A multidimensional lipid fine structure identification method based on the full derivation of carbon-carbon double bonds

Abstract

This application relates to the field of mass spectrometry analysis technology, specifically a method for the multidimensional fine structure identification of lipids based on the full derivatization of carbon-carbon double bonds. The method includes: in-situ epoxidation derivatization of unsaturated lipids to obtain epoxidation products; mass spectrometry analysis of the sodium ion adduct of the unsaturated lipid / epoxidation product to determine the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid; secondary mass spectrometry analysis of the sodium ion adduct of the epoxidation product to obtain daughter ions after neutral loss of the head group; tertiary mass spectrometry analysis of the daughter ions to obtain specific ions at the sn position and carbon-carbon double bond positions; and analysis of the specific ions at the sn position and carbon-carbon double bond positions to determine the sn position and carbon-carbon double bond position of the unsaturated lipid based on the total number of carbon atoms and the degree of unsaturation. This method can simultaneously resolve the sn-linked positions and carbon-carbon double bond positions of fatty acid chains in unsaturated lipids, achieving multidimensional fine structure identification of lipids.

Description

Technical Field

[0001] This application relates to the field of mass spectrometry analysis technology, and mainly to a multidimensional lipid fine structure identification method based on the full derivation of carbon-carbon double bonds. Background Technology

[0002] Lipids play a series of important roles in vital physiological processes such as cell membrane structure formation, energy storage, and signal transduction. Various types of lipids collectively constitute the lipidome of the entire organism or a specific organ, and are finely regulated within the organism to maintain a homeostatic balance between lipid metabolism and synthesis. Increasing research indicates that changes in lipid types, content, and structure are closely related to the occurrence of cancer, diabetes, neurological diseases, and cardiovascular diseases. Therefore, lipidome analysis and lipid structure identification are attracting increasing attention, and the detailed 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 stems from the types and numbers of terminal groups and fatty acid chains. Furthermore, the stereospecific numbering (sn) positions of fatty acid chains, the number and position of carbon-carbon double bonds (C=C), and their cis / trans structures 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 (ESI) has become the preferred ionization technique for lipid analysis. Currently, new analytical techniques based on mass spectrometry are constantly emerging, greatly promoting the development of the field of lipidomics. Mass spectrometry analysis can provide rich structural information in a single analysis by measuring the mass-to-charge ratio of ions, while possessing high specificity and high sensitivity. For example, by combining positive and negative mode ionization with tandem mass spectrometry, it is now possible to determine lipid types and the total number of carbon atoms and degree of unsaturation of fatty acid chains. With further research, it has become possible to identify the sn position of fatty acid chains and the positions of their carbon-carbon double bonds within unsaturated lipids. However, most existing methods can only identify one of the sn or carbon-carbon double bond positions. Using a hybrid dissociation method, such as ozone-induced dissociation (OzID) and ultraviolet light dissociation (UVPD) combined with collision-induced dissociation (CID), can achieve the identification of sn linkages and carbon-carbon double bond positions, but this requires instruments equipped with UVPD functionality or corresponding modifications to the instrument to implement OzID. Therefore, establishing a simple and rapid mass spectrometry method for identifying the fine structure of lipids in multiple dimensions will be of great significance to lipidomics research.

[0004] There are currently two main methods for identifying the positions of sn and carbon-carbon double bonds. One method uses ion fragmentation, such as a hybrid dissociation method combining ozone-induced dissociation (OzID) with collision-induced dissociation (CID), or ultraviolet light dissociation (UVPD) with CID, which can identify the positions of sn and carbon-carbon double bonds. However, this method requires special modifications to the mass spectrometer, making it difficult to apply and promote in ordinary laboratories. The other method involves derivatizing the unsaturated carbon-carbon double bonds in lipids, such as using the Paterno-Büchi reaction. The derivatized lipids can be identified by combining collision-induced dissociation (CID) in a commercial mass spectrometer, thus enabling the identification of the positions of sn and carbon-carbon double bonds. However, the Paterno-Büchi reaction has the disadvantage of low derivatization efficiency, failing to convert all derivatizations into single derivatives. These methods either require complex instrument modifications or have cumbersome and inefficient identification steps, failing to achieve high-throughput structural analysis of the sn and carbon-carbon double bond positions in lipids.

[0005] Therefore, existing technologies still need improvement and development. Summary of the Invention

[0006] In view of the shortcomings 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 product of the unsaturated lipid to be analyzed is fragmented by tandem mass spectrometry, specific ions with high abundance at the sn- position and carbon-carbon double bond position 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:

[0008] This application provides a multidimensional lipid fine structure identification method based on the full derivation of carbon-carbon double bonds, applicable to unsaturated lipids containing a glycerol backbone and a head group, comprising the following steps:

[0009] (1) Dimethyldioxane was used to perform in-situ epoxidation derivatization on the unsaturated lipid to be analyzed, so that all carbon-carbon double bonds in the unsaturated lipid were epoxidized to form an epoxidized structure, and the epoxidized product of the unsaturated lipid was obtained.

[0010] (2) The epoxidation product is injected into a mass spectrometer. After ionization by an ion source, the sodium adduct of the epoxidation product is selected for primary mass spectrometry analysis to obtain the ion mass of the sodium adduct of the epoxidation product. The sodium adduct of unsaturated lipids that have not undergone epoxidation is analyzed by primary mass spectrometry and used as a comparison to determine the total number of carbon atoms and the degree of unsaturation of the unsaturated lipids.

[0011] (3) Perform secondary mass spectrometry analysis on the sodium ion adduct of the epoxidation product, and select the daughter ion obtained after neutral loss of the head group of the sodium ion adduct of the epoxidation product after collision-induced dissociation;

[0012] (4) The daughter ions obtained after the sodium ion adduct of the epoxidation product is neutrally lost from the head group are subjected to third-level mass spectrometry analysis. After collision-induced dissociation, specific ions at the sn position and specific ions at the carbon-carbon double bond position are obtained.

[0013] (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 carbon-carbon double bond position of the unsaturated lipid based on the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid.

[0014] The multidimensional lipid fine structure identification method based on the full derivatization of carbon-carbon double bonds provided in this application is used. After the full derivatization of carbon-carbon double bonds, the unsaturated lipid to be tested forms an epoxide structure in situ. The sodium ion adduct is selected for tandem mass spectrometry analysis. After the fragmentation and loss of the head group in the secondary mass spectrometry analysis, the inferred 1,3-dioxolane ring structure can be formed. Under the fracture tendency of the interaction with the epoxide structure, the characteristic peaks of the sn- position and the carbon-carbon double bond position can be obtained simultaneously after the fragmentation in the tertiary mass spectrometry analysis, which is conducive to realizing multidimensional lipid fine structure identification and analysis.

[0015] The multidimensional lipid fine structure identification method based on the full derivation of carbon-carbon double bonds, wherein step (5), determining the sn position and carbon-carbon double bond position of the unsaturated lipid specifically includes the following steps:

[0016] (51) Determine the sn position of the unsaturated lipid based on the specific ion at the sn position, and determine the type of specific ion at the sn position;

[0017] (52) Determine the fragmentation mode of the epoxidation product based on the specific ion type of the sn position, and determine the distribution of the epoxidation structure in the epoxidation product based on the fragmentation mode;

[0018] (53) The carbon-carbon double bond position of the unsaturated lipid is determined based on the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid, the distribution of the epoxide structure in the daughter ion, the specific ion at the sn position and the specific ion at the carbon-carbon double bond position.

[0019] The multidimensional lipid fine structure identification method based on carbon-carbon double bond full derivatization, wherein in step (3), the daughter ion obtained after the sodium ion adduct of the epoxidation product neutrally loses the head group contains the structure shown in formula (I), where R1 is the fatty acid chain at the sn-1 position and R2 is the fatty acid chain at the sn-2 position.

[0020] Formula (I);

[0021] In step (4), the structure of formula (I) has a tendency to break during the third-level mass spectrometry analysis. During the collision-induced dissociation of the third-level mass spectrometry analysis, a specific ion at the sn position is formed. The specific ion at the sn position includes one or both of the specific ion at the sn-1 position and the specific ion at the sn-2 position.

[0022] The daughter ion forms a specific ion at the carbon-carbon double bond position through the cleavage of the epoxide structure, and the specific ion at the carbon-carbon double bond position contains the structure of formula (I).

[0023] The multidimensional lipid fine structure identification method based on the full derivation of carbon-carbon double bonds, wherein when neither R1 nor R2 contains an epoxide structure, or when R1 contains an epoxide structure but R2 does not, the fracture tendency of the structure of formula (I) is as shown in formula (II), where the dashed line indicates the fracture site. The fracture tends to occur on the 1,3-dioxolane ring structure, and the carbon-oxygen bonds connected at positions 4 and 5 are broken to form a specific ion indicating the sn-1 position.

[0024] Formula (II);

[0025] When R2 contains an epoxide structure while R1 does not, the fracture tendency of the structure of formula (I) is as shown in formula (III), where the dashed line indicates the fracture site. The fracture tends to occur at the carbon-carbon double bond connected by the 1,3-dioxolane ring structure at position 2, forming a specific ion indicating the sn-2 position.

[0026] Formula (III);

[0027] When both R1 and R2 contain epoxide structures, the fracture tendency of the structure of formula (I) is as shown in formula (IV), where the dashed lines indicate fracture sites. Fracture tends to occur on the 1,3-dioxolane ring structure, and the carbon-oxygen bonds connected at positions 4 and 5 break. Fracture also tends to occur on the carbon-carbon double bond connected at position 2 of the 1,3-dioxolane ring structure, simultaneously forming specific ions indicating the sn-1 position and specific ions indicating the sn-2 position.

[0028] Formula (IV).

[0029] The multidimensional lipid fine structure identification method based on the full derivation of carbon-carbon double bonds, wherein step (51) determines the sn position of the unsaturated lipid, including the following methods:

[0030] (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.

[0031] (ii) When a characteristic peak with a phase 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;

[0032] When no characteristic peak with a phase 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.

[0033] The multidimensional lipid fine structure identification method based on carbon-carbon double bond full derivation, wherein in step (4), after three-level mass spectrometry analysis, a specific ion of fatty acid chain containing epoxidation structure at sn position is also obtained. The specific ion of fatty acid chain containing epoxidation structure at sn position is one or two of the specific ion of fatty acid chain containing epoxidation structure at sn-1 position and specific ion of fatty acid chain containing epoxidation structure at sn-2 position.

[0034] 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 the 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 the epoxidized structure at the sn position is the specific ion of the fatty acid chain containing the epoxidized structure at the sn-1 position.

[0035] When the specific ion at the sn position is 30 Da less than the specific ion of the fatty acid chain containing the 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 the epoxidized structure at the sn position is the specific ion of the fatty acid chain containing the epoxidized structure at the sn-2 position.

[0036] The method for identifying fine lipid structures based on the full derivatization of carbon-carbon double bonds, wherein in step (5), the sodium adduct of the unsaturated lipids that have not undergone epoxidation derivatization is subjected to secondary mass spectrometry analysis, and the second daughter ion obtained after the neutral loss of the head group of the sodium adduct of the unsaturated lipids that have not undergone epoxidation derivatization is selected. The second daughter ion obtained after the neutral loss of the head group of the sodium adduct of the unsaturated lipids is subjected to tertiary mass spectrometry analysis to obtain the specific ion of the fatty acid chain containing carbon-carbon double bonds at the sn position. It is compared with the specific ion of the fatty acid chain containing epoxidation structure at the sn position to determine the number of carbons and the number of epoxidation structures of the specific ion of the fatty acid chain containing epoxidation structure at the sn position, thereby determining the number of carbons and the number of epoxidation structures of the specific ion of the specific ion at the sn position.

[0037] 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 ions of the fatty acid chain containing a carbon-carbon double bond at the sn-1 position and the specific ions of the fatty acid chain containing a carbon-carbon double bond at the sn-2 position.

[0038] The multidimensional lipid fine structure identification method based on carbon-carbon double bond full derivatization, wherein in step (1), the in-situ epoxidation derivatization is performed using potassium peroxymonosulfonate and acetone; the in-situ epoxidation derivatization of the unsaturated lipid to be analyzed using dimethyldioxane includes the following steps:

[0039] Take 1 mmol / L of the unsaturated lipid into a container, add 50 μL of 500 mmol / L potassium peroxymonosulfonate, then add 100 μL of acetone, and then add 50 μL of 500 mmol / L sodium bicarbonate solution and mix.

[0040] The reaction was carried out at 60°C for 30 minutes, then extracted with 300 μL of ethyl acetate and dried under nitrogen to obtain the epoxidized product of the unsaturated lipid.

[0041] The reaction process involves ultrasound for 10 minutes.

[0042] The method for identifying the fine structure of lipids based on the full derivatization of carbon-carbon double bonds, wherein the epoxidation product is analyzed by tandem mass spectrometry in positive ion mode.

[0043] The multidimensional lipid fine structure identification method based on carbon-carbon double bond full derivatization, wherein the unsaturated lipid is one of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol and phosphatidic acid;

[0044] Step (3) also includes the following steps:

[0045] The lipid class of the unsaturated lipid to be analyzed is determined based on the mass of the lost neutral head group.

[0046] Beneficial Effects: The multidimensional lipid fine structure identification method based on the full derivatization of carbon-carbon double bonds provided in this application can simultaneously resolve the sn-linked positions of fatty acid chains and the carbon-carbon double bond positions in unsaturated lipids. This technical solution is simple and easy to implement, and can be performed on a commercial mass spectrometer with tertiary mass spectrometry capabilities without requiring modification to the instrument. The in-situ epoxidation derivatization method selected in this application has a fast reaction rate and high reaction yield, which is helpful for the qualitative and quantitative analysis of lipid mixtures. In the identification method provided in this application, the epoxidation product of the lipid to be analyzed can be fragmented to simultaneously obtain high abundance of sn- and carbon-carbon double bond position characteristic ions in a single mass spectrum, thereby achieving multidimensional fine structure identification of lipid sn- and carbon-carbon double bond position isomers. Attached Figure Description

[0047] Figure 1 This application provides a technical roadmap for a multidimensional lipid fine structure identification method based on the full derivation of carbon-carbon double bonds.

[0048] Figure 2 MS3 mass spectra of PC 16:0 / 18:1 (9Z) in Comparative Example 1 of this application were obtained.

[0049] Figure 3 MS3 mass spectra of PC 18:1(9Z) / 16:0 in Comparative Example 2 of this application were obtained.

[0050] Figure 4 The mass spectrum of PC 16:0 / 18:1 (9Z) in Example 1 of this application after epoxidation derivatization is obtained by MS3.

[0051] Figure 5 The mass spectrum of PC 18:1(9Z) / 16:0 in Example 2 of this application after epoxidation derivatization is obtained by MS3.

[0052] Figure 6 The mass spectrum of PC 16:0 / 18:2 (9Z, 12Z) in Example 3 of this application after epoxidation derivatization is obtained by MS3. Detailed Implementation

[0053] This application provides a multidimensional lipid fine structure identification method based on the full derivation of carbon-carbon double bonds. To make the purpose, technical solution, and effects of this application clearer and more explicit, the following provides a more detailed description. It should be understood that the specific embodiments described herein are merely illustrative of this application and are not intended to limit this application.

[0054] Figure 1The technical roadmap provided in this application for a multidimensional lipid fine structure identification method based on carbon-carbon double bond full derivatization is applicable to unsaturated glycerophospholipid lipids containing a glycerol backbone and a head group. First, the intact unsaturated lipid molecule to be analyzed undergoes carbon-carbon double bond full derivatization to obtain an epoxidized product. The sodium ion adduct of the epoxidized product undergoes collision-induced dissociation in secondary mass spectrometry, resulting in the neutral loss of the head group and the formation of a daughter ion. A 1,3-dioxolane ring structure is then formed in the daughter ion. Further fragmentation in tertiary mass spectrometry results in sn-position specific ions and carbon-carbon double bond position specific ions.

[0055] Specifically, this application provides a multidimensional lipid fine structure identification method based on the full derivation of carbon-carbon double bonds, applicable to unsaturated lipids containing a glycerol backbone and a head group, comprising the following steps:

[0056] (1) Dimethyldioxane was used to perform in-situ epoxidation derivatization on the unsaturated lipids to be analyzed, so that all carbon-carbon double bonds in the unsaturated lipids were epoxidized to form epoxidized structures, and epoxidized products of unsaturated lipids were obtained.

[0057] Furthermore, in-situ epoxidation derivatization is performed using potassium peroxide monosulfonate (oxone) and acetone, with potassium peroxide monosulfonate acting as the oxidant and acetone as the catalyst. During the epoxidation derivatization of the carbon-carbon double bond, potassium peroxide monosulfonate and acetone react to generate dimethyldioxane. The generated dimethyldioxane then undergoes in-situ epoxidation derivatization of the carbon-carbon double bond. The oxygen atom of dimethyldioxane attacks the carbon-carbon double bond in the unsaturated lipid, resulting in electrophilic addition and the formation of an intermediate. This intermediate then undergoes hydrogen migration, ultimately yielding an epoxide compound. The resulting byproduct, acetone, is then reused as a reactant to generate dimethyldioxane for further epoxidation derivatization.

[0058] Furthermore, in step (1), the unsaturated lipids to be analyzed are subjected to in-situ epoxidative derivatization using dimethyldioxane, including the following steps:

[0059] Take 1 mmol / L of unsaturated lipid into a container, add 50 μL of 500 mmol / L potassium peroxymonosulfonate, then add 100 μL of acetone, and then add 50 μL of 500 mmol / L sodium bicarbonate solution and mix.

[0060] The reaction was carried out at 60°C for 30 minutes, then extracted with 300 μL of ethyl acetate and dried under nitrogen to obtain the epoxidized product of unsaturated lipids.

[0061] The reaction process involves ultrasound, which lasts for 10 minutes.

[0062] The epoxidation derivatization reaction is also subjected to ultrasound, which accelerates the reaction and improves its efficiency. The sodium bicarbonate solution acts as a buffer during the epoxidation derivatization process, adjusting the pH and preventing ring-opening of the epoxidized product. This protects the epoxidized product and increases its yield.

[0063] This method has a high degree of oxidation and high oxidation efficiency, and can fully derivatize the carbon-carbon double bonds of the unsaturated lipids to be analyzed. It can also form easily broken and easily identifiable epoxidized structures on the carbon-carbon double bonds in situ, which makes it faster to determine the position of the carbon-carbon double bonds in subsequent mass spectrometry analysis.

[0064] (2) The epoxidation product was injected into the mass spectrometer. After ionization by the ion source, the sodium adduct of the epoxidation product was selected for primary mass spectrometry analysis to obtain the ion mass of the sodium adduct of the epoxidation product. The sodium adduct of the unsaturated lipids that had not undergone epoxidation was analyzed by primary mass spectrometry and used as a comparison to determine the total number of carbon atoms and the degree of unsaturation of the unsaturated lipids.

[0065] The degree of unsaturation refers to the degree of unsaturation of the carbon-carbon double bond.

[0066] Furthermore, in this application, the analysis is based on the sodium ion adduct of the epoxide product; therefore, the epoxide product is analyzed by tandem mass spectrometry in positive ion mode. The sodium source can be derived from the atmosphere, and the sodium ion adduct [M+Na] of the epoxide product can be obtained by ionizing the epoxide product sample within the mass spectrometry ion source. + Primary mass spectrometry analysis of the sodium adduct of the epoxidation product yields its ionic mass. Comparison with the ionic mass of the unepoxidized unsaturated lipid not only confirms the number of carbon atoms in the unsaturated lipid but also confirms the occurrence of epoxidation. Each carbon-carbon double bond forms an epoxidized structure after epoxidation, binding an oxygen atom with a mass of 16 Da (Da is one-twelfth the mass of a carbon atom). By comparing the lipid masses before and after epoxidation, the degree of unsaturation of the carbon-carbon double bonds in the unsaturated lipid can be determined. This allows for the determination of the structural information of the unsaturated lipid through the ionic mass of specific ions in subsequent tandem mass spectrometry analysis.

[0067] (3) Secondary mass spectrometry analysis was performed on the sodium ion adduct of the epoxidation product. After collision-induced dissociation, the daughter ion obtained by neutral loss of head group of the sodium ion adduct of the epoxidation product was selected.

[0068] In this application, selecting the sodium adduct of the epoxidation product for collision-induced dissociation can change the lipid fragmentation pathway, and mass spectrometry analysis can obtain better mass spectrometry results. In this step (3), the sodium adduct of the epoxidation product is used as the parent ion for collision-induced dissociation (CID) in secondary mass spectrometry analysis. Sodium ions can not only increase the abundance of fragmented ions in the mass spectrum, but also cause neutral loss of the head group of glycerophospholipids (neutral loss specifically refers to the loss of the head group part which is not positively or negatively charged, but neutral, 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 lipids when they are subjected to collision-induced dissociation in tertiary mass spectrometry analysis.

[0069] Furthermore, the daughter ion obtained after the sodium ion adduct of the epoxidation product in step (3) neutrally loses its head group contains the structure shown in formula (I), where R1 is a fatty acid chain at the sn-1 position and R2 is a fatty acid chain at the sn-2 position.

[0070] Formula (I),

[0071] In the daughter ion, the 1,3-dioxolane ring structure is connected to the fatty acid chain at position sn-1 via a single bond at position 4, and to the remaining fatty acid chain at position sn-2 via a carbon-carbon double bond at position 2.

[0072] Glycerophospholipids are mainly linked to the head group and two fatty acid chains through the glycerol backbone. The head group is mainly linked to different substituents through phosphate groups to form glycerophospholipids of different head group types. Therefore, step (3) may also include the following steps: the lipid class of the unsaturated lipid to be analyzed can be determined based on the mass of the neutral loss head group.

[0073] In this application, the inventors hypothesize that during collision-induced dissociation (CID) in secondary mass spectrometry analysis, the sodium ion adduct of the epoxide product undergoes a change in its glycerol backbone after neutral loss of the head group, and after flipping and recombination, forms a 1,3-dioxolane ring structure as shown in formula (I), as illustrated in the figure below:

[0074]

[0075] Where R represents the substituent portion of the head group of glycerophospholipid connected by a phosphate group, R' represents the fatty acid chain portion remaining from the carbonyl carbon to the end of the fatty acid chain at the sn-1 position, and R” represents the fatty acid chain portion remaining from the carbonyl carbon to the end of the fatty acid chain at the sn-2 position. The dashed line marks the glycerol skeleton, and the values ​​“1, 2, 3” represent the sn- positions on the glycerol skeleton.

[0076] The inventors hypothesize that under the influence of sodium ion addition, during the recombination process, the carbon-oxygen double bond of the carbonyl group in the fatty acid chain at the sn-2 position breaks, transforming from a carbonyl group into a carbon-carbon double bond connected to the remaining fatty acid chain. It is hypothesized that the fatty acid chain at the sn-2 position loses a hydrogen atom (-1 Da), which combines with the head group and undergoes a neutral loss. After the fatty acid chain at the sn-2 position flips, the oxygen on the original carbonyl group connects to the carbon on the glycerol backbone that was originally connected to the head group, forming a 1,3-dioxolane ring structure as shown in formula (I). In the structure shown in formula (I), position 4 of the 1,3-dioxolane ring structure is the carbon atom on the original glycerol backbone that connects to the fatty acid chain at the sn-2 position. Therefore, the 1,3-dioxolane ring structure shown in formula (I) is connected to the fatty acid chain at the sn-1 position via a carbon single bond on the glycerol backbone. Meanwhile, the carbonyl carbon on the fatty acid chain at position 2 of the 1,3-dioxolane structure is the original sn-2 position. Therefore, the 1,3-dioxolane structure shown in formula (I) is connected to the remaining fatty acid chain at position sn-2 through a carbon-carbon double bond.

[0077] In a specific embodiment of this application, taking phosphatidylcholine standard PC 16:0 / 18:1 (9Z) as an example, the ionic mass of the sodium ion adduct of the underrived phosphatidylcholine standard PC 16:0 / 18:1 (9Z) is (m / z 782). In the CID of secondary mass spectrometry analysis, a neutral loss of the phosphatidylcholine head group (-183 Da) occurs, resulting in a daughter ion (m / z 599). This daughter ion (m / z 599) contains a structure as shown in formula (I) with a 1,3-dioxolane ring connected to a carbon-carbon double bond at position 2. The conversion process is shown in the figure below:

[0078]

[0079] In a specific embodiment of this application, taking phosphatidylcholine standard PC 16:0 / 18:1 (9Z) as an example, after epoxidation derivatization using the method provided in this 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 phosphatidylcholine head group (-183 Da) in the CID of secondary mass spectrometry analysis, resulting in a daughter ion (m / z 615). This daughter ion (m / z 615) contains a structure as shown in formula (I) with a 1,3-dioxolane ring connected to a carbon-carbon double bond at position 2, wherein the conversion process is shown in the figure below:

[0080]

[0081] (4) The daughter ions obtained after the sodium ion adduct of the epoxidation product loses the head group in a neutral state are analyzed by a third-order mass spectrometry. After collision-induced dissociation, specific ions at the sn position and specific ions at the carbon-carbon double bond position are obtained.

[0082] In this application, mass spectrometry analysis of the daughter ions obtained after the neutral loss of the head group in the sodium ion adduct of the epoxidation product can simultaneously obtain high-abundance specific ions at the sn position and specific ions at the carbon-carbon double bond position.

[0083] Furthermore, in this application, after multiple experimental investigations, it was found that the daughter ion, due to the structure shown in formula (I), has R1 as a fatty acid chain at the sn-1 position and R2 as a fatty acid chain at the sn-2 position.

[0084] Formula (I),

[0085] In step (4), the structure of formula (I) has a tendency to break during the tertiary mass spectrometry analysis. During the collision-induced dissociation of the tertiary mass spectrometry analysis, a specific ion at the sn position is formed. The specific ion at the sn position includes one or both of the specific ion at the sn-1 position and the specific ion at the sn-2 position.

[0086] When neither R1 (sn-1 position fatty acid chain) nor R2 (sn-2 position fatty acid chain) on either side of the daughter ion to be analyzed contains an epoxidized structure, or when R1 contains an epoxidized structure but R2 does not, the fracture tendency of the structure in formula (I) is as shown in formula (II), with the dashed line indicating the fracture site.

[0087] Equation (II),

[0088] The breakage tends to occur on the 1,3-dioxolane ring structure, and the carbon-oxygen bonds connected at positions 4 and 5 are broken, forming a highly abundant sn-1 position specific ion.

[0089] When R2 contains an epoxide structure while R1 does not, the fracture tendency of the structure in formula (I) is as shown in formula (III), with the dashed line indicating the fracture site.

[0090] Formula (III)

[0091] The breakage tends to occur at the carbon-carbon double bond connected at position 2 in the 1,3-dioxolane structure, forming a highly abundant sn-2 position specific ion;

[0092] When both R1 and R2 contain epoxide structures, the fracture tendency of the structure in formula (I) is shown in formula (IV), with the dashed line indicating the fracture site.

[0093] Formula (IV),

[0094] The breakage tends to occur on the 1,3-dioxolane structure, and the carbon-oxygen bonds connected at positions 4 and 5 break. The breakage also tends to occur on the carbon-carbon double bonds connected at position 2 of the 1,3-dioxolane structure. At this time, a high abundance of specific ions at the sn-1 position and specific ions at the sn-2 position can be formed simultaneously, which has little impact on the sn-position specific ion characterization of unsaturated lipids.

[0095] Furthermore, in step (4), the daughter ions break down the epoxidation structure to form specific ions at the carbon-carbon double bond positions, which include the structure of formula (I). The 1,3-dioxolane structure formed by the daughter ions can alter the lipid fragmentation pathway, giving the epoxidation product a better tendency to fragment. Under the mutual influence of the epoxidation structures, during collision-induced dissociation in tertiary mass spectrometry, some daughter ions will break down the 1,3-dioxolane structure to form high-abundance specific ions at the sn position, while other daughter ions will break down the epoxidation structure to form specific ions at the carbon-carbon double bond positions containing the structure of formula (I). At this time, different fragmentation channels of the ions will occur simultaneously, and the resulting specific ions at the sn position and the specific ions at the carbon-carbon double bond positions can be characterized simultaneously in the mass spectrum of tertiary mass spectrometry, which can effectively improve the identification efficiency of the lipid fine structure.

[0096] In a specific embodiment of this application, taking unsaturated phosphatidylcholine lipids in glycerophospholipids as an example, after carbon-carbon double bond derivatization using the epoxidation derivatization method provided in this application, the epoxidation product undergoes neutral loss (-183 Da) of the phosphatidylcholine head group during secondary mass spectrometry (CID) after sodium ion addition. The daughter ion after neutral head group loss is selected for tertiary mass spectrometry analysis. After collision-induced dissociation, both high-abundance specific ions at the sn position and specific ions at the carbon-carbon double bond position are formed simultaneously.

[0097] (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 carbon-carbon double bond position of the unsaturated lipid based on the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid.

[0098] In this application, when identifying and analyzing unsaturated lipids with a degree of unsaturation not equal to 0, the resulting daughter ions contain the formed 1,3-dioxolane structure, and the fragmentation channels of the 1,3-dioxolane structure and the epoxide structure occur simultaneously. After collision-induced dissociation in tertiary mass spectrometry, the specific ion at the sn position obtained will contain the epoxide structure, and the specific ion at the carbon-carbon double bond position will contain the 1,3-dioxolane structure. Therefore, directly judging based on the specific ion at the carbon-carbon double bond position can only obtain the distance between the carbon-carbon double bond position and the end of the fatty acid chain, and cannot obtain the accurate position information of the carbon-carbon double bond. Therefore, it is necessary to first determine the sn position of the unsaturated lipid. Further, in step (5), determining the sn position and carbon-carbon double bond position of the unsaturated lipid specifically includes the following steps:

[0099] (51) Determine the sn position of unsaturated lipids based on the specific ion at the sn position, and determine the type of specific ion at the sn position;

[0100] (52) Determine the fragmentation mode of the epoxidation product based on the specific ion type of the sn position, and determine the distribution of the epoxidation structure in the fragmentation ion based on the fragmentation mode.

[0101] (53) The carbon-carbon double bond position of unsaturated lipids is determined based on the total number of carbon atoms and degree of unsaturation of unsaturated lipids, the distribution of epoxide structure in daughter ions, the specific ions at the sn position and the specific ions at the carbon-carbon double bond position.

[0102] In this application, the specific ion indicator sn-1 at the sn position indicates that the specific ion at the sn position is the specific ion at the sn-1 position; the specific ion indicator sn-2 at the sn position indicates that the specific ion at the sn position is the specific ion at the sn-2 position.

[0103] In this application, given the established influence of the mutual fracture tendency between the 1,3-dioxolane structure and the epoxide structure, the identification process first requires confirming the type of the highly abundant sn-position specific ion obtained in step (4), specifically whether it indicates the sn-1 or sn-2 position, or whether both sn-1 and sn-2 specific ions were obtained simultaneously. The fracture mode of the daughter ion can be determined based on the sn-position specific ion, and then, based on the fracture mode, it can be further confirmed whether R1 or R2 in the daughter ion contains an epoxide structure. After determining the distribution of the epoxide structure, and given a clear understanding of the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid, the refined structure of the unsaturated lipid can be confirmed based on the ionic mass of the sn-position specific ion and the carbon-carbon double bond specific ion.

[0104] Furthermore, in step (51), determining the sn position of the unsaturated lipid requires first determining whether the specific ion at the obtained sn position is a specific ion at the sn-1 position or a specific ion at the sn-2 position. Specifically, this includes the following methods:

[0105] (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.

[0106] (ii) When a characteristic peak differing by 16 Da is formed near the specific ion at position sn, the specific ion at position sn is the specific ion at position sn-2; when no characteristic peak differing by 16 Da is formed near the specific ion at position sn, the specific ion at position sn is the specific ion at position sn-1. In this application, there is a more intuitive method to determine whether the specific ion at position sn indicates position sn-1 or sn-2. Using the identification method provided in this application, in the CID spectrum of the three-stage mass spectrometry analysis, it was also found that when the specific ion at position sn-2 was characterized, a characteristic peak differing by 16 Da was formed to the left of the specific ion at position sn-2, while the specific ion at position sn-1 did not have such a peak. The inventors hypothesize that in CID (Continuous Ionization) mass spectrometry analysis, when the aliphatic chain at the sn-2 position contains an epoxide structure, due to the tendency for the epoxide structure and the 1,3-dioxolane structure to break, the carbon-carbon double bond connecting the 1,3-dioxolane structure and the aliphatic chain at the sn-2 position breaks. The resulting fragmented ion (R2 aliphatic chain) recombines with an oxygen atom at the break in the carbon-carbon double bond, reforming an aldehyde group and serving as the specific ion for the sn-2 position for characterization. A small portion of the fragmented ions are directly characterized. Therefore, in the CID spectrum of tertiary 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 an oxygen atom is added) nearby. In summary, by analyzing the characterization behavior of the specific ion at the sn position in the spectrum, the sn position of unsaturated lipids can be determined more quickly. Furthermore, since the degree of unsaturation of the unsaturated lipid to be analyzed has been clarified through comparison in the previous steps, and the number of carbons in the specific ion at the sn-2 position is generally less than the number of carbons in the specific ion at the carbon-carbon double bond position, the characteristic peaks formed are also different. Therefore, the additional characteristic peaks with a difference of 16 Da formed here will not cause confusion in the subsequent determination of the carbon-carbon double bond position.

[0107] Furthermore, in step (4), after three-stage mass spectrometry analysis, specific ions of fatty acid chains containing epoxidized structures at the sn position were also obtained. The specific ions of fatty acid chains containing epoxidized structures at the sn position are one or both of the specific ions of fatty acid chains containing epoxidized structures at the sn-1 position and the specific ions of fatty acid chains containing epoxidized structures 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 fatty acid chains containing epoxidized structures 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 fatty acid chains containing epoxidized structures at the sn position is the specific ion of fatty acid chains containing epoxidized structures at the sn-1 position. When the specific ion at the sn position is 30 Da less than the specific ion of fatty acid chains containing epoxidized structures 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 fatty acid chains containing epoxidized structures at the sn position is the specific ion of fatty acid chains containing epoxidized structures at the sn-2 position.

[0108] In this application, when the degree of unsaturation of the unsaturated lipid to be analyzed is 1, after epoxidation derivatization, 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 is neutrally lost by secondary mass spectrometry, the resulting daughter ion is found to have other high abundance specific ions in the CID spectrum of tertiary mass spectrometry. After comparison and calculation, it was found that the ion mass of the high abundance specific ion, after subtracting 1 Da, is exactly equal to the fragmented ion mass of the carbon-oxygen single bond connecting the fatty acid chain containing the epoxidized structure at the sn position to the glycerol backbone. Therefore, the obtained specific ion is the specific ion of the fatty acid chain containing the epoxidized structure at the sn position. By comparing the unsaturated lipids to be analyzed that had not undergone epoxidation derivatization, and following the same steps and operations, other specific ions with a certain abundance were also found in the CID spectrum of the tertiary mass spectrometry analysis. After comparison and calculation, it was found that the ionic mass of this specific ion, after subtracting 1 Da, was exactly equal to the fragmented ion mass of the fatty acid chain containing a carbon-carbon double bond at the sn position, which is broken from the carbon-oxygen single bond on the glycerol backbone. Therefore, the specific ion obtained is the specific ion of the fatty acid chain containing a carbon-carbon double bond at the sn position.

[0109] In this 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 derivatization (this can be determined by judging the number of specific ions at the sn position; under the influence of the epoxidation structure on the breakage of the 1,3-dioxolane structure, the daughter ions can simultaneously obtain specific ions at the sn-1 position and specific ions at the sn-2 position through third-stage mass spectrometry analysis), after the sodium ion adduct of the epoxidation product of the unsaturated lipid to be analyzed loses its head group in neutrality through second-stage mass spectrometry analysis, the daughter ions obtained in the CID spectrum of the third-stage mass spectrometry analysis also show two other high-abundance specific ions. After comparison and calculation, it is found that the ion mass of these high-abundance specific ions, after subtracting 1 Da from each, is exactly equal to the fragmented ion mass of the carbon-oxygen single bond connecting the fatty acid chain containing the epoxidized structure at the sn-1 position and the sn-2 position to the glycerol backbone. Therefore, the obtained specific ions are the specific ions of the fatty acid chain containing the epoxidized structure at the sn position. By comparing the unsaturated lipids to be analyzed without epoxidation derivatization, and following the same steps and operations, two specific ions with a certain abundance were also found in the CID spectrum of the tertiary mass spectrometry analysis. After comparison and calculation, it was found that the ion mass of the two specific ions, after subtracting 1 Da, was exactly equal to the fragmented ion mass of the fatty acid chain containing carbon-carbon double bonds at the sn-1 and sn-2 positions, respectively, which was broken from the carbon-oxygen single bond on the glycerol backbone. Therefore, the specific ions obtained are the specific ions of the fatty acid chain containing carbon-carbon double bonds at the sn position.

[0110] The inventors also discovered that the mass difference between the specific ion indicating a fatty acid chain containing a carbon-carbon double bond at the sn position and the fragment ion of the specific ion indicating a fatty acid chain containing an epoxide structure at the sn position obtained previously is exactly n*16 Da (where n represents the degree of unsaturation and 16 Da represents the combination of an oxygen atom after the carbon-carbon double bond epoxide derivatization). This indicates that, after the identification steps of this application, regardless of whether epoxide derivatization occurs, fatty acid chains at the sn position containing carbon-carbon double bonds or epoxide structures have a certain tendency to break and can be characterized by breakage in tertiary mass spectrometry analysis. In the future, the number of carbons and the number of epoxide structures in the specific ion of the fatty acid chain containing an epoxide structure at the sn position can also be confirmed by comparison.

[0111] Regarding the specific ions of fatty acid chains with epoxide structures at the sn position and the specific ions of fatty acid chains with carbon-carbon double bonds at the sn position, the inventors speculate that after the head group is neutrally lost in the secondary mass spectrometry analysis, not all of the resulting daughter ions form a 1,3-dioxolane ring structure. Some daughter ions still retain the chain structure after the neutral loss of the head group. In the tertiary mass spectrometry analysis affected by the breakage of sodium ion adsorption, fatty acid chains with carbon-carbon double bonds or fatty acid chains with epoxide structures have a higher tendency to break, thus forming specific ions of fatty acid chains with carbon-carbon double bonds at the sn position or specific ions of fatty acid chains with epoxide structures at the sn position.

[0112] The fatty acid chains containing carbon-carbon double bonds or containing epoxidized structures are characterized by adding 1 Da to sodium ions after sodium ion addition, and then being used as specific ions for fatty acid chains containing carbon-carbon double bonds or containing epoxidized structures at the sn position. The inventors speculate that this is because the breakage site is located on the carbon-oxygen single bond connecting the fatty acid chain to the glycerol backbone. After the fatty acid chain containing carbon-carbon double bonds or epoxidized structures breaks, the oxygen at the end of the chain is newly combined with a hydrogen (+1 Da), and is characterized as a highly abundant specific ion.

[0113] When both specific ions at the sn position and specific ions containing an epoxide structure at the sn position are obtained simultaneously, the ionic masses of the specific ions at the sn position and the specific ions containing an epoxide structure at the sn position are correlated due to the breaking effect caused by the interaction between the epoxide structure and the formed formula (I) 1,3-dioxolane structure. The difference between their ionic masses can be used to determine whether the specific ion pair is related to the sn-1 or sn-2 position. This determination method also applies when only specific ions at the sn-1 or sn-2 position are obtained.

[0114] As previously speculated, in the ion fragments analyzed by tertiary mass spectrometry, the specific ion at the sn position is formed by the breakage of a portion of the ion containing the 1,3-dioxolane structure of formula (I); the specific ion of the fatty acid chain containing the epoxidation structure at the sn position is formed by the breakage of a portion of the ion that retains the chain structure (without forming the 1,3-dioxolane structure). In the CID spectrum of tertiary mass spectrometry, both the specific ion at the sn position and the specific ion of the fatty acid chain containing the epoxidation structure at the sn position are obtained with high abundance.

[0115] Under sodium ion addition conditions, and given a defined cleavage pattern, when the cleavage occurs at the 1,3-dioxolane structure of formula (I) in the R1 (sn-1 position fatty acid chain) of the daughter ion, the carbon-oxygen bonds at positions 4 and 5 are broken. Therefore, the specific ion at the sn-1 position has three more carbons and five more hydrogens (+12Da+12Da+12Da+5Da =+41Da) than the fatty acid chain containing the epoxide structure at the sn-1 position, as shown in the figure below. (The values ​​"1, 2, 3" represent the sn-positions on the glycerol backbone, and the dashed lines indicate the carbon chain portion of the glycerol backbone.) Furthermore, because the fatty acid chain containing the epoxide structure at the sn-1 position is characterized by binding a hydrogen atom (1 Da), it is used as a specific ion for characterizing this fatty acid chain. Therefore, in the CID spectrum obtained from the tertiary mass spectrometry analysis, the actual specific ion at the sn-1 position is 40 Da more than the specific ion of the fatty acid chain containing the epoxide structure at the sn-1 position (+41 Da - 1 Da = +40 Da, and indeed, a 40 Da difference in the specific ion pair at the sn-1 position was obtained in the actual spectrum). This corresponds to the investigated breaking and binding patterns, and this 40 Da difference can be used to reversely determine the specific ion pair at the sn-1 position.

[0116] Similarly, under sodium ion addition conditions, and given a defined cleavage pattern, when the epoxide structure is present in the R2 (sn-2 position aliphatic chain) of the daughter ion, the cleavage occurs at the carbon-carbon double bond connected at position 2 in the 1,3-dioxolane structure of formula (I). Therefore, the specific ion at the sn-2 position has one less carbon and two less oxygen atoms (-12 Da -16 Da -16 Da = -44 Da) than the aliphatic chain containing the epoxide structure at the sn-2 position, as shown in the figure below, to form the 1,3-dioxolane structure. (The dashed line indicates the portion of the fatty acid chain at the sn-2 position used to form the 1,3-dioxolane structure.) Furthermore, during the formation of the 1,3-dioxolane structure, the fatty acid chain at the sn-2 position loses a hydrogen atom (-1 Da) when it forms a carbon-carbon double bond from a carbon-oxygen double bond. In addition, as previously explored, during tertiary mass spectrometry analysis, the fragmented ion (R2 fatty chain) formed combines with an oxygen atom at the carbon-carbon double bond breakage site to form an aldehyde group (+16 Da), which is then characterized as a specific ion at the sn-2 position. Since the fatty acid chain containing the epoxide structure at the sn-2 position is characterized by binding a hydrogen atom (1 Da), it is used as a specific ion for characterizing the fatty acid chain containing the epoxide structure at the sn-2 position. Therefore, in the CID spectrum of the third-order mass spectrometry analysis, the specific ion obtained at the sn-2 position is actually 30 Da less than the specific ion of the fatty acid chain containing the epoxide structure at the sn-2 position (-44 Da - 1 Da + 16 Da - 1 Da = -30 Da, and the specific ion pair at the sn-2 position with a difference of 30 Da was indeed obtained in the actual spectrum). This corresponds to the broken and bound patterns found in the investigation. Moreover, the specific ion pair at the sn-2 position can be determined by reverse analysis using this 30 Da difference feature.

[0117] Although unoxidized unsaturated lipids can form specific ions containing carbon-carbon double bonds in fatty acid chains at the sn position during collision-induced dissociation in tertiary mass spectrometry, the lack of epoxidation to form an epoxidized structure does not affect the breakage tendency of the formed 1,3-dioxolane ring structure in tertiary mass spectrometry. Therefore, the sn position of unoxidized unsaturated lipids cannot be determined by this method.

[0118] Under specific breakage and bonding rules, specific ion pairs at the sn-1 position or sn-2 position can be obtained even when only R1 or R2 contains epoxide structures. When both R1 and R2 contain epoxide structures and multiple specific ions are formed in the CID spectrum of the tertiary mass spectrometry analysis, the specific ion pairs at the sn-1 position or sn-2 position can be determined by the difference between the specific ions related to the sn position.

[0119] In a specific embodiment of this application, taking the standard phosphatidylcholine PC 16:0 / 18:1(9Z) as an example, after epoxidation derivatization, the sodium ion adduct (m / z 798) of the epoxidation product of PC 16:0 / 18:1(9Z) was subjected to secondary mass spectrometry analysis (CID). After neutral loss of the head group, a daughter ion (m / z 615) was obtained. Then, the daughter ion (m / z 615) was subjected to tertiary mass spectrometry analysis (CID). High abundance of specific ions (m / z 291) and (m / z 321) were obtained in the spectrum. The specific ion (m / z 291) is a specific ion at the sn-2 position, and the specific ion (m / z 321) is a specific ion of a fatty acid chain containing an epoxidized structure at the sn-2 position. The two specific ions are exactly 30 Da apart.

[0120] In a specific embodiment of this application, taking the standard phosphatidylcholine PC 18:1(9Z) / 16:0 as an example, after epoxidation derivatization, the sodium ion adduct (m / z 798) of the epoxidation product of PC 18:1(9Z) / 16:0 was subjected to secondary mass spectrometry analysis (CID). After neutral loss of the head group, a daughter ion (m / z 615) was obtained. Then, the daughter ion (m / z 615) was subjected to tertiary mass spectrometry analysis (CID). High abundance of specific ions (m / z 321) and (m / z 361) were obtained in the spectrum. The specific ion (m / z 361) is a specific ion at the sn-1 position, and the specific ion (m / z 321) is a specific ion of the fatty acid chain containing the epoxidation structure at the sn-1 position. The difference between them is exactly 40 Da.

[0121] In summary, in step (51), the specific ion at the sn position obtained after epoxidation derivatization can be determined as either the specific ion at the sn-1 position or the specific ion at the sn-2 position, thereby determining the sn position of the unsaturated lipid. Furthermore, based on the characterization of the specific ion at the sn position, three characterization types can be obtained:

[0122] 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.

[0123] (52) Determine the fragmentation mode of the epoxide product's daughter ions based on the specific ion type at the sn position, and determine the distribution of the epoxide structure in the daughter ions based on the fragmentation mode. In step (52), given that the fragmentation tendency has been investigated, and the number and type of specific ions at the sn position have been determined in step (51), the fragmentation mode of the daughter ions can be known. The distribution of the epoxide structure in the daughter ions is determined based on the fragmentation mode, which includes the following distribution types:

[0124] Type 1: Only specific ions at the sn-1 position are obtained. This indicates that in step (4), the daughter ion underwent the cleavage pattern shown in formula (II). The dashed line represents the cleavage site, R1 is the fatty acid chain at the sn-1 position, and R2 is the fatty acid chain at the sn-2 position.

[0125] Equation (II),

[0126] The breakage occurs on the 1,3-dioxolane ring structure, and the carbon-oxygen bonds connected at positions 4 and 5 are broken; this breakage mode indicates that there are two distributions of the epoxide structure in the daughter ion: (A) neither R1 nor R2 contains an epoxide structure; (B) R1 contains an epoxide structure while R2 does not contain an epoxide structure.

[0127] Type 2: Only specific ions at the sn-2 position are obtained. This indicates that in step (4), the daughter ion underwent the breakage pattern shown in formula (III). The dashed line represents the breakage site, R1 is the fatty acid chain at the sn-1 position, and R2 is the fatty acid chain at the sn-2 position.

[0128] Formula (III)

[0129] The breakage occurs at the carbon-carbon double bond connected at position 2 in the 1,3-dioxolane structure; this breakage mode indicates that R2 in the daughter ion contains an epoxide structure, while R1 does not contain an epoxide structure.

[0130] Type 3: When both specific ions at the sn-1 position and specific ions at the sn-2 position are obtained simultaneously. This indicates that in step (4), the daughter ion underwent a breakage pattern as shown in formula (IV). The dashed line represents the breakage site, R1 is the fatty acid chain at the sn-1 position, and R2 is the fatty acid chain at the sn-2 position.

[0131] Formula (IV), The breakage occurs on the 1,3-dioxolane ring structure, and the carbon-oxygen bonds connected at positions 4 and 5 are broken. The breakage also occurs simultaneously on the carbon-carbon double bond connected at position 2 in the 1,3-dioxolane ring structure. This breakage mode indicates that both R1 and R2 in the daughter ion contain epoxide structures.

[0132] Step (53) determines the carbon-carbon double bond position of the unsaturated lipid based on the total number of carbon atoms and degree of unsaturation of the unsaturated lipid, the distribution of the epoxidized structure in the daughter ion, the specific ion at the sn position, and the specific ion at the carbon-carbon double bond position. The daughter ion obtained after neutral head group loss contains the structure shown in formula (I), where R1 is the fatty acid chain at the sn-1 position, and R2 is the fatty acid chain at the sn-2 position.

[0133] Formula (I),

[0134] In the daughter ion, the 1,3-dioxolane ring structure is connected to the fatty acid chain at position sn-1 via a single bond at position 4, and to the remaining fatty acid chain at position sn-2 via a carbon-carbon double bond at position 2.

[0135] Specifically, step (53) includes the following:

[0136] (531) For type 1, only the specific ion at the sn-1 position is obtained, and the ion is distinguished according to the degree of unsaturation of the unsaturated lipid obtained in step (2):

[0137] (a) When the degree of unsaturation of the lipid is 0, neither R1 nor R2 of the daughter ion contains an epoxide structure, nor does it contain a specific ion that forms a carbon-carbon double bond position.

[0138] (b) When the degree of unsaturation of the unsaturated lipid is not 0, the R1 of the daughter ion contains an epoxidation structure while the R2 does not contain an epoxidation structure. The carbon-carbon double bond position of the unsaturated lipid is determined based on the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid, the specific ion at the sn-1 position and the specific ion at the carbon-carbon double bond position.

[0139] (532) For type 2, only the specific ion at the sn-2 position is obtained. The R2 of the daughter ion contains an epoxidation structure, while the R1 does not contain an epoxidation structure. The carbon-carbon double bond position of the unsaturated lipid is determined based on the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid, the specific ion at the sn-2 position, and the specific ion at the carbon-carbon double bond position.

[0140] In (531) and (532), the number of epoxide structures and the number of carbons contained in the specific ion at the sn position can be determined based on the degree of unsaturation of the unsaturated lipid and the ionic mass of the specific ion at the sn position. Based on the total number of carbon atoms in the unsaturated lipid, the number of carbons in the specific ion at the sn position, and the specific ion of the carbon-carbon double bond, the number of carbons, composition, and position of the carbon-carbon double bond of the fatty acid chain at the sn position can be determined. Accordingly, after obtaining the information of the fatty acid chain at the sn position on one side, the information of the fatty acid chain on the other side can also be derived, thereby obtaining the refined structure of the unsaturated lipid.

[0141] (533) For type 3, both the sn-1 position specific ion and the sn-2 position specific ion are obtained. The R1 and R2 of the daughter ion contain epoxide structures. The carbon-carbon double bond position of the unsaturated lipid is determined based on the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid, the sn-1 position specific ion ...

[0142] Furthermore, in step (5) of this application, secondary mass spectrometry analysis can be performed on the sodium adduct of unsaturated lipids that have not undergone epoxidation derivatization. The second daughter ion obtained after the neutral loss of the head group of the sodium adduct of unsaturated lipids that have not undergone epoxidation derivatization is selected. Tertiary mass spectrometry analysis is performed on the second daughter ion obtained after the neutral loss of the head group of the sodium adduct of unsaturated lipids to obtain the specific ion of the fatty acid chain containing a carbon-carbon double bond at the sn position. This specific ion is compared with the specific ion of the fatty acid chain containing an epoxidation structure at the sn position to determine the number of carbons and the number of epoxidation structures of the specific ion of the fatty acid chain containing an epoxidation structure at the corresponding sn position, thereby determining the number of carbons and the number of epoxidation structures of the specific ion at the corresponding 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 ions of the fatty acid chain containing a carbon-carbon double bond at the sn-1 position and the specific ions of the fatty acid chain containing a carbon-carbon double bond at the sn-2 position.

[0143] Since in step (4), specific ions of fatty acid chains containing epoxide structures at the sn position were obtained through tertiary mass spectrometry analysis, and it has been previously explored that fatty acid chains containing carbon-carbon double bonds at the sn position also have a certain tendency to break, the same identification steps were performed on unsaturated lipids that had not undergone epoxide derivatization. In the tertiary mass spectrometry analysis, specific ions of fatty acid chains containing carbon-carbon double bonds at the sn position were formed for comparison. The mass difference between the specific ions and the corresponding specific ions containing epoxide structures at the sn position corresponds to the number of epoxide structures. This can further determine the number of carbons and the number of epoxide structures in the specific ions of fatty acid chains containing epoxide structures at the sn position.

[0144] Furthermore, the number of carbons in the specific ion at the sn-1 position and the number of epoxide structures are determined based on the number of carbons in the specific ion of the fatty acid chain containing the epoxide structure at the sn-1 position and the number of epoxide structures. Similarly, the number of carbons in the specific ion at the sn-2 position and the number of epoxide structures are determined based on the number of carbons in the specific ion of the fatty acid chain containing the epoxide structure at the sn-2 position. Finally, the carbon-carbon double bond position of the unsaturated lipid is determined based on the total number of carbon atoms and the degree of unsaturation, 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.

[0145] In (533), based on the total number of carbon atoms and degree of unsaturation of unsaturated lipids, the number of carbon atoms and number of epoxidation structures of specific ions at the sn-1 position and the sn-2 position, as well as the specific ions of carbon-carbon double bonds, the number of carbon atoms, composition, and position of carbon-carbon double bonds of fatty acid chains at the sn-1 and sn-2 positions can be determined, thereby obtaining the refined structure of unsaturated lipids.

[0146] Furthermore, in primary mass spectrometry analysis, the degree of unsaturation of the unsaturated lipid to be analyzed can be determined 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. Then, the position of the carbon-carbon double bond in the unsaturated lipid is determined based on the degree of unsaturation and the specific ion of the carbon-carbon double bond.

[0147] In unsaturated lipids, when a fatty acid chain contains only one carbon-carbon double bond, after epoxidation derivatization, a single epoxide structure is formed on the chain. This results in a characteristic peak with a 16 Da difference in the CID spectrum of a tertiary mass spectrometer. After determining the sn position of the unsaturated lipid and the number of carbons in the fatty acid chain at that sn position, the distance between the carbon-carbon double bond and the carbonyl terminus can be determined by the mass of the specific ion of the carbon-carbon double bond, thus identifying its position. However, when a fatty acid chain in an unsaturated lipid contains more than one carbon-carbon double bond, after epoxidation derivatization, multiple epoxide structures are formed on the chain. Even when two epoxide structures are far apart (more than three carbons apart), a characteristic peak with a 16 Da difference can still generally be formed for identification. However, if the two epoxide structures are close together (with a separation of three carbons or less between them on an adipose chain), the characteristic peaks that differ by 16 Da in terms of their tendency to break are not as obvious. But this does not affect the confirmation of the carbon-carbon double bond position. In this case, the epoxide structure and the area between the two epoxide structures will break and continue to form a specific ion for 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 determined by reverse deduction based on the ionic mass of the specific ion of the carbon-carbon double bond.

[0148] Furthermore, the identification method provided in this application can also be used to detect unsaturated lipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid. Glycerophospholipids are mainly composed of a glycerol backbone, two fatty acid chains, and a head group consisting of phosphate and its substituents. Different glycerophospholipids are distinguished only by different head group types. The identification method provided in this application is based on the glycerol backbone of glycerophospholipids. After achieving the neutral loss of the head group, the resulting structural transformation of the 1,3-dioxolane ring also occurs on the glycerol backbone. This structure, when analyzed by tandem mass spectrometry, can form high-abundance specific ions at the sn and carbon-carbon double bond positions, enabling multi-dimensional and refined structural identification of unsaturated lipids of different types of glycerophospholipids. The identification method provided in this application is simple in oxidation, easy to reproduce, and highly efficient, making it significant for high-throughput structural identification of lipids.

[0149] In this application, a multidimensional lipid fine structure identification method based on the full derivatization of carbon-carbon double bonds is proposed. The epoxidation derivatization method provided in this application allows for rapid full derivatization of the carbon-carbon double bonds in the unsaturated lipids to be analyzed. By performing mass spectrometry analysis on the sodium ion adduct of the epoxidation product and comparing it with mass spectrometry analysis on unsaturated lipids that have not undergone epoxidation derivatization, the total number of carbon atoms and the degree of unsaturation of the unsaturated lipids can be determined. Secondary mass spectrometry analysis of the sodium ion adduct of the epoxidation product, after neutral loss of the head group, can determine the glycerophospholipid class of the unsaturated lipid to be analyzed. After neutral loss of the head group, the structure of some daughter ions changes. After tertiary mass spectrometry analysis, specific ions at the sn position and carbon-carbon double bond positions are obtained. By determining whether the specific ion at the sn position indicates the sn-1 or sn-2 position, and by analyzing 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 and sn-2 positions can be determined. Then, the specific ion at the carbon-carbon double bond position can be used to further determine the position of the carbon-carbon double bond on the specific fatty acid chain in the unsaturated lipid, thereby determining the fine structure of the unsaturated lipid.

[0150] The following specific examples provide further details.

[0151] The mass spectrometer used in the embodiments and comparative examples of this application is the Qtrap mass spectrometer from Sciex Corporation.

[0152] The method for epoxidative derivatization of the unsaturated lipids to be analyzed in this application embodiment is as follows:

[0153] This application employs dimethyldioxane for in-situ epoxidation derivatization of the carbon-carbon double bonds in the unsaturated lipids to be analyzed. Potassium peroxide monosulfonate is used as the oxidant, and acetone as the catalyst. Dimethyldioxane is generated in situ to fully derivatize the carbon-carbon double bonds in the unsaturated lipids to be analyzed. The specific procedure is as follows: The full derivatization reaction of the carbon-carbon double bonds in the unsaturated lipids to be analyzed is carried out in a 1.5 mL PE tube. 1 mmol / L of the unsaturated lipid to be analyzed is added to the PE tube, followed by 50 μL of 500 mmol / L potassium peroxide monosulfonate, 100 μL of acetone, and 50 μL of 500 mmol / L sodium bicarbonate solution. The entire reaction system is reacted at 60 °C, and the reaction is sonicated for 10 minutes. After 30 minutes of reaction, 300 μL of ethyl acetate is added to extract the epoxidation product of the unsaturated lipid to be analyzed, and then the product is dried under nitrogen. After redissolving in acetonitrile, tandem mass spectrometry analysis is performed.

[0154] Comparative Example 1

[0155] Phosphatidylcholine standard PC 16:0 / 18:1 (9Z) was injected into the mass spectrometer. After ionization by the ion source, the sodium adduct of phosphatidylcholine standard PC 16:0 / 18:1 (9Z) was selected for mass spectrometry analysis (MS), yielding the sodium adduct of PC 16:0 / 18:1 (9Z) [M+Na]. + The ion mass (m / z 782) was subjected to CID fragmentation by secondary mass spectrometry (MS2) to generate a neutral daughter ion (m / z 599) after losing 183 Da (the head group of PC). Then, the neutral daughter ion (m / z 599) was subjected to CID analysis by tertiary mass spectrometry (MS3), as shown in the figure. Figure 2 As shown, the daughter ion (m / z 599) was fragmented by tertiary mass spectrometry to generate a low-abundance specific ion at the sn-1 position (m / z 319), which also formed a fatty acid chain [C18:1+Na] containing a carbon-carbon double bond at the sn-2 position. + The specific ion (m / z 305) can be used to obtain sn position information for PC 16:0 / 18:1 (9Z), such as Figure 2 As 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. Both the specific ion peaks (m / z 319) and (m / z 345) are visible in the same mass spectrum. The lower abundance of the (m / z 345) peak is due to impurities in the sample, specifically the presence of another sn isomer.

[0156] Comparative Example 2

[0157] Phosphatidylcholine standard PC 18:1(9Z) / 16:0 was injected into the mass spectrometer. After ionization by the ion source, the sodium ion adduct of phosphatidylcholine standard PC 18:1(9Z) / 16:0 was selected for mass spectrometry analysis (MS), yielding the sodium ion adduct [M+Na] of PC 16:0 / 18:1(9Z). + The ion mass (m / z 782) was subjected to CID fragmentation by secondary mass spectrometry (MS2) to generate a neutral daughter ion (m / z 599) after losing 183 Da (the head group of PC). Then, the neutral daughter ion (m / z 599) was subjected to CID analysis by tertiary mass spectrometry (MS3), as shown in the figure. Figure 3 As shown, the daughter ion (m / z 599) was fragmented by tertiary mass spectrometry to generate a low-abundance specific ion at the sn-1 position (m / z 345), which also formed a fatty acid chain [C18:1+Na] containing a carbon-carbon double bond at the sn-1 position. + The specific ion (m / z 305) can be used to obtain sn position information for PC 18:1(9Z) / 16:0, such as Figure 3 As shown, 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. Both the specific ion peaks (m / z 319) and (m / z 345) are visible in the same mass spectrum. The lower abundance of the (m / z 319) peak is due to impurities in the sample, specifically the presence of another sn isomer.

[0158] Depend on Figure 2 and Figure 3 It is known that sodium adducts of unsaturated lipids that have not undergone epoxidation are neutrally degraded by MS2 through CID loss of the phosphocholine head group (-183 Da). After MS3 fragmentation, only specific ions with low abundance at the sn- position are obtained, but the carbon-carbon double bond position, another structural information of unsaturated lipids, cannot be obtained.

[0159] Example 1: Phosphatidylcholine standard PC 16:0 / 18:1 (9Z) was selected. The carbon-carbon double bond was fully derivatized in situ using the epoxidation derivatization method described above to obtain the epoxidized product. The epoxidized product was injected into a mass spectrometer, and after ionization, the sodium ion adduct of the epoxidized product was selected for mass spectrometry (MS) analysis, yielding the sodium ion adduct [M+Na] of the PC 16:0 / 18:1 (9Z) epoxidized product. +The ion mass (m / z 798) was neutrally fragmented by CID analysis (MS2) to generate a daughter ion (m / z 615) after losing 183 Da (the head group of PC). This head-fragmented daughter ion (m / z 615) was then subjected to CID analysis (MS3) by tertiary mass spectrometry. Figure 4 As shown, the fragmentation of the daughter ion (m / z 615) by tertiary mass spectrometry analysis generated a high abundance of specific ions at the sn position (m / z 291), which also formed a fatty acid chain [C18:1+O+Na] containing an epoxide structure at the sn-2 position. + The specific ion (m / z 321) was observed, and at the same time, highly abundant specific ions at the carbon-carbon double bond position (m / z 489 and m / z 473) were also obtained in the mass spectrum, indicating that the carbon-carbon double bond position is at position 9 from the carbonyl end. The peaks (m / z 291) and (m / z 361) are both visible in the same mass spectrum; the lower abundance of the (m / z 361) peak is due to impurities in the sample containing another sn isomer.

[0160] It also formed an additional specific ion (m / z 275) with the specific ion at the sn-2 position (m / z 291), which differs by 16 Da. 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) broke, it combined with an oxygen (16 Da) to form an aldehyde group, which was then used as the specific ion (m / z 291) at the sn position for characterization.

[0161] Example 2: Phosphatidylcholine standard PC 18:1(9Z) / 16:0 was selected. The carbon-carbon double bond was fully derivatized in situ using the epoxidation derivatization method described above to obtain the epoxidized product. The epoxidized product was injected into a mass spectrometer, and after ionization, the sodium ion adduct of the epoxidized product was selected for mass spectrometry (MS) analysis, yielding the sodium ion adduct [M+Na] of the PC 18:1(9Z) / 16:0 epoxidized product. + The ion mass (m / z 798) was neutrally fragmented by CID analysis (MS2) to generate a daughter ion (m / z 615) after losing 183 Da (the head group of PC). This head-fragmented daughter ion (m / z 615) was then subjected to CID analysis (MS3) by tertiary mass spectrometry. Figure 5 As shown, the fragmentation of the daughter ion (m / z 615) by tertiary mass spectrometry analysis generated a high abundance of specific ions at the sn position (m / z 361), which also formed a fatty acid chain [C18:1+O+Na] containing an epoxide structure at the sn-1 position. +The specific ion (m / z 321) was observed, and at the same time, highly abundant specific ions at the carbon-carbon double bond position (m / z 489 and m / z 473) were also obtained in the mass spectrum, indicating that the carbon-carbon double bond position is at position 9 from the carbonyl end. The peaks (m / z 291) and (m / z 361) are both visible in the same mass spectrum; the lower abundance of the (m / z 291) peak is due to impurities in the sample containing another sn isomer.

[0162] Example 3: Phosphatidylcholine standard PC 16:0 / 18:2 (9Z, 12Z) was selected. The carbon-carbon double bond was fully derivatized in situ using the epoxidation derivatization method described above to obtain the epoxidized product. The epoxidized product was injected into a mass spectrometer, and after ionization, the sodium ion adduct of the epoxidized product was selected for mass spectrometry (MS) analysis, yielding the sodium ion adduct [M+Na] of the PC 16:0 / 18:2 (9Z, 12Z) epoxidized product. + The ion mass (m / z 812) was neutrally fragmented by CID analysis (MS2) to generate a daughter ion (m / z 629) after losing 183 Da (the head group of PC). This daughter ion (m / z 629) was then subjected to CID analysis (MS3) by tertiary mass spectrometry. Figure 6 As shown, the fragmentation of the daughter ion (m / z 629) by tertiary mass spectrometry analysis generated a high abundance of specific ions at the sn position (m / z 305), which also formed a fatty acid chain [C18:1+2O+Na] containing an epoxide structure at the sn-2 position. + The sample yielded specific ions (m / z 335), and after fragmentation by tertiary mass spectrometry analysis, high abundance of carbon-carbon double bond position-specific ions (m / z 517, m / z 545, and m / z 489) were also obtained, indicating that the carbon-carbon double bond positions were at positions 9 and 12 from the carbonyl end. A lower abundance ion (m / z 375) also appeared in the same mass spectrum, which is attributed to impurities in the sample containing another sn isomer, PC 18:2(9Z, 12Z) / 16:0.

[0163] Since more than one carbon-carbon double bond on the same fatty acid chain underwent epoxidation derivation, the characteristic peaks differing by 16 Da were not used for characterization due to the tendency of the epoxidation structure to break. However, by inversely estimating the mass correspondence of the specific ions formed (m / z 517, m / z 545, and m / z 489), the relevant information of the carbon-carbon double bond can also be obtained after determining the sn position. Here, m / z 545 indicates that one carbon-carbon double bond is located at position 12 from the carbonyl end, m / z 489 indicates that the other carbon-carbon double bond is located at position 9 from the carbonyl end, and m / z 517 indicates that a breakage also occurred between the two epoxidation structures.

[0164] It also formed an additional specific ion (m / z 289) with the specific ion at the sn-2 position (m / z 305), which differed by 16 Da. 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) was broken, it combined with an oxygen (16 Da) to form an aldehyde group, which was then used as the specific ion at the sn position (m / z 291) for characterization.

[0165] from Figures 4-6 It is known that after the unsaturated lipids to be analyzed undergo full derivatization of carbon-carbon double bonds, and after the sodium ion adduct of the epoxidation product of the unsaturated lipids to be analyzed loses the phosphocholine head group (-183 Da) in neutral secondary mass spectrometry analysis, and then undergoes fragmentation analysis in tertiary mass spectrometry analysis, characteristic peaks of high abundance sn- positions and carbon-carbon double bond positions can be obtained simultaneously in the same mass spectrum, thus realizing the simultaneous identification of sn positions and carbon-carbon double bond positions in lipid isomers.

[0166] The multidimensional lipid fine structure identification method based on the full derivation of carbon-carbon double bonds provided in this application is used. After the full derivation of carbon-carbon double bonds, the unsaturated lipid to be tested forms an epoxide structure in situ. Sodium ion addition is selected for tandem mass spectrometry analysis. After the fragmentation and loss of the head group in the secondary mass spectrometry analysis, the inferred 1,3-dioxolane ring structure can be formed. Under the fracture tendency of the interaction with the epoxide structure, after the fragmentation in the tertiary mass spectrometry analysis, the characteristic peaks of high abundance of sn- and carbon-carbon double bond positions can be obtained at the same time, which is conducive to realizing multidimensional lipid fine structure identification and analysis.

[0167] It should be understood that the application of this application is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of this application.

Claims

1. A multidimensional lipid fine structure identification method based on the full derivation of carbon-carbon double bonds, characterized in that, Suitable for unsaturated lipids containing a glycerol backbone and a head group, comprising the following steps: (1) Dimethyldioxane was used to perform in-situ epoxidation derivatization on the unsaturated lipid to be analyzed, so that all carbon-carbon double bonds in the unsaturated lipid were epoxidized to form an epoxidized structure, and the epoxidized product of the unsaturated lipid was obtained. (2) The epoxidation product is injected into a mass spectrometer. After ionization by an ion source, the sodium adduct of the epoxidation product is selected for primary mass spectrometry analysis to obtain the ion mass of the sodium adduct of the epoxidation product. The sodium adduct of unsaturated lipids that have not undergone epoxidation is analyzed by primary mass spectrometry and used 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, and select the daughter ion obtained after neutral loss of the head group of the sodium ion adduct of the epoxidation product after collision-induced dissociation; (4) The daughter ions obtained after the sodium ion adduct of the epoxidation product is neutrally lost from the head group are subjected to third-level mass spectrometry analysis. 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 carbon-carbon double bond position of the unsaturated lipid based on the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid.

2. The method for identifying the fine structure of lipids based on the full derivation of carbon-carbon double bonds according to claim 1, characterized in that, In step (5), determining the sn position and carbon-carbon double bond position of the unsaturated lipid specifically includes the following steps: (51) Determine the sn position of the unsaturated lipid based on the specific ion at the sn position, and determine the type of specific ion at the sn position; (52) Determine the fragmentation mode of the epoxidation product based on the specific ion type of the sn position, and determine the distribution of the epoxidation structure in the epoxidation product based on the fragmentation mode; (53) The carbon-carbon double bond position of the unsaturated lipid is determined based on the total number of carbon atoms and the degree of unsaturation of the unsaturated lipid, the distribution of the epoxide structure in the daughter ion, the specific ion at the sn position and the specific ion at the carbon-carbon double bond position.

3. The method for identifying multidimensional lipid fine structures based on the full derivation of carbon-carbon double bonds according to claim 2, characterized in that, In step (3), the daughter ion obtained after the sodium ion adduct of the epoxidation product neutrally loses its head group contains the structure shown in formula (I), where R1 is a fatty acid chain at the sn-1 position and R2 is a fatty acid chain at the sn-2 position. Formula (I); In step (4), the structure of formula (I) has a tendency to break during the third-level mass spectrometry analysis. During the collision-induced dissociation of the third-level mass spectrometry analysis, a specific ion at the sn position is formed. The specific ion at the sn position includes one or both of the specific ion at the sn-1 position and the specific ion at the sn-2 position. The daughter ion forms a specific ion at the carbon-carbon double bond position through the cleavage of the epoxide structure, and the specific ion at the carbon-carbon double bond position contains the structure of formula (I).

4. The method for identifying the fine structure of lipids based on the full derivation of carbon-carbon double bonds according to claim 3, characterized in that, When neither R1 nor R2 contains an epoxide structure, or when R1 contains an epoxide structure but R2 does not, the fracture tendency of the structure of formula (I) is as shown in formula (II), where the dashed line indicates the fracture site. The fracture tends to occur on the 1,3-dioxolane ring structure, and the carbon-oxygen bonds connected at positions 4 and 5 are broken to form a specific ion indicating the sn-1 position. Formula (II); When R2 contains an epoxide structure while R1 does not, the fracture tendency of the structure of formula (I) is as shown in formula (III), where the dashed line indicates the fracture site. The fracture tends to occur at the carbon-carbon double bond connected by the 1,3-dioxolane ring structure at position 2, forming a specific ion indicating the sn-2 position. Formula (III); When both R1 and R2 contain epoxide structures, the fracture tendency of the structure of formula (I) is as shown in formula (IV), where the dashed lines indicate fracture sites. Fracture tends to occur on the 1,3-dioxolane ring structure, and the carbon-oxygen bonds connected at positions 4 and 5 break. Fracture also tends to occur on the carbon-carbon double bond connected at position 2 of the 1,3-dioxolane ring structure, simultaneously forming specific ions indicating the sn-1 position and specific ions indicating the sn-2 position. Formula (IV).

5. The method for identifying the fine structure of lipids based on the full derivation of carbon-carbon double bonds according to claim 2, characterized in that, In step (51), the sn position of the unsaturated lipid is determined, including 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 phase 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 phase 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 multidimensional lipid fine structures based on the full derivation of carbon-carbon double bonds according to claim 5, characterized in that, In step (4), specific ions of fatty acid chains containing epoxidation structures at the sn position were also obtained through three-stage mass spectrometry analysis. The specific ions of fatty acid chains containing epoxidation structures at the sn position are one or two of the specific ions of fatty acid chains containing epoxidation structures at the sn-1 position and the specific ions of fatty acid chains containing epoxidation structures 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 the 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 the epoxidized structure at the sn position is the specific ion of the fatty acid chain containing the 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 the 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 the epoxidized structure at the sn position is the specific ion of the fatty acid chain containing the epoxidized structure at the sn-2 position.

7. The method for identifying multidimensional lipid fine structures based on the full derivation of carbon-carbon double bonds according to claim 6, characterized in that, It also includes the following steps: Secondary mass spectrometry analysis was performed on the sodium adduct of the unoxidized unsaturated lipids. The second daughter ion obtained after neutral loss of the head group of the sodium adduct of the unoxidized unsaturated lipids was selected. Tertiary mass spectrometry analysis was performed on the second daughter ion obtained after neutral loss of the head group of the sodium adduct of the unsaturated lipids to obtain a specific ion of fatty acid chain containing carbon-carbon double bond at the sn position. This ion was compared with the specific ion of fatty acid chain containing epoxidation structure at the sn position to determine the number of carbons and the number of epoxidation structures of the specific ion of fatty acid chain containing epoxidation structure at the sn position, thereby determining the number of carbons and the number of epoxidation structures of the specific ion of the specific ion at 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 ions of the fatty acid chain containing a carbon-carbon double bond at the sn-1 position and the specific ions of the fatty acid chain containing a carbon-carbon double bond at the sn-2 position.

8. The method for identifying multidimensional lipid fine structures based on the full derivation of carbon-carbon double bonds according to claim 1, characterized in that, In step (1), the in-situ epoxidation derivatization is performed using potassium peroxymonosulfonate and acetone; the in-situ epoxidation derivatization of the unsaturated lipid to be analyzed using dimethyldioxane includes the following steps: Take 1 mmol / L of the unsaturated lipid into a container, add 50 μL of 500 mmol / L potassium peroxymonosulfonate, then add 100 μL of acetone, and then add 50 μL of 500 mmol / L sodium bicarbonate solution and mix. The reaction was carried out at 60°C for 30 minutes, then extracted with 300 μL of ethyl acetate and dried under nitrogen to obtain the epoxidized product of the unsaturated lipid. The reaction process involves ultrasound for 10 minutes.

9. The method for identifying the fine structure of lipids based on the full derivation of carbon-carbon double bonds according to claim 1, characterized in that, The epoxidation products were analyzed by tandem mass spectrometry in positive ion mode.

10. The method for identifying multidimensional lipid fine structures based on the full derivation 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. Step (3) also includes the following steps: The lipid class of the unsaturated lipid to be analyzed is determined based on the mass of the lost neutral head group.

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