Method for analyzing co-elution triglyceride isomeride in liquid chromatography by using CID and EAD secondary mass spectrograms

Abstract

The invention discloses a method for analyzing co-elution triglyceride isomeride in liquid chromatography by using CID and EAD secondary mass spectrograms, which fully analyzes main and derivative fragment ions generated in the secondary mass spectrograms of a triglyceride adduct in CID and EAD modes, further integrates the information of each fragment, synergistically analyzes the fine structure of triglyceride, and improves the quality of the triglyceride isomeride. A prediction-hit strategy is innovatively constructed, the types of triglyceride isomers with different fatty acid compositions are analyzed through a CID secondary mass spectrum, the types of triglyceride isomers with different sn position distributions (sn-2 and sn-1 / 3) are analyzed through an EAD secondary mass spectrum, and finally, the relative content of each isomer of triglyceride is calculated through three specific fragments. According to the method, the identification problem of the co-elution peak of the triglyceride isomeride in LC-MS analysis is solved for the first time.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and in particular relates to a method for analyzing co-eluted triglyceride isomers in liquid chromatography by utilizing CID and EAD secondary mass spectrometry. Background Art

[0002] Analysis of triglyceride composition is crucial in a wide range of fields. It helps assess the nutritional value of foods, verifies compliance with food safety standards, influences flavor and texture, and plays a key role in processing and storage stability. By studying triglyceride changes, processing techniques and storage conditions can be optimized, thereby extending the shelf life of foods. Therefore, triglyceride composition analysis is crucial for nutritional assessment, quality control, and product development.

[0003] Liquid chromatography-mass spectrometry (LC-MS) is an advanced technology for analyzing triglyceride composition in oils and fats, offering high separation efficiency, high sensitivity, excellent selectivity, and robust structural identification capabilities. It is widely used in food chemistry. However, a significant challenge in the analysis of triglycerides using LC-MS is the coelution of isomers. Triglyceride isomers share the same molecular formula but differ in fatty acid chain length, number of double bonds, and Sn position distribution. Due to their highly similar physicochemical properties (such as polarity and hydrophobicity), these isomers have similar retention times during liquid chromatography separation, leading to coelution. Furthermore, during mass spectrometry detection, due to the identical molecular weight of the isomers, their mass spectral signals overlap in mass-to-charge ratio (m / z), making it difficult to effectively distinguish these isomers using primary mass spectrometry. Therefore, addressing the coelution of isomers remains a key technical challenge in the analysis of triglyceride composition using LC-MS. Summary of the Invention

[0004] To address the above problems, the present invention proposes a method for analyzing the types and contents of triglyceride isomers that co-elute in liquid chromatography by combining collision-induced dissociation (CID) and electron-activated dissociation (EAD) mass spectrometry. The method first analyzes triglyceride isomers with different fatty acid compositions (such as fatty acid chain length and number of double bonds) using CID mass spectrometry. Then, the fatty acid composition data obtained by gas chromatography-mass spectrometry (GC-MS) are used to confirm the CID results. Finally, triglyceride isomers with different sn position distributions (such as sn-2 and sn-1 / 3) are analyzed using EAD mass spectrometry, and the types and relative contents of each isomer are calculated based on the EAD data. This method effectively solves the problem of co-elution of triglyceride isomers in LC-MS analysis, providing strong support for scientific research.

[0005] The purpose of this section is to provide a method for analyzing the types and contents of co-eluted triglyceride isomers in liquid chromatography using CID and EAD secondary mass spectrometry, and to briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and in the abstract and title of this application to avoid obscuring the purpose of this section, the abstract, and the title of the invention, and such simplifications or omissions are not intended to limit the scope of the invention.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions (see the overall process Figure 1 ):

[0007] ①LC-MS: [M+NH4] of a certain component + MS2 spectrum in CID mode;

[0008] ②CID: triglyceride-related fragments (right area of the figure, with larger m / z values);

[0009] ③CID: fragments with one FA missing (middle area of the figure, medium m / z value);

[0010] ④CID: fragments of a single FA (left area of the figure, with smaller m / z values);

[0011] ⑤GC-MS: FA composition;

[0012] ⑥Mathematical method: reverse calculation of FA combination;

[0013] ⑦LC-MS: [M+Na] of a certain component + MS2 spectrum in EAD mode;

[0014] ⑧EAD: fragments of a single FA (left area of the figure, with smaller m / z values);

[0015] ⑨Mathematical method: Least square method is used to calculate the relative content of each TG;

[0016] ⑩ Repeat the above steps to obtain the specific composition of all components.

[0017] ①LC-MS: [M+NH4] of a certain component + MS2 spectrum in ClD mode;

[0018] a. Perform LC-MS detection and collect [M+NH4] of a component + MS2 spectrum in CID mode

[0019] ②CID: triglyceride-related fragments (right area of the figure, with larger m / z values);

[0020] a. Triglyceride-related fragments. These fragments are located in the right region of the spectrum (larger m / z values) and appear as a series of fragments with higher intensity in this region.

[0021] b. Use the table below to confirm the presence of triglyceride-related continuous fragments. If present, it indicates that the component is likely a triglyceride. If absent, the component is likely not a triglyceride and another component should be selected.

[0022]

[0023]

[0024] Figure 2 These fragments are also displayed in detail.

[0025] c. Based on the precise m / z value of M obtained above, refer to the table below and use the FormulaCalculator component of SCIEX OS software to reverse-calculate the molecular formula of the component with a relative error of less than 20 ppm.

[0026] element Accurate m / z C 12 H 1.0078250319 O 15.994914619 N 14.003074004 Na 22.98976928 K 38.96370648

[0027] c. Based on the above molecular formula C x H y O z , reconfirm that the component is triglyceride, which is required to contain only C, H and O elements, and the unsaturation is greater than or equal to 3. The calculation formula of unsaturation is (2*x+2-y) / 2, so the triglyceride component is marked as TGx: (2*x+2-y) / 2-3, and the formula removes the unsaturation of the three ester bonds.

[0028] For example, TG 54:8 means that the component contains 54 carbon atoms and 8 unsaturated double bonds.

[0029] ③CID: fragments with one FA missing (middle area of the figure, medium m / z value);

[0030] a. A fragment with one FA missing, i.e. [M+H-FA] + These fragments are located in the middle region of the spectrum (medium m / z value) and appear as a series of fragments with higher intensity in this region.

[0031] b. Collect all [M+H-FA] + Fragments: The minimum strength of the fragments must be ≥ the maximum strength * 5%, and they should be sorted according to the following table.

[0032]

[0033] Where [M+H-FA] + The column shows the m / z value of the fragment; the Intensity column shows the intensity of the fragment, and the entire table is sorted from high to low according to the Intensity value; the NL column shows the m / z value of the neutral loss, which is calculated as NL = m / z ([M + H] + )-m / z([M+H-FA] + ), specifically the m / z value of the missing fatty acid; based on this m / z value, the molecular formula of the fatty acid (FA) can be inversely calculated. a H b O c , further marked as Ca: (2*a+2-b) / 2-1.

[0034] For example, C22:6 means that the fatty acid contains 22 carbon atoms and 6 unsaturated double bonds.

[0035] The FA column displays the calculated fatty acid types using this notation.

[0036] c. Figure 2 As shown, the loss of 1 FA not only produces [M+H-FA] + This fragment ion may also have other derivative fragments. Use the table below to identify fragments from the same source and eliminate false positives.

[0037]

[0038] Figure 2 These fragments are also displayed in detail.

[0039] ④CID: fragments of a single FA (left area of the figure, with smaller m / z values);

[0040] a. Fragments of a single FA, i.e. [FA-OH] + These fragments are located in the left area of the spectrum (smaller m / z values) and appear as a series of fragments with higher intensity in this area.

[0041] b. Collect all [FA-OH] + Fragments: The minimum strength of the fragments must be ≥ the maximum strength * 5%, and they should be sorted according to the following table.

[0042] FA <![CDATA[[FA-OH] + ]]> Intensity (high to low sorting)

[0043] The FA column shows the fatty acid (FA) label Ca: (2*a+2-b) / 2-1, the formula removes the unsaturation of one ester bond; where [FA-OH] + The column shows the m / z value of the fragment; the lntensity column shows the intensity of the fragment, and the entire table is sorted from high to low according to the lntensity value;

[0044] c. Figure 2 As shown, a single FA not only produces [FA-OH] + This fragment ion may also have other derivative fragments. Use the table below to identify fragments from the same source and eliminate false positives.

[0045]

[0046]

[0047] Figure 2 These fragments are also displayed in detail.

[0048] d. Based on the two FA correlation tables obtained in ③ and ④ above, sort them from high to low according to the main fragment intensity. After eliminating FAs that cannot exist based on the molecular formula, all FAs are divided into two categories: one category is FAs with high fragment intensity and confirmed existence, which are considered important and must be explained; the other category is FAs with low fragment intensity and possible existence, which are considered alternative FAs.

[0049] ⑤GC-MS: FA composition;

[0050] a. Detect all FA compositions of the sample by gas chromatography-mass spectrometry and compare them with the FA compositions obtained above to further exclude FAs that are unlikely to exist.

[0051] ⑥Mathematical method: reverse calculation of FA combination;

[0052] a. Using the FA molecular weight from the FA list obtained above as the data source (can be repeated), follow the following formula:

[0053] MW FA1 +MWFA2 +MW FA3 +92-54=The molecular weight of the triglyceride is rounded up to obtain all FA combinations (FA1, FA2, FA3) that meet the formula, that is, the three FAs connected to the TG glycerol, expressed as TG FA1_FA2_FA3.

[0054] b. Sort all FA combinations from high to low according to the sum of the strengths of the three FAs:

[0055] TG FA1_FA2_FA3 Total FA intensity (high to low)

[0056] c. Divide all FA combinations into two categories: one is the FA combination with high fragmentation intensity, which is confirmed to exist and is important and must be explained; the other is the FA combination with low fragmentation intensity, which is possible and is used as an alternative.

[0057] During the screening process, the lower limit requires that important FAs appear at least once, and the upper limit requires that the sum of the number of FA appearances be as small as possible.

[0058] ⑦LC-MS: [M+Na] of a certain component + MS2 spectrum in EAD mode;

[0059] a. Perform LC-MS detection and collect [M+Na] of a component. + MS2 spectrum in EAD mode

[0060] ⑧EAD: fragments of a single FA (left area of the figure, with smaller m / z values);

[0061] a. Based on the FA list obtained above, construct the following table:

[0062]

[0063]

[0064] The FA column shows the type of fatty acid; the MW column shows the molecular weight of the fatty acid; +49, s (sn-2) indicates the m / z value of the fragment that should be produced when the fatty acid is attached to the sn-2 position of glycerol, usually appearing as a single peak; +63, dC (sn-1 / 3) and +65, dO (sn-1 / 3) indicate the m / z value of the fragment that should be produced when the fatty acid is attached to the sn-1 / 3 position of glycerol, usually appearing as a double peak;

[0065] b. Analyze the hits of the above fragments in the EAD mode secondary mass spectrum (single F; A related fragments, left area, small m / z value):

[0066] If the fragment exists, circle the number;

[0067] If the fragments are present and the intensity is high, circle the number twice;

[0068] If the fragments have the same m / z value, they are connected by a straight line;

[0069] Based on the above results, the distribution of FA at the sn-2 position and sn-1 / 3 position can be determined according to the logic in the following table:

[0070]

[0071]

[0072] / FA / indicates that the FA is located at the sn-2 position, and it is possible for several isomers to exist simultaneously.

[0073] c. Obtain more detailed TG profiles, including sn distribution isomers;

[0074] Taking TG 54:8 as an example, the analysis results include the following five isomers:

[0075] TG 22:6_ / 16:1 / _16:1

[0076] TG 20:5_ / 16:1 / _18:2

[0077] TG 20:5_ / 18:3 / _16:0

[0078] TG 20:5_ / 16:0 / _18:3

[0079] TG 22:6_ / 16:0 / _16:2

[0080] Among them, ω1, ω2, ω3, ω4 and ω5 represent the relative contents of the five TG components respectively.

[0081] ⑨Mathematical method: Least square method is used to calculate the relative content of each TG;

[0082] a. Collect the fragment intensities associated with each fatty acid, specifically including the intensities of fragment (sn-2)s, fragment (sn-1 / 3)dC, and fragment (sn-1 / 3)dO).

[0083] b. The general formula is as follows:

[0084] ω1+ω2+…+ωn=100%

[0085] i(FAs)=(ωn+…)×0.754

[0086] i(FAdC)=(ωn+…)×1

[0087] i(FAdO)=(ωn+…)×0.529

[0088] S=∑(i 实验 -i 理论 ) 2

[0089] Taking TG 54:8 as an example, the analysis results include the following five isomers:

[0090] TG 22:6_ / 16:1 / _16:1 ω1

[0091] TG 20:5_ / 16:1 / _18:2 ω2

[0092] TG 20:5_ / 18:3 / _16:0 ω3

[0093] TG 20:5_ / 16:0 / _18:3ω4

[0094] TG 22:6 / 16:0 / _16:2 ω5

[0095] It should be noted that the same FA may appear multiple times, in the same or different triglycerides.

[0096] The formula set for the first stage calculation, representing the total content as 100% and the intensity of the sn-2 fatty acids, is as follows:

[0097] ω1+ω2+ω3+ω4+ω5=100%

[0098] i(16:1s)=(ω1+ω2)×0.754

[0099] i(18:3s)=(ω3)×0.754

[0100] i(16:0s)=(ω4+ω5)×0.754

[0101] The formula group for the second stage calculation, which represents the intensity of the sn-1 / 3 fatty acids (including both dC and dO fragments), is as follows:

[0102] i(22:6dC)=(ω1+ω5)×1

[0103] i(20:5dC)=(ω2+ω3+ω4)×1

[0104] i(16:1dC)=(ω1)×1

[0105] i(18:2dC)=(ω2)×1

[0106] i(16:0dC)=(ω3)×1

[0107] i(18:3dC)=(ω4)×1

[0108] i(16:2dC)=(ω5)×1

[0109] i(22:6dO)=(ω1+ω5)×0.529

[0110] i(20:5dO)=(ω2+ω3+ω4)×0.529

[0111] i(16:1dO)=(ω1)×0.529

[0112] i(18:2dO)=(ω2)×0.529

[0113] i(16:0dO)=(ω3)×0.529

[0114] i(18:3dO)=(ω4)×0.529

[0115] i(16:2dO)=(ω5)×0.529

[0116] The formula for the third stage is as follows:

[0117] S=∑(i 计算 -i 实际 ) 2

[0118] Where ω represents the relative content of TG components, i represents the intensity of fragments, i(FAs), i(FAdC) and i(FAdO) represent the single peaks [FA+Na+C2H2] + (sn-2), double peak (CH2 type) [FA+Na+C3H4] + (sn-1 / 3) and double peak (O type) [FA+Na+C2H2O] + The intensity corresponding to the (sn-1 / 3) fragment, 0.754, 1 and 0.529 are the nominal productivity of the above three fragments respectively.

[0119] c. Use the least squares method to approximate the minimum S value in the formula, and the minimum S value is the end point of the calculation.

[0120] During the actual analysis process, we found that the results of i(FAs) = (ωn + ...) × 0.754 conflicted with those of i(FAdC) = (ωn + ...) × 1 and i(FAdO) = (ωn + ...) × 0.529. Therefore, the calculation formula of the present invention emphasizes a strict priority order. Since the stability of the sn-2 position fragment is higher than that of the sn1 / 3 position, during the calculation process, the formula for the fatty acid-related fragment at the sn-2 position (i.e., i(FAs) = (ωn + ...) × 0.754) has a higher priority than the formula for the fatty acid-related fragment at the sn1 / 3 position (i.e., i(FAdC) = (ωn + ...) × 1 and i(FAdO) = (ωn + ...) × 0.529).

[0121] Therefore, the priority of the first group of formulas above is higher than that of the second and third groups of formulas. If there is a conflict between the formulas, the results of the first group of formulas will be given the first priority.

[0122] The output results are specific values of ω1, ω2 to ωn, which represent the relative content of each isomer, with the total content being 100%.

[0123] ⑩ Repeat the above steps to obtain the specific composition of all components.

[0124] a. Search for [M+NH4] of other components in the LC-MS spectrum + MS2 spectra in CID mode and [M+Na] + In the MS2 spectrum in EAD mode, the above derivation and calculation steps ① to ⑨ were repeated to obtain the specific composition of all components, including the type and content of each isomer in the elution peak.

[0125] The beneficial effects of the present invention are:

[0126] (1) Through long-term experimental research, the main and derivative fragment ions produced by triglyceride adducts in the secondary mass spectra under CID and EAD modes are fully analyzed, and then the information of each fragment is integrated to synergistically analyze the fine structure of triglycerides.

[0127] (2) An innovative prediction-hit strategy was constructed. Specifically, the CID secondary mass spectra were used to analyze the types of triglyceride isomers with different fatty acid compositions (fatty acid chain length and number of double bonds), and the EAD secondary mass spectra were used to analyze the types of triglyceride isomers with different sn position distributions (sn-2 and sn-1 / 3). Finally, the relative content of each triglyceride isomer was calculated using three specific fragments. This solved the problem of identifying co-eluting peaks of triglyceride isomers in LC-MS analysis for the first time. BRIEF DESCRIPTION OF THE DRAWINGS

[0128] 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. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0129] Figure 1 This is a general flow chart of the method for analyzing the types and contents of co-eluted triglyceride isomers in liquid chromatography using CID and EAD secondary mass spectrometry;

[0130] Figure 2 The main and derivative fragment ion patterns of triglyceride adducts produced in the secondary mass spectra under CID and EAD modes;

[0131] Figure 3 This is an example of the isomer resolution process in the triglyceride TG 54:8 elution peak. DETAILED DESCRIPTION

[0132] 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.

[0133] Example:

[0134] In this example, fish oil was used as the sample and fully dissolved in isopropanol solvent. Analysis was performed using a SCIEX ZenoTOF 7600 system. Chromatographic separation was performed using a Waters C18 (100 mm × 3 mm, 3 μm) column at 50°C. The first mobile phase, A, consisted of methanol:acetonitrile:water (1:1:1, 5 mmol / L ammonium acetate, v / v), and mobile phase B was isopropanol (containing 5 mmol / L ammonium acetate). The second mobile phase, A, consisted of methanol:acetonitrile:water (1:1:1, 10 μmol / L sodium formate, v / v), and mobile phase B was isopropanol. The gradient program was as follows: 0–1 min, hold at 20% B; 1–2 min, linear increase from 20% to 40% B; 2–4 min, linear increase from 40% to 60% B; 4–14 min, linear increase from 60% to 98% B; 14–18 min, hold at 98% B; 18–18.5 min, linear decrease from 98% to 20% B. The mobile phase flow rate was set at 0.3 mL / min. The autosampler temperature was 4°C.

[0135] All mass spectrometric data were collected using information-dependent acquisition (IDA) mode. The detector of this system operated in positive electrospray ionization mode. The electron kinetic energy of the CID was 40 eV. The ion source gas 1 (nebulizer gas), gas 2 (heater gas), and curtain gas were set to 55, 55, and 35 psi, respectively. The source temperature was 550°C, and the collision gas (CAD) was set to 7 psi. The low collision energy (CE) of the EAD was 12 V. The electron kinetic energy of the EAD was 15 eV, and the electron beam current was 6000 nA throughout the study. In addition, the trap pulse of the ZenoTOFF was activated with a threshold of 10,000,000 cps. The temperature of the autosampler was 4°C.

[0136] Using the method of the present invention, the isomers in triglyceride TG 54:8 are analyzed as follows: Figure 3 shown.

[0137] ①LC-MS: [M+NH4] of a certain component + MS2 spectrum in CID mode;

[0138] a. Perform LC-MS detection and collect a component (m / z 892.7456, [M+NH4] + )MS2 spectrum in CID mode

[0139] ②CID: triglyceride-related fragments (right area of the figure, with larger m / z values);

[0140] a. Triglyceride-related fragments. These fragments are located in the right region of the spectrum (larger m / z values) and appear as a series of fragments with higher intensity in this region.

[0141] b. Use the table below to confirm the presence of triglyceride-related continuous fragments. If present, it indicates that the component is likely a triglyceride. If absent, the component is likely not a triglyceride and another component should be selected.

[0142] Fragment A Difference Δ Fragment B Difference Δ Fragment C Molecular formula <![CDATA[[M+H-H2O] + ]]> <![CDATA[H2O]]> <![CDATA[[M+H] + ]]> <![CDATA[NH3]]> <![CDATA[[M+NH4] + ]]> m / z value 857.6963 18 875.7042 17 892.7456

[0143] Figure 2 These fragments are also displayed in detail.

[0144] c. Based on the precise m / z value of M obtained above, refer to the table below and use the FormulaCalculator component of SCIEX OS software to reverse-calculate the molecular formula of the component with a relative error of less than 20 ppm.

[0145] element Accurate m / z C 12 H 1.0078250319 O 15.994914619 N 14.003074004 Na 22.98976928 K 38.96370648

[0146] c. Based on the above molecular formula C x H yO z , reconfirm that the component is triglyceride, which is required to contain only C, H and O elements, and the unsaturation is greater than or equal to 3. The calculation formula of unsaturation is (2*x+2-y) / 2, so the triglyceride component is marked as TGx: (2*x+2-y) / 2-3, and the formula removes the unsaturation of the three ester bonds.

[0147] In this embodiment, the component is calculated to be TG 54:8, which means that the component contains 54 carbon atoms and 8 unsaturated double bonds.

[0148] ③CID: fragments with one FA missing (middle area of the figure, medium m / z value);

[0149] a. A fragment with one FA missing, i.e. [M+H-FA] + These fragments are located in the middle region of the spectrum (medium m / z value) and appear as a series of fragments with higher intensity in this region.

[0150] b. Collect all [M+H-FA] + Fragments: The minimum strength of the fragments must be ≥ the maximum strength * 5%, and they should be sorted according to the following table.

[0151]

[0152] The specific results are in the table Figure 3 This is shown in step ③.

[0153] c. Figure 2 As shown, the loss of 1 FA not only produces [M+H-FA] + This fragment ion may also have other derivative fragments. Use the table below to identify fragments from the same source and eliminate false positives.

[0154]

[0155] Figure 2 These fragments are also displayed in detail.

[0156] ④CID: fragments of a single FA (left area of the figure, with smaller m / z values);

[0157] a. Fragments of a single FA, i.e. [FA-OH] + These fragments are located in the left area of the spectrum (smaller m / z values) and appear as a series of fragments with higher intensity in this area.

[0158] b. Collect all [FA-OH] + Fragments: The minimum strength of the fragments must be ≥ the maximum strength * 5%, and they should be sorted according to the following table.

[0159] FA <![CDATA[[FA-OH] + ]]> Intensity (high to low)

[0160] The specific results are in the table Figure 3 This is shown in step ④.

[0161] c. Figure 2 As shown, a single FA not only produces [FA-OH] + This fragment ion may also have other derivative fragments. Use the table below to identify fragments from the same source and eliminate false positives.

[0162]

[0163] Figure 2 These fragments are also displayed in detail.

[0164] d. Based on the two FA correlation tables obtained in ③ and ④ above, sort them from high to low according to the main fragment intensity. After eliminating FAs that cannot exist based on the molecular formula, all FAs are divided into two categories: one category is FAs with high fragment intensity and confirmed existence, which are considered important and must be explained; the other category is FAs with low fragment intensity and possible existence, which are considered alternative FAs.

[0165] ⑤GC-MS: FA composition;

[0166] a. Detect all FA compositions of the sample by gas chromatography-mass spectrometry and compare them with the FA compositions obtained above to further exclude FAs that are unlikely to exist.

[0167] This method is carried out in accordance with the national standard "GB 5009.168-2016 National Food Safety Standard Determination of Fatty Acids in Foods", which is a routine method for determining the fatty acid composition of lipids.

[0168] ⑥Mathematical method: reverse calculation of FA combination;

[0169] a. Using the FA molecular weight from the FA list obtained above as the data source (can be repeated), follow the following formula:

[0170] MW FA1 +MW FA2 +MW FA3 +92-54=The molecular weight of the triglyceride is rounded up to obtain all FA combinations (FA1, FA2, FA3) that meet the formula, that is, the three FAs connected to the TG glycerol, expressed as TG FA1_FA2_FA3.

[0171] b. Sort all FA combinations from high to low according to the sum of the strengths of the three FAs:

[0172] TG FA1_FA2_FA3 Total FA intensity (high to low)

[0173] The specific results are in the table Figure 3 This is shown in step ⑥ of .

[0174] c. Divide all FA combinations into two categories: one is the FA combination with high fragmentation intensity, which is confirmed to exist and is important and must be explained; the other is the FA combination with low fragmentation intensity, which is possible and is used as an alternative.

[0175] During the screening process, the lower limit requires that important FAs appear at least once, and the upper limit requires that the sum of the number of FA appearances be as small as possible.

[0176] ⑦LC-MS: [M+Na] of a certain component + MS2 spectrum in EAD mode;

[0177] a. Perform LC-MS detection and collect [M+Na] of a component. + MS2 spectrum in EAD mode

[0178] ⑧EAD: fragments of a single FA (left area of the figure, with smaller m / z values);

[0179] a. Based on the FA list obtained above, construct the following table:

[0180]

[0181] The specific results are in the table Figure 3 is shown in step ⑧ of .

[0182] The FA column shows the type of fatty acid; the MW column shows the molecular weight of the fatty acid; +49, s (sn-2) indicates the m / z value of the fragment that should be produced when the fatty acid is attached to the sn-2 position of glycerol, usually appearing as a single peak; +63, dC (sn-1 / 3) and +65, dO (sn-1 / 3) indicate the m / z value of the fragment that should be produced when the fatty acid is attached to the sn-1 / 3 position of glycerol, usually appearing as a double peak;

[0183] b. Analyze the hits of the above fragments in the EAD mode secondary mass spectrum (single F; A related fragments, left area, small m / z value):

[0184] If the fragment exists, circle the number;

[0185] If the fragments are present and the intensity is high, circle the number twice;

[0186] If the fragments have the same m / z value, they are connected by a straight line;

[0187] Based on the above results, the distribution of FA at the sn-2 position and sn-1 / 3 position can be determined according to the logic in the following table:

[0188]

[0189]

[0190] / FA / indicates that the FA is located at the sn-2 position, and it is possible for several isomers to exist simultaneously.

[0191] c. Obtain more detailed TG profiles, including sn distribution isomers;

[0192] Taking TG 54:8 as an example, the analysis results include the following five isomers:

[0193] TG 22:6_ / 16:1 / _16:1

[0194] TG 20:5_ / 16:1 / _18:2

[0195] TG 20:5_ / 18:3 / _16:0

[0196] TG 20:5_ / 16:0 / _18:3

[0197] TG 22:6_ / 16:0 / _16:2

[0198] Among them, ω1, ω2, ω3, ω4 and ω5 represent the relative contents of the five TG components respectively.

[0199] ⑨Mathematical method: Least square method is used to calculate the relative content of each TG;

[0200] a. Collect the fragment intensities associated with each fatty acid, specifically including the intensities of fragment (sn-2)s, fragment (sn-1 / 3)dC, and fragment (sn-1 / 3)dO).

[0201] b. The general formula is as follows:

[0202] ω1+ω2+…+ωn=100%

[0203] i(FAs)=(ωn+…)×0.754

[0204] i(FAdC)=(ωn+…)×1

[0205] i(FAdO)=(ωn+…)×0.529

[0206] S=∑(i 实验 -i 理论 ) 2

[0207] Taking TG 54:8 as an example, the analysis results include the following five isomers:

[0208] TG 22:6_ / 16:1 / _16:1 ω1

[0209] TG 20:5_ / 16:1 / _18:2 ω2

[0210] TG 20:5_ / 18:3 / _16:0 ω3

[0211] TG 20:5_ / 16:0 / _18:3ω4

[0212] TG 22:6_ / 16:0 / _16:2 ω5

[0213] It should be noted that the same FA may appear multiple times, in the same or different triglycerides.

[0214] The formula set for the first stage calculation, representing the total content as 100% and the intensity of the sn-2 fatty acids, is as follows:

[0215] ω1+ω2+ω3+ω4+ω5=100%

[0216] i(16:1s)=(ω1+ω2)×0.754

[0217] i(18:3s)=(ω3)×0.754

[0218] i(16:0s)=(ω4+ω5)×0.754

[0219] The formula group for the second stage calculation, which represents the intensity of the sn-1 / 3 fatty acids (including both dC and dO fragments), is as follows:

[0220] i(22:6dC)=(ω1+ω5)×1

[0221] i(20:5dC)=(ω2+ω3+ω4)×1

[0222] i(16:1dC)=(ω1)×1

[0223] i(18:2dC)=(ω2)×1

[0224] i(16:0dC)=(ω3)×1

[0225] i(18:3dC)=(ω4)×1

[0226] i(16:2dC)=(ω5)×1

[0227] i(22:6dO)=(ω1+ω5)×0.529

[0228] i(20:5dO)=(ω2+ω3+ω4)×0.529

[0229] i(16:1dO)=(ω1)×0.529

[0230] i(18:2dO)=(ω2)×0.529

[0231] i(16:0dO)=(ω3)×0.529

[0232] i(18:3dO)=(ω4)×0.529

[0233] i(16:2dO)=(ω5)×0.529

[0234] The formula for the third stage is as follows:

[0235] S=∑(i 计算 -i 实际 ) 2

[0236] Where ω represents the relative content of TG components, i represents the intensity of fragments, i(FAs), i(FAdC) and i(FAdO) represent the single peaks [FA+Na+C2H2] + (sn-2), double peak (CH2 type) [FA+Na+C3H4] + (sn-1 / 3) and double peak (O type) [FA+Na+C2H2O] + The intensity corresponding to the (sn-1 / 3) fragment, 0.754, 1 and 0.529 are the nominal productivity of the above three fragments respectively.

[0237] c. Use the least squares method to approximate the minimum S value in the formula, and the minimum S value is the end point of the calculation.

[0238] The first set of formulas above has a higher priority than the second set of formulas. If there is a conflict between the formulas, the results of the first set of formulas will take precedence.

[0239] The output results are specific values of ω1, ω2 to ωn, which represent the relative content of each isomer, with the total content being 100%.

[0240] The specific results are in the table Figure 3 This is shown in step ⑨.

[0241] ⑩ Repeat the above steps to obtain the specific composition of all components.

[0242] a. Search for [M+N H4] of other components in the LC-MS spectrum + MS2 spectra in CID mode and [M+Na] +In the MS2 spectrum in EAD mode, the above derivation and calculation steps ① to ⑨ were repeated to obtain the specific composition of all components, including the type and content of each isomer in the elution peak.

[0243] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. 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 co-eluted triglyceride isomers in liquid chromatography using CID and EAD secondary mass spectrometry, characterized in that: The following steps are included: (1) Obtaining [M+N H4] of a component by LC-MS + MS2 spectrum in CID mode; (2) CID analysis of triglyceride-related fragments; (3) CID analysis of [M+H-FA] with one missing fatty acid + fragments; (4) CID analysis of [FA-OH] of individual fatty acids + fragments; (5) GC-MS analysis of fatty acid composition; (6) Mathematical methods for calculating fatty acid composition; (7) LC-MS obtains [M+Na] of a certain component + MS2 spectrum in EAD mode; (8) EAD analysis of fragments of a single FA; (9) Calculate the relative content of triglycerides using the least squares method; (10) Repeat steps (1) to (9) to obtain the types and contents of each isomer of all components in the elution peak.

2. The method for co-eluting triglyceride isomers by using CID and EAD secondary mass spectrometry in liquid chromatography according to claim 1, characterized in that: Step (2) includes confirming the presence or absence of triglyceride-related fragments, wherein the triglyceride-related fragments include [M+H-H2O] + 、[M+H] + and [M+NH4] + The molecular formula C is calculated based on the m / z of C = 12, the m / z of H = 1.0078250319, and the m / z of O = 15.994914619. x H y O z ; Then, the triglyceride component is confirmed again based on the unsaturation being greater than or equal to 3, unsaturation = (2*x+2-y) / 2.

3. The method for co-eluting triglyceride isomers by using CID and EAD secondary mass spectrometry in liquid chromatography according to claim 1 or 2, characterized in that: Step (3) involves collecting all [M+H-FA] + Fragments, and meet the following requirements: minimum intensity of fragments ≥ maximum intensity * 5%, listed in order [M+H-FA] + m / z value of fragments, [M+H-FA] + The intensity values of the fragments are sorted from high to low and the m / z values of the missing fatty acids are used to calculate the molecular formula of the fatty acid C a H b O c and marked as Ca: (2*a+2-b) / 2-1; derivative fragments from the same source are identified to exclude false positives, and the derivative fragments include [M+H-FA-C3H6O2-H2O] + 、[M+H-FA-C3H6O2] + 、[M+H-FA-C3H6O2+H2O] + 、[M+H-FA] + At least two of the .

4. The method for co-eluting triglyceride isomers by using CID and EAD secondary mass spectrometry in liquid chromatography according to claim 1 or 2, characterized in that: Step (4) involves collecting all [FA-OH] + Fragments, and meet the following requirements: the lowest intensity of the fragments ≥ the highest intensity * 5%, fatty acids C are listed in order a H b O c Ca: (2*a+2-b) / 2-1, [FA-OH] + m / z value of the fragment, [FA-OH] + Fragments are ranked from high to low intensity; derivative fragments from the same source are identified to exclude false positives, including [FA-OH-H2O] + , [FA-OH] + 、[FA-OH+C3H6O2-H2O] + 、[FA-OH+C3H6O2] + and exclude fatty acids that cannot be present.

5. The method for co-eluting triglyceride isomers by using CID and EAD secondary mass spectrometry in liquid chromatography according to claim 4, characterized in that: Step (5) includes GC-MS detection of fatty acid composition, comparing it with the fatty acids obtained in steps (1) to (4), and eliminating fatty acids that are unlikely to exist.

6. The method for co-eluting triglyceride isomers in liquid chromatography using CID and EAD secondary mass spectrometry according to claim 1 or 2, characterized in that: Step (6) includes following the formula: MW FA1 +MW FA2 +MW FA3 + 92-54 = the molecular weight of the triglyceride, obtain the fatty acid combination that meets the formula, and sort them from high to low according to the sum of the strengths of the three fatty acids in the triglyceride to obtain the fatty acid combination that is confirmed to exist; in the formula, MW FA1 MW FA2 MW FA3 They represent the molecular weight of the three fatty acids in triglycerides.

7. The method for co-eluting triglyceride isomers in liquid chromatography using CID and EAD secondary mass spectrometry according to claim 1 or 2, characterized in that: Step (8) includes establishing a table to respectively display the fatty acid type, fatty acid molecular weight, m / z value of the fragment that should be produced when the fatty acid is connected to the sn-2 position of glycerol, and m / z value of the fragment that should be produced when the fatty acid is connected to the sn-1 / 3 position of glycerol, analyzing the hit situation of the fragment in the EAD mode secondary mass spectrum, judging the distribution of fatty acids at the sn-2 position and the sn-1 / 3 position, and obtaining the isomer analysis results.

8. The method for co-eluting triglyceride isomers in liquid chromatography using CID and EAD secondary mass spectrometry according to claim 1 or 2, characterized in that: Step (9) includes collecting the intensity of each fatty acid-related fragment and calculating the relative content of each triglyceride isomer according to the following formula: ω1+ω2+…+ωn=100% i(FAs)=(ωn+…)×0.754 i(FAdC)=(ωn+…)×1 i(FAdO)=(ωn+…)×0.529 S=∑(i 实验 -i 理论 ) 2 ω represents the relative content of each triglyceride isomer component, i represents the intensity of the fragment, where i(FAs), i(FAdC) and i(FAdO) represent the single peak [FA+Na+C2H2] + (sn-2) fragments, bimodal CH2 type [FA+Na+C3H4] + (sn-1 / 3) fragments and doublet O-type [FA+Na+C2H2O] + (sn-1 / 3) the intensity of the fragment.

9. The method for co-eluting triglyceride isomers in liquid chromatography using CID and EAD secondary mass spectrometry according to claim 8, characterized in that: Through the least squares approximation, the S value in the formula is minimized, and the minimum S value is taken as the calculation end point; The first set of formulas has higher priority than the second and third sets of formulas. If there is a conflict between the formulas, the results of the first set of formulas will take precedence.

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