Multi-dimensional lipid fine structure identification method based on carbon-carbon double bond full derivation

Abstract

The invention relates to the technical field of mass spectrometry, and relates to a multi-dimensional lipid fine structure identification method based on carbon-carbon double bond full derivation, which comprises the following steps: carrying out in-situ epoxidation derivation on unsaturated lipid to obtain an epoxidation product; selecting a sodium ion adduct of the unsaturated lipid / epoxidation product for mass spectrometry, and determining the total number of carbon atoms and unsaturation of the unsaturated lipid; performing secondary mass spectrometry analysis on the sodium ion adduct of the epoxidation product to obtain daughter ions after neutral head group loss; performing three-stage mass spectrometry on the daughter ions to obtain specific ions at sn positions and specific ions at carbon-carbon double bond positions; and analyzing the specific ions at the sn position and the specific ions at the carbon-carbon double bond position, and determining the sn position and the carbon-carbon double bond position of the unsaturated lipid according to the total number of carbon atoms and the unsaturation degree of the unsaturated lipid. According to the method, the sn connection position and the carbon-carbon double bond position of the fatty acid chain in the unsaturated lipid can be analyzed at the same time, and multi-dimensional fine structure identification of the lipid is achieved.

Description

Technical Field

[0001] The present application relates to the technical field of mass spectrometry analysis, and mainly relates to a method for identifying the fine structure of multidimensional lipids based on the full derivatization of carbon-carbon double bonds. Background Art

[0002] Lipids play a series of important roles in important physiological processes of organisms such as cell membrane structure formation, energy storage, and signal transduction. Various different types of lipids together constitute the lipidome of an entire organism or an organ, and are finely regulated in living organisms to form a steady-state balance of lipid metabolism and synthesis. More and more studies have shown that changes in lipid species, content, and structure are closely related to the occurrence of cancers, diabetes, neurological diseases, and cardiovascular and cerebrovascular diseases. Therefore, the analysis of lipidome and the identification of lipid structures have attracted more and more attention, and the fine study of lipid structures has become an important research goal in analytical chemistry and life sciences. Taking glycerophospholipids as an example, their structural diversity mainly comes from the types and numbers of end groups and fatty acid chains. Moreover, the stereospecific numbering (sn) positions where the fatty acid chains are located, as well as the number, position, and cis / trans structure of the carbon-carbon double bonds (C=C) contained therein further increase the complexity of lipid structures and the difficulty of analysis.

[0003] Mass spectrometry (MS) has become a commonly used analytical tool in lipidomics. Electrospray ionization technology has become the preferred ionization technology for lipid analysis. Currently, new analytical techniques and methods based on mass spectrometry are emerging continuously, greatly promoting the development of the lipidomics field. Mass spectrometry analysis can provide rich structural information in one analysis by measuring the mass-to-charge ratio (mass-charge ratio) of ions, and at the same time has high specificity and high sensitivity. For example, by combining ionization in positive and negative modes and tandem mass spectrometry, it has been able to determine the total number of carbon atoms and the degree of unsaturation of lipid types and fatty acid chains. With the in-depth study, it has also become possible to identify the sn positions of fatty acid chains in unsaturated lipids and the positions of the carbon-carbon double bonds contained therein. However, most of the existing methods can only identify one of the sn or carbon-carbon double bond position information. The use of mixed dissociation methods such as ozone-induced dissociation (OzID) and ultraviolet photodissociation (UVPD) combined with collision-induced dissociation (CID) can achieve sn linkage and carbon-carbon double bond position identification, but it requires an instrument equipped with a UVPD function or corresponding modification of the instrument to implement OzID. Therefore, establishing a simple and rapid mass spectrometry analysis method for identifying the fine structure of multidimensional lipids will be of great significance for the study of lipidomics.

[0004] There are mainly two methods for identifying the position of sn and carbon-carbon double bonds. One is the ion fragmentation method. For example, a mixed dissociation method such as ozone-induced dissociation (OzID) combined with collision-induced dissociation (CID), or ultraviolet photodissociation (UVPD) combined with collision-induced dissociation (CID) can be used to identify the position of sn and carbon-carbon double bonds. However, the disadvantage of this method is that it requires special modification of the mass spectrometry instrument, making it difficult to be applied and popularized in ordinary laboratories. Another method is to adopt the strategy of derivatizing the unsaturated carbon-carbon double bonds in lipids, such as using the Paternò-Büchi reaction. The derivatized lipids can be combined with collision-induced dissociation (CID) in a commercial mass spectrometer to identify the position of sn and carbon-carbon double bonds. However, the disadvantage of the Paternò-Büchi reaction is that the derivatization efficiency is relatively low, and not all are converted into a single derivatized product. These methods either require a complex instrument modification process, or the identification steps are cumbersome and the efficiency is not high, and it is impossible to achieve high-throughput structural analysis of the sn position and carbon-carbon double bond position of lipids.

[0005] Therefore, the existing technology still needs to be improved and developed. Summary of the Invention

[0006] In view of the above deficiencies of the prior art, the purpose of this application is to provide a multi-dimensional lipid fine structure identification method based on the full derivatization of carbon-carbon double bonds. After the epoxidation products of the unsaturated lipids to be analyzed are fragmented by tandem mass spectrometry analysis, specific ions with high abundance of sn-position and carbon-carbon double bond positions can be obtained simultaneously in a single mass spectrum, aiming to achieve multi-dimensional fine structure identification of lipid sn- and carbon-carbon double bond position isomers.

[0007] The technical solution of this application is as follows: This application provides a multi-dimensional lipid fine structure identification method based on the full derivatization of carbon-carbon double bonds, which is applicable to unsaturated lipids containing a glycerol backbone and a head group, and includes the following steps: (1) In-situ epoxidation derivatization of the unsaturated lipids to be analyzed using dimethyldioxirane, so that all carbon-carbon double bonds in the unsaturated lipids are epoxidized to form an epoxidized structure, and the epoxidation product of the unsaturated lipids is obtained; (2) Inject the epoxidation product into the mass spectrometer. After ionization by the ion source, select the sodium ion adduct of the epoxidation product for primary mass spectrometry analysis to obtain the ion mass of the sodium ion adduct of the epoxidation product. Perform primary mass spectrometry analysis on the sodium ion adduct of the unsaturated lipids without epoxidation derivatization as a comparison to determine the total number of carbon atoms and the degree of unsaturation of the unsaturated lipids; (3) Perform secondary mass spectrometry analysis on the sodium ion adduct of the epoxidation product. After collision-induced dissociation, select the daughter ion obtained after the sodium ion adduct of the epoxidation product loses the head group neutrally; (4) Perform tandem mass spectrometry analysis on the product ion obtained after neutral loss of the head group from the sodium ion adduct of the epoxidation product. After collision-induced dissociation, specific ions at the sn position and specific ions at the carbon-carbon double bond position are obtained; (5) Analyze the specific ions at the sn position and the specific ions at the carbon-carbon double bond position, and determine the sn position and the carbon-carbon double bond position of the unsaturated lipid according to the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid.

[0008] Using the multi-dimensional lipid fine structure identification method based on full derivatization of carbon-carbon double bonds provided by the present application, the unsaturated lipid to be measured forms an epoxidation structure in situ after full derivatization of the carbon-carbon double bonds. Select the sodium ion adduct for tandem mass spectrometry analysis. After fragmentation and neutral loss of the head group in the second-level mass spectrometry analysis, a putative 1,3-dioxolane structure can be formed. Under the fragmentation tendency that interacts with the epoxidation structure, after fragmentation in the third-level mass spectrometry analysis, characteristic peaks at the sn-position and the carbon-carbon double bond position with high abundance can be obtained simultaneously, which is beneficial to realizing the identification and analysis of the multi-dimensional lipid fine structure.

[0009] In the multi-dimensional lipid fine structure identification method based on full derivatization of carbon-carbon double bonds, in step (5), the determination of the sn position and the carbon-carbon double bond position of the unsaturated lipid specifically includes the following steps: (51) Determine the sn position of the unsaturated lipid according to the specific ion at the sn position, and determine the type of the specific ion at the sn position; (52) Determine the fragmentation mode of the product ion of the epoxidation product according to the type of the specific ion at the sn position, and determine the distribution of the epoxidation structure in the product ion according to the fragmentation mode; (53) Determine the carbon-carbon double bond position of the unsaturated lipid according to the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid, the distribution of the epoxidation structure in the product ion, the specific ion at the sn position, and the specific ion at the carbon-carbon double bond position.

[0010] In the multi-dimensional lipid fine structure identification method based on full derivatization of carbon-carbon double bonds, in step (3), the product ion obtained after neutral loss of the head group from the sodium ion adduct of the epoxidation product contains the structure shown in formula (I), R 1 is the fatty acid chain at the sn-1 position, R 2 is the fatty chain at the sn-2 position, Formula (I); In step (4), the structure of formula (I) has a cleavage tendency in the triple mass spectrometry. During the collision-induced dissociation in the triple mass spectrometry, specific ions at the sn position are formed, and the specific ions at the sn position include one or both of the specific ions at the sn-1 position and the specific ions at the sn-2 position; The daughter ions form specific ions at the carbon-carbon double bond position through the cleavage of the epoxy structure, and the specific ions at the carbon-carbon double bond position contain the structure of formula (I).

[0011] The multi-dimensional lipid fine structure identification method based on the full derivatization of carbon-carbon double bonds, wherein when the R 1 and R 2 both do not contain an epoxy structure, or when the R 1 contains an epoxy structure while the R 2 does not contain an epoxy structure, the cleavage tendency of the structure of formula (I) is as shown in formula (II), the dotted line indicates the cleavage site, the cleavage tends to occur on the 1,3-dioxolane structure, and the carbon-oxygen bonds connected at the 4th and 5th positions are cleaved to form specific ions indicating the sn-1 position, Formula (II); When the R 2 contains an epoxy structure while the R 1 does not contain an epoxy structure, the cleavage tendency of the structure of formula (I) is as shown in formula (III), the dotted line indicates the cleavage site, the cleavage tends to occur on the carbon-carbon double bond connected by the 2nd position of the 1,3-dioxolane structure, and specific ions indicating the sn-2 position are formed, Formula (III); When the R 1 and R 2 both contain an epoxy structure, the cleavage tendency of the structure of formula (I) is as shown in formula (IV), the dotted line indicates the cleavage site, the cleavage tends to occur on the 1,3-dioxolane structure, and the carbon-oxygen bonds connected at the 4th and 5th positions are cleaved. The cleavage also tends to occur on the carbon-carbon double bond connected by the 2nd position of the 1,3-dioxolane structure, and specific ions indicating the sn-1 position and specific ions indicating the sn-2 position are formed simultaneously, Formula (IV).

[0012] The multi-dimensional lipid fine structure identification method based on the full derivatization of carbon-carbon double bonds, wherein in step (51), determining the sn position of the unsaturated lipid includes the following methods: (i) Compare the ion mass of the specific ion at the sn position with the fragmentation characteristic ion mass data to determine whether the specific ion at the sn position is the specific ion at the sn-1 position or the specific ion at the sn-2 position; (ii) When a characteristic peak with a difference of 16 Da is formed near the specific ion at the sn position, the specific ion at the sn position is the specific ion at the sn-2 position; When no characteristic peak with a difference of 16 Da is formed near the specific ion at the sn position, the specific ion at the sn position is the specific ion at the sn-1 position.

[0013] In the multi-dimensional lipid fine structure identification method based on the full derivatization of carbon-carbon double bonds, in step (4), specific ions of fatty acid chains containing an epoxidation structure at the sn position are also obtained through triple mass spectrometry analysis. The specific ions of fatty acid chains containing an epoxidation structure at the sn position are one or both of the specific ions of fatty acid chains containing an epoxidation structure at the sn-1 position and the specific ions of fatty acid chains containing an epoxidation structure at the sn-2 position; In step (5), when the specific ion at the sn position is 40 Da more than the specific ion of the fatty acid chain containing an epoxidation structure at the sn position, the specific ion at the sn position is the specific ion at the sn-1 position, and the specific ion of the fatty acid chain containing an epoxidation structure at the sn position is the specific ion of the fatty acid chain containing an epoxidation structure at the sn-1 position; When the specific ion at the sn position is 30 Da less than the specific ion of the fatty acid chain containing an epoxidation structure at the sn position, the specific ion at the sn position is the specific ion at the sn-2 position, and the specific ion of the fatty acid chain containing an epoxidation structure at the sn position is the specific ion of the fatty acid chain containing an epoxidation structure at the sn-2 position.

[0014] In the multi-dimensional lipid fine structure identification method based on the full derivatization of carbon-carbon double bonds, in step (5), perform a secondary mass spectrometry analysis on the sodium ion adduct of the unsaturated lipid that has not undergone epoxidation derivatization. Select the second daughter ion obtained after neutral loss of the head group from the sodium ion adduct of the unsaturated lipid that has not undergone epoxidation derivatization. Perform a triple mass spectrometry analysis on the second daughter ion obtained after neutral loss of the head group from the sodium ion adduct of the unsaturated lipid to obtain specific ions of fatty acid chains containing carbon-carbon double bonds at the sn position. Compare with the specific ions of fatty acid chains containing an epoxidation structure at the sn position to determine the carbon number and the number of epoxidation structures corresponding to the specific ions of fatty acid chains containing an epoxidation structure at the sn position, thereby determining the carbon number and the number of epoxidation structures corresponding to the specific ions at the sn position; The specific ions of fatty acid chains containing carbon-carbon double bonds at the sn position include one or both of the specific ions of fatty acid chains containing carbon-carbon double bonds at the sn-1 position and the specific ions of fatty acid chains containing carbon-carbon double bonds at the sn-2 position.

[0015] The multi-dimensional lipid fine structure identification method based on full derivatization of carbon-carbon double bonds, wherein, in step (1), the in-situ epoxidation derivatization is carried out using potassium peroxymonosulfate and acetone for epoxidation derivatization; the in-situ epoxidation derivatization of the unsaturated lipid to be analyzed using dimethyldioxirane includes the following steps: Take 1 mmol / L of the unsaturated lipid into a container, add 50 μL of 500 mmol / L potassium peroxymonosulfate, then add 100 μL of acetone, and then add 50 μL of a sodium bicarbonate solution with a concentration of 500 mmol / L and mix; React at 60 °C for 30 minutes, add 300 μL of ethyl acetate for extraction, and dry with nitrogen to obtain the epoxidation product of the unsaturated lipid; During the reaction process of the reaction, ultrasound is carried out, and the ultrasound time is 10 minutes.

[0016] The multi-dimensional lipid fine structure identification method based on full derivatization of carbon-carbon double bonds, wherein the epoxidation product is subjected to tandem mass spectrometry analysis in the positive ion mode.

[0017] The multi-dimensional lipid fine structure identification method based on full derivatization of carbon-carbon double bonds, wherein the unsaturated lipid is one of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid; In step (3), the following steps are further included: Determine the lipid class of the unsaturated lipid to be analyzed according to the lost mass of the neutral loss head group.

[0018] Beneficial effects: The multi-dimensional lipid fine structure identification method based on full derivatization of carbon-carbon double bonds provided by this application can simultaneously analyze the sn-linkage position and carbon-carbon double bond position of fatty acid chains in unsaturated lipids. This technical solution is simple and feasible, can be carried out on a commercial mass spectrometer with triple quadrupole mass spectrometry function, and does not require modification of the mass spectrometer. The in-situ epoxidation derivatization method selected in this application has a relatively fast reaction rate and high reaction yield, which is helpful for qualitative and quantitative analysis of lipid mixtures. In the identification method provided by this application, the epoxidation product of the lipid to be analyzed can obtain high-abundance sn- and carbon-carbon double bond position characteristic ions in a single mass spectrum after fragmentation, so as to realize the multi-dimensional fine structure identification of lipid sn-position and carbon-carbon double bond position isomers. Description of the Drawings

[0019] Figure 1The technical roadmap of the method for identifying the fine structure of multidimensional lipids based on the full derivatization of carbon-carbon double bonds provided by this application.

[0020] Figure 2 The mass spectrum of MS3 for PC 16:0 / 18:1(9Z) in Comparative Example 1 of this application.

[0021] Figure 3 The mass spectrum of MS3 for PC 18:1(9Z) / 16:0 in Comparative Example 2 of this application.

[0022] Figure 4 The mass spectrum of MS3 for PC 16:0 / 18:1(9Z) in Example 1 of this application after epoxidation derivatization.

[0023] Figure 5 The mass spectrum of MS3 for PC 18:1(9Z) / 16:0 in Example 2 of this application after epoxidation derivatization.

[0024] Figure 6 The mass spectrum of MS3 for PC 16:0 / 18:2(9Z,12Z) in Example 3 of this application after epoxidation derivatization. Detailed implementation mode

[0025] This application provides a method for identifying the fine structure of multidimensional lipids based on the full derivatization of carbon-carbon double bonds. To make the purpose, technical solution and effect of this application clearer and more definite, the following further details this application. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0026] Figure 1 The technical roadmap of the method for identifying the fine structure of multidimensional lipids based on the full derivatization of carbon-carbon double bonds provided by this application is applicable to unsaturated glycerophospholipid lipids containing a glycerol backbone and a head group. First, the intact unsaturated lipid molecules to be analyzed are fully derivatized at the carbon-carbon double bonds to obtain an epoxidation product. The sodium ion adduct of the epoxidation product undergoes neutral loss of the head group after collision-induced dissociation in the second-stage mass spectrometry analysis to obtain daughter ions, and a 1,3-dioxolane structure is formed in the daughter ions. After fragmentation by the third-stage mass spectrometry analysis, sn-position-specific ions and carbon-carbon double bond position-specific ions are formed.

[0027] Specifically, this application provides a method for identifying the fine structure of multidimensional lipids based on the full derivatization of carbon-carbon double bonds, which is applicable to unsaturated lipids containing a glycerol backbone and a head group, and includes the following steps: (1) Use dimethyldioxirane to carry out in-situ epoxidation derivatization on the unsaturated lipids to be analyzed, so that all carbon-carbon double bonds in the unsaturated lipids are epoxidized to form an epoxidation structure, and the epoxidation product of the unsaturated lipids is obtained.

[0028] Furthermore, in-situ epoxidation derivatization is carried out using potassium peroxymonosulfate (oxone) and acetone for epoxidation derivatization, where potassium peroxymonosulfate serves as the oxidant and acetone serves as the catalyst. When carrying out epoxidation derivatization on a carbon-carbon double bond, potassium peroxymonosulfate and acetone react to form dimethyldioxirane. The formed dimethyldioxirane performs epoxidation derivatization on the carbon-carbon double bond in-situ at the carbon-carbon double bond. The oxygen atom of dimethyldioxirane attacks the carbon-carbon double bond in the unsaturated lipid to undergo electrophilic addition, generating an intermediate, and then hydrogen migration occurs, finally obtaining an epoxide. The by-product acetone formed is reused as a reaction raw material for generating dimethyldioxirane to continue the epoxidation derivatization.

[0029] Even further, in step (1), dimethyldioxirane is used to carry out in-situ epoxidation derivatization on the unsaturated lipid to be analyzed, including the following steps: Take 1 mmol / L of the unsaturated lipid into a container, add 50 μL of 500 mmol / L potassium peroxymonosulfate, then add 100 μL of acetone, and then add 50 μL of a sodium bicarbonate solution with a concentration of 500 mmol / L and mix; React at 60 °C for 30 minutes, add 300 μL of ethyl acetate for extraction, and dry it with nitrogen to obtain the epoxidation product of the unsaturated lipid; Ultrasound is carried out during the reaction process, and the ultrasound time is 10 minutes.

[0030] Ultrasound is also carried out during the epoxidation derivatization reaction, which can accelerate the epoxidation reaction and is beneficial to improving the reaction efficiency. The sodium bicarbonate solution serves as a buffer during the epoxidation derivatization process to adjust the acidity and alkalinity, playing a buffering role, making the formed epoxidation product not easily undergo ring opening, being beneficial to protecting the epoxidation product and increasing the yield of the epoxidation product.

[0031] This method has a high degree of oxidation and high oxidation efficiency, can carry out full derivatization of the carbon-carbon double bond of the unsaturated lipid to be analyzed, and in-situ forms an epoxidation structure on the carbon-carbon double bond that is easy to break and easy to identify and judge, and can also more quickly determine the position of the carbon-carbon double bond in the subsequent mass spectrometry analysis.

[0032] (2) Inject the epoxidation product into the mass spectrometer. After ionization by the ion source, select the sodium ion adduct of the epoxidation product for first-order mass spectrometry analysis to obtain the ion mass of the sodium ion adduct of the epoxidation product. Carry out first-order mass spectrometry analysis on the sodium ion adduct of the unsaturated lipid without epoxidation derivatization as a comparison to determine the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid.

[0033] The degree of unsaturation therein is the degree of unsaturation of the carbon-carbon double bond.

[0034] Further, in the present application, based on the analysis of the sodium ion adducts of the epoxidation products, the epoxidation products are therefore analyzed by tandem mass spectrometry in the positive ion mode. The sodium source can be derived from the atmosphere, and the sodium ion adducts of the epoxidation products [M+Na] can be obtained by ionizing the epoxidation product sample in the mass spectrometry ion source. + By performing first-order mass spectrometry analysis on the sodium ion adducts of the epoxidation products, the ion mass of the sodium ion adducts of the epoxidation products can be obtained. Comparing it with the ion mass of the un-epoxidized unsaturated lipid can not only confirm the carbon number of the unsaturated lipid to be analyzed, but also confirm the occurrence of epoxidation. After each carbon-carbon double bond undergoes epoxidation, an epoxidation structure is formed, and an oxygen with a mass of 16 Da (Da is one-twelfth of the carbon atom mass) is combined. By comparing the lipid masses before and after epoxidation, the degree of unsaturation of the carbon-carbon double bonds in the unsaturated lipid to be analyzed can be determined, so that the structural information of the unsaturated lipid can be determined by the ion mass of specific ions in the subsequent tandem mass spectrometry analysis.

[0035] (3) Perform second-order mass spectrometry analysis on the sodium ion adducts of the epoxidation products. After collision-induced dissociation, select the daughter ions obtained after the sodium ion adducts of the epoxidation products lose the head group neutrally.

[0036] In the present application, selecting the sodium ion adducts of the epoxidation products for collision-induced dissociation can change the fragmentation pathway of the lipid, and performing mass spectrometry analysis can obtain better mass spectrometry results. In this step (3), the sodium ion adducts of the epoxidation products are used as the parent ions for collision-induced dissociation (CID) in the second-order mass spectrometry analysis. Sodium ions can not only increase the abundance of the fragmented ions in the mass spectrometry diagram, but also cause the glycerophospholipid to undergo neutral loss of the head group (neutral loss specifically refers to the lost head group part being neutral without positive or negative charges, so it is called neutral loss of the head group). The daughter ions after neutral loss of the head group can obtain more refined structural information of the lipid when continuing to perform collision-induced dissociation in the subsequent third-order mass spectrometry analysis.

[0037] Further, the daughter ions obtained after the sodium ion adducts of the epoxidation products in step (3) lose the head group neutrally contain the structure shown in formula (I), R 1 is the fatty acid chain at the sn-1 position, R 2 is the fatty acid chain at the sn-2 position. Formula (I) In the daughter ions, the 1,3-dioxolane structure is connected to the fatty acid chain at the sn-1 position through a single bond at the 4th position, and is connected to the remaining fatty acid chain at the sn-2 position through a carbon-carbon double bond at the 2nd position.

[0038] Glycerophospholipids are mainly connected through a glycerol backbone to a head group part and two fatty acid chain parts. The head group part is mainly connected to different substituents through a phosphate group to form glycerophospholipids of different head group categories. Therefore, in step (3), the following steps can also be included: According to the mass lost by the neutral loss of the head group, the lipid category of the unsaturated lipid to be analyzed can be confirmed.

[0039] In the present application, the inventors speculated that in the collision-induced dissociation (CID) of the secondary mass spectrometry analysis, after the sodium ion adduct of the epoxidation product lost the head group neutrally, the glycerol backbone therein changed and formed a 1,3-dioxolane structure as shown in formula (I) after flipping and recombination. The change process is shown in the following figure:

[0040] Wherein R represents the substituent part connected through the phosphate group in the head group of the glycerophospholipid, R’ represents the remaining fatty chain part of the fatty acid chain at the sn-1 position from the carbonyl carbon to the end of the fatty acid chain at the sn-1 position, R” represents the remaining fatty chain part of the fatty acid chain at the sn-2 position from the carbonyl carbon to the end of the fatty acid chain at the sn-2 position. The dotted part therein is the glycerol backbone, and the numerical values of “1, 2, 3” represent the sn-positions on the glycerol backbone.

[0041] The inventors speculated that under the influence of the sodium ion adduct, the carbon-oxygen double bond of the carbonyl in the fatty acid chain at the sn-2 position was broken during the recombination process, changing from a carbonyl to a carbon-carbon double bond connecting to the remaining part of the fatty acid chain. It is speculated that a hydrogen (-1Da) is lost from the fatty acid chain at the sn-2 position, and this hydrogen combines with the head group part to achieve neutral loss. After the fatty acid chain at the sn-2 position is flipped, the oxygen on the original carbonyl is connected to the carbon that originally connected the head group part on the glycerol backbone, enclosing to form a 1,3-dioxolane structure as shown in formula (I). In the structure shown in formula (I), the carbon at the 4th position of the 1,3-dioxolane structure is the carbon on the original glycerol backbone connecting the fatty acid chain at the sn-2 position. Therefore, the 1,3-dioxolane structure shown in formula (I) is connected to the fatty acid chain at the sn-1 position through a carbon single bond on the glycerol backbone. At the same time, the carbonyl carbon on the original fatty acid chain at the sn-2 position is at the 2nd position of the 1,3-dioxolane structure. Therefore, the 1,3-dioxolane structure shown in formula (I) is connected to the remaining fatty chain part at the sn-2 position through a carbon-carbon double bond.

[0042] In a specific embodiment of the present application, taking phosphatidylcholine standard PC 16:0 / 18:1 (9Z) as an example, the ionic mass of the sodium ion adduct of the underivatized phosphatidylcholine standard PC 16:0 / 18:1 (9Z) is (m / z 782). In the CID of the secondary mass spectrometry analysis, after the neutral loss of the phosphocholine head group (-183 Da), a daughter ion (m / z 599) is obtained. This daughter ion (m / z 599) contains a structure in which a 1,3-dioxolane is connected to a carbon-carbon double bond at the 2nd position as shown in formula (I), and the conversion process is shown in the following figure:

[0043] In a specific embodiment of the present application, taking phosphatidylcholine standard PC 16:0 / 18:1 (9Z) as an example, after epoxidation derivatization by the epoxidation derivatization method provided in the present application, an epoxidation product of PC 16:0 / 18:1 (9Z) is obtained. The sodium ion adduct (m / z 798) of the epoxidation product of PC 16:0 / 18:1 (9Z) undergoes a neutral loss of the phosphocholine head group (-183 Da) in the CID of the secondary mass spectrometry analysis to obtain a daughter ion (m / z 615). This daughter ion (m / z 615) contains a structure in which a 1,3-dioxolane is connected to a carbon-carbon double bond at the 2nd position as shown in formula (I), and the conversion process is shown in the following figure:

[0044] (4) Perform tertiary mass spectrometry analysis on the daughter ion obtained after the neutral loss of the head group of the sodium ion adduct of the epoxidation product. Specific ions at the sn position and specific ions at the carbon-carbon double bond position are obtained after collision-induced dissociation.

[0045] In the present application, performing mass spectrometry analysis of collision-induced dissociation on the daughter ion obtained after the neutral loss of the head group of the sodium ion adduct of the epoxidation product can simultaneously obtain specific ions at the sn position with high abundance and specific ions at the carbon-carbon double bond position.

[0046] Furthermore, in the present application, after multiple experimental explorations, it is found that since the daughter ion contains the structure shown in formula (I), R 1 is the fatty acid chain at the sn-1 position, R 2 is the fatty chain at the sn-2 position, Formula (I). In step (4), the structure of formula (I) has a cleavage tendency in the tertiary mass spectrometry analysis. During the collision-induced dissociation in the tertiary mass spectrometry analysis, specific ions at the sn position are formed. The specific ions at the sn position include one or both of the specific ions at the sn-1 position and the specific ions at the sn-2 position.

[0047] When R on both sides of the daughter ion to be analyzed1 either the fatty acid chain at the sn-1 position) or R 2 when neither the fatty acid chain at the sn-2 position nor R 1 contains an epoxidized structure, or when R 2 contains an epoxidized structure while R Formula (II), the cleavage tends to occur at the 1,3-dioxolane structure, and the carbon-oxygen bonds connected at the 4th and 5th positions are cleaved, forming a specific ion at the sn-1 position with high abundance; When R 2 contains an epoxidized structure while R 1 does not contain an epoxidized structure, the cleavage tendency of the structure of formula (I) is as shown in formula (III), and the dashed line indicates the cleavage site, Formula (III), the cleavage tends to occur at the carbon-carbon double bond connected to the 1,3-dioxolane structure through the 2nd position, forming a specific ion at the sn-2 position with high abundance; When R 1 and R 2 both contain an epoxidized structure, the cleavage tendency of the structure of formula (I) is as shown in formula (IV), and the dashed line indicates the cleavage site, Formula (IV), the cleavage tends to occur at the 1,3-dioxolane structure, and the carbon-oxygen bonds connected at the 4th and 5th positions are cleaved. The cleavage also tends to occur at the carbon-carbon double bond connected to the 1,3-dioxolane structure through the 2nd position. At this time, specific ions at both the sn-1 position and the sn-2 position with high abundance can be formed, and it has little effect on the characterization of the sn-position specific ions of unsaturated lipids.

[0048] Further, in step (4), the daughter ions form specific ions at the positions of carbon-carbon double bonds through the cleavage of the epoxy structure. The specific ions at the positions of carbon-carbon double bonds contain the structure of formula (I). The formed 1,3-dioxolane structure in the daughter ions can change the cleavage pathway of lipids, endowing the epoxy products with better cleavage tendency. Under the mutual influence of the epoxy structures, during the collision-induced dissociation in the tandem mass spectrometry analysis, some daughter ions will cleave through the 1,3-dioxolane structure to form specific ions at the sn positions with high abundance, and the other part of the daughter ions will cleave through the epoxy structure to form specific ions at the positions of carbon-carbon double bonds containing the structure of formula (I). At this time, different fragmentation channels of the ions will occur simultaneously, and the specific ions at the sn positions and the specific ions at the positions of carbon-carbon double bonds obtained can be characterized simultaneously in the mass spectrum of the tandem mass spectrometry analysis, which can effectively improve the identification efficiency of the fine structure of lipids.

[0049] In a specific embodiment of the present application, taking the unsaturated phosphatidylcholine lipid in glycerophospholipids as an example, after the full derivatization of carbon-carbon double bonds by the epoxy derivatization method provided in the present application, when the epoxy product is subjected to CID in the second-stage mass spectrometry after sodium ion adduction, a neutral loss (-183 Da) of the phosphocholine head group of phosphatidylcholine will occur. The daughter ions after selecting the neutral loss head group are subjected to tandem mass spectrometry analysis, and specific ions at the sn positions with high abundance and specific ions at the positions of carbon-carbon double bonds are simultaneously formed after collision-induced dissociation.

[0050] (5) Analyze the specific ions at the sn positions and the specific ions at the positions of carbon-carbon double bonds, and determine the sn positions and the positions of carbon-carbon double bonds of the unsaturated lipid according to the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid.

[0051] In the present application, when identifying and analyzing unsaturated lipids with a degree of unsaturation not equal to 0, since the formed 1,3-dioxolane structure is included in the obtained daughter ions, and the fragmentation channels of the 1,3-dioxolane structure and the epoxy structure will occur simultaneously, the specific ions at the sn positions obtained after the collision-induced dissociation in the tandem mass spectrometry analysis will contain the epoxy structure, and the specific ions at the positions of carbon-carbon double bonds obtained will contain the 1,3-dioxolane structure. Therefore, directly judging according to the specific ions at the positions of carbon-carbon double bonds can only obtain the distance between the position of the carbon-carbon double bond and the end of the fatty acid chain, and the accurate position information of the carbon-carbon double bond cannot be obtained. Therefore, it is necessary to first determine the sn position of the unsaturated lipid. Further, in step (5), determining the sn position and the position of the carbon-carbon double bond of the unsaturated lipid specifically includes the following steps: (51) Determine the sn position of the unsaturated lipid according to the specific ions at the sn positions, and determine the type of the specific ions at the sn positions; (52) Determine the fragmentation mode of the daughter ions of the epoxidation product according to the specific ion type at the sn position, and determine the distribution of the epoxidation structure in the daughter ions according to the fragmentation mode; (53) Determine the positions of the carbon-carbon double bonds of the unsaturated lipid according to the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid, the distribution of the epoxidation structure in the daughter ions, the specific ions at the sn position, and the specific ions at the carbon-carbon double bond positions.

[0052] In the present application, the specific ion at the sn position indicating the sn-1 position means that the specific ion at the sn position is the specific ion at the sn-1 position; the specific ion at the sn position indicating the sn-2 position means that the specific ion at the sn position is the specific ion at the sn-2 position.

[0053] In the present application, when the influence of the fragmentation tendency between the 1,3-dioxolane structure and the epoxidation structure has been explored, during the identification process, it is first necessary to confirm the type of the specific ion at the sn position with high abundance obtained in step (4), specifically indicating the sn-1 position or the sn-2 position, or whether the specific ions at the sn-1 position and the sn-2 position are obtained simultaneously. According to the specific ion at the sn position, the fragmentation mode of the daughter ions can be determined, and based on the fragmentation mode, it is further possible to inversely confirm the R in the daughter ions 1 or R 2 Whether it contains an epoxidation structure. After determining the distribution of the epoxidation structure, when the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid are clear, the refined structure of the unsaturated lipid can be confirmed according to the ion masses of the specific ions at the sn position and the carbon-carbon double bond positions.

[0054] Further, in step (51), to determine the sn position of the unsaturated lipid, it is first necessary to determine whether the specific ion at the sn position obtained is the specific ion at the sn-1 position or the specific ion at the sn-2 position. Specifically, it includes the following methods: (i) Compare the ion mass of the specific ion at the sn position with the fragmentation characteristic ion mass data to determine whether the specific ion at the sn position is the specific ion at the sn-1 position or the specific ion at the sn-2 position; (ii)When a characteristic peak with a difference of 16 Da is formed near the specific ion at the sn position, the specific ion at the sn position is the specific ion at the sn-2 position; when no characteristic peak with a difference of 16 Da is formed near the specific ion at the sn position, the specific ion at the sn position is the specific ion at the sn-1 position. In the present application, there is a more intuitive method to determine whether the specific ion at the sn position indicates the sn-1 or sn-2 position. Through the identification method provided by the present application, in the CID spectrum of the triple mass spectrometry analysis, it is also found that when the specific ion at the sn-2 position is characterized, a characteristic peak with a difference of 16 Da is formed on the left side of the specific ion at the sn-2 position, while there is no such peak for the specific ion at the sn-1 position. The inventor speculates that in the CID of the triple mass spectrometry analysis, when the fatty chain at the sn-2 position contains an epoxidized structure, due to the cleavage tendency between the epoxidized structure and the 1,3-dioxolane structure, the carbon-carbon double bond connecting the 1,3-dioxolane structure and the fatty chain at the sn-2 position breaks, and the formed fragment ion (R 2 fatty chain) newly combines an oxygen at the cleavage site of the carbon-carbon double bond to form an aldehyde group and is characterized as the specific ion at the sn-2 position, and a small part of the fragment ions are directly characterized. Therefore, on the CID spectrum of the triple mass spectrometry analysis, the specific ion at the sn-2 position often forms a characteristic peak with a difference of 16 Da (the carbon-carbon double bond breaks and combines an oxygen) nearby. In summary, by analyzing the characterization behavior of the specific ion at the sn position in the spectrum, the sn position of the unsaturated lipid can be determined more quickly. Moreover, since the degree of unsaturation of the unsaturated lipid to be analyzed is clarified through comparison in the previous steps, and the number of carbons of the specific ion at the sn-2 position is generally less than that of the specific ion at the carbon-carbon double bond position, and the formed characteristic peak patterns are also different, the additional characteristic peak with a difference of 16 Da formed here will not cause confusion in the subsequent determination of the carbon-carbon double bond position.

[0055] Furthermore, in step (4), specific ions of fatty acid chains with an epoxidized structure at the sn position were also obtained through three-stage mass spectrometry analysis. The specific ions of fatty acid chains with an epoxidized structure at the sn position are one or both of the specific ions of fatty acid chains with an epoxidized structure at the sn-1 position and the specific ions of fatty acid chains with an epoxidized structure at the sn-2 position; in step (5), when the specific ions at the sn position are 40 Da more than the specific ions of fatty acid chains with an epoxidized structure at the sn position, the specific ions at the sn position are the specific ions at the sn-1 position, and the specific ions of fatty acid chains with an epoxidized structure at the sn position are the specific ions of fatty acid chains with an epoxidized structure at the sn-1 position; when the specific ions at the sn position are 30 Da less than the specific ions of fatty acid chains with an epoxidized structure at the sn position, the specific ions at the sn position are the specific ions at the sn-2 position, and the specific ions of fatty acid chains with an epoxidized structure at the sn position are the specific ions of fatty acid chains with an epoxidized structure at the sn-2 position.

[0056] In the present application, when the degree of unsaturation of the unsaturated lipid to be analyzed is 1, after epoxidation derivation, one of the fatty acid chains contains an epoxidized structure. After the sodium ion adduct of the epoxidation product of the unsaturated lipid to be analyzed loses the head group through secondary mass spectrometry analysis, in the CID spectrum of the three-stage mass spectrometry analysis of the resulting daughter ions, other highly abundant specific ions were also found. After comparison and calculation, it was found that after subtracting 1 Da from the ion mass of the highly abundant specific ions, it was exactly equal to the fragmentation ion mass of the carbon-oxygen single bond connecting the fatty acid chain with an epoxidized structure at the sn position to the glycerol backbone. Therefore, the obtained specific ions are the specific ions of the fatty acid chain with an epoxidized structure at the sn position. By selecting the unsaturated lipid to be analyzed without epoxidation derivation for comparison, after the same steps and operations, in the CID spectrum of the three-stage mass spectrometry analysis, specific ions with a certain abundance were also found. After comparison and calculation, it was found that after subtracting 1 Da from the ion mass of the specific ions, it was exactly equal to the fragmentation ion mass of the carbon-oxygen single bond of the fatty acid chain with a carbon-carbon double bond at the sn position breaking from the glycerol backbone. Therefore, the obtained specific ions are the specific ions of the fatty acid chain with a carbon-carbon double bond at the sn position.

[0057] In the present application, when the degree of unsaturation of the unsaturated lipid to be analyzed is greater than or equal to 2 and both fatty acid chains contain epoxidized structures after epoxidation derivation (which can be determined by the number of specific ions at the sn position obtained by judgment. Under the influence of the cleavage of the 1,3-dioxolane structure by the epoxidized structure, specific ions at the sn-1 position and specific ions at the sn-2 position can be obtained simultaneously through the tertiary mass spectrometry analysis of the daughter ions), after the sodium ion adduct of the epoxidation product of the unsaturated lipid to be analyzed undergoes neutral loss of the head group through the secondary mass spectrometry analysis, two other highly abundant specific ions are also found in the CID spectrum of the tertiary mass spectrometry analysis of the obtained daughter ions. After comparison and calculation, it is found that after subtracting 1 Da from the ion masses of the highly abundant specific ions, they are exactly equal to the fragmentation ion masses of the carbon-oxygen single bonds connecting the fatty acid chains containing epoxidized structures at the sn-1 position and the sn-2 position to the glycerol backbone respectively. Therefore, the obtained specific ions are the specific ions of the fatty acid chains containing epoxidized structures at the sn position. By selecting the unsaturated lipid to be analyzed without epoxidation derivation for comparison, through the same steps and operations, two specific ions with a certain abundance are also found in the CID spectrum of the tertiary mass spectrometry analysis. After comparison and calculation, it is found that after subtracting 1 Da from the ion masses of the two specific ions, they are exactly equal to the fragmentation ion masses of the carbon-oxygen single bonds of the fatty acid chains containing carbon-carbon double bonds at the sn-1 position and the sn-2 position cleaved from the glycerol backbone respectively. Therefore, the obtained specific ions are the specific ions of the fatty acid chains containing carbon-carbon double bonds at the sn position.

[0058] The inventors also found that the fragmentation ion masses of the specific ions indicating the fatty acid chains containing carbon-carbon double bonds at the sn position and the specific ions indicating the fatty acid chains containing epoxidized structures at the sn position obtained previously just differ by n * 16 Da (n represents the degree of unsaturation, and 16 Da represents the addition of one oxygen after the epoxidation derivation of the carbon-carbon double bond). This indicates that through the identification steps of the present application, regardless of whether epoxidation derivation occurs, the fatty acid chains at the sn position containing carbon-carbon double bonds or epoxidized structures have a certain cleavage tendency and are characterized by cleavage in the tertiary mass spectrometry analysis. Subsequently, the carbon number of the specific ions of the fatty acid chains containing epoxidized structures at the sn position and the number of epoxidized structures can also be confirmed by comparison.

[0059] Regarding the specific ions of fatty acid chains with an epoxidized structure at the sn position and the specific ions of fatty acid chains with a carbon-carbon double bond at the sn position, the inventors speculated that after the head group underwent a neutral loss in the secondary mass spectrometry analysis, not all of the resulting product ions formed a 1,3-dioxolane structure. Instead, there were also some product ions that retained the chain structure after the neutral loss of the head group. In the tertiary mass spectrometry analysis affected by the cleavage of the sodium ion adduct, the fatty acid chains containing a carbon-carbon double bond or an epoxidized structure had a higher tendency to break, thus forming the specific ions of fatty acid chains with a carbon-carbon double bond at the sn position or the specific ions of fatty acid chains with an epoxidized structure at the sn position.

[0060] After the fatty acid chains containing a carbon-carbon double bond or an epoxidized structure were adducted with a sodium ion and then increased by 1 Da, they were characterized as the specific ions of fatty acid chains with a carbon-carbon double bond at the sn position or the specific ions of fatty acid chains with an epoxidized structure at the sn position. The inventors speculated that this was because the cleavage site was located at the carbon-oxygen single bond connecting the fatty acid chain to the glycerol backbone. After the fatty acid chain containing a carbon-carbon double bond or an epoxidized structure broke, the terminal oxygen newly bound a hydrogen (+1 Da) and was characterized as a specific ion with high abundance.

[0061] When the specific ions at the sn position and the specific ions containing an epoxidized structure at the sn position were obtained simultaneously, due to the cleavage effect brought about by the interaction between the epoxidized structure and the formed 1,3-dioxolane structure of formula (I), the ion masses of the specific ions at the sn position and the specific ions containing an epoxidized structure at the sn position were correlated. The specific ion pair related to the sn-1 position or the sn-2 position could be determined by the difference in ion mass between the two. This determination method was also applicable when only the specific ions at the sn-1 or sn-2 position were obtained.

[0062] As previously speculated, among the product ions in the tertiary mass spectrometry analysis, the specific ions at the sn position were formed by the cleavage of some product ions containing the 1,3-dioxolane structure of formula (I); the specific ions of the fatty acid chain containing an epoxidized structure at the sn position were formed by the cleavage of some product ions that retained the chain structure (without forming a 1,3-dioxolane structure). In the CID spectrum of the tertiary mass spectrometry analysis, the specific ions at the sn position and the specific ions of the fatty acid chain containing an epoxidized structure at the sn position with high abundance were obtained simultaneously.

[0063] Under the condition of sodium ion adduction, when a definite cleavage mode was obtained, due to the R in the product ion 1When the epoxidized structure is included in the fatty acid chain at the sn-1 position and the cleavage occurs at the 1,3-dioxolane structure of formula (I), the carbon-oxygen bonds connected at the 4th and 5th positions are cleaved. Therefore, the specific ion at the sn-1 position obtained has three carbons and five hydrogens in the glycerol backbone part as shown in the following figure more than the fatty acid chain with an epoxidized structure at the sn-1 position (+12Da + 12Da + 12Da + 5Da = +41Da): (The numerical values of "1, 2, 3" therein represent the sn-positions on the glycerol backbone, and the part marked with a dotted line represents the carbon chain part on the glycerol backbone). Moreover, since the fatty acid chain with an epoxidized structure at the sn-1 position combines a hydrogen (1Da) as the specific ion of the fatty acid chain with an epoxidized structure at the sn-1 position for characterization during characterization, in the CID spectrum of the tandem mass spectrometry analysis, the actually obtained specific ion at the sn-1 position is 40Da more than the specific ion of the fatty acid chain with an epoxidized structure at the sn-1 position (+41Da - 1Da = +40Da, and in the actual spectrum, a pair of specific ions at the sn-1 position with a difference of 40Da is indeed obtained), which also corresponds to the cleavage rule and binding rule explored, and the specific ion pair at the sn-1 position can be reversely determined through this characteristic with a difference of 40Da.

[0064] Similarly, under the condition of sodium ion adduction, when the determined cleavage mode is obtained, due to the R in the daughter ion 2 When the epoxidized structure is included in the fatty acid chain at the sn-2 position and the cleavage occurs at the carbon-carbon double bond connected through the 2nd position of the 1,3-dioxolane structure of formula (I), the specific ion at the sn-2 position obtained is less than the fatty acid chain with an epoxidized structure at the sn-2 position by one carbon and two oxygens for forming the 1,3-dioxolane structure part as shown in the following figure (-12 Da - 16 Da - 16 Da = -44 Da): (The part marked with a dotted line represents the part of the fatty acid chain at the sn-2 position used to form the 1,3-dioxolane structure), and when the 1,3-dioxolane structure is formed, the fatty acid chain at the sn-2 position loses a hydrogen (-1Da) when the carbon-oxygen double bond forms a carbon-carbon double bond. Additionally, as explored above during the tandem mass spectrometry analysis, the fragmented ions formed (R 2At the cleavage site of the carbon-carbon double bond in the fatty acid chain, a new oxygen atom is incorporated to form an aldehyde group (+16 Da), which is characterized as a specific ion at the sn-2 position. Since the fatty acid chain containing an epoxidized structure at the sn-2 position incorporates a hydrogen atom (1 Da) during characterization as a specific ion for the fatty acid chain with an epoxidized structure at the sn-2 position, in the CID spectrum of the tandem mass spectrometry analysis, the actual specific ion at the sn-2 position is 30 Da less than the specific ion for the fatty acid chain with an epoxidized structure at the sn-2 position (-44 Da - 1 Da + 16 Da - 1 Da = -30 Da, and in the actual spectrum, a pair of specific ions at the sn-2 position with a 30 Da difference is indeed obtained). This also corresponds to the cleavage and binding rules explored, and the specific ion pair at the sn-2 position can be determined inversely through this 30 Da difference feature.

[0065] Although unsaturated lipids without epoxidation can form specific ions of fatty acid chains containing carbon-carbon double bonds at the sn position during collision-induced dissociation in tandem mass spectrometry analysis, since no epoxidation-derived epoxidized structure is formed, it has no effect on the cleavage tendency of the formed 1,3-dioxolane structure in tandem mass spectrometry analysis. Therefore, unsaturated lipids without epoxidation cannot be used to determine the sn position by this method.

[0066] Under specific cleavage and binding rules, corresponding specific ion pairs at the sn-1 position or sn-2 position can be obtained only when the epoxidized structure is contained in R 1 or R 2 ; when the epoxidized structure is contained in both R 1 and R 2 and multiple specific ions are formed in the CID spectrum of tandem mass spectrometry analysis, the specific ion pair at the sn-1 position or sn-2 position can be determined by the difference between the obtained specific ions related to the sn position.

[0067] In a specific embodiment of the present application, taking the standard phosphatidylcholine PC 16:0 / 18:1(9Z) as an example, after epoxidation derivatization, CID of the sodium ion adduct (m / z 798) of the epoxidation product of PC 16:0 / 18:1(9Z) was performed by second-order mass spectrometry. After neutral loss of the head group, a daughter ion (m / z 615) was obtained. Then, specific ions with high abundances of (m / z 291) and (m / z 321) were obtained in the spectrum after performing CID of the third-order mass spectrometry on the daughter ion (m / z 615). Among them, the specific ion (m / z 291) is the specific ion at the sn-2 position, and the specific ion (m / z 321) is the specific ion of the fatty acid chain containing an epoxidation structure at the sn-2 position. The difference between the two specific ions is exactly 30 Da.

[0068] In a specific embodiment of the present application, taking the standard phosphatidylcholine PC 18:1(9Z) / 16:0 as an example, after epoxidation derivatization, CID of the sodium ion adduct (m / z 798) of the epoxidation product of PC 18:1(9Z) / 16:0 was performed by second-order mass spectrometry. After neutral loss of the head group, a daughter ion (m / z 615) was obtained. Then, specific ions with high abundances of (m / z 321) and (m / z 361) were obtained in the spectrum after performing CID of the third-order mass spectrometry on the daughter ion (m / z 615). Among them, the specific ion (m / z 361) is the specific ion at the sn-1 position, and the specific ion (m / z 321) is the specific ion of the fatty acid chain containing an epoxidation structure at the sn-1 position. The difference between them is exactly 40 Da.

[0069] In summary, in step (51), by the above method, it can be determined whether the specific ion at the sn position obtained after epoxidation derivatization is the specific ion at the sn-1 position or the specific ion at the sn-2 position, so as to determine the sn position of the unsaturated lipid. And after judgment according to the characterization of the specific ion at the sn position, three characterization types can be obtained: Type 1: Only the specific ion at the sn-1 position is obtained; Type 2: Only the specific ion at the sn-2 position is obtained; Type 3: Both the specific ion at the sn-1 position and the specific ion at the sn-2 position are obtained.

[0070] (52) Determine the fragmentation mode of the daughter ion of the epoxidation product according to the type of the specific ion at the sn position, and determine the distribution of the epoxidation structure in the daughter ion according to the fragmentation mode. In step (52), in the case where the fragmentation tendency has been explored, and the number and type of the specific ions at the sn position have been determined in step (51), the fragmentation mode of the daughter ion can be known, and the distribution of the epoxidation structure in the daughter ion can be determined according to the fragmentation mode. Correspondingly, the following distribution types are included: Type 1: Only specific ions at the sn-1 position are obtained. It means that in step (4), the fragmentation mode shown in formula (II) occurs in the daughter ions. The dotted line indicates the fragmentation site, and R 1 is the fatty acid chain at the sn-1 position, and R 2 is the fatty chain at the sn-2 position. For formula (II), the fragmentation occurs on the 1,3-dioxolane structure, and the carbon-oxygen bonds connected at the 4th and 5th positions are broken; this fragmentation mode indicates two distribution cases of the epoxidized structure in the daughter ions: (A) Neither R 1 nor R 2 contains an epoxidized structure; (B) R 1 contains an epoxidized structure while R 2 does not contain an epoxidized structure.

[0071] Type 2: Only specific ions at the sn-2 position are obtained. It means that in step (4), the fragmentation mode shown in formula (III) occurs in the daughter ions. The dotted line indicates the fragmentation site, and R 1 is the fatty acid chain at the sn-1 position, and R 2 is the fatty chain at the sn-2 position. For formula (III), the fragmentation occurs on the carbon-carbon double bond connected by the 2nd position of the 1,3-dioxolane structure; this fragmentation mode indicates that R 2 in the daughter ions contains an epoxidized structure, while R 1 does not contain an epoxidized structure.

[0072] Type 3: When specific ions at both the sn-1 position and the sn-2 position are obtained simultaneously. It means that in step (4), the fragmentation mode shown in formula (IV) occurs in the daughter ions. The dotted line indicates the fragmentation site, and R 1 is the fatty acid chain at the sn-1 position, and R 2 is the fatty chain at the sn-2 position. For formula (IV), the fragmentation occurs on the 1,3-dioxolane structure, and the carbon-oxygen bonds connected at the 4th and 5th positions are broken, and the fragmentation also occurs simultaneously on the carbon-carbon double bond connected by the 2nd position of the 1,3-dioxolane structure; this fragmentation mode indicates that both R 1 and R 2 in the daughter ions contain an epoxidized structure.

[0073] Step (53) determines the positions of carbon-carbon double bonds of the unsaturated lipid based on the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid, the distribution of the epoxidized structure in the daughter ions, the specific ions at the sn positions, and the specific ions at the positions of the carbon-carbon double bonds. The daughter ions obtained after losing the head group by neutral loss contain a structure shown in formula (I), where R 1 is the fatty acid chain at the sn-1 position, and R 2 is the fatty chain at the sn-2 position. Formula (I) In the daughter ions, the 1,3-dioxolane structure is connected to the fatty acid chain at the sn-1 position through a single bond at the 4th position and is connected to the remaining fatty chain at the sn-2 position through a carbon-carbon double bond at the 2nd position.

[0074] Specifically, step (53) includes the following cases: (531) For the specific ions only at the sn-1 position in type 1, they are distinguished according to the degree of unsaturation of the unsaturated lipid obtained in step (2): (a) When the degree of unsaturation of the lipid is 0, neither R 1 nor R 2 in the daughter ions contains an epoxidized structure, and no specific ions at the positions of the carbon-carbon double bonds are formed.

[0075] (b) When the degree of unsaturation of the unsaturated lipid is not 0, R 1 in the daughter ions contains an epoxidized structure while R 2 does not contain an epoxidized structure. The positions of the carbon-carbon double bonds of the unsaturated lipid are determined based on the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid, the specific ions at the sn-1 position, and the specific ions at the positions of the carbon-carbon double bonds.

[0076] (532) For the specific ions only at the sn-2 position in type 2, R 2 in the daughter ions contains an epoxidized structure while R 1 does not contain an epoxidized structure. The positions of the carbon-carbon double bonds of the unsaturated lipid are determined based on the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid, the specific ions at the sn-2 position, and the specific ions at the positions of the carbon-carbon double bonds.

[0077] In (531) and (532), based on the degree of unsaturation of the unsaturated lipid and the ion masses of the specific ions characterizing the sn positions therein, the number of epoxy structures and the number of carbons contained in the sn-position specific ions can be determined. Based on the total number of carbon atoms of the unsaturated lipid, the number of carbons of the sn-position specific ions, and the specific ions of the carbon-carbon double bonds, the number of carbons, composition, and the position of the carbon-carbon double bonds of the fatty acid chains at the sn positions can be determined. Correspondingly, after obtaining the information of the fatty acid chains at one sn position, the information of the fatty acid chains at the other side can also be deduced, and then the refined structure of the unsaturated lipid can be obtained.

[0078] (533) For the type 3 where the specific ions at the sn-1 position and the specific ions at the sn-2 position are obtained simultaneously, the R of the daughter ions 1 and R 2 both contain epoxy structures. Based on the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid, the specific ions at the sn-1 position, the specific ions at the sn-2 position, and the specific ions of the carbon-carbon double bond positions, the carbon-carbon double bond positions of the unsaturated lipid are determined.

[0079] Further, in step (5) of the present application, secondary mass spectrometry analysis can also be performed on the sodium ion adduct of the unsaturated lipid that has not undergone epoxy derivatization. Select the second daughter ion obtained after neutral loss of the head group of the sodium ion adduct of the unsaturated lipid that has not undergone epoxy derivatization. Perform tertiary mass spectrometry analysis on the second daughter ion obtained after neutral loss of the head group of the sodium ion adduct of the unsaturated lipid to obtain the specific ions of the fatty acid chains containing carbon-carbon double bonds at the sn positions. Compare with the specific ions of the fatty acid chains containing epoxy structures at the corresponding sn positions to determine the number of carbons and the number of epoxy structures of the specific ions of the fatty acid chains containing epoxy structures at the corresponding sn positions, thereby determining the number of carbons and the number of epoxy structures of the specific ions at the corresponding sn positions; the specific ions of the fatty acid chains containing carbon-carbon double bonds at the sn positions include one or both of the specific ions of the fatty acid chains containing carbon-carbon double bonds at the sn-1 position and the specific ions of the fatty acid chains containing carbon-carbon double bonds at the sn-2 position.

[0080] Since in step (4), specific ions of the fatty acid chains containing epoxy structures at the sn positions are also obtained through tertiary mass spectrometry analysis, and it has been explored above that the fatty acid chains containing carbon-carbon double bonds at the sn positions also have a certain tendency to break for characterization. Therefore, the same identification steps are performed on the unsaturated lipid that has not undergone epoxy derivatization. The specific ions of the fatty acid chains containing carbon-carbon double bonds at the sn positions are formed as a comparison in the tertiary mass spectrometry analysis. The mass difference between them and the specific ions of the corresponding epoxy structure-containing fatty acid chains at the sn positions corresponds to the number of epoxy structures, and the number of carbons and the number of epoxy structures of the specific ions of the fatty acid chains containing epoxy structures at the corresponding sn positions can be further determined.

[0081] Further, the carbon number and the number of epoxy structures of the specific ion corresponding to the sn-1 position are determined according to the carbon number and the number of epoxy structures of the fatty acid chain containing an epoxy structure at the sn-1 position, the carbon number and the number of epoxy structures of the specific ion corresponding to the sn-2 position are determined according to the carbon number and the number of epoxy structures of the fatty acid chain containing an epoxy structure at the sn-2 position, and the carbon-carbon double bond position of the unsaturated lipid is determined according to the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid, the specific ion at the sn-1 position, the specific ion at the sn-2 position, and the specific ion at the carbon-carbon double bond position.

[0082] In (533), according to the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid, the carbon number and the number of epoxy structures of the specific ions at the sn-1 position and the sn-2 position respectively, and the specific ion of the carbon-carbon double bond, the carbon number, composition, and the position of the carbon-carbon double bond of the fatty acid chains at the sn-1 position and the sn-2 position can be determined, and then the refined structure of the unsaturated lipid can be obtained.

[0083] Further, in the first-stage mass spectrometry analysis, the degree of unsaturation of the unsaturated lipid to be analyzed can be judged by mass comparison. After determining the sn position of the unsaturated lipid, the characteristic peak of 16 Da related to the specific ion at the sn position is excluded, and then the position of the carbon-carbon double bond in the unsaturated lipid is determined according to the degree of unsaturation and the specific ion of the carbon-carbon double bond.

[0084] Among them, when only one carbon-carbon double bond is contained in a fatty acid chain of an unsaturated lipid, after epoxidation derivation, a single epoxidation structure is formed on the chain, and characteristic peaks with a difference of 16 Da are correspondingly formed in the CID spectrum of the triple mass spectrometry analysis. After determining the sn position of the unsaturated lipid and the number of carbons in the fatty acid chain at the sn position, the distance between the carbon-carbon double bond and the carbonyl carbon terminus can be judged by the mass of the specific ion of the carbon-carbon double bond, and thus the position of the carbon-carbon double bond can be determined. When more than one carbon-carbon double bond is contained in a fatty acid chain of an unsaturated lipid, after epoxidation derivation, more than one epoxidation structure is formed on the chain. When the two epoxidation structures are far apart (the distance between the two epoxidation structures on the fatty acid chain is more than three carbon numbers), generally characteristic peaks with a difference of 16 Da can still be formed for judgment. If the two epoxidation structures are close to each other (the distance between the two epoxidation structures on the fatty acid chain is three carbon numbers or less), at this time, the formation of characteristic peaks with a difference of 16 Da for the cleavage tendency by the two epoxidation structures is not so obvious, but this does not affect the confirmation of the carbon-carbon double bond position. At this time, the epoxidation structure and the part between the two epoxidation structures will break to continue to form specific ions at the carbon-carbon double bond position. After determining the sn position of the unsaturated lipid and the number of carbons in the fatty acid chain at the sn position, the corresponding carbon-carbon double bond position can still be judged by back-calculating according to the ion mass of the specific ion of the carbon-carbon double bond.

[0085] Furthermore, the identification method provided by the present application can also be used to detect unsaturated lipids in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidic acid, etc. Glycerophospholipids are mainly composed of a glycerol backbone, two fatty acid chains, and a head group of phosphoric acid and its substituted groups. Different glycerophospholipids are only distinguished by different head group categories, and the identification method provided by the present application is based on the glycerol backbone in glycerophospholipids. After realizing the neutral loss of the head group part, the structural transformation of the formed 1,3-dioxolane also occurs on the glycerol backbone. When this structure is analyzed by tandem mass spectrometry, specific ions at the sn position and the carbon-carbon double bond position with high abundance can be formed, and the multi-dimensional fine structure of unsaturated lipids in different types of glycerophospholipids can be identified. The oxidation method provided by the present application is simple, easy to reproduce, and has high identification efficiency, which is of great significance for the high-throughput structure identification of lipids.

[0086] In the present application, according to the multi-dimensional lipid fine structure identification method based on the full derivation of carbon-carbon double bonds proposed in the present application, the full derivation of carbon-carbon double bonds of the unsaturated lipid to be analyzed can be rapidly carried out through the epoxidation derivation method provided by the present application. By performing mass spectrometry analysis on the sodium ion adduct of the epoxidation product and using the mass spectrometry analysis of the unsaturated lipid without epoxidation derivation as a comparison, the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid can be determined. By performing secondary mass spectrometry analysis on the sodium ion adduct of the epoxidation product, the glycerophospholipid category of the unsaturated lipid to be analyzed can be determined after the neutral loss of the head group. After the neutral loss of the head group, the partial ion structure changes. Through tertiary mass spectrometry analysis, the specific ions at the sn position and the specific ions at the carbon-carbon double bond position are obtained. By determining whether the specific ions at the sn position indicate the sn-1 position or the sn-2 position and analyzing in combination with the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid, the composition of the two fatty acid chains in the unsaturated lipid and the connection relationship between the two fatty acid chains and the sn-1 position and the sn-2 position can be determined. Then, the position of the carbon-carbon double bond on the specific fatty acid chain in the unsaturated lipid can be further determined through the specific ions at the carbon-carbon double bond position, so that the fine structure of the unsaturated lipid can be determined.

[0087] The following is a further illustration through specific examples.

[0088] The mass spectrometer used in the examples and comparative examples of the present application is the Qtrap mass spectrometer of Sciex company.

[0089] The method for epoxidation derivation of the unsaturated lipid to be analyzed in the examples of the present application is as follows: In the present application, dimethyldioxirane is used for in-situ epoxidation derivation of the carbon-carbon double bonds in the unsaturated lipid to be analyzed. Among them, potassium peroxymonosulfate is used as the oxidant and acetone is used as the catalyst to in-situ generate dimethyldioxirane for full derivation of the carbon-carbon double bonds in the unsaturated lipid to be analyzed. The specific operation is as follows: The full derivation reaction of the carbon-carbon double bonds of the unsaturated lipid to be analyzed is carried out in a 1.5 mL PE tube. Take 1 mmol / L of the unsaturated lipid to be analyzed into the PE tube, then add 50 μL of 500 mmol / L potassium peroxymonosulfate, then add 100 μL of acetone, and then add 50 μL of 500 mmol / L sodium bicarbonate solution. The entire reaction system reacts at 60 °C. During the reaction, ultrasound is carried out, and the ultrasound time is 10 minutes. After reacting for 30 minutes, 300 μL of ethyl acetate is added to extract the epoxidation product of the unsaturated lipid to be analyzed, and then it is dried with nitrogen. After re-dissolving with acetonitrile, tandem mass spectrometry analysis is carried out.

[0090] Comparative Example 1 Inject the phosphatidylcholine standard PC 16:0 / 18:1(9Z) into the mass spectrometer. After ionization by the ion source, select the sodium adduct of the phosphatidylcholine standard PC 16:0 / 18:1(9Z) for mass spectrometry analysis (MS) to obtain the sodium adduct [M+Na] of PC 16:0 / 18:1(9Z). + The ionic mass (m / z 782) of + is obtained. After CID fragmentation in the second-stage mass spectrometry analysis (MS2), a neutral loss of 183 Da (the head group of PC) generates a daughter ion (m / z 599). Then, perform CID analysis (MS3) of the third-stage mass spectrometry on the daughter ion (m / z 599) after neutral loss of the head group. As Figure 2 shown, the daughter ion (m / z 599) generates a specific ion with low abundance at the sn-1 position (m / z 319) after fragmentation in the third-stage mass spectrometry analysis. Among them, a fatty acid chain containing a carbon-carbon double bond at the sn-2 position [C18:1+Na] + of the specific ion (m / z 305) is formed, which can be used to obtain the sn position information of PC 16:0 / 18:1(9Z). As Figure 2 shown, for PC 16:0 / 18:1(9Z), the specific ion (m / z 319) indicates that the fatty acid chain C16:0 is at the sn-1 position. Among them, the two peaks of the specific ions (m / z 319) and (m / z 345) are both visible in the same mass spectrum. The peak with lower abundance (m / z 345) among these two peaks is due to the impurity of the sample mixed with another sn isomer.

[0091] Comparative Example 2 Inject the phosphatidylcholine standard PC 18:1(9Z) / 16:0 into the mass spectrometer. After ionization by the ion source, select the sodium adduct of the phosphatidylcholine standard PC 18:1(9Z) / 16:0 for mass spectrometry analysis (MS) to obtain the sodium adduct [M+Na] of PC 16:0 / 18:1(9Z). + The ionic mass (m / z 782) of + is obtained. After CID fragmentation in the second-stage mass spectrometry analysis (MS2), a neutral loss of 183 Da (the head group of PC) generates a daughter ion (m / z 599). Then, perform CID analysis (MS3) of the third-stage mass spectrometry on the daughter ion (m / z 599) after neutral loss of the head group. As Figure 3 shown, the daughter ion (m / z 599) generates a specific ion with low abundance at the sn-1 position (m / z 345) after fragmentation in the third-stage mass spectrometry analysis. Among them, a fatty acid chain containing a carbon-carbon double bond at the sn-1 position [C18:1+Na] + of the specific ion (m / z 305) is formed, which can be used to obtain the sn position information of PC 18:1(9Z) / 16:0.Figure 3 As shown in the figure, for PC 18:1(9Z) / 16:0, the specific ion (m / z 345) indicates that the fatty acid chain C18:1 is at the sn-1 position. The specific ion (m / z 319) and (m / z 345) peaks are both visible in the same mass spectrum. The lower abundance (m / z 319) peak is caused by the sample being impure and mixed with another sn isomer.

[0092] Depend on Figure 2 and Figure 3 It can be seen that the sodium ion adduct of unsaturated lipids that have not undergone epoxidation derivatization loses the phosphorylcholine head group (-183 Da) through MS2 CID neutralization, and then only obtains low-abundance sn-position specific ions through MS3 fragmentation, while the carbon-carbon double bond position, another structural information of unsaturated lipids, cannot be obtained.

[0093] Example 1 A phosphatidylcholine standard substance PC 16:0 / 18:1(9Z) was selected, and the above-mentioned epoxidation derivatization method was used to perform full derivatization of the carbon-carbon double bond in situ to obtain an epoxidation product. The epoxidation product was injected into a mass spectrometer, and after ionization by an ion source, the sodium ion adduct of the epoxidation product was selected for mass spectrometry analysis (MS) to obtain the sodium ion adduct of the epoxidation product of PC 16:0 / 18:1(9Z) [M+Na] + The ion mass (m / z 798) of the second-level mass spectrometry analysis (MS2) is neutrally fragmented by CID and loses 183 Da (the head group of PC) to generate a daughter ion (m / z 615). Then, the daughter ion (m / z 615) of the head group is subjected to CID analysis (MS3) of the third-level mass spectrometry, such as Figure 4 As shown in the figure, the daughter ion (m / z 615) was fragmented by triple mass spectrometry to generate a highly abundant sn-specific ion (m / z 291), in which a fatty acid chain containing an epoxidized structure [C18:1+O+Na] at the sn-2 position was also formed. + At the same time, the specific ions of the high-abundance carbon-carbon double bond position in the mass spectrum (m / z 489 and m / z 473) can also be obtained, indicating that the position of the carbon-carbon double bond is 9 positions away from the carbonyl carbon end. The two peaks (m / z 291) and (m / z 361) are both visible in the same mass spectrum. The lower abundance (m / z 361) peak is due to the impurity of the sample mixed with another sn isomer.

[0094] In addition, a specific ion (m / z 275) with a 16 Da difference was additionally formed with the specific ion (m / z 291) at the sn-2 position. It is speculated that the specific ion (m / z 275) was originally connected to the 1,3-dioxolane structure through a carbon-carbon double bond. After the carbon-carbon double bond of the specific ion (m / z 275) was broken, it combined with an oxygen (16 Da) to form an aldehyde group, and then was characterized as the specific ion (m / z 291) at the sn position.

[0095] Example 2: Phosphatidylcholine standard PC 18:1(9Z) / 16:0 was selected. After the carbon-carbon double bonds were fully derivatized in situ by the above epoxidation derivatization method, the epoxidation product was obtained. The epoxidation product was injected into a mass spectrometer. After ionization by the ion source, the sodium ion adduct of the epoxidation product was selected for mass spectrometry analysis (MS), and the sodium ion adduct [M+Na] + of the epoxidation product of PC 18:1(9Z) / 16:0 with an ion mass of (m / z 798) was obtained. After secondary mass spectrometry analysis (MS2) CID fragmentation, 183 Da (the head group of PC) was neutrally lost to generate a daughter ion (m / z 615). Then, CID analysis (MS3) of the tertiary mass spectrometry was performed on the daughter ion (m / z 615) without the head group. As Figure 5 shown, the daughter ion (m / z 615) generated a specific ion (m / z 361) at the sn position with high abundance after tertiary mass spectrometry analysis and fragmentation. Among them, a specific ion (m / z 321) of the fatty acid chain containing an epoxidation structure at the sn-1 position [C18:1+O+Na] + was also formed. At the same time, specific ions (m / z 489 and m / z 473) at the carbon-carbon double bond position with high abundance in the mass spectrum were also obtained, indicating that the position of its carbon-carbon double bond was the 9th position from the carbonyl carbon end. Among them, the two peaks (m / z 291) and (m / z 361) were both visible in the same mass spectrum. The lower abundance peak (m / z 291) among these two peaks was due to the sample being impure and mixed with another sn isomer.

[0096] Example 3: Phosphatidylcholine standard PC 16:0 / 18:2(9Z, 12Z) was selected. After the carbon-carbon double bonds were fully derivatized in situ by the above epoxidation derivatization method, the epoxidation product was obtained. The epoxidation product was injected into a mass spectrometer. After ionization by the ion source, the sodium ion adduct of the epoxidation product was selected for mass spectrometry analysis (MS), and the sodium ion adduct [M+Na] +The ion mass (m / z 812) loses 183 Da (the head group of PC) neutrally after CID fragmentation by secondary mass spectrometry (MS2) to generate a daughter ion (m / z 629). Then, CID analysis (MS3) of the tertiary mass spectrometry is performed on the daughter ion (m / z 629) without the head group. As Figure 6 shown, after fragmentation by tertiary mass spectrometry, the daughter ion (m / z 629) generates a specific ion with high abundance at the sn position (m / z 305), and a fatty acid chain [C18:1+2O+Na] containing an epoxidized structure at the sn-2 position forms a specific ion (m / z 335). At the same time, specific ions with high abundance at the carbon-carbon double bond positions (m / z 517, m / z 545, and m / z 489) can also be obtained after fragmentation by tertiary mass spectrometry, indicating that the positions of the carbon-carbon double bonds are at the 9th and 12th positions from the carbonyl carbon end. The lower-abundance (m / z 375) also appears in the same mass spectrum, which is due to sample impurity and contamination with another sn isomer PC 18:2(9Z,12Z) / 16:0. + Since more than one carbon-carbon double bond on the same fatty acid chain has undergone epoxidation derivatization, under the influence of the cleavage tendency of the epoxidized structure, it is not characterized by characteristic peaks with a difference of 16 Da. However, by back-calculating based on the mass correspondence of the formed specific ions (m / z 517, m / z 545, and m / z 489), after determining the information at the sn position, the relevant information of the carbon-carbon double bonds can also be obtained. Among them, m / z 545 indicates that one of the carbon-carbon double bond positions is at the 12th position from the carbonyl carbon end, m / z 489 indicates that the other carbon-carbon double bond position is at the 9th position from the carbonyl carbon end, and m / z 517 indicates that cleavage also occurs between the two epoxidized structures.

[0097] In addition, a specific ion (m / z 289) with a difference of 16 Da is additionally formed with the specific ion (m / z 305) at the sn-2 position. It is speculated that the specific ion (m / z 289) was originally connected to the 1,3-dioxolane structure through a carbon-carbon double bond. After the carbon-carbon double bond of the specific ion (m / z 275) breaks, it combines with an oxygen (16 Da) to form an aldehyde group, and then is characterized as a specific ion (m / z 291) at the sn position.

[0098] From

[0099] From Figures 4 - 6It can be seen that after the unsaturated lipid to be analyzed undergoes full derivatization of carbon-carbon double bonds, and after the sodium ion adduct of the epoxidation product of the unsaturated lipid to be analyzed loses the phosphocholine head group (-183 Da) in the second-stage mass spectrometry analysis and then undergoes fragmentation in the third-stage mass spectrometry analysis, characteristic peaks of the sn-position and the carbon-carbon double bond position with high abundance can be obtained simultaneously in the same mass spectrum, thereby realizing the simultaneous identification of the sn-position and the carbon-carbon double bond position in lipid isomers.

[0100] Using the multi-dimensional lipid fine structure identification method based on full derivatization of carbon-carbon double bonds provided by the present application, the unsaturated lipid to be measured forms an epoxidation structure in situ after full derivatization of carbon-carbon double bonds. Sodium ion adduct is selected for tandem mass spectrometry analysis. After the fragmentation in the second-stage mass spectrometry analysis loses the head group, a putative 1,3-dioxolane structure can be formed. Under the cleavage tendency that interacts with the epoxidation structure, after fragmentation in the third-stage mass spectrometry analysis, characteristic peaks of the sn- and carbon-carbon double bond positions with high abundance can be obtained simultaneously, which is conducive to realizing the identification and analysis of multi-dimensional lipid fine structures.

[0101] It should be understood that the application of the present application is not limited to the above examples. For those of ordinary skill in the art, improvements or transformations can be made according to the above description, and all such improvements and transformations should fall within the protection scope of the present application.

Claims

1. A method for identifying the fine structure of multi-dimensional lipids fully derived from carbon-carbon double bonds, characterized in that it is applicable to unsaturated lipids containing a glycerol backbone and a head group, and includes the following steps: (1) In-situ epoxidation derivatization of the unsaturated lipid to be analyzed is carried out using dimethyldioxirane, so that all carbon-carbon double bonds in the unsaturated lipid are epoxidized to form an epoxidized structure, and an epoxidized product of the unsaturated lipid is obtained; (2) Inject the epoxidized product into a mass spectrometer. After ionization by an ion source, select the sodium ion adduct of the epoxidized product for first-order mass spectrometry analysis to obtain the ion mass of the sodium ion adduct of the epoxidized product. Perform first-order mass spectrometry analysis on the sodium ion adduct of the unsaturated lipid without epoxidation derivatization as a comparison to determine the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid; (3) Perform second-order mass spectrometry analysis on the sodium ion adduct of the epoxidized product. After collision-induced dissociation, select the daughter ion obtained after the sodium ion adduct of the epoxidized product loses the head group; (4) Perform third-order mass spectrometry analysis on the daughter ion obtained after the sodium ion adduct of the epoxidized product loses the head group. After collision-induced dissociation, obtain the specific ions at the sn position and the specific ions at the carbon-carbon double bond position; (5) Analyze the specific ions at the sn position and the specific ions at the carbon-carbon double bond position, and determine the sn position and the carbon-carbon double bond position of the unsaturated lipid according to the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid.

2. The method for identifying the fine structure of multi-dimensional lipids fully derived from carbon-carbon double bonds according to claim 1, characterized in that in step (5), the determination of the sn position and the carbon-carbon double bond position of the unsaturated lipid specifically includes the following steps: (51) Determine the sn position of the unsaturated lipid according to the specific ions at the sn position, and determine the type of the specific ions at the sn position; (52) Determine the cleavage mode of the daughter ion of the epoxidized product according to the type of the specific ions at the sn position, and determine the distribution of the epoxidized structure in the daughter ion according to the cleavage mode; (53) Determine the carbon-carbon double bond position of the unsaturated lipid according to the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid, the distribution of the epoxidized structure in the daughter ion, the specific ions at the sn position, and the specific ions at the carbon-carbon double bond position.

3. The method for identifying the fine structure of multi-dimensional lipids fully derived from carbon-carbon double bonds according to claim 2, characterized in that In step (3), the daughter ion obtained by neutral loss of the head group from the sodium ion adduct of the epoxidation product contains the structure shown in formula (I), where R 1 is the fatty acid chain at the sn-1 position, and R 2 is the fatty chain at the sn-2 position. Formula (I); in step (4), the structure of formula (I) has a cleavage tendency in the third-order mass spectrometry analysis. During the collision-induced dissociation in the third-order mass spectrometry analysis, specific ions at the sn position are formed, and the specific ions at the sn position include one or both of the specific ions at the sn-1 position and the specific ions at the sn-2 position; The daughter ion forms specific ions at the carbon-carbon double bond position through the cleavage of the epoxidized structure, and the specific ions at the carbon-carbon double bond position contain the structure of formula (I).

4. The method for identifying the multi-dimensional lipid fine structure fully derived from carbon-carbon double bonds according to claim 3, characterized in that, When neither R 1 nor R 2 contains an epoxy structure, or when R 1 contains an epoxy structure while R 2 does not contain an epoxy structure, the cleavage tendency of the structure of formula (I) is as shown in formula (II), the dashed line indicates the cleavage site, the cleavage tends to occur at the 1,3-dioxolane structure, and the carbon-oxygen bonds connected at the 4th and 5th positions are cleaved to form specific ions indicating the sn-1 position, Formula (II); When the R 2 contains an epoxy structure while R 1 does not contain an epoxy structure, the cleavage tendency of the structure of formula (I) is as shown in formula (III), the dashed line indicates the cleavage site, and the cleavage tends to occur at the carbon-carbon double bond where the 1,3-dioxolane structure is connected through the 2-position, forming a specific ion indicating the sn-2 position. Formula (III); When both R 1 and R 2 contain an epoxy structure, the cleavage tendency of the structure of formula (I) is as shown in formula (IV), the dotted line indicates the cleavage site, the cleavage tends to occur on the 1,3-dioxolane structure, and the carbon-oxygen bonds connected at the 4th and 5th positions are cleaved, and the cleavage also tends to occur on the carbon-carbon double bond connected by the 1,3-dioxolane structure through the 2nd position, and at the same time, specific ions indicating the sn-1 position and specific ions indicating the sn-2 position are formed. Formula (IV).

5. The method for identifying the multi-dimensional lipid fine structure fully derived from carbon-carbon double bonds according to claim 2, characterized in that, In step (51), determining the sn position of the unsaturated lipid includes the following methods: (i) Comparing the ion mass of the specific ion at the sn position with the fragmentation characteristic ion mass data to determine whether the specific ion at the sn position is the specific ion at the sn-1 position or the specific ion at the sn-2 position; (ii) When a characteristic peak with a difference of 16 Da is formed near the specific ion at the sn position, the specific ion at the sn position is the specific ion at the sn-2 position; When no characteristic peak with a difference of 16 Da is formed near the specific ion at the sn position, the specific ion at the sn position is the specific ion at the sn-1 position.

6. The method for identifying the multi-dimensional lipid fine structure fully derived from carbon-carbon double bonds according to claim 5, characterized in that, In step (4), specific ions of fatty acid chains containing an epoxidized structure at the sn position are also obtained through triple mass spectrometry analysis. The specific ions of fatty acid chains containing an epoxidized structure at the sn position are one or both of the specific ions of fatty acid chains containing an epoxidized structure at the sn-1 position and the specific ions of fatty acid chains containing an epoxidized structure at the sn-2 position; In step (5), when the specific ion at the sn position is 40 Da more than the specific ion of the fatty acid chain containing an epoxidized structure at the sn position, the specific ion at the sn position is the specific ion at the sn-1 position, and the specific ion of the fatty acid chain containing an epoxidized structure at the sn position is the specific ion of the fatty acid chain containing an epoxidized structure at the sn-1 position; When the specific ion at the sn position is 30 Da less than the specific ion of the fatty acid chain containing an epoxidized structure at the sn position, the specific ion at the sn position is the specific ion at the sn-2 position, and the specific ion of the fatty acid chain containing an epoxidized structure at the sn position is the specific ion of the fatty acid chain containing an epoxidized structure at the sn-2 position.

7. The method for identifying the multi-dimensional lipid fine structure fully derived from carbon-carbon double bonds according to claim 6, characterized in that, It further includes the following steps: Perform secondary mass spectrometry analysis on the sodium ion adduct of the unepoxidized and derivatized unsaturated lipid, select the second daughter ion obtained after neutral loss of the head group of the sodium ion adduct of the unepoxidized and derivatized unsaturated lipid, perform tertiary mass spectrometry analysis on the second daughter ion obtained after neutral loss of the head group of the sodium ion adduct of the unsaturated lipid, obtain the specific ion of the fatty acid chain containing a carbon-carbon double bond at the sn position, compare it with the specific ion of the fatty acid chain containing an epoxidized structure at the sn position, determine the carbon number and the number of epoxidized structures of the specific ion corresponding to the fatty acid chain containing an epoxidized structure at the sn position, so as to determine the carbon number and the number of epoxidized structures of the specific ion corresponding to the sn position; The specific ion of the fatty acid chain containing a carbon-carbon double bond at the sn position includes one or both of the specific ion of the fatty acid chain containing a carbon-carbon double bond at the sn-1 position and the specific ion of the fatty acid chain containing a carbon-carbon double bond at the sn-2 position.

8. The multi-dimensional lipid fine structure identification method based on the full derivatization of carbon-carbon double bonds according to claim 1, characterized in that, In step (1), the in-situ epoxidation derivatization is carried out using potassium peroxymonosulfate and acetone for epoxidation derivatization; the in-situ epoxidation derivatization of the unsaturated lipid to be analyzed using dimethyldioxirane includes the following steps: Take 1 mmol / L of the unsaturated lipid into a container, add 50 μL of 500 mmol / L potassium peroxymonosulfate, then add 100 μL of acetone, and then add 50 μL of a sodium bicarbonate solution with a concentration of 500 mmol / L and mix; React at 60 °C for 30 minutes, add 300 μL of ethyl acetate for extraction, and dry with nitrogen to obtain the epoxidation product of the unsaturated lipid; During the reaction process, ultrasound is performed, and the ultrasound time is 10 minutes.

9. The multi-dimensional lipid fine structure identification method based on the full derivatization of carbon-carbon double bonds according to claim 1, characterized in that, The epoxidation product is subjected to tandem mass spectrometry analysis in the positive ion mode.

10. The multi-dimensional lipid fine structure identification method based on the full derivatization of carbon-carbon double bonds according to claim 1, characterized in that, The unsaturated lipid is one of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid; In step (3), the following steps are further included: Determine the lipid class of the unsaturated lipid to be analyzed according to the mass lost by neutral loss of the head group.

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