Method for identifying unsaturated lipid multilevel structure based on asymmetric epoxidation in combination with parallel liquid chromatography-mass spectrometry

Abstract

The invention relates to a method for identifying a multi-level structure of unsaturated lipid based on asymmetric epoxidation combined with parallel liquid chromatography-mass spectrometry, which comprises the following steps: carrying out epoxidation reaction on unsaturated lipid and an oxidant under the action of a Salen-Mn catalyst to obtain an epoxidation product; analyzing the epoxidized product by adopting a parallel liquid chromatography-mass spectrometry technology; determining the double-bond cis-trans configuration of the unsaturated lipid according to the chromatographic behavior of the epoxidized product; and determining the number of double bonds of the unsaturated lipid, the positions of the double bonds and / or the positions of fatty acyl chains sn where the double bonds are located according to diagnosis ion pairs generated by the epoxidation product in the mass spectrum. Compared with the prior art, the method has the advantages that asymmetric epoxidation is combined with free radical induced dissociation, and comprehensive structural analysis can be performed on FA type lipid double-bond positions and cis-trans isomerism and PC type lipid double-bond positions, sn positions and cis-trans isomerism under the condition that no standard substance exists.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, specifically to a method for identifying the multilevel structure of unsaturated lipids based on asymmetric epoxidation combined with parallel liquid chromatography-mass spectrometry (LC-MS). Background Technology

[0002] Lipids are a class of hydrophobic biomolecules that perform a variety of biological functions in organisms, playing crucial roles in cell structure, energy storage, signal transduction, and biological activity. Unsaturated lipids are a subclass of lipids, typically containing one or more C=C double bonds. The double bonds between the carbon atoms of unsaturated lipids can exist in cis or trans configurations; cis-trans isomerism can alter the spatial conformation and hydrophilicity of lipid molecules, thus affecting their role and function in cell membranes. Glycerophospholipids (GPLs) are key components of cell membranes, consisting of a glycerol backbone connecting two fatty acyl chains (located at sn-1 and sn-2 positions, respectively). The length of the fatty acyl chain and its position on the glycerol backbone (sn position) affect cell membrane fluidity and interfere with cell signal transduction. Therefore, studying the positional isomers and cis-trans isomers of the C=C double bond, as well as the sn positions of the fatty acyl chains containing the carbon-carbon double bonds, is essential for understanding the absorption, transport, and metabolism of lipids in organisms.

[0003] Due to the diversity of lipids, the development of lipidomics analysis methods is challenging. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) have become effective methods for lipid analysis due to their powerful analytical capabilities and good separation capabilities. These methods can provide structural information of unsaturated lipids, such as the length of fatty acyl chains and the degree of unsaturation. However, unsaturated lipids have a large number of isomers with similar structures, making direct chromatographic separation of isomers difficult. Furthermore, collision-induced dissociation (CID) in mass spectrometry is insufficient to break the target compound and generate structure-related characteristic ions, making it difficult to identify the C=C double bond position and the sn position of the fatty acyl chain. Therefore, current lipid research still has significant limitations in the comprehensive structural resolution of unsaturated lipids, and cannot simultaneously identify the double bond position of carbon-carbon double bonds, cis-trans isomerism, and the sn position of the fatty acyl chain containing the carbon-carbon double bond. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to overcome the defects or deficiencies of the prior art and provide a method for identifying the multilevel structure of unsaturated lipids based on asymmetric epoxidation combined with parallel liquid chromatography-mass spectrometry.

[0005] This invention discovers that the Salen-Mn catalyst can catalyze the oxidation of unsaturated lipids by MMPP. Due to the steric effect of the Salen ligand, this reaction exhibits significant differences for cis-unsaturated and trans-unsaturated lipids. Specifically, cis-unsaturated lipids mainly generate cis-epoxides in the reaction, with only a small portion generating trans-epoxides; while trans-unsaturated lipids almost completely and selectively generate trans-epoxides. Because of the differences in the spatial structure of the cis- and trans-epoxides, their elution times in chromatography also differ. Cis-epoxides, due to the increased polarity caused by the bending of the fatty acyl chain, exhibit shorter retention times in reversed-phase chromatography; trans-epoxides, on the other hand, have longer retention times. This difference in chromatographic behavior between the epoxides generated from the cis- and trans-isomers allows for the inference of the cis- and trans-configurations of the epoxides based on these differences, even in the absence of standards. When analyzing samples, the difference in content between cis- and trans-configured unsaturated lipids leads to the generation of stereoisomers (cis-epoxidation products and trans-epoxidation products) in varying proportions after the reaction. Therefore, qualitative and quantitative analysis of the cis- and trans-epoxidation products can be performed to identify the cis- and trans-isomers of unsaturated lipids.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] A method for identifying the multilevel structure of unsaturated lipids based on asymmetric epoxidation combined with parallel liquid chromatography-mass spectrometry (LC-MS) includes:

[0008] S1, unsaturated lipids undergo epoxidation with an oxidant under the action of Salen-Mn catalyst to obtain epoxidized products;

[0009] S2. The epoxidation products of step S1 were separated by parallel liquid chromatography. Based on the differences in epoxidation products of unsaturated lipids and their chromatographic behavior, the cis-trans configuration of the double bonds of unsaturated lipids was identified.

[0010] S3. Perform mass spectrometry analysis on the epoxidized product separated by chromatography in step S2: The epoxidized product is subjected to mass spectrometry collision-induced dissociation and fragmentation to generate diagnostic ions. The position of the double bond of the unsaturated lipid and the sn position of the fatty acyl chain containing the double bond are determined based on the diagnostic ions.

[0011] Compared to existing technologies, this invention employs a Salen-Mn catalyst to catalyze an asymmetric epoxidation reaction between unsaturated lipids and an oxidant. Due to the steric effect of the Salen ligand, the cis and trans isomers exhibit differences in the reaction: cis-configured unsaturated lipids mainly generate cis-epoxidation products, while trans-configured unsaturated lipids almost entirely generate trans-epoxidation products. This results in cis and trans isomers of unsaturated lipids generating epoxidation derivatives with differences in configuration and relative content. By utilizing the polarity differences between the different configurational derivatives, cis and trans epoxidation products can be distinguished chromatographically. Furthermore, by combining their chromatographic peaks and peak areas, qualitative and quantitative analysis of the cis and trans isomerism of unsaturated lipid double bonds can be achieved. Further, collision-induced dissociation (CID) of the epoxidation derivatives, and by analyzing their characteristic diagnostic ion pairs, the double bond positions of the unsaturated lipids can be accurately identified, thereby achieving multi-level structural analysis of unsaturated lipids.

[0012] Furthermore, the Salen-Mn catalyst is (R,R)-(-)-N,N′-bis(3,5-di-tert-butylsalicylyl)-1,2-cyclohexanediamine manganese chloride(III).

[0013] Furthermore, the oxidant is selected from any one of sodium hypochlorite (NaClO), m-chloroperoxybenzoic acid (m-CPBA), monoperoxyphthalic acid (MMPP), and hydrogen peroxide (H2O2).

[0014] Furthermore, the molar ratio of the oxidant to the unsaturated lipid is 1-100:1; the molar ratio of the catalyst to the substrate is 1%-10%.

[0015] Furthermore, step S1 also includes the extraction of the epoxidation product: the reaction solution after the epoxidation reaction is purified by using an aqueous solution of ethylenediaminetetraacetic acid (EDTA) and a saturated aqueous solution of sodium thiosulfate to remove excess oxidant and catalyst, and then extracted with methyl tert-butyl ether (MTBE) to obtain the epoxidation product.

[0016] Furthermore, the parallel liquid chromatography includes alternating analysis of two samples using a first parallel liquid phase and a second parallel liquid phase. By using parallel liquid chromatography, mutual interference between different types of lipid detection is reduced. Lipids susceptible to buffer salt interference (such as fatty acids) are separated from lipids that need to be detected under buffer salt conditions. Analyzing them under different chromatographic conditions avoids mutual interference between different types of lipid detection and improves detection sensitivity.

[0017] Furthermore, the mobile phases RA and RB used in the first parallel liquid chromatography in the parallel liquid chromatography contain 5-10 mmol / L of ammonium bicarbonate. The ammonium bicarbonate reacts with the sn-position isomer of the epoxidation product to form an adduct. Based on the characteristic diagnostic ion ammonium bicarbonate produced by the adduct in the mass spectrometer, the sn position of the double bond of the unsaturated lipid corresponding to the adduct is determined.

[0018] Furthermore, the parallel liquid chromatography in step S2 is performed using a Shimadzu Nexera MX system:

[0019] The conditions for the first parallel liquid chromatography were as follows: column: Waters ACQUITY BEH C18; flow rate: 0.25 mL / min; column temperature: 45℃; injection volume: 2 μL; mobile phase A: acetonitrile:deionized water = 50:50, containing 5-10 mM ammonium bicarbonate; mobile phase B: isopropanol:acetonitrile:deionized water = 80:15:5, containing 5-10 mM ammonium bicarbonate: gradient elution.

[0020] The conditions for the second parallel liquid chromatography were as follows: column: Waters ACQUITY BEH C18; flow rate: 0.3 mL / min; column temperature: 45℃; injection volume: 2 μL; mobile phase A: deionized water; mobile phase B: acetonitrile; gradient elution.

[0021] Further, in step S2, the identification of the double bond cis-trans configuration of unsaturated lipids includes: identification by detecting the number of epoxidized products and their retention time distribution characteristics on the chromatogram, wherein the epoxidized products of unsaturated lipids include fully epoxidized products, the fully epoxidized products of cis-configured unsaturated lipids include both cis-configured and trans-configured products, which are shown as multiple peaks with different retention times on the chromatogram; the fully epoxidized products generated by trans-unsaturated lipids in the epoxidation reaction only contain trans-configured products, which are shown as a single product peak on the chromatogram.

[0022] Furthermore, among the products generated by the epoxidation reaction of cis-configured unsaturated lipids, the relative content of cis-configured products is higher than that of their corresponding trans-configured products. Moreover, under the same initial concentration conditions, the overall relative content of epoxidation products generated from cis-configured unsaturated lipids is higher than that of epoxidation products generated from trans-configured unsaturated lipids.

[0023] Furthermore, step S2 also includes: obtaining the peak areas of the cis-configuration product and the trans-configuration product based on the liquid chromatogram of the obtained epoxidation product, and performing relative quantitative analysis of the double bond cis-trans configuration of the unsaturated lipid.

[0024] Furthermore, the unsaturated lipids include at least one of fatty acids, glycerides, phospholipids, and sphingolipids.

[0025] This invention targets unsaturated lipids with sn-position isomers, such as unsaturated phosphatidylcholine. It investigates a method for identifying the multilevel structure of unsaturated lipids based on asymmetric epoxidation combined with parallel liquid chromatography-mass spectrometry (LC-MS). The study reveals that unsaturated phosphatidylcholine can form an adduct with ammonium bicarbonate, which, upon fragmentation, produces a characteristic diagnostic ion representing the sn-1 chain, allowing identification of the sn position of the fatty acyl chain. Combining sn position identification with the aforementioned methods for identifying cis-trans isomers and carbon-carbon double bond position isomers allows for comprehensive structural analysis of phosphatidylcholine simultaneously.

[0026] Based on this, the double bond positions of phosphatidylcholine-based unsaturated lipids and the sn position of the fatty acyl chain containing the double bond are determined by the following method: collision-induced dissociation is used to fragment the adduct formed by lipid and bicarbonate, and the sn position and the position of the carbon-carbon double bond are deduced by analyzing the characteristic fragment ions produced.

[0027] Furthermore, the mass spectrometer used is a quadrupole time-of-flight mass spectrometer equipped with an electrospray ionization source (ESI), and ultra-high performance liquid chromatography-mass spectrometry is used to analyze the analytes in both positive and negative ion modes. Attached Figure Description

[0028] Figure 1(a) is a chromatogram of the epoxidized products generated by the Salen-Mn catalytic asymmetric epoxidation reaction of fatty acid lipid isomers FA18:1(9Z / 9E) and FA18:1(11Z / 11E) in Example 1.

[0029] Figure 1(b) shows the diagnostic ion diagrams of the double bond positions generated after CID treatment of the epoxidation products of FA18:1(9Z / 9E) and FA18:1(11Z / 11E) in Example 1.

[0030] Figure 1(c) shows the differences in the epoxidation products of different contents of the cis-trans isomers FA18:1(Δ9) in Example 1 after derivatization treatment. The curves from top to bottom represent the molar ratios of FA18:1(9Z) and FA18:1(9E) as 1:9, 1:7, 1:5, 1:3, 1:1, 3:1, 5:1, 7:1, and 9:1, respectively. Furthermore, a linear relationship was established between the molar ratio of the cis-trans isomers of FA18:1(Δ9) and FA18:1(Δ11) and the peak area ratio (left peak / right peak) of their corresponding epoxidation products.

[0031] Figure 2(a) is a chromatogram of the monocyclic oxidation products generated by derivatization of polyunsaturated fatty acid lipid isomers FA18:2(9Z,12Z) and FA18:2(9E,12E) in Example 2.

[0032] Figure 2(b) is a chromatogram of the polyepoxide products generated by derivatization of the polyunsaturated fatty acid lipid isomers FA18:2(9Z,12Z) and FA18:2(9E,12E) in Example 2.

[0033] Figure 2(c) Diagnostic ion diagrams of the double bond positions generated after CID fragmentation of the fully epoxidized products of FA18:2(9Z, 12Z) and FA18:2(9E, 12E) in Example 2.

[0034] Figure 3(a) shows the difference in chromatographic behavior of the products generated by the sn-position isomers PC (16:0 / 18:1) and PC (18:1 / 16:0) of the phosphatidylcholine lipids after Salen-Mn catalytic asymmetric epoxidation reaction in Example 3 of the present invention.

[0035] Figure 3(b) shows the diagnostic ion map of the sn position obtained by CID fragmentation of the epoxide products of PC (16:0 / 18:1) and PC (18:1 / 16:0) in Example 3 at an energy of CE = 30 eV.

[0036] Figure 3(c) shows the diagnostic ion diagram of the double bond positions generated by the CID fragmentation of the epoxidation products of PC (16:0 / 18:1) and PC (18:1 / 16:0) in Example 3 at an energy of CE = 80 eV. Detailed Implementation

[0037] Based on the above research, this invention provides a method for identifying the multilevel structure of unsaturated lipids based on asymmetric epoxidation combined with parallel liquid chromatography-mass spectrometry.

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0039] Example 1

[0040] This embodiment 1 provides a method for identifying the multilevel structure of unsaturated lipids based on asymmetric epoxidation combined with parallel liquid chromatography-mass spectrometry, including the following steps:

[0041] S1. Asymmetric epoxidation reaction of unsaturated lipids: A lipid standard solution (50 μL, 20 μM) containing two pairs of cis-trans isomers of unsaturated fatty acids FA18:1 (9Z / 9E) and FA18:1 (11Z / 11E) was added to 50 μL of an aqueous solution containing Salen-Mn catalyst (catalyst amount is 10 mol% of the substrate, i.e., unsaturated fatty acids) and 8 mM MMPP. After mixing evenly, the mixture was reacted at 10 °C for 10 min to generate epoxidized products of unsaturated lipids. The epoxidized products were extracted to obtain the sample to be tested.

[0042] Preferably, the extraction method for the epoxidation product is as follows: 200 μL of 0.5 M sodium bicarbonate aqueous solution and 200 μL of 0.1 M EDTA aqueous solution are added sequentially to the reaction solution after the epoxidation reaction, and the mixture is vortexed for 1 minute to remove residual oxidant and metal catalyst. Then, 400 μL of MTBE is added for extraction, vortexed for 1 minute, centrifuged for 3 minutes to separate the phases, the upper phase is collected, evaporated to dryness, and then reconstituted to obtain the epoxidation product, which is the sample to be tested.

[0043] S2. Parallel liquid chromatography analysis was performed on the epoxidized products extracted in step S1. Based on the peaks and peak areas of the epoxidized products in the chromatogram, the cis-trans configurations and ratios of the corresponding unsaturated lipids were determined. Referring to Figure 1(a), when the ion chromatogram of the extracted products was 297.24 in negative ion mode, significant differences were found between the cis-trans isomers of the epoxidized products. Specifically, the epoxidation products of cis-configured unsaturated lipids (FA18:1(9Z) and FA18:1(11Z)) exhibit two peaks in chromatography (i.e., cis-configured and trans-configured products, with the relative content of the cis-configured epoxidation product being higher than that of its corresponding trans-configured epoxidation product). In contrast, the derivatization of trans-configured unsaturated lipids (FA18:1(9E) and FA18:1(11E)) results in only a single peak (i.e., the trans-configured product). Furthermore, under the same initial concentration conditions, the overall relative content of the epoxidation products generated from cis-configured unsaturated lipids is higher than that generated from trans-configured unsaturated lipids. This chromatographic difference in the epoxidation products generated from cis- and trans-configured unsaturated lipids allows for the inference of lipid cis- and trans-configurations even in the absence of standards.

[0044] S3. Mass spectrometry analysis of lipid epoxidation products was performed using CID technology. The epoxidation products underwent CID fragmentation to generate diagnostic ion pairs related to the double bond position. The double bond position of the unsaturated lipid was determined based on these diagnostic ion pairs. Referring to Figure 1(b), fragmentation analysis of lipid epoxidation products using CID technology, by analyzing the diagnostic ion pairs generated from the epoxidation products (m / z 171.1 and m / z 155.1), the double bond position in the unsaturated lipid was determined to be at position 9; while the diagnostic ion pairs (m / z 183.0 and m / z 199.1) confirmed that the double bond position in the unsaturated lipid was at position 11.

[0045] Referring to Figure 1(c), when the two isomers FA 18:1 (9Z) and FA 18:1 (9E) were mixed at molar ratios ranging from 1:9 to 9:1, a good linear relationship (R0) was observed between the ratio of FA 18:1 (9E) to FA 18:1 (9Z) content and the peak area ratio of the two peaks (left / right) of the epoxidation product in the extractive ion chromatography (EIC). 2>0.98), thus the linear relationship can be used for relative quantitative analysis of cis-trans isomers in mixed samples.

[0046] Example 2

[0047] This embodiment 2 provides a method for identifying the multilevel structure of unsaturated lipids based on asymmetric epoxidation combined with parallel liquid chromatography-mass spectrometry, including the following steps:

[0048] S1. Asymmetric epoxidation reaction of unsaturated lipids: 50 μL (20 μM) of a lipid standard solution (FA18:2(9Z,12Z) and FA18:2(9E,12E) containing two double bonds was added to 50 μL of an aqueous solution containing Salen-Mn catalyst (catalyst amount equal to 10 mol% of the substrate) and 8 mM MMPP. After mixing thoroughly, the mixture was reacted at 10 °C for 10 min to generate epoxidized products of unsaturated lipids. The epoxidized products were then extracted to obtain the sample to be tested.

[0049] Preferably, the extraction method for the epoxidation product is as follows: 200 μL of 0.5 M sodium bicarbonate aqueous solution and 200 μL of 0.1 M EDTA aqueous solution are added sequentially to the reaction solution after the epoxidation reaction, and the mixture is vortexed for 1 minute to remove residual oxidant and metal catalyst. Then, 400 μL of MTBE is added for extraction, vortexed for 1 minute, centrifuged for 3 minutes to separate the phases, the upper phase is collected, evaporated to dryness, and then reconstituted to obtain the epoxidation product, which is the sample to be tested.

[0050] S2. Parallel liquid chromatography analysis was performed on the epoxidized products extracted in step S1 to identify the cis-trans configuration of the unsaturated lipid double bonds based on the chromatographic behavior of the epoxidized products. Referring to Figure 2(a), the ion chromatogram of the monoepoxidized products of polyunsaturated lipids extracted in negative ion mode, with m / z 295.23 as the characteristic ion, was obtained. For a pair of polyunsaturated lipids containing two double bonds, after derivatization, the monoepoxidized product of the cis-configured unsaturated lipid showed three chromatographic peaks. In contrast, the trans-configured unsaturated lipid produced only two chromatographic peaks after derivatization, and its retention time was longer than that of the cis-configured unsaturated lipid epoxidized product peak.

[0051] Referring to Figure 2(a), the ion chromatogram of the fully epoxidized polyunsaturated lipid product corresponding to m / z 311.22 is extracted in negative ion mode. After derivatization, the cis-configured unsaturated lipids generate two chromatographic peaks. The peak with the higher response corresponds to the epoxidized product with a bicis configuration, while the peak with the lower response corresponds to the epoxidized product containing one cis and one trans configuration. The trans-configured unsaturated lipids generate only one product peak, corresponding to the epoxidized product with a bitrans configuration.

[0052] S3. Mass spectrometry analysis of lipid epoxidation products was performed using CID technology. The epoxidation products underwent CID fragmentation to generate diagnostic ion pairs related to the double bond positions. The double bond positions of the unsaturated lipids were determined based on these diagnostic ion pairs. Referring to Figure 2(b), fragmentation analysis of polyunsaturated lipid epoxidation products using CID technology was performed. By detecting the characteristic diagnostic ion pairs (m / z 171.1 and m / z 155.1), it was determined that a double bond exists at position C9 in the unsaturated lipids; while another set of diagnostic ion pairs (m / z 211.1 and m / z 227.1) indicated that the second double bond was located at position C12.

[0053] Therefore, by combining the chromatographic behavior analysis of step S2 and the fragmentation characteristics analysis of step S3 of the polyunsaturated lipid epoxidation products, the positions of double bonds and cis-trans isomer configurations of polyunsaturated fatty acids can be identified simultaneously.

[0054] Example 3

[0055] This embodiment 3 provides a method for identifying the multilevel structure of unsaturated lipids based on asymmetric epoxidation combined with parallel liquid chromatography-mass spectrometry (LC-MS), including the following steps:

[0056] S1. Asymmetric epoxidation reaction of unsaturated lipids: 50 μL (20 μM) of a lipid standard solution (18:1(9Z) / 16:0) and PC(16:0 / 18:1(9Z)) containing a pair of unsaturated phosphatidylcholine PC (18:1(9Z) / 16:0) and PC (16:0 / 18:1(9Z)) containing sn-position isomers were added to an aqueous solution containing 50 μL of Salen-Mn catalyst (catalyst amount is 10 mol% of substrate) and 8 mM MMPP. After mixing thoroughly, the mixture was reacted at 10 °C for 10 min to generate epoxidized products of unsaturated lipids. The epoxidized products were extracted to obtain the sample to be tested.

[0057] Preferably, the extraction method for the epoxidation product is as follows: 200 μL of 0.5 M sodium bicarbonate aqueous solution and 200 μL of 0.1 M EDTA aqueous solution are added sequentially to the reaction solution after the epoxidation reaction, and the mixture is vortexed for 1 minute to remove residual oxidant and metal catalyst. Then, 400 μL of MTBE is added for extraction, vortexed for 1 minute, centrifuged for 3 minutes to separate the phases, the upper phase is collected, evaporated to dryness, and then reconstituted to obtain the epoxidation product, which is the sample to be tested.

[0058] S2. Parallel liquid chromatography analysis was performed on the epoxidized product extracted in step S1. Based on the chromatographic behavior of the epoxidized product on the chromatogram, the cis-trans configuration of the corresponding unsaturated lipid was determined. Referring to Figure 3(a), in negative ion mode, the ion chromatogram (m / z 836.56) of the extracted derivatized product and bicarbonate adduct showed that both lipid configurations produced two characteristic peaks after derivatization, thus identifying both lipids as cis configuration. Compared to PC (16:0 / 18:1(9Z)), its sn-position isomer PC (18:1(9Z) / 16:0) exhibited a longer retention time.

[0059] S3. Mass spectrometry analysis of lipid epoxidation products was performed using CID technology. The epoxidation products underwent CID fragmentation to generate characteristic diagnostic ion pairs related to the double bond position and the sn position of the fatty acyl chain. The double bond position and sn position of the unsaturated lipid were determined based on these diagnostic ion pairs. Referring to Figure 3(b), MS / MS fragmentation analysis of the two isomers of the epoxidation products was performed using collision-induced dissociation (CID) technology at CE = 30 eV. The results showed that the epoxidation product of PC (18:1(9Z) / 16:0) generated a characteristic fragment ion of m / z 461.2, while the epoxidation product of PC (16:0 / 18:1(9Z)) generated a characteristic fragment of m / z 419.2. The difference between these characteristic ions (m / z 461.2 and m / z 419.2) stems from the different structures of the fatty acyl chain at the sn-1 position. Therefore, the mass difference of these characteristic fragment ions can be used to identify the sn-position isomers of lipid molecules.

[0060] Referring to Figure 3(c), under the condition of CE = 80 Ev, the position of the double bond in the unsaturated lipid was determined to be at position 9 based on the diagnostic ion pair (m / z 171.1 and m / z 155.1) generated by the epoxidation product.

[0061] Therefore, by combining the method of identifying cis-trans isomers in step S31 with the method of identifying carbon-carbon double bond positional isomers and the positional isomers of the fatty acyl chain in step S32, a comprehensive structural analysis of phosphatidylcholine can be performed simultaneously.

[0062] In summary, the present invention has the following advantages compared with the prior art:

[0063] (1) This invention overcomes the shortcomings of existing technologies that cannot perform multidimensional analysis of unsaturated lipids. It combines asymmetric epoxidation with free radical-induced dissociation to achieve multi-level structural analysis of the double bond position and cis-trans isomerism of FA lipids and the double bond position, sn position and cis-trans isomerism of PC lipids.

[0064] (2) By introducing Salen-Mn catalysts, particularly (R,R)-(-)-N,N′-bis(3,5-di-tert-butylsalicylene)-1,2-cyclohexanediamine manganese chloride (III), cis-unsaturated and trans-unsaturated lipids are induced to generate different epoxidized products. Furthermore, by utilizing the difference in elution times between cis- and trans-epoxidized products in chromatography, qualitative and quantitative analysis of the cis- and trans-configurations of the epoxidized products can be achieved even in the absence of standards, thereby enabling qualitative and quantitative analysis of the cis- and trans-configurations of unsaturated lipids.

[0065] (3) By combining parallel liquid chromatography, the ion inhibition of FAs detection by buffer salts can be eliminated, and the full structure of PC can be analyzed. In addition, the mutual interference between lipids can be eliminated, thus improving the sensitivity of lipid detection.

[0066] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for identifying the multilevel structure of unsaturated lipids based on asymmetric epoxidation combined with parallel liquid chromatography-mass spectrometry, characterized in that, include: S1, unsaturated lipids undergo epoxidation with an oxidant under the action of Salen-Mn catalyst to obtain epoxidized products; S2. The epoxidation products of step S1 were separated by parallel liquid chromatography. Based on the differences in epoxidation products of unsaturated lipids and their chromatographic behavior, the cis-trans configuration of the double bonds of unsaturated lipids was identified. S3. Perform mass spectrometry analysis on the epoxidized product separated by chromatography in step S2: The epoxidized product is subjected to mass spectrometry collision-induced dissociation and fragmentation to generate diagnostic ions. The position of the double bond of the unsaturated lipid and the sn position of the fatty acyl chain containing the double bond are determined based on the diagnostic ions.

2. The method according to claim 1, characterized in that, The Salen-Mn catalyst is (R,R)-(-)-N,N′-bis(3,5-di-tert-butylsalicylyl)-1,2-cyclohexanediamine manganese chloride(III).

3. The method according to claim 1, characterized in that, The oxidant is selected from any one of sodium hypochlorite, m-chloroperoxybenzoic acid, monoperoxyphthalic acid, and hydrogen peroxide.

4. The method according to claim 1, characterized in that, The molar ratio of the oxidant to the unsaturated lipid is 1-100:1; the molar ratio of the catalyst to the substrate is 1%-10%.

5. The identification method according to claim 1, characterized in that, Step S2, the parallel liquid chromatography, includes alternating analysis of two samples using a first parallel liquid phase and a second parallel liquid phase. The conditions for the first parallel liquid chromatography were: column: Waters ACQUITY BEH C18; flow rate: 0.25 mL / min; column temperature: 45℃; mobile phase A: Acetonitrile:deionized water = 50:50, containing 5-10mM ammonium bicarbonate; Mobile phase B: isopropanol:acetonitrile:deionized water = 80:15:5, containing 5-10mM ammonium bicarbonate; The conditions for the second parallel liquid chromatography were as follows: column: Waters ACQUITY BEH C18; flow rate: 0.3 mL / min; column temperature: 45℃; mobile phase A: deionized water; mobile phase B: acetonitrile.

6. The method according to claim 1, characterized in that, In step S2, the identification of the cis-trans configuration of the double bond of unsaturated lipids includes: identification by detecting the number of epoxidized products and their retention time distribution characteristics on the chromatogram, wherein the epoxidized products of unsaturated lipids include fully epoxidized products, the fully epoxidized products of cis-configured unsaturated lipids include both cis-configured and trans-configured products, which are shown as multiple peaks with different retention times on the chromatogram; the fully epoxidized products generated by trans-unsaturated lipids in the epoxidation reaction only contain trans-configured products, which are shown as a single product peak on the chromatogram.

7. The method according to claim 6, characterized in that, In the products generated by the epoxidation reaction of cis-configured unsaturated lipids, the relative content of cis-configured products is higher than that of their corresponding trans-configured products. Furthermore, under the same initial concentration conditions, the overall relative content of epoxidation products generated from cis-configured unsaturated lipids is higher than that of epoxidation products generated from trans-configured unsaturated lipids.

8. The method according to claim 1, characterized in that, Step S2 further includes: obtaining the peak areas of the cis-configuration product and the trans-configuration product based on the liquid chromatogram of the obtained epoxidation product, and performing relative quantitative analysis of the double bond cis-trans configuration of the unsaturated lipid.

9. The method according to any one of claims 1-8, characterized in that, The unsaturated lipids include at least one of fatty acids, glycerides, phospholipids, and sphingolipids.

10. The method according to claim 9, characterized in that, The unsaturated lipids are phosphatidylcholine-based unsaturated lipids. The positions of the double bonds and the sn position of the fatty acyl chain containing the double bonds are determined by the following method: collision-induced dissociation is used to fragment the adduct formed by the lipid and bicarbonate ions, and the sn position and the position of the carbon-carbon double bond are deduced by analyzing the characteristic fragment ions produced.

Images