Glyceride structure non-targeted analysis method and system based on chromatography-mass spectrometry

Abstract

The invention relates to the field of glyceride structure analysis, in particular to a non-targeted analysis method and system for a glyceride structure based on a chromatography-mass spectrometry technology. The method comprises the following steps: constructing a molecular total composition database, a chain composition identification database and a carbon-carbon double bond position identification database; performing primary mass spectrometric detection on the glyceride sample to obtain a substance molecular weight, and comparing the substance molecular weight with the lipid molecular weight in the database to obtain total composition data; performing secondary mass spectrometric detection on the total composition data, comparing lipid chain hierarchy diagnosis ions with a database, and determining chain composition data and chain sn-position; performing photochemically derived secondary mass spectrometry on the chain composition data, and comparing lipid C = C diagnosis ions with a database to obtain C = C position information; in combination with total composition, chain composition, chain sn-position and C = C position information of glyceride, a biomedical research process is guided. According to the invention, non-targeted, rapid, comprehensive and high-throughput identification of total composition, chain composition, chain sn-position and C = C position of a complex glyceride sample is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of glyceride structure analysis, and in particular to a non-targeted analysis method and system for glyceride structure based on chromatography-mass spectrometry technology. Background Art

[0002] Glycerides (GL) include monoacylglycerol (MG), diacylglycerol (DG) and triacylglycerol (TG). Among them, TG is a key component of adipocytes and plays an important role in energy storage and cellular fatty acid delivery. Studies have shown that glycerides are an important component of cellular lipid homeostasis, and their metabolic disorders are often associated with the occurrence or progression of metabolic diseases. Therefore, detailed structural analysis of glycerides is of great significance for disease phenotype research.

[0003] Traditional mass spectrometry cannot effectively distinguish the structural isomers of glycerides, especially in the analysis of carbon-carbon double bonds (C=C) at different positions on the fatty acid chain. However, the combination of photochemical derivatization and chromatography-tandem mass spectrometry makes it possible to identify the fine structure of glycerides (i.e., sn-position, C=C position). Photochemical derivatization includes online derivatization and offline derivatization, and commonly used derivatization reagents include keto reagents and pyridine reagents. Photochemical derivatization can not only achieve C=C position identification, but also improve sensitivity.

[0004] In conventional analysis, the fine structural analysis of glycerides is often limited to the scope of targeted substances, that is, identification of known targets. However, the complex mass spectrometry analysis tasks and the wide variety of glycerides have severely restricted the expansion of glyceride analysis into the field of multi-component, comprehensive omics analysis, especially the goal of achieving non-targeted, high-coverage screening and identification. This series of challenges has led to a significant reduction in the ease of use of current glyceride analysis methods, with problems such as long time consumption, low accuracy, and limited identification numbers. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a non-targeted analysis method and system for glyceride structure based on chromatography-mass spectrometry technology, the method comprising the following steps:

[0006] S1: construct the molecular composition database, chain composition identification database and carbon-carbon double bond position identification database respectively;

[0007] S2: performing primary mass spectrometry detection on the glyceride sample to obtain the molecular weight of the substance, and comparing the molecular weight of the substance with the lipid molecular weight in the molecular total composition database to obtain the total composition data of the glyceride;

[0008] S3: performing secondary mass spectrometry detection on the total composition data of the glyceride to obtain lipid chain-level diagnostic ions, and comparing the lipid chain-level diagnostic ions with lipid chain-level diagnostic ions in the chain composition identification database to obtain chain composition data and chain sn-positions of the glyceride;

[0009] S4: performing photochemically derived secondary mass spectrometry analysis on the chain composition data of the glyceride to obtain lipid carbon-carbon double bond diagnostic ions, and comparing the lipid carbon-carbon double bond diagnostic ions with lipid carbon-carbon double bond diagnostic ions in the carbon-carbon double bond position identification database to obtain carbon-carbon double bond position information of the glyceride;

[0010] S5: Obtain non-targeted analysis results of glyceride structure according to the total composition data, the chain composition data, the chain sn-position and the carbon-carbon double bond position information, and guide the biomedical research process of related components based on the non-targeted analysis results of glyceride structure.

[0011] In one embodiment of the present invention, in S2, the method for obtaining the total composition data of glycerides is as follows:

[0012] The glyceride sample is detected by liquid chromatography separation-primary mass spectrometry to obtain a primary mass spectrum, and the primary mass spectrum is analyzed to obtain a molecular mass-to-charge ratio Y of the lipid primary mass spectrum;

[0013] Based on the molecular composition database, the analysis mode is selected as positive ion and adduct ion, and the theoretical molecular mass-to-charge ratio of the glyceride molecule detected in the first-stage mass spectrometry full scan mode is obtained as [M+X], wherein M is the molecular weight of the glyceride and X is the adduct ion;

[0014] A first mass matching tolerance and a first peak finding threshold are set, the error between the molecular mass-to-charge ratio Y and the theoretical molecular mass-to-charge ratio [M+X] is calculated, and first mass spectrum data is obtained in which the error is less than the first mass matching tolerance and the peak threshold exceeds the first peak finding threshold, and the total composition data of glyceride is obtained based on the first mass spectrum data.

[0015] In one embodiment of the present invention, in S3, the method for obtaining the chain composition data and chain sn-position of glycerides is as follows:

[0016] The total composition data of the glycerides are detected by liquid chromatography separation-secondary mass spectrometry to obtain lipid chain-level diagnostic ions, i.e., actual fragment ion molecular mass-to-charge ratios;

[0017] Based on the chain composition identification database, the analysis mode is selected as positive ion, adduct ion and fragmentation energy to obtain the theoretical fragment ion molecular mass-to-charge ratio of the glyceride molecule detected in the secondary mass spectrometry full scan mode;

[0018] A second mass matching tolerance and a second peak finding threshold are set, and the error between the mass-to-charge ratio of the actual fragment ion molecule and the mass-to-charge ratio of the theoretical fragment ion molecule is calculated to obtain second mass spectrum data in which the error is less than the second mass matching tolerance and whether the peak threshold exceeds the second peak finding threshold; and based on the second mass spectrum data, the chain composition data and chain sn-position of the glyceride are obtained.

[0019] In one embodiment of the present invention, in S4, the method for obtaining the carbon-carbon double bond position information of the glyceride is as follows:

[0020] The chain composition data of the glyceride are detected by liquid chromatography separation-photochemical derivatization-secondary mass spectrometry to obtain a photochemical derivatization-secondary mass spectrum, and the molecular mass-to-charge ratio of the lipid carbon-carbon double bond diagnostic ion is obtained according to the photochemical derivatization-secondary mass spectrum;

[0021] Based on the carbon-carbon double bond position identification database, the analysis mode is selected as positive ion, adduct ion and fragmentation energy to obtain the theoretical fragment ion molecular mass-to-charge ratio of the glyceride molecule detected in the secondary mass spectrometry mode;

[0022] A third mass matching tolerance and a third peak finding threshold are set, and the error between the molecular mass-to-charge ratio of the lipid carbon-carbon double bond diagnostic ion and the molecular mass-to-charge ratio of the theoretical fragment ion is calculated to obtain third mass spectrum data in which the error is less than the third mass matching tolerance and whether the peak threshold exceeds the third peak finding threshold; based on the third mass spectrum data, the carbon-carbon double bond position information of glyceride is obtained.

[0023] In one embodiment of the present invention, in S1, the method for constructing a molecular composition database is:

[0024] The molecular formula of the fatty acid chain is defined as C n H (2n+1-2db) , n is the total carbon number of the fatty acid chain, db is the degree of unsaturation of the fatty acid chain;

[0025] Connecting the glyceride backbone and at least one fatty acid chain, and generating the molecular formula of the substance by calculation;

[0026] The molecular weight of the corresponding glyceride structure is calculated based on the mass of each element in the molecular formula of the substance.

[0027] In one embodiment of the present invention, in S1, the chain composition identification database includes backbone fragment ion molecular weight information and chain ion molecular weight information, and the construction method thereof is:

[0028] The molecular weight information of the skeleton fragment ion is expressed as the molecular weight of the glyceride + adduct ion - ammonia molecule - missing chain, i.e., M+X–NH3-C n H (2n-2db) O2;

[0029] The chain ion molecular weight information is expressed as the missing chain-hydroxyl group, i.e., C n H (2n-2db) O2–OH;

[0030] Where n is the total number of carbon atoms in the fatty acid chain and db is the number of carbon-carbon double bonds in the fatty acid chain.

[0031] In one embodiment of the present invention, in S1, the carbon-carbon double bond position identification database is established based on a carbonyl-containing photochemical derivatization reagent, wherein the construction method is different depending on whether the photochemical derivatization reagent is charged, as follows:

[0032] If the photochemical derivatization reagent is an uncharged carbonyl-containing compound, the carbon-carbon double bond position identification database includes molecular weight information of acyl ions after PB reaction and molecular weight information of carbonyl ions after PB reaction;

[0033] If the photochemical derivatization reagent is a charged carbonyl-containing compound, the carbon-carbon double bond position identification database includes the molecular weight information of the skeleton fragment ions and the molecular weight information of the chain ions where the carbon-carbon double bond is located.

[0034] In one embodiment of the present invention, if the photochemical derivatization reagent is an uncharged carbonyl-containing compound, the molecular weight information of the acyl ion after the PB reaction is expressed as the molecular weight of the glyceride + the adduct ion + the molecular weight of the photochemical derivatization reagent - a randomly lost fatty acid chain - the hydroxyl group - the chain fragment lost after the double bond of the fatty chain that undergoes the PB reaction is broken, that is, [M+X+photochemical derivatization reagent]-C n H (2n-2db) O2–OH–C n-x H 2(n-x-fdb) ;

[0035] The molecular weight information of the carbonyl ion after the PB reaction is expressed as the molecular weight of the glyceride + the adduct ion + the molecular weight of the photochemical derivatization reagent - a randomly lost fatty acid chain - hydroxyl - the chain fragment lost after the double bond of the fatty chain that undergoes the PB reaction is broken + methylene, that is, [M+X+photochemical derivatization reagent]-C n H (2n-2db) O2–OH–C n-x H 2(n-x-fdb) +C2H2;

[0036] Among them, db is the number of carbon-carbon double bonds in the fatty acid chain, n is the total carbon number of the fatty acid chain undergoing PB reaction, x is the position of the carbon-carbon double bond of the fatty chain undergoing PB reaction, and fdb is the unsaturation on the chain fragment lost after the double bond of the fatty chain undergoing PB reaction is broken.

[0037] In one embodiment of the present invention, if the photochemical derivatization reagent is a charged carbonyl-containing compound, the molecular weight information of the skeleton fragment ion is expressed as the molecular weight of the glyceride + the molecular weight of the photochemical derivatization reagent + the adduct ion - the chain fragment lost after the double bond of the fatty chain that undergoes the PB reaction is broken - the oxygen atom, that is, M+PB+XC n-x H 2(n-x-fdb) -O;

[0038] The molecular weight information of the chain ion where the carbon-carbon double bond is located is expressed as the molecular weight of the photochemical derivatization reagent + the adducted ion + the chain fragment lost after the double bond of the fatty chain in the PB reaction is broken - the oxygen atom, that is, PB+X+C n-x H 2(n-x-fdb) -O;

[0039] Among them, db is the number of carbon-carbon double bonds in the fatty acid chain, n is the total carbon number of the fatty acid chain undergoing PB reaction, x is the position of the carbon-carbon double bond of the fatty chain undergoing PB reaction, and fdb is the unsaturation on the chain fragment lost after the double bond of the fatty chain undergoing PB reaction is broken.

[0040] Based on the same inventive concept, the present invention also provides a non-targeted analysis system of glyceride structure based on chromatography-mass spectrometry technology, which is used to implement the steps of the non-targeted analysis method of glyceride structure based on chromatography-mass spectrometry technology, and the system includes the following modules:

[0041] A database construction module is used to construct a molecular composition database, a chain composition identification database, and a carbon-carbon double bond position identification database respectively;

[0042] A total composition acquisition module is used to perform a primary mass spectrometry test on the glyceride sample to obtain the molecular weight of the substance, and compare the molecular weight of the substance with the lipid molecular weight in the molecular total composition database to obtain the total composition data of the glyceride;

[0043] A chain composition acquisition module, for performing secondary mass spectrometry detection on the total composition data of the glyceride to obtain lipid chain level diagnostic ions, and comparing the lipid chain level diagnostic ions with the lipid chain level diagnostic ions in the chain composition identification database to obtain the chain composition data and chain sn-position of the glyceride;

[0044] A carbon-carbon double bond position information acquisition module is used to perform photochemically derived secondary mass spectrometry analysis on the chain composition data of the glyceride to obtain lipid carbon-carbon double bond diagnostic ions, and compare the lipid carbon-carbon double bond diagnostic ions with lipid carbon-carbon double bond diagnostic ions in the carbon-carbon double bond position identification database to obtain carbon-carbon double bond position information of the glyceride;

[0045] An application guidance module is used to obtain a non-targeted analysis result of the glyceride structure according to the total composition data, the chain composition data, the chain sn-position and the carbon-carbon double bond position information, and to guide the biomedical research process of related components based on the non-targeted analysis result of the glyceride structure.

[0046] The above technical solution of the present invention has the following advantages compared with the prior art:

[0047] The present invention deeply integrates photochemical derivatization technology and tandem mass spectrometry, and constructs a glyceride structure identification database by innovatively establishing a characteristic ion calculation algorithm. On this basis, a non-targeted data-dependent de novo identification analysis method is further developed, aiming to achieve non-targeted, rapid, comprehensive, and high-throughput identification of the total composition, chain composition, chain sn-position, and carbon-carbon double bond (C=C) position of glycerides in complex samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0049] Figure 1 This is a flow chart of the non-targeted analysis method of glyceride structure based on chromatography-mass spectrometry technology provided in Example 1 of the present invention;

[0050] Figure 2 This is a specific flow chart of the non-targeted analysis method of glyceride structure based on chromatography-mass spectrometry technology provided in Example 1 of the present invention;

[0051] Figure 3 Schematic diagram of the structure of the non-targeted analysis system of glyceride structure based on chromatography-mass spectrometry technology provided in Example 2 of the present invention;

[0052] Description of the accompanying drawings in the specification: 100, database construction module; 200, total component acquisition module; 300, chain component acquisition module; 400, carbon-carbon double bond position information acquisition module; 500, application guidance module. DETAILED DESCRIPTION

[0053] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0054] Embodiment 1

[0055] See also Figure 1 and Figure 2As shown, the present invention provides a non-targeted analysis method and system for glyceride structure based on chromatography-mass spectrometry technology, and the method comprises the following steps:

[0056] S1: construct the molecular composition database, chain composition identification database and carbon-carbon double bond position identification database respectively;

[0057] S2: performing primary mass spectrometry detection on the glyceride sample to obtain the molecular weight of the substance, and comparing the molecular weight of the substance with the lipid molecular weight in the molecular total composition database to obtain the total composition data of the glyceride;

[0058] S3: performing secondary mass spectrometry detection on the total composition data of the glyceride to obtain lipid chain-level diagnostic ions, and comparing the lipid chain-level diagnostic ions with lipid chain-level diagnostic ions in the chain composition identification database to obtain chain composition data and chain sn-positions of the glyceride;

[0059] S4: performing photochemically derived secondary mass spectrometry analysis on the chain composition data of the glyceride to obtain lipid carbon-carbon double bond diagnostic ions, and comparing the lipid carbon-carbon double bond diagnostic ions with lipid carbon-carbon double bond diagnostic ions in the carbon-carbon double bond position identification database to obtain carbon-carbon double bond position information of the glyceride;

[0060] S5: Obtaining a non-targeted analysis result of the glyceride structure according to the total composition data, the chain composition data and the carbon-carbon double bond position information, and guiding the biomedical research process of related components based on the non-targeted analysis result of the glyceride structure.

[0061] It can be concluded from the above technical scheme that by constructing a molecular total composition database, a chain composition identification database and a carbon-carbon double bond position identification database, these databases provide rich reference information for subsequent mass spectrometry data comparison, thereby improving the accuracy and coverage of identification; the primary mass spectrometry in step S2 is used for preliminary screening to quickly obtain the total composition data of glycerides, greatly shortening the analysis time, and further analyzing the chain composition of glycerides through the secondary mass spectrometry in step S3, using the comparison of lipid chain-level diagnostic ions with the database to improve the identification accuracy of the chain composition, and introducing photochemically derived secondary mass spectrometry analysis in step S4, using lipid carbon-carbon double bond diagnostic ions and database matching, to achieve accurate identification of the carbon-carbon double bond position of glycerides. The entire analysis process is highly automated, from sample processing to data comparison, reducing the error and time cost of manual operation.

[0062] Furthermore, in step S1, the method for constructing the molecular composition database is:

[0063] The molecular formula of fatty acid chain (FA) is defined as C n H (2n+1-2db), n is the total carbon number of the fatty acid chain, db is the degree of unsaturation of the fatty acid chain (C = number of C), for example, the molecular formula of FA (18:1) is C 18 H 35 ,This database includes 111 FAs;

[0064] Connect the glyceride backbone and at least one fatty acid chain, and calculate the molecular formula of the substance through the formula; for example: the backbone molecular formulas of triglycerol (TG), diglycerol (DG) and monoglycerol (MG) are C3O6-H, C3H2O5, and C3H5O4, respectively, and can be connected to 3, 2, and 1 fatty acid chains respectively. According to the rehearsal combination of 111 fatty acid chains, the glyceride molecular formula can be fused through field merging. The backbone molecular formula of TG is C3O6-H, and the molecular formula of FA (16:0) is C 16 H 33 TG head group and three FA (16:0) chains can form TG 48:0, whose molecular formula is C3O6-H+3C 16 H 33 =C 51 H 98 O6.

[0065] According to the mass of each element in the molecular formula of the substance, the molecular weight of the corresponding glyceride structure is calculated, where the exact mass of C is 12, the exact mass of H is 1.0078, and the exact mass of O is 15.9949. The molecular weight M can be calculated as x*C+y*H+z*O. For example, the molecular formula of TG (48:0) is C 51 H 98 O6, M=51*12+98*1.0078+6*15.9949=806.7363.

[0066] Further, in step S1, the chain composition identification database includes backbone fragment ion molecular weight information and chain ion molecular weight information, and its construction method is:

[0067] The molecular weight information of the skeleton fragment ion is expressed as the molecular weight of the glyceride + adduct ion - ammonia molecule - missing chain, i.e., M+X–NH3-C n H (2n-2db) O2;

[0068] The chain ion molecular weight information is expressed as the missing chain-hydroxyl group, i.e., C n H (2n-2db) O2–OH;

[0069] Where n is the total number of carbon atoms in the fatty acid chain and db is the number of carbon-carbon double bonds in the fatty acid chain.

[0070] Further, in step S1, the carbon-carbon double bond position identification database is established based on a carbonyl-containing photochemical derivatization (PB) reagent, wherein the construction method is different depending on whether the photochemical derivatization reagent is charged, as follows:

[0071] If the photochemical derivatization reagent is an uncharged carbonyl-containing compound, the carbon-carbon double bond position identification database includes molecular weight information of acyl ions after PB reaction and molecular weight information of carbonyl ions after PB reaction;

[0072] If the photochemical derivatization reagent is a charged carbonyl-containing compound, the carbon-carbon double bond position identification database includes the molecular weight information of the skeleton fragment ions and the molecular weight information of the chain ions where the carbon-carbon double bond is located.

[0073] Specifically, if the photochemical derivatization reagent is an uncharged carbonyl-containing compound, the molecular weight information of the acyl ion after the PB reaction is expressed as the molecular weight of the glyceride + the adduct ion + the molecular weight of the photochemical derivatization reagent - a randomly lost fatty acid chain - hydroxyl - the chain fragment lost after the double bond of the fatty chain that undergoes the PB reaction is broken, that is, [M+X+photochemical derivatization reagent]-C n H (2n-2db) O2–OH–C n-x H 2(n-x-fdb) ;

[0074] The molecular weight information of the carbonyl ion after the PB reaction is expressed as the molecular weight of the glyceride + the adduct ion + the molecular weight of the photochemical derivatization reagent - a randomly lost fatty acid chain - hydroxyl - the chain fragment lost after the double bond of the fatty chain that undergoes the PB reaction is broken + methylene, that is, [M+X+photochemical derivatization reagent]-C n H (2n-2db) O2–OH–C n-x H 2(n-x-fdb) +C2H2;

[0075] Among them, db is the number of carbon-carbon double bonds in the fatty acid chain, n is the total carbon number of the fatty acid chain undergoing PB reaction, x is the position of the carbon-carbon double bond of the fatty chain undergoing PB reaction, and fdb is the unsaturation on the chain fragment lost after the double bond of the fatty chain undergoing PB reaction is broken.

[0076] Specifically, if the photochemical derivatization reagent is a charged carbonyl-containing compound, the molecular weight information of the skeleton fragment ion is expressed as the molecular weight of the glyceride + the molecular weight of the photochemical derivatization reagent + the adduct ion - the chain fragment lost after the double bond of the fatty chain that undergoes the PB reaction is broken - the oxygen atom, that is, M+PB+XC n-x H 2(n-x-fdb) -O;

[0077] The molecular weight information of the chain ion where the carbon-carbon double bond is located is expressed as the molecular weight of the photochemical derivatization reagent + the adducted ion + the chain fragment lost after the double bond of the fatty chain in the PB reaction is broken - the oxygen atom, that is, PB+X+C n-x H 2(n-x-fdb) -O;

[0078] Among them, db is the number of carbon-carbon double bonds in the fatty acid chain, n is the total carbon number of the fatty acid chain undergoing PB reaction, x is the position of the carbon-carbon double bond of the fatty chain undergoing PB reaction, and fdb is the unsaturation on the chain fragment lost after the double bond of the fatty chain undergoing PB reaction is broken.

[0079] In the above technical scheme, there are two nomenclatures for the double bond positions of lipids, namely, Δ nomenclature and n-nomenclature. In the Δ nomenclature, the C=C is counted from the acyl head, for example, Δ9 means that there is a double bond on the ninth carbon atom starting from the acyl head; while in the n-nomenclature, the C=C is counted from the methyl end of the fatty acid chain, for example, n-6 means that there is a double bond on the sixth carbon atom starting from the methyl end.

[0080] Further, in step S2, the method for obtaining the total composition data of glycerides is as follows:

[0081] The glyceride sample is detected by liquid chromatography separation-primary mass spectrometry to obtain a primary mass spectrum, and the primary mass spectrum is analyzed to obtain a molecular mass-to-charge ratio Y of the lipid primary mass spectrum;

[0082] Based on the molecular composition database, the analysis mode is selected as positive ion and adduct ion, and the theoretical molecular mass-to-charge ratio of the glyceride molecule detected in the first-stage mass spectrometry full scan mode is obtained as [M+X], wherein M is the molecular weight of the glyceride and X is the adduct ion;

[0083] The first mass matching tolerance (tol1=0.005) and the first peak finding threshold (th1=40000) are set, and the error Δ=|M+XY| between the molecular mass-to-charge ratio Y and the theoretical molecular mass-to-charge ratio [M+X] is calculated, and the first mass spectrum data is obtained in which the error Δ is less than the first mass matching tolerance tol1 and the peak threshold exceeds the first peak finding threshold th1, and the total composition data of glyceride is obtained according to the first mass spectrum data.

[0084] Further, in step S3, the method of obtaining the chain composition data and chain sn-position of glycerides is as follows:

[0085] The total composition data of the glycerides are detected by liquid chromatography separation-secondary mass spectrometry to obtain lipid chain-level diagnostic ions, that is, actual fragment ion molecular mass-to-charge ratios (a, b, c...);

[0086] Based on the chain composition identification database, the analysis mode is selected as positive ion, adduct ion and fragmentation energy (CID), and the theoretical fragment ion molecular mass-to-charge ratio (m, n, o...) of the glyceride molecule detected in the secondary mass spectrometry full scan mode is obtained;

[0087] The second mass matching tolerance (tol2=0.005) and the second peak search threshold (th2=200) are set, and the error Δ=|ma| between the actual fragment ion molecular mass-to-charge ratio and the theoretical fragment ion molecular mass-to-charge ratio is calculated, and the second mass spectrum data is obtained, and the error is less than the second mass matching tolerance tol2 and the peak threshold exceeds the second peak search threshold th2. According to the second mass spectrum data, the chain composition data of glyceride is obtained. In addition, according to the chain level diagnostic ion ratio, the chain sn-position can be identified.

[0088] Further, in step S4, the method for obtaining the carbon-carbon double bond position information of the glyceride is as follows:

[0089] The chain composition data of the glyceride are detected by using liquid chromatography separation-secondary mass spectrometry and a photochemical derivatization reagent to obtain a photochemical derivatization-secondary mass spectrum, and the molecular mass-to-charge ratio (A, B, C...) of the lipid carbon-carbon double bond diagnostic ion is obtained according to the photochemical derivatization-secondary mass spectrum;

[0090] Based on the carbon-carbon double bond position identification database, the analysis mode is selected as positive ion, adduct ion and fragmentation energy (CID), and the theoretical fragment ion molecular mass-to-charge ratio (M, N, O...) of the glyceride molecule detected in the secondary mass spectrometry mode is obtained;

[0091] The third mass matching tolerance (tol3=0.01) and the third peak finding threshold (th3=100) are set, and the error Δ=|MA| between the molecular mass-to-charge ratio of the lipid carbon-carbon double bond diagnostic ion and the molecular mass-to-charge ratio of the theoretical fragment ion is calculated, and the third mass spectrum data is obtained in which the error is less than the third mass matching tolerance tol3 and whether the peak threshold exceeds the third peak finding threshold th3, and the carbon-carbon double bond position information of glyceride is obtained according to the third mass spectrum data.

[0092] In the above technical solution, when the photochemical derivatization reagent used is an uncharged carbonyl-containing compound, it is mixed in the mobile phase to achieve online derivatization and detection; when the photochemical derivatization reagent is a charged carbonyl-containing compound, it needs to be derivatized offline before detection. In addition, the wavelength of photochemical derivatization is 190-760nm, and the molar ratio of the photochemical derivatization reagent to the glyceride sample can be 1×10 -9 ~1×10 9 ; The light reaction time is 0.1~1200s.

[0093] Furthermore, the basic information of the liquid chromatography column used in the above analysis process includes: pore size: Particle size: 1.7μm, inner diameter × length: 2.1mm × 150mm, packaging quantity: 1 / pk.

[0094] It should be noted that the applicable chromatographic scope of the present invention includes but is not limited to chromatographic analysis methods such as normal phase chromatography (NPLC), reverse phase chromatography (RPLC), ion exchange chromatography (IEC) and supercritical fluid chromatography (SFC).

[0095] The analysis process uses either the automatic mode or the assisted manual mode. In the automatic mode, all operations in the technical solution can be automatically implemented, including the automatic creation and triggering of the full process method of sample detection, to the precise control of the instrument. At the same time, this mode also has the function of automatic data analysis, including automatic comparison of results, in-depth analysis, clear judgment and final output. This series of automated steps ensures the consistency and efficiency of the analysis process.

[0096] The assisted manual mode provides more flexible operation options. It supports the full-process guidance function, can assist in generating the precise method parameters required for secondary mass spectrometry acquisition, and also has the ability to automatically analyze data. However, in this mode, only the creation and triggering of the process method require manual operation. When choosing to use the assisted manual mode, the full-process guidance system will provide detailed key parameter information of the process method, including but not limited to key data such as the precursor ion of the secondary mass spectrometry, which greatly facilitates the operation of staff when creating process methods.

[0097] Embodiment 2

[0098] Based on the same inventive concept as that of Example 1, the present invention also provides a non-targeted analysis system for glyceride structure based on chromatography-mass spectrometry, which is used to implement the steps of the non-targeted analysis method for glyceride structure based on chromatography-mass spectrometry described in Example 1. Figure 3 As shown, the system includes the following modules:

[0099] A database construction module 100 is used to construct a molecular composition database, a chain composition identification database, and a carbon-carbon double bond position identification database respectively;

[0100] The total composition acquisition module 200 is used to perform a primary mass spectrometry test on the glyceride sample to obtain the molecular weight of the substance, and compare the molecular weight of the substance with the lipid molecular weight in the molecular total composition database to obtain the total composition data of the glyceride;

[0101] The chain composition acquisition module 300 is used to perform secondary mass spectrometry detection on the total composition data of the glyceride to obtain lipid chain level diagnostic ions, compare the lipid chain level diagnostic ions with the lipid chain level diagnostic ions in the chain composition identification database, and obtain the chain composition data and chain sn-position of the glyceride;

[0102] The carbon-carbon double bond position information acquisition module 400 is used to perform photochemically derived secondary mass spectrometry analysis on the chain composition data of the glyceride to obtain lipid carbon-carbon double bond diagnostic ions, and compare the lipid carbon-carbon double bond diagnostic ions with lipid carbon-carbon double bond diagnostic ions in the carbon-carbon double bond position identification database to obtain the carbon-carbon double bond position information of the glyceride;

[0103] The application guidance module 500 is used to obtain the non-targeted analysis results of the glyceride structure according to the total composition data, the chain composition data, the chain sn-position and the carbon-carbon double bond position information, and guide the biomedical research process of related components based on the non-targeted analysis results of the glyceride structure.

[0104] A mass spectrometry-based non-targeted analysis system for glyceride structure proposed in this embodiment is used to implement the aforementioned non-targeted analysis method for glyceride structure based on chromatography-mass spectrometry technology. Therefore, the specific implementation method of the synchronous response single-point displacement monitoring system can be seen in the embodiment part of the aforementioned non-targeted analysis method for glyceride structure based on chromatography-mass spectrometry technology. For example, the database construction module 100, the total component acquisition module 200, the chain component acquisition module 300, the carbon-carbon double bond position information acquisition module 400 and the application guidance module 500 are respectively used to correspond to the implementation of steps S1, S2, S3, S4 and S5 in the mass spectrometry-based non-targeted analysis method for glyceride structure in Example 1. Therefore, its specific implementation method can refer to the description of the corresponding each part of the embodiment. In order to avoid redundancy, it will not be repeated here.

[0105] Those skilled in the art will appreciate that the embodiments of the present application may be provided as methods, systems, or computer program products. Therefore, the present application may adopt the form of a complete hardware embodiment, a complete software embodiment, or an embodiment in combination with software and hardware. Moreover, the present application may adopt the form of a computer program product implemented in one or more computer-usable storage media (including but not limited to disk storage, CD-ROM, optical storage, etc.) that include computer-usable program code.

[0106] The present application is described with reference to the flowcharts and / or block diagrams of the methods, devices (systems), and computer program products according to the embodiments of the present application. It should be understood that each process and / or box in the flowchart and / or block diagram, as well as the combination of the processes and / or boxes in the flowchart and / or block diagram, can be implemented by computer program instructions. These computer program instructions can be provided to a processor of a general-purpose computer, a special-purpose computer, an embedded processor, or other programmable data processing device to generate a machine, so that the instructions executed by the processor of the computer or other programmable data processing device generate instructions for implementing the processes in the flowchart and / or block diagram. Figure 1 A process or multiple processes and / or boxes Figure 1 A device that provides the functions specified in a block or multiple blocks.

[0107] These computer program instructions may also be stored in a computer readable memory capable of directing a computer or other programmable data processing device to operate in a specific manner, so that the instructions stored in the computer readable memory produce an article of manufacture including an instruction device, which implements the process Figure 1 A process or multiple processes and / or boxes Figure 1 A function specified in one or more boxes.

[0108] These computer program instructions can also be loaded onto a computer or other programmable data processing device so that a series of operating steps are executed on the computer or other programmable device to produce a computer-implemented process, thereby providing instructions for implementing the process in the computer or other programmable device. Figure 1 A process or multiple processes and / or boxes Figure 1 The steps for the functions specified in one or more boxes.

[0109] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A non-targeted analysis method of glyceride structure based on chromatography-mass spectrometry, characterized in that: The following steps are involved: S1: construct the molecular composition database, chain composition identification database and carbon-carbon double bond position identification database respectively; S2: performing primary mass spectrometry detection on the glyceride sample to obtain the molecular weight of the substance, and comparing the molecular weight of the substance with the lipid molecular weight in the molecular total composition database to obtain the total composition data of the glyceride; S3: performing secondary mass spectrometry detection on the total composition data of the glyceride to obtain lipid chain-level diagnostic ions, and comparing the lipid chain-level diagnostic ions with lipid chain-level diagnostic ions in the chain composition identification database to obtain chain composition data and chain sn-positions of the glyceride; S4: performing photochemically derived secondary mass spectrometry analysis on the chain composition data of the glyceride to obtain lipid carbon-carbon double bond diagnostic ions, and comparing the lipid carbon-carbon double bond diagnostic ions with lipid carbon-carbon double bond diagnostic ions in the carbon-carbon double bond position identification database to obtain carbon-carbon double bond position information of the glyceride; S5: Obtain non-targeted analysis results of glyceride structure according to the total composition data, the chain composition data, the chain sn-position and the carbon-carbon double bond position information, and guide the biomedical research process of related components based on the non-targeted analysis results of glyceride structure.

2. The non-targeted analysis method of glyceride structure based on chromatography-mass spectrometry according to claim 1, characterized in that: In S2, the method for obtaining the total composition data of glycerides is as follows: The glyceride sample is detected by liquid chromatography separation-primary mass spectrometry to obtain a primary mass spectrum, and the primary mass spectrum is analyzed to obtain a molecular mass-to-charge ratio Y of the lipid primary mass spectrum; Based on the molecular composition database, the analysis mode is selected as positive ion and adduct ion, and the theoretical molecular mass-to-charge ratio of the glyceride molecule detected in the first-stage mass spectrometry full scan mode is obtained as [M+X], wherein M is the molecular weight of the glyceride and X is the adduct ion; A first mass matching tolerance and a first peak finding threshold are set, the error between the molecular mass-to-charge ratio Y and the theoretical molecular mass-to-charge ratio [M+X] is calculated, and first mass spectrum data is obtained in which the error is less than the first mass matching tolerance and the peak threshold exceeds the first peak finding threshold, and the total composition data of glyceride is obtained based on the first mass spectrum data.

3. The non-targeted analysis method of glyceride structure based on chromatography-mass spectrometry according to claim 1, characterized in that: In S3, the method for obtaining the chain composition data and chain sn-position of glycerides is as follows: The total composition data of the glycerides are detected by liquid chromatography separation-secondary mass spectrometry to obtain lipid chain-level diagnostic ions, i.e., actual fragment ion molecular mass-to-charge ratios; Based on the chain composition identification database, the analysis mode is selected as positive ion, adduct ion and fragmentation energy to obtain the theoretical fragment ion molecular mass-to-charge ratio of the glyceride molecule detected in the secondary mass spectrometry full scan mode; A second mass matching tolerance and a second peak finding threshold are set, and the error between the mass-to-charge ratio of the actual fragment ion molecule and the mass-to-charge ratio of the theoretical fragment ion molecule is calculated to obtain second mass spectrum data in which the error is less than the second mass matching tolerance and whether the peak threshold exceeds the second peak finding threshold; and based on the second mass spectrum data, the chain composition data and chain sn-position of the glyceride are obtained.

4. The non-targeted analysis method of glyceride structure based on chromatography-mass spectrometry according to claim 1, characterized in that: In S4, the method for obtaining the carbon-carbon double bond position information of glyceride is as follows: The chain composition data of the glyceride are detected by liquid chromatography separation-photochemical derivatization-secondary mass spectrometry to obtain a photochemical derivatization-secondary mass spectrum, and the molecular mass-to-charge ratio of the lipid carbon-carbon double bond diagnostic ion is obtained according to the photochemical derivatization-secondary mass spectrum; Based on the carbon-carbon double bond position identification database, the analysis mode is selected as positive ion, adduct ion and fragmentation energy to obtain the theoretical fragment ion molecular mass-to-charge ratio of the glyceride molecule detected in the secondary mass spectrometry mode; A third mass matching tolerance and a third peak finding threshold are set, and the error between the molecular mass-to-charge ratio of the lipid carbon-carbon double bond diagnostic ion and the molecular mass-to-charge ratio of the theoretical fragment ion is calculated to obtain third mass spectrum data in which the error is less than the third mass matching tolerance and whether the peak threshold exceeds the third peak finding threshold; based on the third mass spectrum data, the carbon-carbon double bond position information of glyceride is obtained.

5. The non-targeted analysis method of glyceride structure based on chromatography-mass spectrometry according to claim 1, characterized in that: In S1, the method for constructing the molecular composition database is: The molecular formula of the fatty acid chain is defined as C n H (2n+1-2db) , n is the total carbon number of the fatty acid chain, db is the degree of unsaturation of the fatty acid chain; Connecting the glyceride backbone and at least one fatty acid chain, and generating the molecular formula of the substance by calculation; The molecular weight of the corresponding glyceride structure is calculated based on the mass of each element in the molecular formula of the substance.

6. The non-targeted analysis method of glyceride structure based on chromatography-mass spectrometry according to claim 1, characterized in that: In S1, the chain composition identification database includes backbone fragment ion molecular weight information and chain ion molecular weight information, and its construction method is: The molecular weight information of the skeleton fragment ion is expressed as the molecular weight of the glyceride + adduct ion - ammonia molecule - missing chain, i.e., M+X–NH3-C n H (2n-2db) O2; The chain ion molecular weight information is expressed as the missing chain-hydroxyl group, i.e., C n H (2n-2db) O2–OH; Where n is the total number of carbon atoms in the fatty acid chain and db is the number of carbon-carbon double bonds in the fatty acid chain.

7. The non-targeted analysis method of glyceride structure based on chromatography-mass spectrometry according to claim 1, characterized in that: In S1, the carbon-carbon double bond position identification database is established based on a carbonyl-containing photochemical derivatization reagent, wherein the construction method is different depending on whether the photochemical derivatization reagent is charged, as follows: If the photochemical derivatization reagent is an uncharged carbonyl-containing compound, the carbon-carbon double bond position identification database includes molecular weight information of acyl ions after PB reaction and molecular weight information of carbonyl ions after PB reaction; If the photochemical derivatization reagent is a charged carbonyl-containing compound, the carbon-carbon double bond position identification database includes the molecular weight information of the skeleton fragment ions and the molecular weight information of the chain ions where the carbon-carbon double bond is located.

8. The non-targeted analysis method of glyceride structure based on chromatography-mass spectrometry according to claim 7, characterized in that: If the photochemical derivatization reagent is an uncharged carbonyl-containing compound, the molecular weight information of the acyl ion after the PB reaction is expressed as the molecular weight of the glyceride + the adduct ion + the molecular weight of the photochemical derivatization reagent - a randomly lost fatty acid chain - hydroxyl - the chain fragment lost after the double bond of the fatty chain that undergoes the PB reaction is broken, that is, [M+X+photochemical derivatization reagent]-C n H (2n-2db) O2–OH–C n-x H 2(n-x-fdb) ; The molecular weight information of the carbonyl ion after the PB reaction is expressed as the molecular weight of the glyceride + the adduct ion + the molecular weight of the photochemical derivatization reagent - a randomly lost fatty acid chain - hydroxyl - the chain fragment lost after the double bond of the fatty chain that undergoes the PB reaction is broken + methylene, that is, [M+X+photochemical derivatization reagent]-C n H (2n-2db) O2–OH–C n-x H 2(n-x-fdb) +C2H2; Among them, db is the number of carbon-carbon double bonds in the fatty acid chain, n is the total carbon number of the fatty acid chain undergoing PB reaction, x is the position of the carbon-carbon double bond of the fatty chain undergoing PB reaction, and fdb is the unsaturation on the chain fragment lost after the double bond of the fatty chain undergoing PB reaction is broken.

9. The non-targeted analysis method of glyceride structure based on chromatography-mass spectrometry according to claim 7, characterized in that: If the photochemical derivatization reagent is a charged carbonyl-containing compound, the molecular weight information of the skeleton fragment ion is expressed as the molecular weight of the glyceride + the molecular weight of the photochemical derivatization reagent + the adduct ion - the chain fragment lost after the double bond of the fatty chain that undergoes the PB reaction is broken - the oxygen atom, that is, M+PB+XC n-x H 2(n-x-fdb) -O; The molecular weight information of the chain ion where the carbon-carbon double bond is located is expressed as the molecular weight of the photochemical derivatization reagent + the adducted ion + the chain fragment lost after the double bond of the fatty chain in the PB reaction is broken - the oxygen atom, that is, PB+X+C n-x H 2(n-x-fdb) -O; Among them, db is the number of carbon-carbon double bonds in the fatty acid chain, n is the total carbon number of the fatty acid chain undergoing PB reaction, x is the position of the carbon-carbon double bond of the fatty chain undergoing PB reaction, and fdb is the unsaturation on the chain fragment lost after the double bond of the fatty chain undergoing PB reaction is broken.

10. A non-targeted analysis system of glyceride structure based on chromatography-mass spectrometry, characterized in that: The system is used to implement the steps of the non-targeted analysis method of glyceride structure based on chromatography-mass spectrometry technology as described in any one of claims 1 to 9, and comprises the following modules: A database construction module is used to construct a molecular composition database, a chain composition identification database, and a carbon-carbon double bond position identification database respectively; A total composition acquisition module is used to perform a primary mass spectrometry test on the glyceride sample to obtain the molecular weight of the substance, and compare the molecular weight of the substance with the lipid molecular weight in the molecular total composition database to obtain the total composition data of the glyceride; A chain composition acquisition module, for performing secondary mass spectrometry detection on the total composition data of the glyceride to obtain lipid chain level diagnostic ions, and comparing the lipid chain level diagnostic ions with the lipid chain level diagnostic ions in the chain composition identification database to obtain the chain composition data and chain sn-position of the glyceride; A carbon-carbon double bond position information acquisition module is used to perform photochemically derived secondary mass spectrometry analysis on the chain composition data of the glyceride to obtain lipid carbon-carbon double bond diagnostic ions, and compare the lipid carbon-carbon double bond diagnostic ions with lipid carbon-carbon double bond diagnostic ions in the carbon-carbon double bond position identification database to obtain carbon-carbon double bond position information of the glyceride; An application guidance module is used to obtain non-targeted analysis results of glyceride structure based on the total composition data, the chain composition data, the chain sn-position and the carbon-carbon double bond position information, and guide biomedical research of related components based on the non-targeted analysis results of glyceride structure.

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