Method for detecting triglyceride double bond structural isomers

Abstract

The application discloses a method for detecting triglyceride double bond structural isomers. The method comprises the following steps: based on the molecular ion peak [M+N]+, the fragment ion [M+N-R]+ and the fragment ion [M+N-2R]+ in the primary mass spectrum result and the secondary mass spectrum result of a triglyceride sample, comparing with a lipid compound database, so as to obtain the structure type of each branched chain of the triglyceride sample and determine the mass-to-charge ratio range of the fragment ion [M+N-R]+; based on the abundance of the acyl chain fragment ion between the molecular ion peak [M+N]+ and the fragment ion [M+N-R]+, the ratio of the abundance and the difference from the adjacent mass-to-charge ratio of the characteristic acyl chain fragment ion, so as to determine the position and cis-trans structure of each branched chain double bond. The method discloses the fine fragmentation rule of TAG double bond isomers, and constructs a comprehensive TAG double bond isomer characterization strategy.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical chemistry, in particular, to a method for detecting triglyceride double bond structural isomers. BACKGROUND

[0002] Triglycerides (TAG) are an important class of physiological function lipids generated by esterification of three fatty acids (saturated fatty acids and unsaturated fatty acids) and one molecule of glycerol. Unsaturated triglyceride acyl chains usually have one or more -C=C- groups, thus forming double bond position isomers and cis-trans isomers, which can have great differences in biological functions. For example, the number of C=C groups determines the degree of unsaturation of the lipid, and the position of C=C groups can affect the permeability and thickness of the cell membrane, the plasticity of cancer cells, etc. For example, the two isomers of linolenic acid, alpha-linolenic acid and gamma-linolenic acid, participate in different cellular pathways in the process of inhibiting tumor cell growth; cis-3-hexenol can induce the expression of defense genes in plants, while trans-2-hexenol is a component of insect pheromones. Therefore, fine structure characterization of the position of C=C groups and cis-trans isomers in the acyl chain of unsaturated triglycerides helps to understand the energy conversion relationship between triglyceride structural information and biological action, and plays an important role in the fields of clinical diagnosis, life sciences and lipid biology.

[0003] Therefore, the method for fine structure characterization of unsaturated triglycerides needs to be studied. SUMMARY

[0004] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, one object of the present application is to provide a method for detecting triglyceride double bond structural isomers, which reveals the fine fragmentation rules of TAG double bond isomers by studying the mass spectrometry fragmentation characteristics of double bond position isomers, cis-trans isomers, fragment abundance and abundance ratio, etc., constructs a comprehensive characterization strategy for TAG double bond isomers, and improves the efficiency and accuracy of double bond structure characterization, thereby providing support for in-depth exploration of lipid structure.

[0005] According to an aspect of the present application, the present application provides a method for detecting triglyceride double bond structural isomers. According to an embodiment of the present application, the method comprises: providing a triglyceride sample to be detected; mixing the triglyceride sample with a salt compound to obtain a test solution; detecting the test solution by using a high-resolution mass spectrometer to obtain a primary mass spectrum result and a secondary mass spectrum result; based on the molecular ion peak [M+N]+, the fragment ion [M+N-R]+ and the fragment ion [M+N-2R]+ in the primary mass spectrum result and the secondary mass spectrum result, comparing with a lipid compound database to obtain the structural type of each branched chain of the triglyceride sample and determine the mass-to-charge ratio range of the fragment ion [M+N-R]+, wherein M represents a triglyceride, N represents a cation combined with the ionized triglyceride, R represents a fatty acid acyl chain of the triglyceride sample, and the carboxyl carbon at the end of the fatty acid acyl chain is marked as C1; and based on the abundance of the acyl chain fragment ion between the molecular ion peak [M+N]+ and the fragment ion [M+N-R]+, the ratio of the abundance, and the difference from the mass-to-charge ratio of the adjacent characteristic acyl chain fragment ion, to determine the position and cis-trans structure of each branched chain double bond.

[0006] The method for detecting triglyceride double bond structural isomers according to the embodiment of the present application reveals the fine fragmentation rules of TAG double bond isomers by studying the mass spectrum fragmentation characteristics of double bond position isomers, cis-trans isomers, fragment abundance and abundance ratio, constructs a comprehensive characterization strategy of TAG double bond isomers, and improves the efficiency and accuracy of double bond structure characterization, thereby providing support for in-depth exploration of lipid structure.

[0007] In addition, the method for detecting triglyceride double bond structural isomers according to the above-mentioned embodiments of the present application can also have the following additional technical features:

[0008] According to an embodiment of the present application, the detection conditions of the high-resolution mass spectrometer are as follows: working solution flow rate: 30 uL / min; monitoring mode: high-resolution multiple reaction monitoring; electrospray ionization: ESI(+); electron kinetic energy of EAD: 8-17 eV; electron beam current: 7000 nA; electrospray voltage: +5500 V; ion source temperature: 300 DEG C; atomization gas: 30 psi; heating gas: 30 psi; gas curtain gas: 30 psi. In this way, the triglyceride compound is detected, and the accuracy and sensitivity of the detection are high.

[0009] According to an embodiment of the present application, the method for determining the double bond position of each branched chain of the triglyceride sample comprises: determining the molecular ion peak [M+N] + and the fragment ion [M+N-R] +the acyl chain fragment ions between which the carbon atoms are located; determining the position of the double bond based on the mass number difference between the branched chain adjacent to the acyl chain fragment ions.

[0010] According to an embodiment of the present application, the mass number difference between the branched chain adjacent to the acyl chain fragment ions is 14.0±0.1, and a single bond exists between the two carbon atoms.

[0011] According to an embodiment of the present application, the mass number difference between the branched chain adjacent to the acyl chain fragment ions is 26.0±0.1, and it is set that the carbon atom corresponding to the acyl chain fragment ion adjacent to the fatty acid acyl chain carbonyl end is located at the m-1th position, and the carbon atom corresponding to the other acyl chain fragment ion is located at the m+1th position, and the carbon atom at the mth position does not generate the acyl chain fragment ion, and a double bond exists between the carbon atoms at the mth position and the m+1th position.

[0012] According to an embodiment of the present application, the branched chain contains one double bond, and the branched chain determines that the ion with the highest abundance in each of the acyl chain fragment ions is generated by the single bond breaking between the carbon atom at the m+2th position and the carbon atom at the m+3th position.

[0013] According to an embodiment of the present application, the branched chain contains one double bond, and the method for determining the cis-trans structure of the double bond comprises: determining the position of the double bond, and setting that the double bond is located between the carbon atom at the nth position and the carbon atom at the n+1th position; determining the abundance of the even-electron product ion fragment of the carbon atom at the n+1th position and the abundance of the odd-electron product ion; and calculating the I value of the carbon atom at the n+1th position based on formula (1), wherein when the I value <1.0, the double bond is cis, and when the I value >1.0, the double bond is trans.

[0014] wherein I=abundance of even-electron product ion fragment ÷abundance of odd-electron product ion (1).

[0015] According to an embodiment of the present application, the branched chain contains one double bond, and the method for determining the cis-trans structure of the double bond comprises: determining the position of the double bond, and setting that the double bond is located between the carbon atom at the nth position and the carbon atom at the n+1th position; and calculating the relative abundance of the acyl chain fragment ion fragment of the carbon atom at the n-1th position and the carbon atom at the n+1th position based on formula (2), wherein when the relative abundance I of the acyl chain fragment ions of the carbon atom at the n-1th position and the carbon atom at the n+1th position are both greater than 0.2, the double bond is cis, and when the relative abundance I of the acyl chain fragment ions of the carbon atom at the n-1th position and the carbon atom at the n+1th position are both less than 0.2, the double bond is trans.

[0016] wherein I=abundance of acyl chain fragment ion fragment ÷abundance of molecular ion peak [M+N] + (2).

[0017] Additional aspects and advantages of the application will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following and the attendant drawings or can be learned by practice of the application. BRIEF DESCRIPTION OF DRAWINGS

[0018] The foregoing and / or additional aspects and advantages of the present application are achieved by providing a method for quantifying triglycerides in a sample, comprising:

[0019] Figure 1 shows the secondary mass spectrum of TAG 17:1 / 17:1 / 17:1 (10Z) in EAD cleavage mode (a), and the enlarged view of the high m / z fragment region (b) according to one embodiment of the present application;

[0020] Figure 2 shows the enlarged view of the high m / z fragment region of five monounsaturated compounds according to one embodiment of the present application;

[0021] Figure 3 shows the comparison of the secondary mass spectrum of TAG 18:1 / 18:1 / 18:1 (9Z) and TAG 18:1 / 18:1 / 18:1 (9E) in EAD cleavage mode (double bond region) according to one embodiment of the present application;

[0022] Figure 4 shows the comparison of the relative abundance of the characteristic fragment ions produced by the sequential cleavage of acyl chains -CH2 in TAG 18:1 / 18:1 / 18:1 (9Z) and TAG 18:1 / 18:1 / 18:1 (9E) according to one embodiment of the present application, wherein the relative intensity is expressed as the ratio of the characteristic fragment ion to the mass spectrum response value of the parent ion;

[0023] Figure 5 shows the secondary mass spectrum of TAG 22:6 / 22:6 / 22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z) in EAD cleavage mode (a), and the enlarged view of the high m / z fragment region (b) according to one embodiment of the present application;

[0024] Figure 6 shows the quantification of oleic acid triglyceride C18:1 cis / trans isomers based on the function relationship between the relative molar concentration ratio of cis C18:1 / trans C18:1 and the relative abundance ratio of the characteristic EIEIO fragment ions (m / z 793.6 and 794.6) in solution according to one embodiment of the present application. DETAILED DESCRIPTION

[0025] Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein the same or like component have the same or similar designations. The embodiments described below are presented by way of example only and are not intended to limit the present application as defined by the appended claims and their equivalents.

[0026] It should be noted that the terms "first", "second" are used only for descriptive purposes and should not be construed as indicating or implying relative importance or an indicated number of technical features. Thus, features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise stated, the meaning of "a plurality" is two or more.

[0027] According to one aspect of the present application, the present application provides a method for detecting triglyceride double bond structural isomers. According to the method for detecting triglyceride double bond structural isomers of the embodiments of the present application, by studying the double bond position isomer, the mass spectrometry cracking characteristic fragment of cis-trans isomer, the fragment abundance and the abundance ratio, etc., the fine fragmentation rule of TAG double bond isomer is revealed, a comprehensive characterization strategy of TAG double bond isomer is constructed, and the efficiency and accuracy of double bond structure characterization are improved, which provides support for in-depth exploration of lipid structure.

[0028] In order to facilitate the connection of the method for detecting triglyceride double bond structural isomers of the embodiments of the present application, the method is explained and described here, which comprises:

[0029] S100 providing a sample

[0030] According to the embodiments of the present application, a triglyceride sample to be tested is provided. The sample is a purified triglyceride sample.

[0031] S200 preparing a sample solution

[0032] According to the embodiments of the present application, the triglyceride sample is mixed with a salt compound to obtain a sample solution. TAG is a non-polar compound and is not easy to ionize, so some salt (such as sodium or ammonium cation) must be added to form an adduct for all analysis work.

[0033] According to the embodiments of the present application, the triglyceride sample is mixed with sodium salt. Sodium TAG is a more easily cracked target parent ion than ammoniated TAG, because the sodium parent ion is easy to lose double bonds. In contrast, ammoniated TAG tends to crack at the NH4 + group, resulting in no fragment loss inside the fatty acid chain. Therefore, it is preferred to add sodium acetate to form a sodium TAG ion.

[0034] S300 mass spectrometry detection

[0035] According to an embodiment of the present application, the to-be-tested liquid is detected by using a high-resolution mass spectrometer, so as to obtain a primary mass spectrum result and a secondary mass spectrum result.

[0036] According to an embodiment of the present application, the detection condition of the high-resolution mass spectrometer is as follows: working solution flow rate: 30 uL / min; monitoring mode: high-resolution multiple reaction monitoring; electro-spray ionization: ESI(+); electron kinetic energy of EAD: 8-17 eV; electron beam current: 7000 nA; electro-spray voltage: +5500 V; ion source temperature: 300 DEG C; atomization gas: 30 psi; heating gas: 30 psi; gas curtain gas: 30 psi.

[0037] S400 determining structural formula range

[0038] According to an embodiment of the present application, based on the molecular ion peak [M+N] + , fragment ion [M+N-R] + and fragment ion [M+N-2R] + in the primary mass spectrum result and the secondary mass spectrum result, by comparison with a lipid compound database, the structural type of each branch of the triglyceride sample is obtained, and the mass-to-charge ratio range of the fragment ion [M+N-R]+ is determined, wherein M represents a triglyceride, N represents a cation combined with the ionized triglyceride, and R represents a fatty acid acyl chain of the triglyceride sample, specifically, one fatty acid acyl chain is broken, that is, one R group is reduced, to produce the fragment ion [M+N-R] + , two fatty acid acyl chains are broken, that is, two R groups are reduced, to produce the fragment ion [M+N-2R] + ; the carboxyl carbon at the end of the fatty acid acyl chain is denoted as C1, further, the nth carbon atom is the nth carbon atom denoted as 1 at the carboxyl end, and so on, n-1 is the n-1th carbon atom denoted as 1 at the carboxyl end.

[0039] According to an embodiment of the present application, based on the mass-to-charge ratio of the ion peak [M+N] + , by comparison with a lipid compound database, the possible composition of a fatty acid is obtained. The molecular weight of TAG can be obtained by the mass-to-charge ratio of the ion peak [M+N] + , and by comparison with a lipid compound database, the R group molecular composition of the possible TAG of the molecular weight is obtained, based on the possible composition of the fatty acid, the mass-to-charge ratio range of the fragment ion [M+N-R] + and the mass-to-charge ratio range of the fragment ion [M+N-2R] + are determined respectively.

[0040] S500 determining position isomer

[0041] According to an embodiment of the present application, the abundance of acyl chain fragment ions between the molecular ion peak [M+N] + and the fragment ion [M+N-R] + , the ratio of the abundance, and the difference of the mass number between adjacent acyl chain fragment ions of the characteristic acyl chain fragment ions, so as to determine the position of each branched double bond and the cis-trans structure. That is, the position of double bond and the cis-trans structure of each fatty acid acyl chain are determined.

[0042] (I) Double bond position

[0043] The EAD mass spectrum of unsaturated TAGs can be observed, in the mass region between the TAG molecular ion [M+Na] + and the product ion [M+Na-R1] + losing a single acyl chain, a series of complete acyl chain fragments can be found, indicating that the fatty acid chain can produce continuous de-CH2 fragments, and the position of the double bond connection does not break. The inventors found the following method for determining the position of the double bond by studying the EAD mass spectrum:

[0044] According to an embodiment of the present application, the method for determining the position of the branched double bond of the triglyceride sample comprises: determining the acyl chain fragment ions of each carbon atom between the molecular ion peak [M+N] + and the fragment ion [M+N-R] + , determining the position of the double bond based on the mass number between adjacent acyl chain fragment ions of the branched chain. It should be noted that this method is suitable for fatty acid chains with only one double bond, and also suitable for fatty acid chains with multiple double bonds.

[0045] Specifically, according to an embodiment of the present application, the mass number difference between adjacent acyl chain fragment ions of the branched chain is 14.0±0.1, indicating that there is a single bond between two carbon atoms, that is, the position of de-CH2 fragments.

[0046] Specifically, according to an embodiment of the present application, the mass number difference between adjacent acyl chain fragment ions of the branched chain is 26.0±0.1, and it is assumed that the carbon atom corresponding to the acyl chain fragment ion adjacent to the carbonyl end of the fatty acid acyl chain is located at the m-1 position, and the carbon atom corresponding to the other acyl chain fragment ion is located at the m+1 position, and the carbon atom at the m position does not produce the acyl chain fragment ion, indicating that there is a double bond between the carbon atoms at the m and m+1 positions. That is, for the C=C double bond, for each double bond in the fragment part of the acyl chain, the m / z of the radical fragment will move +2H, resulting in a fragment peak with a mass number difference of 26 Da. Since the double bond is not a favorable cleavage site, the fragment ion abundance forms a "V" shape at the double bond position, clearly indicating the position of the C=C double bond.

[0047] Further, in order to verify the accuracy of the above method, according to an embodiment of the present application, the branched chain contains one double bond, and the ion with the highest abundance in each of the acyl chain fragment ions of the branched chain should be the ion generated by breaking the single bond between the m+2 carbon atom and the m+3 carbon atom.

[0048] Further, the method can be understood as follows: each monounsaturated compound will generate C x-2 fragment peaks (x represents the total number of carbon atoms in the fatty acyl chain), and the internal fragmentation of the acyl chain has the same double bond identification method, that is, the double bond position does not contain a fragment peak, and the C-C single bond between the two C atoms is broken to generate a fragment with the highest abundance.

[0049] (B) cis-trans structure

[0050] Due to the presence of double bonds, unsaturated triglycerides can form cis-trans isomers with different spatial structures, and the cis-trans isomers can affect the physical properties (such as melting point, solubility), chemical properties (such as reactivity) and biological functions (such as metabolic pathways, cell signaling, nutritional value) of the triglycerides, so it is of great significance to accurately characterize the cis-trans isomers. It should be noted that the following two methods are only for fatty acid chains with one double bond.

[0051] Method one:

[0052] According to an embodiment of the present application, the branched chain contains one double bond, and the method for determining the cis-trans structure of the double bond comprises: determining the position of the double bond, setting the double bond between the n th and n+1 th carbon atoms; determining the abundance of the even-electron product ion fragment of the n+1 th carbon atom and the abundance of the odd-electron product ion; based on formula (1), the I value of the n+1 th carbon atom is calculated, when I value <1.0, the double bond is cis; when I value >1.0, the double bond is trans,

[0053] wherein I = abundance of even-electron product ion fragment ÷ abundance of odd-electron product ion (1).

[0054] This method explores the abundance ratio of free radical fragments (even-electron product ion fragments) and hydrogen loss non-free radical fragments (odd-electron product ion fragments), and the trans structure should appear stronger abundance than the cis structure.

[0055] Method two:

[0056] According to the embodiment of the present application, the branched chain contains one double bond, and the method for determining the cis-trans structure of the double bond comprises: determining the position of the double bond, setting the double bond between the n th and (n+1) th carbon atoms; calculating the relative abundance of the acyl chain fragment ion fragments of the n-1 th and (n+1) th carbon atoms based on formula (2), when the relative abundance I of the acyl chain fragment ions of the n-1 th and (n+1) th carbon atoms is greater than 0.2, the double bond is cis, and when the relative abundance I is less than 0.2, the double bond is trans,

[0057] wherein I = abundance of acyl chain fragment ion fragments ÷ abundance of molecular ion peak [M+N]+ (2).

[0058] The method directly compares the abundance of fragment peaks generated by the internal rupture-CH2 of the acyl chain, and the C-C bond near the double bond shows stronger abundance of fragment peaks of cis structure than that of trans structure

[0059] The present application will be described below with reference to specific examples. It should be noted that these examples are merely illustrative and should not be construed as limiting the scope of the present application.

[0060] The scheme of the present application will be explained below with reference to examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be purchased, for example, from Sigma Company.

[0061] Example 1

[0062] In this embodiment, the method for detecting the double bond structural isomers of triglyceride according to the embodiment of the present application is used for verification, and the specific process is as follows:

[0063] 1. Instruments and reagents

[0064] Instruments: Zeno-TOF7600 high-resolution mass spectrometer and SCIEX 3.0 data processing software (AB Sciex Company, USA), equipped with ESI ion source; Milli-Q ultrapure water system (Millipore Company, USA); HarvardApparatus injection pump Pump11Elite;

[0065] Reagents: n-hexane, isopropyl alcohol (chromatographic pure, ThermoFisher Company, USA); formic acid, ammonium formate, sodium acetate (chromatographic pure, Sigma-Aldrich Company, USA);

[0066] Lipid standards: 17 kinds of triglyceride standards were purchased from ZZSTANDARD, the lipid structure nomenclature proposed by Liebisch et al.

[24] was used in this study: the position of double bond in fatty acyl chain was indicated in a pair of parentheses according to the Delta nomenclature, the position of double bond was counted from the carboxyl end, using C-1, C-2, C-3, … symbols (where C-1 is the carbon of carboxyl group), for example, FA 18:1, if the double bond is between C-11 and C-12 from the carboxyl end, it is represented as FA 18:1 (D11); the double bond geometry was defined using Z / E designation (Z for cis, E for trans). For example, TAG 18:1 / 18:1 / 18:1 (9Z) indicates that the triglyceride contains three unsaturated C18 fatty acyl chains, containing one cis double bond at the D-9 position.

[0067] The detailed information of standards is shown in Table 1;

[0068] Table 1. Basic information of 17 kinds of double bond isomer compounds

[0069]

[0070]

[0071]

[0072] 2. Preparation of standard solution

[0073] Preparation of standard stock solution: accurately weigh each triglyceride standard, dissolve in n-hexane solvent, prepare 0.5 mg / mL standard stock solution, store at -20℃, and use as needed;

[0074] Preparation of standard working solution: transfer 1 mL of standard stock solution, dilute with isopropyl alcohol: water (9:1 v / v) mixed solution containing 0.5 mM sodium acetate, prepare 0.05 mg / ml standard working solution, store at -20℃, and use as needed.

[0075] 3. Mass spectrometry method

[0076] All qualitative data were collected by high resolution multiple reaction monitoring (MRM HR ) mode; the detector of the system was operated under positive ion mode (ESI+); the atomizing gas, heating gas and gas curtain gas were all set to 30 psi; the ion source temperature was 300℃, and the electrospray voltage was 5500V; the electron kinetic energy (KE) of EAD was set to 8-17eV, and the electron beam current was 7000nA; the working solution was directly injected into the ion source at a flow rate of 30μL / min by needle pump sampling, and the collection time was 6min. The raw data were processed using SCIEX OS software 3.3 with browser and analysis module.

[0077] 4. Results and discussion

[0078] Four representative TAGs double bond isomers were selected in this example, including cis-mono unsaturated TAGs (5 kinds), cis-polyunsaturated TAGs (8 kinds), mono unsaturated cis-trans isomer TAGs (3 groups) and 1 group of polyunsaturated cis-trans isomer compounds, a total of 17 kinds. The mono unsaturated TAGs are TAG 14:1 / 14:1 / 14:1(9Z), TAG 16:1 / 16:1 / 16:1(9Z), TAG 18:1 / 18:1 / 18:1(9Z), TAG 17:1 / 17:1 / 17:1(10Z), TAG 20:1 / 20:1 / 20:1(11Z), respectively; 8 kinds of polyunsaturated TAGs are TAG 18:2 / 18:2 / 18:2(9Z, 12Z), TAG 18:3 / 18:3 / 18:3(9Z, 12Z, 15Z), TAG 20:2 / 20:2 / 20:2(11Z, 14Z), TAG 20:3 / 20:3 / 20:3(8Z, 11Z, 14Z), TAG 20:4 / 20:4 / 20:4(5Z, 8Z, 11Z, 14Z), TAG 20:5 / 20:5 / 20:5(5Z, 8Z, 11Z, 14Z, 17Z), TAG 22:2 / 22:2 / 22:2(13Z, 16Z), TAG 22:6 / 22:6 / 22:6(4Z, 7Z, 10Z, 13Z, 16Z, 19Z); 3 groups of mono unsaturated cis-trans isomer compounds are TAG 16:1 / 16:1 / 16:1(9Z) and TAG 16:1 / 16:1 / 16:1(9E), TAG 18:1 / 18:1 / 18:1(9Z) and TAG 18:1 / 18:1 / 18:1(9E), and TAG 20:1 / 20:1 / 20:1(11Z) and TAG 20:1 / 20:1 / 20:1(11E); 1 group of polyunsaturated cis-trans isomer compounds is TAG 18:2 / 18:2 / 18:2(9Z, 12Z) and TAG 18:2 / 18:2 / 18:2(9E, 12E).

[0079] 4.1 Fine characterization of double bonds of mono unsaturated compounds

[0080] 4.1.1 Characterization of double bond position

[0081] From the EAD mass spectrum of the 5 kinds of mono unsaturated TAGs, it can be observed that the TAG molecular ion [M+Na] + and the product ion [M+Na-R1] +In the mass region between m / z = 865.73 and 597.49, a series of acyl chain fragments can be found, indicating that the fatty acid chain can produce continuous de-CH2fragments, while the position of the double bond connection does not break. Taking the mass spectrum of TAG 17:1 / 17:1 / 17:1(10Z) as an example Figure 1 ), the [M+Na] + m / z = 865.73; in the [M+Na-R] + range, the parent ion [M+Na] + loses one C17:1 fatty acyl to form one fragment ion [M+Na-RxCOOH] + m / z = 597.49, which can be the loss of C17:1 fatty acyl at any of sn-1, sn-2, sn-3; between m / z = 865.73 and 597.49, there is a series of acyl chain fragment ions, and compared with the fragment ion [M+Na-2R]+losing the double acyl chain, the relative abundance of these diagnostic peaks is usually 10.1% of it. The typical fragmentation pattern of the double bond in the EAD spectrum is C-C single bond fragmentation, while the C=C double bond does not break, and each cleavage site produces one odd electron radical fragment and one even electron non-radical fragment. For C-C single bond, the radical fragment appears as a series of evenly spaced 14.01 Da ions (i.e. methylene or -CH2), and for C=C double bond, the m / z of the radical fragment moves +2H for each double bond appearing in the fragment part of the acyl chain, resulting in fragment peaks with a mass number difference of 26 Da. Since the double bond is not a favorable cleavage site, the fragment ion abundance forms a "V" shape at the double bond position, clearly indicating the position of the C=C double bond. By zooming in on the high m / z fragment region Figure 1 b) it can be observed that the fragment peak group with a mass number difference of 26 Da, whose abundance is "V" shape, can determine that there is a double bond at this position, i.e. there is a C=C double bond at C10-C11, and accordingly, it can be inferred that the unsaturated C17 fatty acyl chain contains one double bond at C-10 position.

[0082] By zooming in on the high m / z fragment region Figure 1 b) it can be observed that there is a very low abundance of fragment interference at C-10 position, and the characteristics of the "V" shape distribution of fragment ion abundance are not easy to observe, in order to explore a more accurate method for identifying the position of the double bond, the relative abundance of the fragment ions produced by de-CH2within the fatty acyl chain is further compared. Taking the mass spectrum of TAG 17:1 / 17:1 / 17:1(10Z) as an example, as shown in Figure 1Figure 1R shows that the highest abundance of fragment ions is found to be produced by the cleavage of the C-C bond at two carbons away from the methyl end, i.e. if there is a C=C double bond at the C10 position, the highest abundance of fragments is produced by the cleavage of the C-C single bond at the C12 position, two carbons away from the two carbons; the reason for this phenomenon can be that the C-C configuration at the C12 position is different from that at other positions, and is more prone to cleavage; the same rule is found in the secondary mass spectra of TAG 14:1 / 14:1 / 14:1 (9Z), TAG 16:1 / 16:1 / 16:1 (9Z), TAG 18:1 / 18:1 / 18:1 (9Z), TAG 20:1 / 20:1 / 20:1 (11Z), etc. (see Figure 1S), i.e. Figure 2 Figure 1T shows that, by comparing five monounsaturated triglycerides (X axis), it is found that the internal cleavage of the fatty acyl chain has the same characteristic fragmentation rule, i.e. continuous -CH2is removed, and the C=C double bond is not cleaved (Y axis), and each monounsaturated compound will produce C n-2 fragments (n represents the total number of carbon atoms in the fatty acyl chain); the internal cleavage of the acyl chain has the same double bond recognition method, i.e. the double bond position does not contain a fragment peak, and the C-C single bond cleavage at two carbons produces the highest abundance of fragments (Z axis).

[0083] 4.1.2 Double bond cis-trans characterization

[0084] Due to the presence of double bonds, unsaturated triglycerides can form cis-trans isomers with different spatial structures, which can affect the physical properties (such as melting point, solubility), chemical properties (such as reactivity) and biological functions (such as metabolic pathways, cell signaling, nutritional value) of triglycerides, so it is of great significance to accurately characterize cis-trans isomers. Taking the mass spectra of the double bond region of TAG 18:1 / 18:1 / 18:1 (9Z) and TAG 18:1 / 18:1 / 18:1 (9E) as examples, as shown in Figure 3 Figure 1U, it is observed that the two spectra have similar abundance curves, and the C-C single bond in the acyl chain cleaves to produce two fragment ions: an even-electron (non-radical) product ion and its corresponding odd-electron (radical) product ion; however, there is a significant difference in the abundance of the radical peak m / z = 794.638 and the hydrogen loss non-radical peak m / z = 793.639 produced by the cleavage of the C-C single bond at the C10 position adjacent to the double bond, which is that the abundance of the "hydrogen loss" even-electron fragment produced by the cleavage of the C-C bond at the C10 position of the trans isomer is higher, and the abundance of the odd-electron radical fragment produced by the cleavage of the C-C bond at the C10 position of the cis isomer is higher.

[0085] The ratio (I) of the abundance of the [hydrogen lost peak intensity at x position]+ and the [radical peak intensity at x position]+ characteristic fragments was further compared. This example analyzes the I values of three groups of monounsaturated cis-trans isomer compounds, namely TAG 16:1 / 16:1 / 16:1(9Z) and TAG 16:1 / 16:1 / 16:1(9E), TAG 18:1 / 18:1 / 18:1(9Z) and TAG 18:1 / 18:1 / 18:1(9E), and TAG 20:1 / 20:1 / 20:1(11Z) and TAG 20:1 / 20:1 / 20:1(11E).

[0086]

[0087] As can be seen from the results in Table 2, the I values of the cis isomer glycerol triesters such as TAG 16:1 / 16:1 / 16:1(9Z), TAG 18:1 / 18:1 / 18:1(9Z), and TAG 20:1 / 20:1 / 20:1(11Z) are in the range of 0.67-0.96, and the I values of the trans isomer glycerol triesters such as TAG 16:1 / 16:1 / 16:1(9E), TAG 18:1 / 18:1 / 18:1(9E), and TAG 20:1 / 20:1 / 20:1(11E) are in the range of 1.12-1.44. In summary, it can be considered that the I value of the cis isomer TAGs is <1.0, and the I value of the trans isomer TAGs is >1.0, and accordingly, the cis and trans double bond isomer TAGs can be identified by comparing the ratio of the abundance of the radical fragment and the hydrogen lost non-radical fragment ion, which is one of the very important identification bases. This method solves the problem of needing a group of cis-trans compounds for comparison and differentiation, and makes up for the shortage of expensive and scarce lipid standards that are not easy to purchase.

[0088] Table 2. Relative abundance ratio of radical ion and hydrogen lost non-radical fragment ion under EAD cleavage mode

[0089]

[0090] I [1、2、3、4、5、6] I represents the ratio of the mass spectrometry response values in 6 time periods (0-1 min, 1-2 min, 2-3 min, 3-4 min, 4-5 min, 5-6 min) within 6 min; I [1、2、3、4、5、6] I represents I [非自由基]+ I represents I [自由基]+ I* represents the average of the mass spectrometry response ratio values of the 6 time periods;

[0091] To explore the more subtle differences between cis-trans isomers, the relative abundance of fragment peaks generated by the internal breakage of the fatty acyl chain was further compared. Specifically, within the mass range of the quasi-molecular ion [M+Na]+ and the [M+Na-R]+ of the lost fatty acyl chain, the relative abundance of fragment peaks generated by the breakage of the C-C single bond was compared. Taking the compounds TAG 18:1 / 18:1 / 18:1(9Z) and TAG 18:1 / 18:1 / 18:1(9E) as examples, ... Figure 4 As shown, green bars represent the cis configuration and orange bars represent the trans configuration. The acyl chains break sequentially within the -CH2 group, with fragments produced by C-18 removal (m / z = 892.77), C-17 removal (m / z = 878.84), C-16 removal (m / z = 864.87), C-15 removal (m / z = 850.76), C-14 removal (m / z = 836.71), C-13 removal (m / z = 822.72), C-12 removal (m / z = 808.67), C-11 removal (m / z = 794.65), and C9-... There is an unbroken double bond at C10. Removing C=C produces a fragment with m / z = 768.64, removing C-8 produces a fragment with m / z = 754.62, removing C-7 produces a fragment with m / z = 740.54, removing C-6 produces a fragment with m / z = 726.61, removing C-5 produces a fragment with m / z = 712.58, removing C-4 produces a fragment with m / z = 698.56, removing C-3 produces a fragment with m / z = 684.54, removing C-2 produces a fragment with m / z = 670.52, and C-1 is a carbonyl carbon, which is removed together with -O- and is not included in this comparison. Therefore, a total of 16 characteristic fragments are generated. Except for the C8 and C10 C-C bond breaks, the abundance of fragment peaks generated by C-C bond breaks at other positions is not significantly different in cis / trans compounds. The fragment peaks generated by C8 and C10 C-C bond breaks near the double bond show a stronger abundance in the cis structure than in the trans structure. Therefore, the cis and trans double bond isomers TAG can be identified by directly comparing the relative abundance of characteristic fragment ions. Figure 4 As shown, the relative abundance of fragments generated by the breakage of the C8 and C10 C-C bonds in the cis configuration is >0.2, while the relative abundance of fragments in the trans configuration is <0.2. The same fragmentation pattern and trend of relative abundance were also found in the secondary spectra of two groups of monounsaturated cis-trans isomer triglycerides, namely TAG 16:1 / 16:1 / 16:1(9Z) and TAG 16:1 / 16:1 / 16:1(9E), TAG 20:1 / 20:1 / 20:1(11Z) and TAG 20:1 / 20:1 / 20:1(11E), which can be used as an auxiliary basis for the identification of cis-trans isomers.

[0092] 4.2 Characterization of Double Bond Positions in Polyunsaturated Compounds

[0093] From the EAD mass spectrum of the eight polyunsaturated TAGs, it can be observed that a series of intact acyl chain fragment ions can be found in the mass region between the TAG molecular ion [M+Na] + and the loss of a single acyl chain product ion [M+Na-R1] + The mass spectrum of TAG 22:6 / 22:6 / 22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z) is taken as an example Figure 5 ), the [M+Na]+m / z = 1045.71 is observed; within the [M+Na-R]+range, the parent ion [M+Na]+loses one C22:6 fatty acyl to form one fragment ion [M+Na-RxCOOH] + m / z = 717.47, this fragment ion can be the loss of C22:6 fatty acyl at sn-1, sn-2, sn-3 position; between m / z = 1045.71 and 717.47, there is a series of acyl chain fragment ions, the fatty acid chain can produce continuous de-CH2fragmentation, while the position of double bond linkage does not break, resulting in a group of fragment peaks with a mass number difference of 26 Da, the abundance of fragment ions forms a "V" shape at the position of double bond, clearly indicating the position of C=C double bond. By zooming in the high m / z fragment region Figure 5 b) it can be observed that there are C=C double bonds at C4-C5, C7-C8, C10-C11, C13-C14, C16-C17, C19-C20, from which it can be deduced that the unsaturated C22 fatty acyl chain contains 6 double bonds at C4, C7, C10, C13, C16, C19 positions, respectively. This method has the ability to identify the number and position of multiple double bonds.

[0094] Example 2

[0095] For all the unsaturated triglycerides analyzed in Example 1, the distinction between cis and trans isomers shows a trend that trans (trans triglycerides) exhibits a higher ratio than cis (cis triglycerides) as the electron beam energy (Ke) increases, this difference indicates that the relative abundance ratio of the two fragment ions near the double bond of the fatty acyl chain plays an important role in the determination of the content of triglyceride cis and trans isomers.

[0096] To verify this hypothesis, this example analyzes the mixture of cis C18:1 and trans C18:1 standards prepared at different molar ratios, and monitors the relative abundance of the non-radical dehydrogenation fragment m / z = 793.6 and the radical fragment m / z = 794.6 ion. As Figure 6 shown, the relative ratio of specific fragment ions increases linearly as the relative molar concentration ratio of cis C18:1 in the solution increases.

[0097] Further, cis C18:1 and trans C18:1 mixture with concentration ratio of 1:1 was also prepared in the present example and the ratio obtained was 0.24073 (0.24036 was obtained from standard curve calculation) and the relative error was 0.15%. Thus, the method of the present example can be used to determine the relative proportion of cis and trans isomers in a lipid mixture.

[0098] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0099] Although the embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and variations can be made thereto without departing from the principles and spirit of the present application, and the scope of the present application is defined by the claims and their equivalents.

Claims

1. A method of detecting triglyceride double bond structural isomers, characterized by, The method comprises the following steps: providing a triglyceride sample to be detected; mixing the triglyceride sample with a salt compound to obtain a sample to be detected; detecting the sample to be detected by using a high-resolution mass spectrometer to obtain a primary mass spectrum result and a secondary mass spectrum result; based on the molecular ion peak [M+N] in the primary mass spectrum result and the molecular ion peak [M+N] in the secondary mass spectrum result + , the fragment ion [M+N-R] + , and the fragment ion [M+N-2R] + , by comparing with a lipid compound database, so as to obtain the structure type of each branched chain of the triglyceride sample and determine the mass-to-charge ratio range of the fragment ion [M+N-R] + , wherein M represents a triglyceride, N represents a cation combined with the ionized triglyceride, R represents a fatty acid acyl chain of the triglyceride sample, and the carboxyl carbon at the end of the fatty acid acyl chain is marked as C1; and based on the abundance of acyl chain fragment ions between the molecular ion peak [M+N] and the fragment ion [M+N-R] and the difference in mass-to-charge ratios of characteristic acyl chain fragment ions + + to determine the position and cis-trans configuration of each double bond in the branched chain.​ 2. The method of claim 1, wherein, the detection conditions of the high-resolution mass spectrometer are as follows: working solution flow rate: 30 uL / min; monitoring mode: high-resolution multiple reaction monitoring; electrospray ionization: ESI(+); EAD electron kinetic energy: 8-17 eV; electron beam current: 7000 nA; electrospray voltage: +5500 V; ion source temperature: 300 DEG C; atomization gas: 30 psi; heating gas: 30 psi; gas curtain gas: 30 psi.

3. The method of claim 1, wherein, The method for determining the position of the branched double bond of the triglyceride sample comprises the following steps: determining the acyl chain fragment ion of each carbon atom between the molecular ion peak [M+N] + and the fragment ion [M+N-R] + ​ determining the position of the double bond based on the mass number difference between the branched chain adjacent acyl chain fragment ions.

4. The method of claim 3, wherein, If the mass number difference between the branched chain adjacent acyl chain fragment ions is 14.0±0.1, then the two carbon atoms are single-bonded.

5. The method of claim 3, wherein, If the mass number difference between the branched chain adjacent acyl chain fragment ions is 26.0±0.1, and it is determined that the carbon atom corresponding to the acyl chain fragment ion adjacent to the carbonyl end of the fatty acid acyl chain is located at the m-1 position, then the carbon atom corresponding to the other acyl chain fragment ion is located at the m+1 position, and the carbon atom at the m position does not generate the acyl chain fragment ion, so that the carbon atoms at the m position and the m+1 position are double-bonded.

6. The method of claim 5, wherein, If the branched chain contains one double bond, then the ion with the highest abundance in each of the acyl chain fragment ions of the branched chain is generated by single-bond breaking between the m+2 position carbon atom and the m+3 position carbon atom.

7. The method of claim 1, wherein, If the branched chain contains one double bond, the method for determining the cis-trans structure of the double bond comprises the following steps: determining the position of the double bond, and determining that the double bond is located between the n position carbon atom and the n+1 position carbon atom; determining the abundance of the even-electron product ion fragment of the n+1 position carbon atom and the abundance of the odd-electron product ion; calculating the I value of the n+1 position carbon atom based on formula (1), wherein when the I value <1.0, the double bond is cis; and when the I value >1.0, the double bond is trans. I = abundance of even-electron product ion fragment ÷ abundance of odd-electron product ion (1).

8. The method of claim 1, wherein, If the branched chain contains one double bond, the method for determining the cis-trans structure of the double bond comprises the following steps: determining the position of the double bond, and determining that the double bond is located between the n position carbon atom and the n+1 position carbon atom; calculating the relative abundance of the acyl chain fragment ion fragments of the n-1 position carbon atom and the n+1 position carbon atom based on formula (2), wherein when the relative abundance I of the acyl chain fragment ions of the n-1 position carbon atom and the n+1 position carbon atom are both greater than 0.2, the double bond is cis; and when the relative abundance I of the acyl chain fragment ions of the n-1 position carbon atom and the n+1 position carbon atom are both less than 0.2, the double bond is trans. I = relative abundance of acyl chain fragment ion fragments of n-1 position carbon atom and n+1 position carbon atom (2). wherein I = abundance of acyl chain fragment ion fragments ÷ molecular ion peak [M + N] + abundance (2).

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