Method for analyzing fine structure of oxidized triglyceride based on mass spectrum data

Abstract

The invention discloses a method for analyzing a fine structure of oxidized triglyceride based on mass spectrum data, which comprises the following steps: integrating CID and EAD complementary mass spectrum data, collecting four spectrograms of CID MS2 ([M + Na] < + >), CID MS2 ([M + NH4] < + >), EAD MS2 ([M + Na] < + >) and EAD MS2 ([M + NH4] < + >) of components, strictly arranging the spectrograms according to the sequence, designing a new prediction-hit strategy, and gradually deducing, analyzing and verifying the structure. The strategy successfully identifies fine structures of oxidized triglycerides such as hydroxyl, ketone group, epoxy group and peroxy group, and more completely describes information such as types, quantity and positions of oxidized groups, positions of unsaturated double bonds, sn distribution of fatty acid and the like. By performing cross validation on the information of each region in the four spectrograms of each substance, the method remarkably improves the identification accuracy.

Description

Technical Field

[0001] The invention belongs to the field of food detection, and in particular relates to a method for analyzing the fine structure of oxidized triglycerides based on mass spectrometry data. Background Art

[0002] Edible oils are prone to oxidation during heating or long-term storage, generating a variety of oxidation products, including oxidized triglycerides containing oxidized groups such as epoxy, hydroxyl, and aldehyde groups. These products have high reactivity in the body, and long-term or high-dose intake may pose multiple risks to health. First, some oxidized triglycerides exhibit strong inflammatory and cytotoxic properties, which may induce oxidative stress and damage cell membranes and their functional structures. This process may cause cell apoptosis or necrosis, thereby increasing the risk of chronic diseases. In addition, the decomposition products of oxidized triglycerides, such as aldehyde compounds, may react with proteins and nucleic acids in the body to form covalent adducts, potentially inducing chronic diseases such as cancer and cardiovascular disease. Therefore, in-depth research on the fine structure and generation mechanism of oxidized triglycerides is of great significance for assessing their health risks and guiding the safe use of edible oils.

[0003] There are many methods for detecting oxidized triglycerides, each with its own characteristics and limitations. Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy preliminarily determine the presence of oxidized groups in oxidized triglycerides by detecting infrared absorption or Raman scattering of specific functional groups. However, due to the low resolution, these methods cannot provide specific structural information and are difficult to meet the needs of accurate analysis of complex oxides. Gas chromatography-mass spectrometry (GC-MS) indirectly analyzes oxidized triglycerides by separating and identifying the decomposition products of oxidized triglycerides (such as aldehydes, ketone fatty acids, etc.), but this method is difficult to directly analyze the complete structure of oxidized triglycerides and has limited applicability to non-volatile or thermally unstable oxidation products. Liquid chromatography-mass spectrometry (LC-MS) based on collision-induced dissociation (CID) is suitable for analyzing the complete molecular structure of oxidized triglycerides, and can detect a few oxidation sites and groups (such as epoxides, peroxides, etc.). It is a common method for studying the structural changes of oxidized triglycerides. However, in actual samples, the fine structural analysis of oxidized triglycerides, such as the type, number, and SN distribution of oxidized groups, still faces certain challenges.

[0004] In practical applications, the secondary mass spectra obtained in EAD mode are accompanied by many interfering fragments, especially [M+H-FA] + The fragments produced by further cleavage of unstable oxidized fatty acids can lead to structural misjudgment (such as the unstable diagnostic ion of the sn-2 fatty acid) and false positive problems (such as when the m / z values ​​of two fatty acids differ by 16Da or 18Da, they will be mistakenly judged as the coexistence of multiple oxidized triglycerides).

[0005] Different from the previous methods, the present invention integrates the complementary mass spectrometry data of CID and EAD to collect the CIDMS2 ([M+Na] + )、CID MS2([M+NH4] + )、EAD MS2([M+Na] + ) and EAD MS2([M+NH4] + ) and arranged the spectra strictly in this order, and designed a new prediction-hit strategy to gradually deduce, analyze and verify the structure. This strategy successfully identified the fine structure of oxidized triglycerides such as hydroxyl, keto, epoxy and peroxy groups, and more completely described the type, number, position, position of unsaturated double bonds and sn distribution of fatty acids of the oxidized groups. By cross-validating the information of each region in the four spectra of each substance, this method significantly improved the accuracy of identification. This new method not only provides new application potential for the study of oxidized triglycerides, but also is expected to promote technological progress in the field of lipidomics. Summary of the invention

[0006] The purpose of this section is to provide a method for analyzing the fine structure of oxidized triglycerides based on mass spectrometry data and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the present application to avoid blurring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In view of the structural misjudgment and false positive problems existing in the mass spectrometry data processing process of identifying the structure of oxidized triglycerides by the above-mentioned and / or existing methods, the present invention is proposed.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] (1) A total of 4 batches of parallel experiments were performed to obtain LC-MS data (CID and EAD modes); (2) preliminary processing of mass spectrometry data to obtain a list of screened precursor ions; (3) collection of MS2 spectra of sodium and ammonium adduct fragmentation of individual components; (4) analysis of CID MS2 ([M+Na] + ) spectrum; (5) analysis of CID MS2 ([M+NH4] + ) spectrum; (6) analysis of EAD MS2 ([M+Na] + ) spectrum; (7) analysis of EADMS2([M+NH4] + ) spectrum; (8) complete the fine structure analysis of this oxidized triglyceride; (9) repeat steps (3) to (8) to obtain a list of structures of all components.

[0010] (1) A total of 4 batches of experiments were performed to obtain LC-MS data (CID and EAD modes):

[0011] Dissolve 10 mg of oil sample in 10 mL of isopropanol, vortex to dissolve, filter with filter membrane and then inject.

[0012] The lipid samples were analyzed and data was collected by LC-MS. The m / z values ​​were identified by high-resolution instruments and the accurate molecular formula of the ions was calculated for subsequent structural analysis.

[0013] The complementarity of the CID and EAD modes that fragment the parent ion in mass spectrometry lies in the different fragmentation principles. CID technology is based on gas-mediated fragmentation technology. By controlling the flow rate and movement speed of the gas, the gas collides with the compound molecules to cause fragmentation; EAD technology is based on electron-mediated fragmentation technology. By controlling the density and movement energy of the electron beam, the electrons combine with the compound molecules to induce their fragmentation. Because the fragmentation principles of EAD and CID are different, their fragments are also different, and the two are complementary in MS2 information.

[0014] The analysis was performed using a SCIEX ZenoTOF 7600 instrument. The HPLC was performed using a Kinetex XB-C18 column (100 mm × 4.6 mm, 2.6 μm; Phenomenex) for separation of oxidized triglycerides using gradient elution. Mobile phase A consisted of methanol, acetonitrile, and water (1:1:3, v / v / v), and mobile phase B was isopropanol. The elution gradient was: 0-0.5 min, 20% B; 0.5-1.5 min, 20% B to 40% B; 1.5-3.0 min, 40% B to 60% B; 3.0-14.0 min, 60% B to 98% B; 14.0-14.1 min, 98% B to 20% B. The electrospray ionization parameters were set as follows: ion source temperature 550 °C, spray voltage 5500 V, curtain gas 35 psi, nebulizer gas 50 psi, auxiliary gas 55 psi, and declustering potential 60 V. In TOF MS mode, the accumulation time was 150 ms and the mass range was m / z 50-1200. For MS / MS acquisition, information-dependent acquisition was used for 10 cycles with an accumulation time of 35 ms and a mass range of m / z 50-1200. The collision energy for CID was 40 eV, while the collision energy for EAD mode was 12 eV. The structural identification of oxidized triglycerides was performed using SCIEX OS 3.0 software, and the molecular formula of the compound was determined by mass accuracy (error <10 ppm) and isotope pattern deviation (<10%).

[0015] The energy of the CID mode is set to 40 eV, and the energy of the EAD mode is set to 12 eV. This energy range can retain an appropriate amount of parent ions in the MS2 spectrum while ensuring sufficient abundance of fragment ions.

[0016] Data acquisition of LC-MS data (CID and EAD modes) was performed for a total of 4 batches of experiments.

[0017] Experiment No. Buffer salt added to mobile phase A and B MS2 fragmentation pattern 1 Sodium formate (5 μmol / L) CID 2 Ammonium acetate (5mmol / L) CID 3 Sodium formate (5 μmol / L) EAD 4 Ammonium acetate (5mmol / L) EAD

[0018] (2) Preliminary processing of mass spectrometry data to obtain a list of screened precursor ions

[0019] S1. Use the SCIEX OS software equipped with the instrument to extract all mass spectrometric features with a signal-to-noise ratio (S / N) greater than 3 and obtain a list of parent ions, including retention time and accurate m / z values:

[0020] S2. Compare the parent ion lists obtained from the above LC-MS experiments 1 and 2, and compare the parent ion lists obtained from the above LC-MS experiments 3 and 4, and look for parent ion pairs with similar retention times (deviation <0.5min) and a precise m / z value difference of 4.9553951484Da, to obtain a screened parent ion list that can simultaneously form sodium adducts and ammonium adducts.

[0021] This m / z difference is calculated as follows, [M+Na] + That is, M+22.98976928, [M+NH4] + That is, M+18.0343741316, 22.98976928-18.0343741316=4.9553951484).

[0022] S3. Use the Formula plug-in of the SCIEX OS software equipped with the instrument to calculate the molecular formula of the compound based on the accurate m / z value of the above parent ion, which must meet the C x H y O 7-12 The final candidate list of precursor ions of oxidized triglycerides is obtained.

[0023] (3) Collect MS2 spectra of sodium and ammonium adduct fragmentation of individual components

[0024] Using the SCIEX OS software equipped with the instrument, the four spectra corresponding to each component are extracted as follows:

[0025] S1、CID MS2([M+Na] + ) spectrum, taken from LC-MS experiment 1.

[0026] S2、CID MS2([M+NH4] + ) spectrum, taken from LC-MS experiment 2.

[0027] S3, EAD MS2 ([M+Na] +) spectrum, taken from LC-MS experiment 3.

[0028] S4, EAD MS2 ([M+NH4] + ) spectrum, taken from LC-MS experiment 4.

[0029] (4) Analysis of CID MS2 ([M+Na] + ) Spectrum

[0030] S1. Collect the fragment ions with the highest abundance in the middle area of ​​the MS2 spectrum (near the m / z value after the parent ion loses one fatty acid (FA)) (sort all fragment ions from high to low according to the detected abundance. If the abundance of adjacent fragment ions differs by more than 3 times, stop obtaining the fragment ions with lower abundance).

[0031] S2. Among these fragment ions, search for ion pairs with a difference of 21.98194425Da, among which the ion with the larger m / z value is [M+Na-FA] + , the ion with a smaller m / z value is [M+H-FA] + .

[0032] S3, according to the above [M+H-FA] + The fragment ions of , calculated to obtain a list of all candidate fatty acids, based on which [M+Na] + The predicted table of relevant fatty acid fragment ions under CID fragmentation conditions is as follows:

[0033] <![CDATA[FA-OH-H2O]]> FA-OH FA+Na FA(m / z) FA-35 FA-17 FA+23 282 247 265 305 … … … …

[0034] Taking the fatty acid of m / z 282 as an example, the predicted fragment ions related to this fatty acid include m / z 247, m / z 265 and m / z 305, and the prediction tables for other fatty acids are similar.

[0035] S4. In the m / z 100-400 region of the MS2 spectrum, the hits in the above fatty acid prediction table are verified with the fragment ions actually detected. This is one of the manifestations of the prediction-hit strategy. If more than two fragment ions in the above fatty acid prediction table are hit, it means that the fatty acid does exist. Repeat the above fatty acid prediction-hit steps to obtain n fatty acids present in the spectrum.

[0036] S5. Select 3 fatty acids from n fatty acids. A single fatty acid can be repeated up to 3 times. List all combinations.

[0037] S6. Find all fatty acid combinations that meet the formula m / z(FA1)+m / z(FA2)+m / z(FA3)+92-54=m / z(M), thereby obtaining the fatty acid composition of the component as TG FA1_FA2_FA3.

[0038] S7. Summarize the above information, and obtain the precise m / z values ​​of the three fatty acids connected to glycerol based on the spectrum, and calculate the molecular formulas of the three fatty acids based on the precise m / z values.

[0039] (5) Analysis of CID MS2 ([M+NH4] + ) Spectrum

[0040] S1. According to the FA obtained in step (4) above, a list of all candidate fatty acids is calculated, and based on this, [M+NH4] is constructed. + The predicted table of relevant fatty acid fragment ions under CID fragmentation conditions is as follows:

[0041] <![CDATA[FA-OH-H2O]]> FA-OH <![CDATA[FA-OH+C3H6O2-H2O]]> <![CDATA[FA-OH+C3H6O2]]> FA(m / z) FA-35 FA-17 FA+39 FA+57 282 247 265 321 339 … … … … …

[0042] Taking the fatty acid of m / z 282 as an example, the predicted fragment ions related to this fatty acid include m / z 247, m / z 265, m / z 321 and m / z 339, and the prediction tables for other fatty acids are similar.

[0043] S2, in the m / z 100-400 region of the MS2 spectrum, the hits in the above fatty acid prediction table are verified again with the fragment ions actually detected, which is the application of the prediction-hit strategy again. If more than two fragment ions in the above fatty acid prediction table are hit, it means that the fatty acid does exist. Thus, the verification of the analytical results of the above step (4) is completed from another perspective.

[0044] S3, [M+NH4] in MS2 spectrum + In the vicinity of the parent ion, look for [M+H] + 、[M+H-H2O] + 、[M+H-2H2O] + and [M+H-3H2O] + The derivative fragment ions of , thus losing ammonium and continuously losing water, confirmed the existence of triglyceride structure.

[0045] Special cases that require attention: if a high abundance of [M+H-H2O2] is detected in this area + Fragment ions indicate the presence of peroxide groups, while other oxidized groups have no characteristics here.

[0046] S4, in the middle area of ​​the MS2 spectrum (near the m / z value after the parent ion loses one fatty acid (FA), i.e. [M+H-FA]+ ) and collect the fragment ions with the highest abundance (sort all the fragment ions from high to low according to the detected abundance. If the abundance of adjacent fragment ions differs by more than 3 times, stop obtaining the fragment ions with lower abundance).

[0047] S5. These fragment ions are divided into two categories: [M+H-FA] + Main fragment ions and derived fragment ions. First, based on the FA obtained in step (4) above, calculate and confirm [M+H-FA] + The main fragment ion is the main fragment ion, and the others are determined as derivative fragment ions; then the type of oxidation group is confirmed based on the dehydration and oxygen loss of the main fragment ion and the relative abundance of the derivative fragment ions. The judgment rules are as follows:

[0048]

[0049] Note: If the keto group is at the end of the fatty acid, it can also be called an aldehyde group.

[0050] S6. In addition to the above single fatty acid related fragment ion region, parent ion region and fragment ion region with one fatty acid missing, if a difference of 15.9949Da is found in other regions, that is, a difference of one oxygen atom, it indicates the presence of peroxy and epoxy groups. In the cleavage process of CID mode, the peroxy group loses one water to form an epoxy group, so the diagnosis process of peroxy and epoxy groups is partially similar. Further, based on the m / z value of the characteristic ion pair with a difference of one oxygen atom, combined with the information obtained in other regions, the exact position of the peroxy or epoxy group on the fatty acid can be diagnosed.

[0051] S7. Summarizing the above information, the type of oxidation group (hydroxyl, keto, epoxy and peroxy) of the oxidized fatty acid to which the oxidized triglyceride is attached can be determined based on this spectrum. For peroxy and epoxy groups, the exact location of the peroxy or epoxy group on the fatty acid can be diagnosed based on the characteristic ion pairs that differ by one oxygen atom. However, the above two spectra still cannot provide information on the distribution of the double bonds and sn positions of hydroxyl, keto and fatty acids.

[0052] (6) Analysis of EAD MS2 ([M+Na] + ) Spectrum

[0053] S1. According to the FA obtained in step (5) above, a list of all candidate fatty acids is calculated, and based on this, [M+Na] is constructed. + The predicted table of related fatty acid fragment ions under EAD fragmentation conditions is as follows:

[0054]

[0055]

[0056] As shown in the table above, the glycerol of triglycerides is connected to three fatty acids. Depending on whether the fatty acids are the same, a total of 6 situations are listed. A, B and C represent different fatty acids. In this format, the sn-2 fatty acid is specially marked and represented by a double slash symbol.

[0057] The fatty acid-related fragment ions, the inventors have found through extensive experiments that oxidized triglycerides are only present in [M+Na] + The fragmentation pattern of [M+NH4] generates specific fragments indicating the distribution of fatty acids at the sn-2 position or sn-1 / 3 position of glycerol. + There are no such specific fragments in the fragmentation spectrum of [M+Na] + In the fragmentation spectrum, [FA+Na+C2H2] + Used to indicate the sn-2 fatty acid, [FA+Na+C3H4] + and [FA+Na+C2H2O] + Used to indicate sn-1 / 3 fatty acids.

[0058] S2. In the m / z 100-400 region of the MS2 spectrum, the hits in the above fatty acid prediction table are verified again with the actually detected fragment ions. If the predicted fragment actually exists, a check mark is marked; if the predicted fragment does not actually exist, a cross is marked. All information is summarized, and based on the above table, it can be specifically determined which fatty acid is located at the sn-2 position of the triglyceride.

[0059] The nomenclature of oxidized triglycerides mainly follows the LipidMaps classification system. OOH, OH, oxo and Ep represent the oxidized groups of peroxy, hydroxyl, keto and epoxy groups, respectively. For example, TG 18:1_ / 9,10-Ep-18:0 / _18:2 indicates that the oxidized fatty acid with epoxy groups at the C9 and C10 positions is located at the sn-2 position of the triglyceride.

[0060] S3, [M+Na-FA] in MS2 spectrum + To the region of the parent ion, first determine the position of the carbon-carbon double bond according to the rule that "the carbon-carbon single bond is broken but the carbon-carbon double bond is not broken", then look for the diagnostic ion and identify the type and position of the oxidized group according to the following rules:

[0061]

[0062] (7) Analysis of EAD MS2 ([M+NH4] + ) Spectrum

[0063] S1. According to the FA obtained in step (6) above, a list of all candidate fatty acids is calculated, and based on this, [M+NH4] is constructed. +The predicted table of related fatty acid fragment ions under EAD fragmentation conditions is as follows:

[0064] <![CDATA[FA-OH-H2O]]> FA-OH <![CDATA[FA-OH+C3H6O2-H2O]]> <![CDATA[FA-OH+C3H6O2]]> FA(m / z) FA-35 FA-17 FA+39 FA+57 282 247 265 321 339 … … … … …

[0065] Taking the fatty acid of m / z 282 as an example, the predicted fragment ions related to this fatty acid include m / z 247, m / z 265, m / z 321 and m / z 339, and the predicted tables for other fatty acids are similar.

[0066] S2, in the m / z 100-400 region of the MS2 spectrum, the hits in the above-mentioned fatty acid prediction table are verified again with the fragment ions actually detected, which is the application of the prediction-hit strategy again. If more than two fragment ions in the above-mentioned fatty acid prediction table are hit, it means that the fatty acid does exist. Thus, the verification of the analytical results of the above-mentioned step (6) is completed from another angle.

[0067] S3, [M+NH4] in MS2 spectrum + In the vicinity of the parent ion, look for [M+H] + 、[M+H-H2O] + 、[M+H-2H2O] + and [M+H-3H2O] + The derivative fragment ions of , thus losing ammonium and continuously losing water, confirmed the existence of triglyceride structure.

[0068] Special cases that require attention: if a high abundance of [M+H-H2O2] is detected in this area + Fragment ions indicate the presence of peroxide groups, while other oxidized groups have no characteristics here.

[0069] S4, [M+NH4-FA] in MS2 spectrum + To the region of the parent ion, first determine the position of the carbon-carbon double bond according to the rule that "the carbon-carbon single bond is broken but the carbon-carbon double bond is not broken", then look for the diagnostic ion and identify the type and position of the oxidized group according to the following rules:

[0070]

[0071] Different from the [M+Na] in step (6) above + The identification process in step (7) is based on [M+NH4] + The same oxidation group type and position are verified through the fragmentation process of different adducts, which has the effect of cross-verification.

[0072] (8) Complete the detailed structural analysis of this oxidized triglyceride

[0073] The information of the above four spectra is summarized and analyzed step by step to obtain the fine structure of this component.

[0074] For example, TG 10-oxo-10:1(Δ8)_ / 18:1(Δ9) / _9,10-Ep-18:0 contains three fatty acids, 10-oxo-10:1(Δ8), 18:1(Δ9) and 9,10-Ep-18:0, among which 18:1(Δ9) is located at the sn-2 position of the triglyceride, while the two fatty acids 10-oxo-10:1(Δ8) and 9,10-Ep-18:0 are located at the sn-1 / 3 position of the triglyceride; 10-oxo-10:1(Δ8) indicates that the keto group is located at the C10 position, containing 10 carbons and 1 carbon-carbon unsaturated double bond (the double bond is located at the C8 position); 18:1(Δ9) indicates oleic acid, containing 18 carbons and 1 carbon-carbon unsaturated double bond (the double bond is located at the C9 position); 9,10-Ep-18:0 indicates that the epoxy group is located at the C9 and C10 positions.

[0075] (9) Repeat steps (3) to (8) to obtain a list of structures of all components.

[0076] Based on the parent ion list obtained in step (2), steps (3) to (8) are repeated to complete the fine structure analysis of each component and finally obtain the structure list of all components.

[0077] The beneficial effects of the present invention are:

[0078] (1) Different from using only CID MS2 ([M+NH4] + ) and EAD MS2([M+NH4] + ) two spectra, the present invention integrates the complementary mass spectrometry data of CID and EAD, and collects the CID MS2 ([M+Na] + )、CID MS2([M+NH4] + )、EAD MS2([M+Na] + ) and EAD MS2([M+NH4] + ) were arranged strictly in this order (the analysis order could not be changed, otherwise false positives might result), and a new prediction-hit strategy was designed to gradually deduce, analyze and verify the structure, effectively avoiding the false positive problem of misjudging the coexistence of multiple oxidized triglycerides when the m / z values ​​of two fatty acids differed by 16Da or 18Da.

[0079] (2) Establish a new fatty acid-related prediction table and evaluate the hits of the predicted fragment ions in the actual spectra, thereby cross-confirming the specific composition of fatty acids across multiple spectra.

[0080] (3) A new determination table for sn-2 fatty acids was established to evaluate the hits of the predicted fragment ions in the actual spectra, thereby cross-confirming the distribution of fatty acids at the sn-2 and sn-1 / 3 positions.

[0081] (4) Innovatively construct a prediction-hit strategy. The specific operation is to establish a prediction table based on existing information and theoretical knowledge, then analyze the hit status of the experimental data of the actual mass spectrum in the table, and finally observe the global distribution of fragment ions in the table to comprehensively analyze the fatty acid type of triglycerides, the distribution of fatty acids at the sn-2 position and sn-1 / 3 position of glycerol, and the position of unsaturated double bonds. This method of analyzing mass spectra under the macro-guidance of the prediction table can significantly reduce the subjective errors or misjudgments caused by manual search. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0083] Figure 1 It is a schematic diagram of the technical solution flow of the present invention;

[0084] Figure 2 The present invention is used to identify the co-eluting components of TG 10-oxo-10:1(Δ8)_ / 18:1(Δ9) / _9,10-Ep-18:0 and TG18:1(Δ9)_ / 10-oxo-10:1(Δ8) / _9,10-Ep-18:0;

[0085] Figure 3 The identification process of TG 18:1(Δ9)_ / 18:1(Δ9) / _9-OH-18:1(Δ10) was carried out by applying the method of the present invention;

[0086] Figure 4 The identification process of TG 18:1(Δ9)_ / 18:1(Δ9) / _10-oxo-18:0 using the method of the present invention;

[0087] Figure 5 The identification process of TG 18:1(Δ9)_ / 18:1(Δ9) / _9,10-Ep-18:0 using the method of the present invention;

[0088] Figure 6 The present invention is used to identify TG 18:1(Δ9)_ / 18:1(Δ9) / _11-OOH-18:1(Δ9). DETAILED DESCRIPTION

[0089] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with specific embodiments.

[0090] Example:

[0091] High oleic sunflower oil fried for 12 hours was used as the sample.

[0092] (1) A total of 4 batches of experiments were performed to obtain LC-MS data (CID and EAD modes); (2) preliminary processing of mass spectrometry data to obtain a list of screened precursor ions; (3) collection of MS2 spectra of sodium and ammonium adduct fragmentation of individual components; (4) analysis of CID MS2 ([M+Na] + ) spectrum; (5) analysis of CID MS2 ([M+NH4] + ) spectrum; (6) analysis of EAD MS2 ([M+Na] + ) spectrum; (7) analysis of EAD MS2 ([M+NH4] + ) spectrum; (8) complete the fine structure analysis of this oxidized triglyceride; (9) repeat steps (3) to (8) to obtain a list of structures of all components.

[0093] Figure 2 The identification process of the co-eluting components of TG 10-oxo-10:1(Δ8)_ / 18:1(Δ9) / _9,10-Ep-18:0 and TG18:1(Δ9)_ / 10-oxo-10:1(Δ8) / _9,10-Ep-18:0 using the method of the present invention is as follows:

[0094] Figure 2 (A) shows m / z 825.6212 (RT 13.29 min, [M+Na] + , CID 40eV) parent ion MS2 spectrum. In the area near the parent ion, the oxidized TG parent ion did not undergo significant dehydration, indicating that it has high stability as a sodium adduct. This stability effectively avoids water loss and the generation of interfering ions, thereby supporting the accurate identification of normal or oxidized fatty acids. In the fragment ion region where a fatty acid was lost, highly abundant fragment ions of m / z 641.5095, 619.5276, 543.3647, 521.3848, 527.3680 and 505.3875 were observed. Analysis showed that the 22Da difference between these fragment ions suggested [M+Na-FA] + With [M+H-FA] +Based on the m / z values ​​of 641.5095, 543.3647, and 527.3680, the molecular weights of neutral FA were determined to be 184, 282, and 298 Da, respectively, corresponding to the molecular formula C 10 H 16 O2、C 18 H 34 O2 and C 18 H 36 O3. In the region of individual fatty acid-related fragment ions, fragment ions at m / z 207, 265, and 321 further confirmed the presence of these fatty acids, consistent with previous findings. No additional significant information was provided in other regions. Overall, [M+Na] + The MS2 spectrum in CID mode supported the compound as TG 184_282_298.

[0095] Figure 2 (B) shows m / z 820.6646 (RT 13.29 min, [M+NH4] + , CID 40eV) parent ion MS2 spectrum. In this analysis, ammonium adducts become the main form, which is mainly attributed to the addition of ammonium acetate in the mobile phase. In the area near the parent ion, continuous dehydration events were observed, indicating that the component structure contains oxidized groups and ester bonds. These dehydration phenomena are attributed to the presence of oxidized groups and ester bonds, further supporting the structural characteristics of oxidized triglycerides. In the fragment ion region where a fatty acid is lost, mass spectrometry reveals fragment ions corresponding to the loss of three FAs, which appear at m / z619.5292, 521.3882 and 505.3890, respectively, supporting the previous discovery of sodium adducts. In addition, additional derivative peaks were detected at m / z603.5399, 601.5184 and 503.3737. Further analysis showed that the fatty acid with a molecular weight of 298Da suffered from the loss of water and oxygen, while the fatty acids with molecular weights of 184Da and 282Da did not show such losses. Through molecular formula calculation and the judgment table described above, it is speculated that FA with a molecular weight of 184Da may contain a keto group, while FA with a molecular weight of 298Da contains an epoxy group. In the region of single fatty acid-related fragment ions, the observed ions successfully match the theoretical values ​​of fatty acids. In addition, a difference of 16Da appeared between the paired ions in other regions (such as m / z 677.4949 and 661.5049, m / z 493.3899 and 477.3945, m / z 395.2427 and 379.2476), further confirming that the epoxy group of the fatty acid with a molecular weight of 298Da is located on the 9th and 10th carbons. Overall, [M+NH4] +The MS2 spectrum of the ion in CID mode confirmed that the compound was TG 184(1OXO)_282_298(9,10-Ep).

[0096] Figure 2 (C) shows m / z 825.6212 (RT 13.29 min, [M+Na] + , EAD 12eV) parent ion MS2 spectrum. This analysis takes advantage of the stability of triglyceride sodium adducts. First, the molecular formulas of the high-abundance fragment ions in the range of m / z 527 to 825 were calculated and classified based on the number of oxygen atoms. The analysis started with fragmentation of the parent ion, gradually removing the FA unit. Fragment ions containing eight oxygen atoms were identified in the range of m / z 543.3657 to 825.6215; subsequently, fragment ions containing seven oxygen atoms were divided into two groups: m / z 642.4840 to 825.6215 and m / z 527.3703 to 825.6215. The consecutive fragment ions at m / z 670.4838, 683.4827, 699.4794 and 711.4740 indicated the presence of epoxy groups located on carbons 9 and 10, further confirming this structural feature. In addition, the significant fragment ion at m / z 796.6316 supports the judgment that the fatty acid with a molecular weight of 184Da has a keto group at the C10 position. In the region of individual fatty acid-related fragment ions, a table was constructed to match the observed fragment ions, especially the ions associated with FAs with molecular weights of 184Da, 282Da, and 298Da. The fragment ion at m / z 233 indicates that the fatty acid with a molecular weight of 184Da is located at the sn-2 position, while the fragment at m / z 311 indicates that the fatty acid with a molecular weight of 282Da is also located at the sn-2 position. By comparing the intensities of the fragment ions at m / z 347 and 345, the possibility that the fatty acid with a molecular weight of 298Da is located at the sn-2 position can be ruled out. Overall, [M+Na] + The MS2 spectrum of the ions in EAD mode confirmed that they were co-eluting components of TG 10-oxo-10:1(Δ8)_ / 18:1(Δ9) / _9,10-Ep-18:0 and TG 18:1(Δ9)_ / 10-oxo-10:1(Δ8) / _9,10-Ep-18:0.

[0097] Figure 2 (D) shows m / z 820.6646 (RT 13.29 min, [M+NH4] +, EAD 12eV) parent ion MS2 spectrum. The spectrum reconfirmed the type and position of the oxidized groups through diagnostic ions. Specifically, the peaks at m / z 665.5331, 678.5327, 694.5203 ​​and 706.5303 indicate the presence of epoxy groups at carbon positions 9 and 10. In addition, the high intensity ion at m / z 791.6634 further confirmed the presence of a keto group at the C10 position. Overall, this spectrum reconfirmed the type and position of the key oxidized groups within the TG molecule.

[0098] Following a similar approach, Figure 3 , Figure 4 , Figure 5 and Figure 6 The method of the present invention was used to complete the identification process of TG 18:1(Δ9)_ / 18:1(Δ9) / _9-OH-18:1(Δ10), TG 18:1(Δ9)_ / 18:1(Δ9) / _10-oxo-18:0, TG 18:1(Δ9)_ / 18:1(Δ9) / _9,10-Ep-18:0 and TG 18:1(Δ9)_ / 18:1(Δ9) / _11-OOH-18:1(Δ9).

[0099] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for analyzing the fine structure of oxidized triglycerides based on mass spectrometry data, characterized in that: It consists of the following steps: (1) Divide the oil sample to be tested into 4 groups of parallel experiments, and obtain LC-MS data in CID and EAD modes respectively; (2) Preprocess the mass spectrometry data to obtain a list of screened precursor ions; (3) Collect the MS2 spectra of the sodium and ammonium adducts of individual components; (4) Analysis of CID MS2 ([M+Na] + ) spectrum; (5) Analysis of CID MS2 ([M+NH4] + ) spectrum; (6) Analysis of EAD MS2 ([M+Na] + ) spectrum; (7) Analysis of EAD MS2 ([M+NH4] + ) spectrum; (8) Complete the fine structure analysis of the oxidized triglycerides in the oil sample; (9) Repeat steps (3) to (8) to obtain the structure lists of all components.

2. The method for analyzing the fine structure of oxidized triglycerides based on mass spectrometry data according to claim 1, characterized in that: In step (1), dissolve the oil sample to be tested in isopropanol. Use a Kinetex XB-C18 column for chromatography. The mobile phase A for chromatography consists of methanol, acetonitrile and water in a volume ratio of 1:1:3, and the mobile phase B is isopropanol; in the 4 groups of parallel experiments, 5 μmol / L sodium formate is added to both the mobile phase A and the mobile phase B in experiment 1, 5 mmol / L ammonium acetate is added to both the mobile phase A and the mobile phase B in experiment 2, 5 μmol / L sodium formate is added to both the mobile phase A and the mobile phase B in experiment 3, and 5 mmol / L ammonium acetate is added to both the mobile phase A and the mobile phase B in experiment 4; the elution gradient is: 0 - 0.5 minutes, 20% B; 0.5 - 1.5 minutes, 20% B to 40% B; 1.5 - 3.0 minutes, 40% B to 60% B; 3.0 - 14.0 minutes, 60% B to 98% B; 14.0 - 14.1 minutes, 98% B to 20% B.

3. The method for analyzing the fine structure of oxidized triglycerides based on mass spectrometry data according to claim 2, characterized in that: In step (1), the electrospray ionization parameters are set as follows: the ion source temperature is 550 °C, the spray voltage is 5500 V, the curtain gas is 35 psi, the nebulizing gas is 50 psi, the auxiliary gas is 55 psi, and the declustering potential is 60 V; in the TOF MS mode, the accumulation time is 150 ms, and the mass range is m / z 50 - 1200; information-dependent acquisition is adopted, for 10 cycles, the accumulation time is 35 ms, and the mass range is m / z 50 - 1200; the collision energy for CID is 40 eV, and the collision energy for the EAD mode is 12 eV; the molecular formula of the compound is determined by the mass accuracy and the isotope pattern deviation, where the mass accuracy error < 10 ppm and the isotope pattern deviation < 10%; CID mode data are collected in experiments 1 and 2, and EAD mode data are collected in experiments 3 and 4.

4. The method for analyzing the fine structure of oxidized triglycerides based on mass spectrometry data according to claim 3, characterized in that: In step (2), extract all mass spectrometry features with a signal-to-noise ratio S / N greater than 3 to obtain a list of precursor ions; compare the precursor ion lists of experiments 1 and 2, compare the precursor ion lists of experiments 3 and 4, and find the retention time deviation < 0.5 min and the difference in the exact m / z value The parent ion pair of 4.9553951484Da was used to obtain the parent ion list that can form sodium adduct and ammonium adduct at the same time, and the molecular formula of the compound was calculated. x H y O 7-12 The required precursor ion list is used to obtain the final candidate list of precursor ions of oxidized triglycerides.

5. The method for analyzing the fine structure of oxidized triglycerides based on mass spectrometry data according to claim 4, characterized in that: In step (3), CID MS2 ([M+Na] + ) spectrum, and extracted CID MS2 ([M+NH4] + ) spectrum, and extracted EAD MS2 ([M+Na] + ) spectrum, and extracted EAD MS2 ([M+NH4] + ) spectrum.

6. The method for analyzing the fine structure of oxidized triglycerides based on mass spectrometry data according to claim 5, characterized in that: In step (4), the fragment ions with the highest abundance are collected in the middle region of the MS2 spectrum. If the abundance of adjacent fragment ions differs by more than 3 times, the fragment ions with lower abundance are stopped. Among the fragment ions with the highest abundance, ion pairs with a difference of 21.98194425 Da are searched, and the ion with the larger m / z value is [M+Na-FA] + , the ion with a smaller m / z value is [M+H-FA] + , according to the obtained [M+H-FA] + The fragment ions of [M+Na] + A prediction table of fatty acid fragment ions under CID fragmentation conditions; using a prediction-hit strategy: in the m / z 100-400 region of the MS2 spectrum, the hit situation of the above fatty acid prediction table is verified with the actually detected fragment ions, if more than two fragment ions in the prediction table of the fatty acid fragment ions are hit, it means that the fatty acid does exist; repeating the prediction-hit strategy to obtain n fatty acids existing in the MS2 spectrum; selecting 3 of the n fatty acids, looking for all fatty acid combinations that meet the formula of m / z(FA1)+m / z(FA2)+m / z(FA3)+92-54=m / z(M), thereby obtaining the fatty acid composition of the component as TG FA1_FA2_FA3, obtaining the precise m / z values ​​of the three fatty acids connected to glycerol and calculating the three fatty acid molecular formulas.

7. The method for analyzing the fine structure of oxidized triglycerides based on mass spectrometry data according to claim 6, characterized in that: In step (5), according to the fatty acid molecular formula obtained in step (4), [M+NH4] + Under CID fragmentation conditions, the predicted fatty acid fragment ions are used to verify the hits in the predicted fatty acid fragment ions table again in the m / z 100-400 region of the MS2 spectrum with the actually detected fragment ions. If more than two fragment ions in the predicted fatty acid fragment ions table are hit, it means that the fatty acid does exist. In the [M+NH4] + In the vicinity of the parent ion, look for [M+H] + 、[M+H-H2O] + 、[M+H-2H2O] + and [M+H-3H2O] + The derivative fragment ions of triglyceride were confirmed by the loss of ammonium and continuous water loss. The fragment ions with the highest abundance were collected in the middle area of ​​the MS2 spectrum. If the abundance of adjacent fragment ions differed by more than 3 times, the fragment ions with lower abundance were stopped. These fragment ions with the highest abundance were divided into two categories: [M+H-FA] + The main fragment ions and derived fragment ions are calculated and confirmed based on the fatty acid molecular formula obtained in step (4) [M+H-FA] + The main fragment ion and the remaining fragment ions are determined to be derived fragment ions. The type of oxidized group is confirmed according to the dehydration and deoxygenation of the main fragment ion and the relative abundance of the derived fragment ions. Except for the fragment ion region related to a single fatty acid, the parent ion region and the fragment ion region with one fatty acid lost, if ion pairs with a difference of 15.9949Da are found in other regions, it indicates the presence of peroxy or epoxy groups. Further, according to the m / z value of the characteristic ion pair with a difference of one oxygen atom, the exact position of the peroxy or epoxy group on the fatty acid is diagnosed, and the type of oxidized group of the oxidized fatty acid connected to the oxidized triglyceride is determined, including hydroxyl, keto, epoxy or peroxy groups.

8. The method for analyzing the fine structure of oxidized triglycerides based on mass spectrometry data according to claim 7, characterized in that: In step (6), according to step (5), construct [M+Na] + Prediction table of related fatty acid fragment ions under EAD fragmentation conditions, in [M+Na] + In the fragmentation spectrum, [FA+Na+C2H2] + Used to indicate the sn-2 fatty acid, [FA+Na+C3H4] + and [FA+Na+C2H2O] + Used to indicate the sn-1 / 3 fatty acid; in the m / z 100-400 region of the MS2 spectrum, the actual detected fragment ions are used to verify the hits in the fatty acid fragment ion prediction table to specifically determine which fatty acid is located at the sn-2 position of the triglyceride; in the [M+Na-FA] + To the region of the parent ion, the position of the carbon-carbon double bond is first determined according to the rule of "the carbon-carbon single bond is broken but the carbon-carbon double bond is not broken", and then the diagnostic ion is searched to identify the type and position of the oxidized group.

9. The method for analyzing the fine structure of oxidized triglycerides based on mass spectrometry data according to claim 8, characterized in that: In step (7), according to step (6), [M+NH4] + Under EAD fragmentation conditions, the relevant fatty acid fragment ion prediction table is used to verify the hits in the fatty acid fragment ion prediction table in the m / z 100-400 region of the MS2 spectrum with the actual detected fragment ions. If more than two fragment ions in the fatty acid fragment ion prediction table are hit, it means that the fatty acid does exist. + In the vicinity of the parent ion, look for [M+H] + 、[M+H-H2O] + 、[M+H-2H2O] + and [M+H-3H2O] + The derivative fragment ions of the triglyceride structure are confirmed by the loss of ammonium and continuous water loss. If the high abundance [M+H-H2O2] + Fragment ions indicate the presence of peroxide groups; [M+NH4-FA] in the MS2 spectrum + To the region of the parent ion, the position of the carbon-carbon double bond is first determined according to the rule of "the carbon-carbon single bond is broken but the carbon-carbon double bond is not broken", and then the diagnostic ion is searched to identify the type and position of the oxidized group.

10. The method for analyzing the fine structure of oxidized triglycerides based on mass spectrometry data according to any one of claims 1 to 9, characterized in that: In step (8), summarize the spectral information in steps (4) to (7) to obtain the fine structure of the oxidized triglycerides in the oil sample.

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