomeric structure is unsaturated fatty acid, glyceride or glycerophospholipid.
In one or more embodiments of the present invention, in step 1), the carbonyl-containing reactant has the structural formula:
wherein, R1 is phenyl, pyridyl or other aromatic ring type derivatives, and R2 is alkyl, alkoxy, phenyl, pyridyl or other aromatic ring type derivatives.
Preferably, the carbonyl-containing reactant is methyl benzoylformate.
In one or more embodiments of the invention, in step 1), the photocatalyst is an iridium catalyst, preferably, the iridium catalyst is Ir [ dFppy ]2(dtbbpy) PF6, Ir [ dF (me) ppy ]2(dtbbpy) PF6, Ir [ dF (CF3) ppy ]2(dtbbpy) PF6, Ir [ Fppy ]2(dtbbpy) PF6, or Ir [ ppy ]2(dtbbpy) PF6, and the like.
In one or more embodiments of the present invention, in the step 1), the solvent is one or more selected from water, methanol, ethanol, acetonitrile, and chloroform.
In one or more embodiments of the present invention, in the step 1), the molar ratio of the reaction reagent containing a carbonyl group to the substrate containing an unsaturated double bond and a cis-trans isomeric structure in the solution to be detected is (0.01-100): 1, preferably, the molar ratio of the reaction reagent containing the carbonyl group to the substrate containing the unsaturated double bond and the cis-trans isomeric structure is (8-15): 1, preferably, the content of the photocatalyst is 0.1 to 10 (w/w)%.
In one or more embodiments of the invention, in the step 2), the visible light wavelength is 380-500 nm; preferably, the visible light wavelength is 440-460 nm.
In one or more embodiments of the present invention, in the step 2), the light reaction time is 0.5 to 60min, preferably 6 to 10 min.
In one or more embodiments of the invention, in the step 4), analyzing the characteristic fragment ions includes analyzing a theoretical molecular formula of the secondary fragment ions according to a secondary mass spectrum of the product obtained in the step 2), comparing the theoretical molecular formula of the secondary fragment ions with a molecular formula of a substrate containing an unsaturated double bond and a cis-trans isomeric structure or a reaction product obtained in the step 2), and combining the unsaturation degree of the characteristic ions to reversely deduce the position of the double bond.
In one or more embodiments of the present invention, the analyzing and comparing in step 5) includes detecting a substrate containing an unsaturated double bond and a cis-trans isomeric structure to obtain a first chromatogram, detecting the reaction product obtained in step 2) to obtain a second chromatogram, and comparing the retention time of the substrate containing an unsaturated double bond and a cis-trans isomeric structure with the retention time of a newly generated isomer corresponding to the substrate containing an unsaturated double bond and a cis-trans isomeric structure, thereby determining the cis-trans isomeric structure of the substrate containing an unsaturated double bond and a cis-trans isomeric structure.
The principle of the invention is as follows:
the invention firstly proposes that a lipid compound containing an ortho-dicarbonyl can be excited by triplet state energy transfer of a catalyst under the visible light catalysis condition and carry out a photocycloaddition reaction with olefin through processes such as electron transfer and the like, and the lipid compound containing an unsaturated double bond and a cis-trans isomeric structure respectively reacts with methyl benzoylformate under the visible light catalysis system condition to form an oxygen-containing quaternary heterocyclic structure; secondly, ionizing the obtained reaction product by a mass spectrum ion source, and performing secondary fragmentation (such as collision induced dissociation) in the mass spectrum to generate characteristic fragments for judging the position of the unsaturated double bond, thereby realizing effective limitation of the position of the carbon-carbon double bond; and simultaneously extracting lipid compounds with unsaturated double bonds and photoisomerization products thereof in a reaction system, and comparing and analyzing the generation quantity of isomers and the retention time of a liquid phase after the reaction, thereby determining the carbon-carbon double bond cis-trans configuration.
In the lipid compound containing the unsaturated double bond and the cis-trans isomeric structure, the hydrophilic action of the cis-structure and the chromatographic column is weaker than that of the trans-structure and the chromatographic column, so when a substrate containing the unsaturated double bond and the cis-trans isomeric structure is separated by the column, the cis-structure generates a peak earlier, and the trans-structure generates a peak later.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the invention firstly proposes that benzoyl methyl formate and lipid compound containing unsaturated double bond and cis-trans isomeric structure are subjected to [2+2] cycloaddition reaction under a visible light catalytic system, and a tandem mass spectrometry method is applied to detect and analyze reaction products so as to judge the position of double bonds; the related photo cycloaddition reaction has simple condition, less side reaction, fast reaction and high yield, and can effectively solve the problems of low yield of the existing photochemical reaction for judging the unsaturated double bond position of the lipid compound and the like.
2. The visible light catalytic system adopted by the invention can simultaneously realize the conversion of the cis-trans configuration of the compound containing carbon-carbon double bonds, and further can be combined with liquid chromatography-mass spectrometry to identify double-bond cis-trans isomers.
3. The invention adopts a photo-cycloaddition-photo-isomerization dual-function reaction system, and can simultaneously realize the identification of carbon-carbon double bond positions and cis-trans isomerization.
4. According to the invention, the benzoyl methyl formate and the lipid compound containing unsaturated double bonds and cis-trans isomeric structures are subjected to [2+2] cycloaddition reaction under a visible light catalytic system, and the reaction can be carried out under 440-460 nm visible light, so that the reaction is safe and reliable, and the operation is convenient.
Drawings
FIG. 1 is a first order mass spectrum of the reaction of fatty acid C18:1(9Z) with methyl benzoylformate of example 1;
FIG. 2 is a secondary mass spectrum obtained by reacting fatty acid C18:1(9Z) with methyl benzoylformate of example 1;
FIG. 3 is a graph of extracted ion flow before and after the reaction of fatty acids C18:1(9Z) and C18:1(9E) with methyl benzoylformate in example 1; wherein, 9Z-K is an ion flow diagram before fatty acid C18:1(9Z) reacts with methyl benzoylformate; 9Z-PB is an ion flow diagram of fatty acid C18:1(9Z) after reaction with methyl benzoylformate; 9E-K is an ion flow diagram of fatty acid C18:1(9E) before reaction with methyl benzoylformate; 9E-PB is an ion flow diagram of fatty acid C18:1(9E) after reaction with methyl benzoylformate;
FIG. 4 is a first order mass spectrum of fatty acid C18:2(9Z, 12Z) reacted with methyl benzoylformate of example 2;
FIG. 5 is a secondary mass spectrum obtained by reacting fatty acid C18:2(9Z, 12Z) with methyl benzoylformate from example 2;
FIG. 6 is a graph of extracted ion flow before and after the reaction of fatty acids C18:2(9Z, 12Z) and C18:2(9E,12E) with methyl benzoylformate in example 2; wherein FA 18:2_ Z-K is an ion flow diagram of fatty acid C18:2(9Z, 12Z) before reaction with methyl benzoylformate; FA 18:2_ Z-PB is an ion flow diagram of fatty acid C18:2(9Z, 12Z) after reaction with methyl benzoylformate; FA 18:2_ E-K is an ion flow diagram of fatty acid C18:2(9Z, 12Z) before reaction with methyl benzoylformate; FA 18:2_ E-PB is an ion flow diagram of fatty acid C18:2(9Z, 12Z) after reaction with methyl benzoylformate;
FIG. 7 is a first order mass spectrum of phosphatidylcholine PC 18:1(9Z) _18:1(9Z) reacted with methyl benzoylformate from example 3;
FIG. 8 is a secondary mass spectrum obtained by reacting phosphatidylcholine PC 18:1(9Z) _18:1(9Z) with methyl benzoylformate from example 3;
FIG. 9 is a graph of extracted ion streams of phosphatidylcholine PC 18:1(9Z) _18:1(9Z) and PC 18:1(9E) _18:1(9E) before and after reaction with methyl benzoylformate from example 3; wherein, PC 36: 2-9Z-K is an ion flow diagram before fatty acid C18:1(9Z) reacts with methyl benzoylformate; PC 36:2_9Z-PB is an ion flow diagram of fatty acid C18:1(9Z) after reaction with methyl benzoylformate; PC 36:2_ Z-K is an ion flow diagram of fatty acid C18:1(9E) before reaction with methyl benzoylformate; PC 36:2_ Z-PB is an ion flow diagram of fatty acid C18:1(9E) after reaction with methyl benzoylformate.
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The methods used are conventional methods known in the art unless otherwise specified, and the consumables and reagents used are commercially available unless otherwise specified. Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
Example 1
A method for simultaneously identifying the position of double bonds and cis-trans isomerism of a carbon-carbon double bond isomer comprises the following steps:
1) respectively adding a solution of a fatty acid compound FA (C18:1(9Z)) and a solution of a fatty acid compound FA (C18:1(9E)) containing an unsaturated double bond and a cis-trans isomeric structure into a liquid phase vial, blowing the solution with a nitrogen stream, dissolving methyl benzoylformate in methanol, dissolving a photocatalyst in an acetonitrile solution, mixing the two solutions in the liquid phase vial (the total volume is 200 mu L, wherein the concentration of the fatty acid compound containing the unsaturated double bond is 10mM, the solubility of the methyl benzoylformate is 100mM, and the concentration of the photocatalyst is 5mM), blowing the nitrogen stream for 20s by using a 2mL syringe needle, irradiating the reaction with visible light (450nm), terminating the reaction for 10min, detecting and analyzing the obtained reaction product by mass spectrometry, detecting a substrate FA and the reaction product in a negative ion mode, considering that the ionization efficiency of the FA and the reaction product in the negative ion mode is only related to carboxyl, the high yield of the system is illustrated by the fact that the FA content after the reaction is significantly lower than the product content, as shown in figure 1;
2) simultaneously, a Na ion peak of a reaction product is cracked by utilizing a Collision Induced Dissociation (CID) technology to obtain corresponding characteristic fragment ions, and a corresponding secondary mass spectrogram (figure 2) is obtained;
3) comparing the secondary mass spectrum obtained in step 2) with the theoretical secondary fragment ions (as shown in FIG. 2), compound P1a (C) was detected 18 H 24 O 4 + Na) and the compound P2a (C) 9 H 16 O 3 + Na) from the products P1 and P2, respectively, indicating that the fatty acid reacted with methyl benzoylformate to form an oxygen-containing quaternary heterocyclic structure;
4) the diagnostic fragment ion P1a obtained by step 3) was compared with the molecular formula of the theoretical PB product (C) 27 H 42 O 5 Na) and the number of the carbon is different by 9, indicating that the unsaturated double bond is positioned at the 9(18-9) position; or the characteristic ion P2a generated by pathway II, compared with the molecular formula of the reaction substrate FA C18:1 (C) 18 H 24 O 3 Na), the number of carbons differs by 9, and it can also be judged that the unsaturated double bond is at the 9(18-9) position.
5) By comparing FA extraction ion flow diagrams before and after a reaction system (as shown in FIG. 3), FA 18:1(9Z) generates a new isomer after the reaction (mass spectrum data is the same as FA 18:1(9Z)), and the theory shows that FA 18:1(9Z) generates a peak earlier than FA 18:1(9E), so that the fatty acid compound containing an unsaturated double bond and a cis-trans isomeric structure is in a cis-structure. While FA 18:1(9E)) also produces an isomer (mass spectrum data is the same as FA 18:1(9E)), and the FA 18:1(9Z) peaks earlier than FA 18:1(9E) according to theory, so that fatty acid compounds containing an unsaturated double bond and cis-trans isomerism are in trans structure, and detection results also show that cis-form and trans-form FA are in isomeric transformation in the reaction system, but the transformation ratio is different, and the ratio of 9E to 9Z is only 1.71%, as shown in FIG. 3.
Example 2
A method for identifying the position of a double bond of a carbon-carbon double bond isomer comprises the following steps:
1) respectively adding fatty acid compounds FA (C18:2(9Z, 12Z)) and FA (C18:2(9E,12E)) containing two unsaturated double bonds and a cis-trans isomeric structure into a liquid phase vial, blowing dry by nitrogen flow, dissolving methyl benzoylformate in methanol, dissolving a photocatalyst in acetonitrile solution, mixing the two in the liquid phase vial (the total volume is 200 mu L, wherein the concentration of the fatty acid compounds containing the unsaturated double bonds is 10mM, the solubility of the methyl benzoylformate is 100mM, and the concentration of the photocatalyst is 5mM), blowing nitrogen flow for 20s by using a 2mL syringe needle, irradiating the reaction by visible light (450nm), stopping the reaction for 10min, and detecting and analyzing the obtained reaction product by mass spectrometry, as shown in FIG. 4;
2) simultaneously, the protonation peak of the reaction product is cracked by using a CID technology to obtain corresponding characteristic fragment ions and a corresponding secondary mass spectrogram (figure 5);
3) comparing the secondary mass spectrum obtained in step 2) with the theoretical secondary fragment ions (as shown in FIG. 6), the ion pair compound P1a (C) is detected 21 H 28 O 4 + Na) and the compound P2a (C) 12 H 20 O 3 +Na)，P1a(C 18 H 24 O 4 + Na) and the compound P2a (C) 9 H 16 O 3 + Na) indicating that both double bonds in the fatty acid react with methyl benzoylformate to form an oxygen-containing quaternary heterocyclic structure;
4) the diagnostic fragment ion P1a obtained by step 3) was compared with the molecular formula of the theoretical PB product (C) 27 H 42 O 5 Na), the number of carbons differs by 9 and 6, respectively, indicating that the unsaturated double bond is located at the 9(18-9) position and the 12(18-6) position; or the characteristic ion P2a generated by pathway II, compared with the molecular formula of the reaction substrate FA C18:1 (C) 18 H 22 O 3 Na), the number of carbons differs by 9 and 6, respectively, and it can also be judged that the unsaturated double bond is located at the 9(18-9) and 12(18-6) positions.
5) By comparing fatty acid extraction ion flow diagrams before and after a reaction system (as shown in figure 6), two new isomers (mass spectrum data is the same as FA 18:2(9Z, 12Z)) are generated after the reaction of a detected sample FA 18:2(9Z,12Z), the theory shows that FA 18:2(9Z,12Z) generates a peak earlier than FA 18:2(9E,12E), and the peak of the isomer generated by the reaction is relatively late, so that the fatty acid compound containing two unsaturated double bonds and a cis-trans isomeric structure is in a cis-structure; the chromatographic peak of the other isomer is located in the middle, and it is assumed that one of the double bonds of the isomer undergoes cis-trans transformation, as shown in FIG. 6. Correspondingly, an isomer is generated after the test sample FA 18:2(9E,12E) reacts (mass spectrum data are the same as those of FA 18:2(9E,12E)), and the peak of the isomer generated by the reaction is relatively early, so that the fatty acid compound containing two unsaturated double bonds and a cis-trans isomeric structure is known to be in a trans structure; however, the trans-cis isomer FA 18:2(9Z,12Z) which is converted into the cis isomer is not detected in the reaction system of the trans form, and the trans form is more compact and therefore more stable, so that it is difficult to convert the trans form into the cis isomer. The photocatalysis system can also analyze the position of double bonds of lipid with double unsaturated double bonds and cis-trans isomerism.
Example 3
A method for identifying the position of a double bond of a carbon-carbon double bond isomer comprises the following steps:
1) respectively adding a phosphatidylcholine compound P (C18:1(9Z) _18:1(9Z)) solution and a phosphatidylcholine compound P (C18:1(9E) _18:1(9E)) solution containing two unsaturated double bonds and a cis-trans isomeric structure into a liquid phase vial, blowing a nitrogen gas flow for drying, dissolving methyl benzoylformate in methanol, dissolving a photocatalyst in an acetonitrile solution, mixing the two solutions in the liquid phase vial (the total volume is 200 mu L, wherein the concentration of the fatty acid compound of the unsaturated double bonds is 10mM, the solubility of the methyl benzoylformate is 100mM, and the concentration of the photocatalyst is 5mM), blowing a nitrogen gas flow for 20s by using a 2mL syringe needle, irradiating the reaction by visible light (450nm), stopping the reaction for 10min, and detecting and analyzing the obtained reaction product by a mass spectrum, wherein the reaction product is shown in FIG. 7;
2) simultaneously, the protonation peak of the reaction product is cracked by using a CID technology to obtain corresponding characteristic fragment ions and a corresponding secondary mass spectrogram (figure 8);
3) comparing the secondary mass spectrum obtained in step 2) with the theoretical secondary fragment ions (as shown in FIG. 6), compound P1a (C) was detected 18 H 24 O 3 ) And compound P2a (C) 12 H 16 O 3 ) The PC reacts with methyl benzoylformate to form an oxygen-containing quaternary heterocyclic structure;
4) comparing the fragment ions P1a obtained in step 3) with the secondary fragment ions of the theoretical PB reaction product, and finding that one of them is mainly fragmented to generate the compound a (C) as shown in FIG. 8 44 H 75 NO 10 P), another predominantly leads to the compound b (C) 35 H 67 NO 9 P); comparison of the formula of Compound a with the theoretical PB product (C) 53 H 92 NO 11 P), the difference of the number of carbon is 9; or the compound b and PC (C) 44 H 84 NO 8 P) and the number of carbons also differs by 9, indicating that the unsaturated double bond is at the 9(18-9) position; the methyl benzoylformate can react with the PC compound under the irradiation of visible light catalysis, and can be applied to judging the position of a carbon-carbon double bond.
5) By comparing the extracted ion flow diagrams of PC 18:1(9Z) _18:1(9Z) and PC 18:1(9E) _18:1(9E) before and after the reaction system (as shown in FIG. 9), the PC 18:1(9Z) _18:1(9Z) generates two new isomers after the reaction (mass spectrum data is the same as that of PC 18:1(9Z) _18:1(9Z)), and the theory shows that the peak appearance of the PC 18:1(9Z) _18:1(9Z) is earlier than that of PC 18:1(9E) _18:1(9E), and the peak appearance of the isomer generated by the reaction is relatively late, so that the phosphatidylcholine compound containing two unsaturated double bonds and a cis-trans-isomeric structure is in a cis-structure; the retention time of one was the same as that of PC (9E), and the other isomer was in an intermediate position, presumably due to cis-trans isomeric conversion of one of the double bonds of the isomer, as shown in FIG. 9. Whereas PC 18:1(9E) -18: 1(9E) detected only one isomer via the photocatalytic system (mass spectra data identical to PC 18:1(9E) -18: 1 (9E)). The peak of the isomer generated by the reaction is relatively early, so that the phosphatidylcholine compound containing two unsaturated double bonds and a cis-trans isomeric structure is in a trans-structure; the photocatalysis system can also analyze the double bond position and cis-trans isomerism of the PC lipid at the same time.
Comparative example 1
A method for simultaneously identifying the position of a double bond and a cis-trans configuration of a carbon-carbon double bond isomer comprises the following steps: dissolving fatty acid compound FA (C18:1(9Z)) containing an unsaturated double bond in methanol (10mM), preparing methyl benzoylformate into solution (100mM) with a certain concentration, mixing the two solutions (the total volume is 200 μ L), and respectively reacting for 1min, 10min and 30min by exciting irradiation with visible light (450 nm); carrying out mass spectrum detection and analysis on the obtained reaction product; first, by extracting an ion flow diagram, a considerable product signal cannot be extracted, which indicates that a PB product with high yield cannot be obtained by short-time irradiation of visible light with 450nm in a system without adding a photocatalyst.
Comparative example 2
A method for simultaneously identifying the position of double bonds and the configuration of double bonds of a carbon-carbon double bond isomer comprises the following steps: preparing reagents such as acetone, benzophenone, anthraquinone and the like with a certain concentration (100mM), respectively mixing the reagents with fatty acid solution FA (C18: 1(9)) (10mM) (the total volume is 200 muL, the concentration of the reagents such as benzophenone is 100mM, carrying out reaction, wherein the reaction time is 1-30min, the reaction is carried out under the excitation of 450nm visible light, PB products can not be detected, and the result shows that other carbonyl substrates can not be excited by the photocatalytic system to carry out PB reaction, so that the double bond position can be identified.
Although the embodiments of the present invention have been shown and described, it is understood that the above embodiments are illustrative and not restrictive, and that those skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention and that the present invention also includes the modifications and changes.
Claims (7)
1. A method for simultaneously identifying the position of double bonds of carbon-carbon double bond isomers and cis-trans isomerism is characterized by comprising the following steps:
1) dissolving a substrate containing unsaturated double bonds and cis-trans isomeric structures, a reaction reagent containing carbonyl and a photocatalyst in a solvent, and uniformly stirring to obtain a solution to be detected;
2) carrying out illumination reaction on the solution to be detected obtained in the step 1) under the excitation of visible light to obtain a reaction product;
3) injecting the reaction product obtained in the step 2) into a mass spectrum, and further performing CID fragmentation to obtain characteristic fragment ions after chromatographic analysis and ionization of an ion source;
4) analyzing the characteristic fragment ions obtained in the step 3), and reversely deducing the position of the unsaturated double bond;
5) analyzing and comparing the chromatographic behaviors of the substrate containing the unsaturated double bond and the cis-trans isomeric structure in the step 1) and the reaction product obtained in the step 2) to deduce the cis-trans isomeric structure of the unsaturated double bond;
in the step 1), the substrate containing the unsaturated double bond and the cis-trans isomeric structure is a lipid compound containing the unsaturated double bond and the cis-trans isomeric structure; the reaction reagent containing carbonyl is methyl benzoylformate; the visible light wavelength is 450 nm.
2. The method for simultaneously identifying the double bond positions and the cis-trans isomerism of a carbon-carbon double bond isomer according to claim 1, wherein in the step 1), the photocatalyst is an iridium catalyst.
3. The method for simultaneously identifying the double bond positions and the cis-trans isomerism of a carbon-carbon double bond isomer according to claim 1, wherein in the step 1), the solvent is one or more selected from water, methanol, ethanol, acetonitrile and chloroform.
4. The method for simultaneously identifying the double bond positions and the cis-trans isomerism of a carbon-carbon double bond isomer according to claim 1, wherein in the step 1), the molar ratio of the reaction reagent containing the carbonyl group to the substrate containing the unsaturated double bond and the cis-trans isomerism structure in the solution to be detected is 10: 1, the concentration of the photocatalyst is 5 mM.
5. The method for simultaneously identifying the double bond positions and the cis-trans isomerism of a carbon-carbon double bond isomer according to claim 1, wherein in the step 2), the light reaction time is 10 min.
6. The method for simultaneously identifying the position of the double bond and the cis-trans isomerism of a carbon-carbon double bond isomer according to claim 1, wherein in the step 4), the characteristic fragment ions are analyzed, and the position of the double bond is deduced reversely by comparing the molecular formula of the theoretical secondary fragment ions with the molecular formula of the substrate containing the unsaturated double bond and the cis-trans isomerism structure or the reaction product obtained in the step 2) according to the secondary mass spectrum of the product obtained in the step 2).
7. The method for simultaneously identifying double bond positions and cis-trans isomerism of isomers with carbon-carbon double bonds as claimed in claim 1, wherein in said step 5), analysis and comparison are performed, comprising detecting a substrate containing unsaturated double bonds and cis-trans isomerism to obtain a first chromatogram, detecting a reaction product obtained in step 2) to obtain a second chromatogram, and comparing the retention time of the substrate containing unsaturated double bonds and cis-trans isomerism with the retention time of a newly formed isomer corresponding to said substrate containing unsaturated double bonds and cis-trans isomerism, thereby determining the cis-trans isomerism of said substrate containing unsaturated double bonds and cis-trans isomerism.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| GB201801992D0 (en) * | 2014-04-17 | 2018-03-28 | Micromass Ltd | Method of localizing lipid double bonds |
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| CN111060584A (en) * | 2019-12-31 | 2020-04-24 | 武汉大学 | Method for identifying position of double bonds of carbon-carbon double bond isomer |
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| AU2007211893B2 (en) * | 2007-06-04 | 2013-05-16 | The University Of Wollongong | A method for the determination of the position of unsaturation in a compound |
| US10043645B2 (en) * | 2014-04-17 | 2018-08-07 | Micromass Uk Limited | Method of localizing lipid double bonds |
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| CN110441383A (en) * | 2019-07-05 | 2019-11-12 | 清华大学 | Method based on epoxidation and Mass Spectrometric Identification unsaturated lipids carbon-carbon double bond position |
| CN110632222A (en) * | 2019-09-23 | 2019-12-31 | 清华大学 | Method for analyzing sn isomer in phosphatidylcholine based on mass spectrum and application thereof |
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