Method for detecting triglyceride double-bond structure isomer

Abstract

The invention discloses a method for detecting a triglyceride double-bond structure isomer. The method comprises the following steps: on the basis of molecular ion peaks [M + N] < + >, fragment ions [M + N-R] < + > and fragment ions [M + N-2R] < + > in a primary mass spectrum result and a secondary mass spectrum result of a triglyceride sample, comparing with a lipid compound database so as to obtain the structure type of each branch chain of the triglyceride sample and determine the mass-to-charge ratio range of the fragment ions [M + N-R] < + >; based on the abundance of acyl chain fragment ions between a molecular ion peak [M + N] < + > and a fragment ion [M + N-R] < + >, the ratio of the abundance and the difference value of the mass-to-charge ratio of the adjacent characteristic acyl chain fragment ions, the positions and cis-trans structures of all branched chain double bonds are determined. According to the method, the fine fragmentation law of the TAG double-bond isomer is disclosed, and a comprehensive TAG double-bond isomer characterization strategy is constructed.

Description

Technical Field

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

[0002] Triglycerides (TAGs) are a class of important physiological lipids generated by the esterification reaction of three fatty acids (saturated fatty acids and unsaturated fatty acids) and one molecule of glycerol. There are usually one or more -C=C- in the acyl chain of unsaturated triglycerides, so a series of double bond position isomers and cis-trans configuration isomers are formed, and these isomers may have great differences in biological functions. For example, the number of C=C determines the degree of unsaturation of lipids, and the position of C=C can affect the permeability and thickness of cell membranes, the plasticity of cancer cells, etc. For example, the two isomers of linolenic acid, α-linolenic acid and γ-linolenic acid, participate in different cell 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 pheromone component of insects. Therefore, achieving the fine structural characterization of the C=Cs position and cis-trans isomers on the acyl chain of unsaturated triglycerides helps to understand the energy conversion relationship between triglyceride structural information and biological effects, and plays an important role in clinical diagnosis, life sciences, and lipid biology.

[0003] Therefore, methods for fine structural characterization of unsaturated triglycerides need to be studied. Summary of the invention

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

[0005] According to one aspect of the present invention, the present invention provides a method for detecting triglyceride double bond structural isomers. According to an embodiment of the present invention, the method comprises: providing a triglyceride sample to be tested; mixing the triglyceride sample with a salt compound to obtain a test liquid; detecting the test liquid using a high-resolution mass spectrometer to obtain a primary mass spectrometry result and a secondary mass spectrometry result; based on the molecular ion peaks [M+N]+, fragment ions [M+NR]+ and fragment ions [M+N-2R]+ in the primary mass spectrometry result and the secondary mass spectrometry result, comparing with a lipid compound database to obtain the structural classes of each branch chain of the triglyceride sample. The method comprises the following steps: first, determining the mass-to-charge ratio range of the fragment ion [M+NR]+, wherein M represents triglyceride, N represents the cation bound to the ionized triglyceride, R represents the fatty acid acyl chain of the triglyceride sample, and the carboxyl carbon at the end of the fatty acid acyl chain is recorded 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+NR]+, the abundance ratio, and the difference in mass-to-charge ratio of the adjacent characteristic acyl chain fragment ion, so as to determine the position of each branch double bond and the cis-trans structure.

[0006] According to the method for detecting triglyceride double bond structural isomers in an embodiment of the present invention, the fine fragmentation rules of TAG double bond isomers are revealed by studying double bond position isomers, cis-trans isomer mass spectrometry fragmentation characteristic fragments, fragment abundance and abundance ratio, etc., and a comprehensive TAG double bond isomer characterization strategy is constructed, which improves the efficiency and accuracy of double bond structure characterization and provides support for in-depth exploration of lipid structure.

[0007] In addition, the method for detecting triglyceride double bond structural isomers according to the above embodiment of the present invention may also have the following additional technical features:

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

[0009] According to an embodiment of the present invention, the method for determining the double bond position of the branched chain of a triglyceride sample comprises: determining the molecular ion peak [M+N] + and the fragment ion [M+NR] +The acyl chain fragment ions of each carbon atom between the branches; and determining the position of the double bond based on the mass number between the adjacent acyl chain fragment ions of the branch chain.

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

[0011] According to an embodiment of the present invention, the mass number difference between adjacent acyl chain fragment ions of the branch chain is 26.0±0.1, and the carbon atom corresponding to the acyl chain fragment ion adjacent to the carbonyl end of the fatty acid acyl chain is assumed to be located at the m-1 position, and the carbon atom corresponding to another acyl chain fragment ion is located at the m+1 position. If the carbon atom at the m position does not produce the acyl chain fragment ion, there is a double bond between the carbon atoms at the m position and the m+1 position.

[0012] According to an embodiment of the present invention, the side chain contains a double bond, and the side chain determines that the ion with the highest abundance among the acyl chain fragment ions is produced by the breakage of the single bond between the carbon atom at the m+2 position and the carbon atom at the m+3 position.

[0013] According to an embodiment of the present invention, the side chain contains a double bond, and the method for determining the cis-trans structure of the double bond includes: determining the position of the double bond, setting the double bond to be located between the nth and n+1th carbon atoms; determining the abundance of even-electron product ion fragments and the abundance of odd-electron product ions of the n+1th carbon atom; calculating the I value of the n+1th carbon atom based on formula (1), when the I value is <1.0, the double bond is cis; when the I value is >1.0, the double bond is trans.

[0014] Here, I = abundance of even-electron product ion fragments ÷ abundance of odd-electron product ions (1).

[0015] According to an embodiment of the present invention, the side chain contains a 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 to be located between the nth and n+1th carbon atoms; calculating the relative abundance of the acyl chain fragment ion fragments of the n-1th and n+1th carbon atoms based on formula (2), when the relative abundance I of the acyl chain fragment ions of the n-1th and n+1th carbon atoms are both 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.

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

[0017] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0019] Figure 1 Shown is a secondary mass spectrum (a) of TAG 17:1 / 17:1 / 17:1(10Z) in EAD fragmentation mode and a zoomed-in view of the high m / z fragment region (b) according to one embodiment of the present invention;

[0020] Figure 2 Shows an enlarged view of high m / z fragment regions of five monounsaturated compounds according to one embodiment of the present invention;

[0021] Figure 3 The figure shows a comparison of secondary mass spectra (double bond region) of TAG 18:1 / 18:1 / 18:1(9Z) and TAG 18:1 / 18:1 / 18:1(9E) in EAD fragmentation mode according to one embodiment of the present invention;

[0022] Figure 4 The figure shows a comparison of the relative abundances of characteristic fragment ions -CH2 generated by the acyl chain breaking in sequence, taking TAG 18:1 / 18:1 / 18:1(9Z) and TAG 18:1 / 18:1 / 18:1(9E) as examples according to an embodiment of the present invention, wherein the relative intensity is expressed as the ratio of the mass spectrometry response value of the characteristic fragment ion to the parent ion;

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

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

[0025] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0026] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

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

[0028] In order to facilitate the connection of the method for detecting triglyceride double bond structural isomers in the embodiment of the present invention, the method is explained here, and the method comprises:

[0029] S100 Samples Available

[0030] According to an embodiment of the present invention, a triglyceride sample to be tested is provided, which is a purified triglyceride sample.

[0031] S200 Preparation of test solution

[0032] According to an embodiment of the present invention, the triglyceride sample is mixed with a salt compound to obtain a test solution. TAG is a non-polar compound that is not easily ionized, and all analytical work must add some salt (such as sodium or ammonium cations) to form an adduct.

[0033] According to an embodiment of the present invention, the triglyceride sample is mixed with sodium salt. Sodium-containing TAG is a target precursor ion that is easier to cleave than aminated TAG because the sodium-containing precursor ion is prone to lose the double chain. In contrast, aminated TAG tends to be more susceptible to cleavage in NH4 + The cleavage at the group results in no fragment loss within the fatty acid chain. Therefore, it is preferred to add sodium acetate to form sodium TAG ions.

[0034] S300 Mass Spectrometry

[0035] According to an embodiment of the present invention, a high-resolution mass spectrometer is used to detect the test liquid so as to obtain a primary mass spectrometry result and a secondary mass spectrometry result.

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

[0037] S400 determines the range of structural formula

[0038] According to an embodiment of the present invention, based on the molecular ion peak [M+N] in the primary mass spectrometry result and the secondary mass spectrometry result + 、Fragment ions [M+NR] + and fragment ions [M+N-2R] + , by comparing with the lipid compound database, in order to obtain the structural type of each branch chain of the triglyceride sample and determine the mass-to-charge ratio range of the fragment ion [M+NR]+, wherein M represents triglyceride, N represents the cation bound to the ionized triglyceride, and R represents the fatty acid acyl chain of the triglyceride sample. Specifically, the breakage of a fatty acid acyl chain, that is, the reduction of an R group, produces the fragment ion [M+NR] + , the two fatty acid acyl chains are broken, that is, two R groups are reduced, and the fragment ion [M+N-2R] is produced. + ; The carboxyl carbon at the end of the fatty acid acyl chain is denoted as C1. Furthermore, the carbon atom at the nth position is the nth carbon atom with the carbon at the carboxyl end being denoted as 1, and so on. n-1 is the n-1th carbon atom with the carbon at the carboxyl end being denoted as 1.

[0039] According to an embodiment of the present invention, based on the ion peak [M+N] + The mass-to-charge ratio of the lipid compound database was compared to obtain the possible composition of fatty acids. + The molecular weight of TAG can be obtained by comparing it with the lipid compound database to obtain the possible R-based molecular composition of TAG with this molecular weight. Based on the possible composition of the fatty acid, the fragment ions [M+NR] are determined respectively. + The mass-to-charge ratio range and the fragment ions [M+N-2R] + mass-to-charge ratio range.

[0040] S500 determination of positional isomers

[0041] According to an embodiment of the present invention, based on the molecular ion peak [M+N] + and fragment ions [M+NR] + The abundance of the acyl chain fragment ions, the ratio of the abundance, and the difference in mass-to-charge ratio between the adjacent and characteristic acyl chain fragment ions are used to determine the position and cis-trans structure of the double bonds of each branch chain, that is, to determine the double bond position and cis-trans structure of each fatty acid acyl chain.

[0042] (I) Double bond position

[0043] The EAD mass spectrum of unsaturated TAGs shows that in the TAG molecular ion [M+Na] + and loss of a single acyl chain product ion [M+Na-R1] + In the mass region between , a series of complete acyl chain fragments can be found, indicating that the fatty acid chain can produce continuous de-CH2 fragments, while the double bond connection position will not be broken. The inventor studied the EAD mass spectrum and found the following method to determine the double bond position:

[0044] According to an embodiment of the present invention, the method for determining the double bond position of the branched chain of a triglyceride sample comprises: determining the molecular ion peak [M+N] + and the fragment ion [M+NR] + The acyl chain fragment ions of each carbon atom between the branches are determined; and the position of the double bond is determined based on the mass number between the adjacent acyl chain fragment ions of the branch chain. It should be noted that this method is applicable to both fatty acid chains with only one double bond and fatty acid chains with multiple double bonds.

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

[0046] Specifically, according to an embodiment of the present invention, the mass number difference between the adjacent acyl chain fragment ions of the branch chain is 26.0±0.1, and the carbon atom corresponding to the acyl chain fragment ion adjacent to the carbonyl end of the fatty acid acyl chain is assumed to be 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 produce the acyl chain fragment ion, and there is a double bond between the carbon atoms at the mth position and the m+1th position. That is to say, for the C=C double bond, for each double bond that appears in the fragment part of the acyl chain, the m / z of the radical fragment will move +2H, generating a fragment peak with a mass number difference of 26Da. Since the double bond is not a favorable cleavage site, the abundance of the fragment ion 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 invention, the side chain contains a double bond, then the ion with the highest abundance among the acyl chain fragment ions of the side chain should be produced by the rupture of the single bond between the carbon atom at the m+2 position and the carbon atom at the m+3 position.

[0048] Further, the method can be understood that each monounsaturated compound will produce C x-2 The fragment peaks (x represents the total carbon number of the fatty acyl chain) are shown in Figure 2. The internal rupture of the acyl chain has the same double bond recognition method, that is, there is no fragment peak at the double bond position, and the fragment abundance generated by the rupture of the CC single bond at two Cs is the highest.

[0049] (II) Cis-trans structure

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

[0051] Method 1:

[0052] According to an embodiment of the present invention, the side chain contains a double bond, and the method for determining the cis-trans structure of the double bond includes: determining the position of the double bond, setting the double bond to be located between the nth and n+1th carbon atoms; determining the abundance of even-electron product ion fragments and the abundance of odd-electron product ions of the n+1th carbon atom; calculating the I value of the n+1th carbon atom based on formula (1), when the I value is <1.0, the double bond is cis; when the I value is >1.0, the double bond is trans.

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

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

[0055] Method 2:

[0056] According to an embodiment of the present invention, the side chain contains a 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 to be located between the nth and n+1th carbon atoms; calculating the relative abundance of the acyl chain fragment ion fragments of the n-1th and n+1th carbon atoms based on formula (2), when the relative abundance I of the acyl chain fragment ions of the n-1th and n+1th carbon atoms are both 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] Here, I = the abundance of the acyl chain fragment ion fragments ÷ the abundance of the molecular ion peak [M+N]+ (2).

[0058] This method directly compares the abundance of the fragment peaks produced by the internal break of the acyl chain -CH2. The fragment peaks produced by the break of the CC bond near the double bond show a stronger abundance in the cis structure than in the trans structure.

[0059] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and should not be construed as limiting the present invention.

[0060] Below in conjunction with embodiment, the scheme of the present invention will be explained.It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.Unrepresented specific techniques or conditions in the embodiment, according to the technology or conditions described in the document in this area or according to the product specification sheet.Reagents used or instruments that do not indicate manufacturers are conventional products that can be obtained commercially, for example, can be purchased from Sigma company.

[0061] Example 1

[0062] In this embodiment, the method for detecting the triglyceride double bond structural isomers according to the embodiment of the present invention was used for verification, as follows:

[0063] 1. Instruments and reagents

[0064] Instruments: Zeno-TOF7600 high-resolution mass spectrometer and SCIEX3.0 data processing software (ABSciex, USA), equipped with ESI ion source; Milli-Q ultrapure water system (Millipore, USA); Harvard Apparatus syringe pump Pump11Elite;

[0065] Reagents: n-hexane, isopropanol (chromatographic grade, ThermoFisher, USA); formic acid, ammonium formate, sodium acetate (chromatographic grade, Sigma-Aldrich, USA);

[0066] Lipid standards: 17 triglyceride standards were purchased from ZZSTANDARD. This study used the lipid structure nomenclature proposed by Liebisch et al.

[24] : the position of the double bond in the fatty acyl chain is indicated in a pair of brackets according to the Δ nomenclature. The position of the double bond is counted from the carboxyl end and is indicated using symbols such as C-1, C-2, C-3, ... (where C-1 is the carbon of the carboxyl group). For example, FA18:1, if counted from the carboxyl end, has a double bond between C-11 and C-12, it is expressed as FA 18:1 (Δ11). The Z / E designation (Z for cis and E for trans) is used to define the double bond geometry. For example, TAG 18:1 / 18:1 / 18:1(9Z) indicates that the triglyceride contains three unsaturated C18 fatty acyl chains and a cis double bond at the Δ-9 position.

[0067] Detailed information of the standards is shown in Table 1;

[0068] Table 1. Basic information of 17 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 it in n-hexane solvent, prepare a 0.5 mg / mL standard stock solution, store it at -20°C until use;

[0074] Preparation of standard working solution: Pipette 1 mL of the standard stock solution and dilute it with a mixed solution of isopropanol: water (9:1 v / v) containing 0.5 mM sodium acetate to prepare a 0.05 mg / ml standard working solution, and store it at -20°C until use.

[0075] 3. Mass spectrometry

[0076] All qualitative data were obtained by high-resolution multiple reaction monitoring (MRM HR ) mode; the detector of the system was operated in electrospray ionization positive mode (ESI+); the nebulizer gas, heating gas and curtain gas were all set to 30psi; the ion source temperature was 300℃, 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 injection, and the acquisition time was 6min. The raw data were processed using SCIEX OS software 3.3 with browser and analysis modules.

[0077] 4. Results and Discussion

[0078] In this example, four types of representative TAGs double bond isomers were selected, including cis-monounsaturated triglycerides (5 types), cis-polyunsaturated triglycerides (8 types), monounsaturated cis-trans isomerized triglycerides (3 groups) and 1 group of polyunsaturated cis-trans isomerized compounds, totaling 17 types. The monounsaturated triglycerides 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); the 8 polyunsaturated triglycerides 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); three groups of monounsaturated cis-trans isomers 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 TAG 20:1 / 20:1 / 20:1(11Z) and TAG 20:1 / 20:1 / 20:1(11E); 1 group of polyunsaturated cis-trans isomers are TAG 18:2 / 18:2 / 18:2(9Z, 12Z) and TAG18:2 / 18:2 / 18:2(9E, 12E).

[0079] 4.1 Detailed characterization of double bonds in monounsaturated compounds

[0080] 4.1.1 Characterization of double bond position

[0081] From the EAD mass spectra of the five monounsaturated TAGs, it can be observed that in the TAG molecular ion [M+Na] + and loss of a single acyl chain product ion [M+Na-R1] +In the mass region between , a series of complete acyl chain fragments can be found, indicating that the fatty acid chain can produce continuous de-CH2 fragments, while the double bond connection position will not be broken. Take the mass spectrum of TAG 17:1 / 17:1 / 17:1(10Z) as an example ( Figure 1 ), observed [M+Na] + m / z = 865.73; in [M+Na-R] + In the range, the parent ion [M+Na] + 1 fragment ion formed by loss of a C17:1 fatty acyl group [M+Na-RxCOOH] + m / z = 597.49, this fragment ion can be the loss of C17:1 fatty acyl at any position of sn-1, sn-2, sn-3; between m / z = 865.73 and 597.49, there are a series of acyl chain fragment ions, and the relative abundance of these diagnostic peaks is usually 10.1% compared with the fragment ion [M+Na-2R]+ of the loss of the double acyl chain. The typical fragmentation pattern of double bonds in the EAD spectrum is CC single bond fragmentation, while the C=C double bond does not fragment, and each cleavage site produces an odd-electron free radical fragment and an even-electron non-free radical fragment. For CC single bonds, the free radical fragments appear as a series of ions (i.e., methylene or -CH2) evenly spaced at 14.01Da. For C=C double bonds, the m / z of the free radical fragment will move +2H for each double bond in the fragment part of the acyl chain, producing fragment peaks with a mass number difference of 26Da. Since the double bond is not a favorable cleavage site, the abundance of the fragment ions forms a "V" shape at the double bond position, clearly indicating the location of the C=C double bond. The spectrum is enlarged by the high m / z fragment region ( Figure 1 b) It can be observed that the fragment peak group with a mass number difference of 26Da and its abundance is "V" type, which can determine the presence of a double bond at this position, that is, there is a C=C double bond at C10-C11. Based on this, it can be inferred that the unsaturated C17 fatty acyl chain contains a double bond at the C-10 position.

[0082] Zoom in on the high m / z fragment region ( Figure 1 b) It can be observed that there is a fragment interference with very low abundance at the C-10 position, and the "V"-shaped distribution of the fragment ion abundance is not easy to observe. In order to explore a more accurate method for identifying the double bond position, the relative abundance of the fragment ions generated by the desorption of -CH2 inside the fatty acyl chain was further compared. Take the mass spectrum of TAG 17:1 / 17:1 / 17:1(10Z) as an example. Figure 1As shown in Figure 1b, it is found that the abundance of fragment ions generated by the breakage of the CC bond two Cs away from the double bond position at the methyl end is the highest, that is, if there is a C=C double bond at the C10 position, then the abundance of fragment ions generated by the breakage of the CC single bond at the C12 position two Cs away is the highest; the possible reason for this phenomenon is that the CC configuration at the C12 position is different from that at other positions, making it easier to break; the same pattern is also found in the secondary spectra of four monounsaturated triglycerides, such as TAG 14:1 / 14:1 / 14:1(9Z), 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) (see Figure 1S), as shown in Figure 1S. Figure 2 As shown in the figure, by comparing the five monounsaturated triglycerides (X axis), it is found that the internal cleavage of the fatty acyl chain has the same characteristic cleavage law, that is, the continuous removal of -CH2, and the C=C double bond does not break (Y axis). Each monounsaturated compound will produce C n-2 The fragment peaks (n represents the total carbon number of the fatty acyl chain); the internal break of the acyl chain has the same double bond recognition method, that is, the double bond position does not contain a fragment peak, and the fragment abundance generated by the break of the CC single bond at two Cs is the highest (Z axis).

[0083] 4.1.2 Characterization of double bond cis-trans

[0084] Unsaturated triglycerides may form cis-trans isomers with different spatial structures due to the presence of double bonds. Cis-trans isomers may affect the physical properties (such as melting point and solubility), chemical properties (such as reactivity) and biological functions (such as metabolic pathways, cell signal transduction, nutritional value) of triglycerides. Therefore, it is of great significance to accurately characterize cis-trans isomers. Take the double bond region mass spectra of TAG 18:1 / 18:1 / 18:1(9Z) and TAG 18:1 / 18:1 / 18:1(9E) as an example. Figure 3 As shown, it was observed that the two spectra had similar abundance curves, and the cleavage of the CC single bond in the acyl chain produced two fragment ions: an even-electron (non-radical) product ion and its corresponding odd-electron (radical) product ion; however, at the C10 site adjacent to the double bond, the abundances of the radical peak m / z=794.638 and the hydrogen loss non-radical peak m / z=793.639 produced by the CC single bond cleavage were significantly different, which was manifested in that the "hydrogen loss" even-electron fragment formed by the CC cleavage at the C10 position of the trans isomer was higher in abundance, and the odd-electron radical fragment formed by the cis isomer was higher in abundance.

[0085] The abundance ratios (I) of the characteristic fragments [hydrogen lost peak intensity at x position]+ and [radicalpeak intensity at x position]+ were further compared. In this example, three groups of monounsaturated cis-trans isomers were analyzed, 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] From the results in Table 2, we can see that the I values ​​of cis-configuration triglycerides 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 to 0.96, and the I values ​​of trans-configuration triglycerides 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 to 1.44. In summary, it can be considered that the I value of cis-configuration TAGs is <1.0, while the I value of trans-configuration TAG is >1.0. Based on this, the cis- and trans-double bond isomers of TAG can be identified by comparing the ratio of the abundance of free radical fragments and hydrogen loss non-free radical fragment ions, which is one of the very important identification bases. This method solves the problem of needing a set of cis- and trans-compounds for comparison and differentiation, and makes up for the shortcomings of lipid standards being expensive, scarce, and difficult to purchase.

[0088] Table 2. Relative abundance ratios of radical ions and hydrogen loss non-radical fragment ions in EAD fragmentation mode

[0089]

[0090] I [1、2、3、4、5、6] It is expressed as the mass spectrometer response values ​​of 6 time periods (0-1min, 1-2min, 2-3min, 3-4min, 4-5min, 5-6min) within 6min. [1、2、3、4、5、6] Represented as I [非自由基]+ :I [自由基]+ , I* is represented by the mean of the mass spectrometer response ratios in six time periods;

[0091] In order to explore the more detailed differences between cis and trans isomers, the relative abundance of the fragment peaks produced by the internal breakage of the fatty acyl chain was further compared, that is, the relative abundance of the fragment peaks produced by the breakage of the CC single bond was compared within the mass range of the quasi-molecular ion [M+Na]+ and the loss of a fatty acyl chain [M+Na-R]+. Taking TAG 18:1 / 18:1 / 18:1(9Z) and TAG 18:1 / 18:1 / 18:1(9E) compounds as examples, Figure 4 As shown, the green column represents the cis structure, the orange column represents the trans structure, and the -CH2 inside the acyl chain is broken in sequence, namely, the fragments generated by removing C-18 are m / z = 892.77, the fragments generated by removing C-17 are m / z = 878.84, the fragments generated by removing C-16 are m / z = 864.87, the fragments generated by removing C-15 are m / z = 850.76, the fragments generated by removing C-14 are m / z = 836.71, the fragments generated by removing C-13 are m / z = 822.72, the fragments generated by removing C-12 are m / z = 808.67, the fragments generated by removing C-11 are m / z = 794.65, and the fragments generated by removing C-9 are m / z = 836.71. There is an unbroken double bond at C10, and the removal of C=C produces a fragment with m / z=768.64, the removal of C-8 produces a fragment with m / z=754.62, the removal of C-7 produces a fragment with m / z=740.54, the removal of C-6 produces a fragment with m / z=726.61, the removal of C-5 produces a fragment with m / z=712.58, the removal of C-4 produces a fragment with m / z=698.56, the removal of C-3 produces a fragment with m / z=684.54, the removal of C-2 produces a fragment with m / z=670.52, C-1 is a carbonyl carbon, which is detached together with -O- and is not included in this comparison, so a total of 16 characteristic fragments are generated. Except for the CC bond breakage at C8 and C10, the abundance of the fragment peaks produced by the CC bond breakage at other positions has no significant difference in cis / trans compounds. The cis structure of the fragment peak produced by the CC bond breakage at C8 and C10 near the double bond shows a stronger abundance than the trans structure. Based on this, the cis and trans double bond isomers of TAG can be identified by directly comparing the relative abundance of characteristic fragment ions. Figure 4 As shown in the figure, it can be considered that the relative abundance values ​​of the fragments produced by the breakage of the CC bonds at positions C8 and C10 of the cis configuration are >0.2, while the relative abundance values ​​of the trans configuration fragments are <0.2. The same fragmentation rules and trends in relative abundance values ​​were also found in the secondary spectra of two groups of monounsaturated cis- and trans-isomerized triglycerides, 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 TAG20:1 / 20:1 / 20:1(11E), which can serve as an auxiliary basis for the identification of cis- and trans-isomers.

[0092] 4.2 Characterization of double bond positions in polyunsaturated compounds

[0093] From the EAD mass spectra of 8 polyunsaturated TAGs, it can be observed that in the TAG molecular ion [M+Na] + and loss of a single acyl chain product ion [M+Na-R1] + In the mass region between , a series of complete acyl chain fragment ions can be found. Take the mass spectrum of TAG 22:6 / 22:6 / 22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z) as an example ( Figure 5 ), [M+Na]+ m / z = 1045.71 was observed; in the [M+Na-R]+ range, the parent ion [M+Na]+ lost a C22:6 fatty acyl group to form a fragment ion [M+Na-RxCOOH] + m / z = 717.47, this fragment ion can be the loss of C22:6 fatty acyl at any position of sn-1, sn-2, sn-3; between m / z = 1045.71 and 717.47, there are a series of acyl chain fragment ions, the fatty acid chain can produce continuous de-CH2 fragments, and the position of the double bond connection will not break, producing a fragment peak group with a mass number difference of 26Da, and the abundance of the fragment ion forms a "V" shape at the double bond position, clearly indicating the position of the C=C double bond. By enlarging the spectrum in the high m / z fragment area ( 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, and C19-C20, from which it can be inferred that the unsaturated C22 fatty acyl chain contains 6 double bonds, respectively at C4, C7, C10, C13, C16, and C19. This method has the ability to identify the number and position of multiple double bonds.

[0094] Example 2

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

[0096] To test this hypothesis, this example analyzed mixtures of cis C18:1 and trans C18:1 standards prepared at different molar ratios and monitored the relative abundance of the non-radical hydrogen loss fragment m / z = 793.6 and the radical fragment m / z = 794.6 ions. Figure 6 As shown, with the increase of the relative molar concentration ratio of cis-C18:1 in the solution, the relative ratio of specific fragment ions increased linearly.

[0097] In addition, this embodiment also prepared a mixture with a concentration ratio of 1:1 between cis C18:1 and trans C18:1. The experimentally obtained ratio was 0.24073 (0.24036 calculated from the standard curve), and the relative error was 0.15%. Therefore, the method of the embodiment of the present invention can be used to determine the relative ratio of cis and trans isomers in a lipid mixture.

[0098] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

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

Claims

1. A method for detecting triglyceride double bond structural isomers, characterized in that: include: Providing a triglyceride sample to be tested; Mixing the triglyceride sample with a salt compound to obtain a test solution; Using a high-resolution mass spectrometer to detect the test liquid to obtain a primary mass spectrometry result and a secondary mass spectrometry result; Based on the molecular ion peaks [M+N] in the primary mass spectrometry results and the secondary mass spectrometry results + 、Fragment ions [M+NR] + and fragment ions [M+N-2R] + , by comparing with the lipid compound database, so as to obtain the structural type of each branch chain of the triglyceride sample and determine the fragment ion [M+NR] + wherein M represents triglyceride, N represents a cation bound to 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 denoted as C1; and Based on molecular ion peak [M+N] + and fragment ions [M+NR] + The abundance of the acyl chain fragment ions and the difference in the mass-to-charge ratio of the characteristic acyl chain fragment ions can be used to determine the position of the double bonds of each branch chain and the cis-trans structure.

2. The method according to claim 1, characterized in that The detection conditions of the high-resolution mass spectrometer are: Working solution flow rate: 30uL / min; Monitoring mode: High-resolution multiple reaction monitoring; Electrospray ionization: ESI(+); Electron kinetic energy of EAD: 8-17 eV; Electron beam current: 7000nA; Electrospray voltage: +5500 V; Ion source temperature: 300°C; Atomizing gas: 30psi; Heating gas: 30psi; Curtain gas: 30psi.

3. The method according to claim 1, characterized in that The method for determining the double bond position of the branched chain of a triglyceride sample comprises: Determine the molecular ion peak [M+N] + and the fragment ion [M+NR] + the acyl chain fragment ions between each carbon atom; The position of the double bond is determined based on the mass number between adjacent acyl chain fragment ions of the branched chain.

4. The method according to claim 3, characterized in that The mass number difference between adjacent acyl chain fragment ions of the branched chain is 14.0±0.1, and there is a single bond between the two carbon atoms.

5. The method according to claim 3, characterized in that: The mass number difference between adjacent acyl chain fragment ions of the branch chain is 26.0±0.

1. 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 another acyl chain fragment ion is located at the m+1 position. If the carbon atom at the m position does not produce the acyl chain fragment ion, there is a double bond between the carbon atoms at the m position and the m+1 position.

6. The method according to claim 5, characterized in that The branch chain contains a double bond, and the ion with the highest abundance among the acyl chain fragment ions of the branch chain is produced by the cleavage of the single bond between the carbon atom at the m+2 position and the carbon atom at the m+3 position.

7. The method according to claim 1, characterized in that The side chain contains a double bond, and the method for determining the cis-trans structure of the double bond comprises: Determine the position of the double bond, setting the double bond to be between the nth and n+1th carbon atoms; Determine the abundance of even-electron product ion fragments and the abundance of odd-electron product ions of the n+1th carbon atom; The I value of the carbon atom at position n+1 is calculated based on formula (1). When the I value is <1.0, the double bond is cis; when the I value is >1.0, the double bond is trans. Here, I = abundance of even-electron product ion fragments ÷ abundance of odd-electron product ions (1).

8. The method according to claim 1, characterized in that The side chain contains a double bond, and the method for determining the cis-trans structure of the double bond comprises: Determine the position of the double bond, setting the double bond to be between the nth and n+1th carbon atoms; The relative abundance of the acyl chain fragment ion fragments at the n-1th and n+1th carbon atoms is calculated based on formula (2). When the relative abundance I of the acyl chain fragment ions at the n-1th and n+1th carbon atoms is greater than 0.2, the double bond is cis; when the relative abundance I is less than 0.2, the double bond is trans. Where I = abundance of acyl chain fragment ion fragments ÷ molecular ion peak [M+N] + abundance (2).

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