Method for detecting triglyceride position isomers

Abstract

The invention discloses a method for detecting a triglyceride position isomer. The method comprises the following steps: providing a triglyceride sample to be detected; mixing a triglyceride sample with a salt compound to obtain a to-be-detected solution; detecting the liquid to be detected by using a high-resolution mass spectrometer so as to obtain a primary mass spectrum result and a secondary mass spectrum result; the method comprises the steps that a triglyceride sample is obtained, the structure types of all branch chains of the triglyceride sample are obtained on the basis of a first-level mass spectrum result and a second-level mass spectrum result, the triglyceride sample comprises three fatty acid chains sn1, sn2 and sn3 in sequence, and the structure types of the triglyceride sample comprise the AAA type, the AAB type, the ABA type and the ABC type according to the structural difference of sn1, sn2 and sn3. According to the method, first-level and second-level mass spectrum fragment modes with rich information and a fine splitting rule of a TGA position isomer are obtained through a high-resolution mass spectrometer, and a comprehensive sn position isomer characterization strategy of the TGA is constructed.

Description

Technical Field

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

[0002] The development of lipidomics requires standards with clear structure and purity, which are used in the development of lipid analysis methods and the quality control of analysis results. Triglycerides (TAGs) are an important class of lipids with physiological functions. They are produced by esterification of three fatty acids (FA) and one molecule of glycerol. According to the position of FA on the glycerol backbone, they are named sn-1, sn-2 and sn-3. The arrangement order of the fatty acyl chains connected to the glycerol backbone is different, resulting in a large number of fatty acyl chain position isomers (sn position isomers). The position difference of fatty acyl chains has an important influence on the physical properties, biochemical properties and nutritional value of triglycerides. For example, palmitic acid C16.0 at the sn-2 position of the glycerol backbone has the effect of promoting the absorption of minerals by infants and young children, while palmitic acid at the sn-1 and sn-3 positions is easily hydrolyzed by intestinal lipase into free palmitic acid, which undergoes saponification reaction with minerals such as calcium and magnesium, reducing the absorption and utilization efficiency of fat by the human body.

[0003] Therefore, identification of sn-positional isomers of triglycerides in lipid structural characterization remains to be investigated. 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 object of the present invention is to provide a method for detecting triglyceride positional isomers, wherein electron activation dissociation (EAD) is to apply electron beams of different energies to positively charged lipid ions so that the spectrum shows a fragmentation pattern rich in information, thereby realizing the identification of the fatty acyl position in the lipid structure.

[0005] According to one aspect of the present invention, the present invention provides a method for detecting triglyceride positional 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 liquid to be tested; detecting the liquid to be tested using a high-resolution mass spectrometer to obtain a primary mass spectrometry result and a secondary mass spectrometry result; and obtaining the structural type of each branch chain of the triglyceride sample based on the primary mass spectrometry result and the secondary mass spectrometry result, wherein the three fatty acid chains contained in the triglyceride sample are sn1, sn2 and sn3 in sequence, and according to the structural differences of sn1, sn2 and sn3, the structural types of the triglyceride sample include AAA type, AAB type, ABA type and ABC type.

[0006] According to the method for detecting triglyceride positional isomers of an embodiment of the present invention, primary and secondary mass spectral fragments with rich information and fine fragmentation rules of TGA positional isomers are obtained by a high-resolution mass spectrometer, a comprehensive TGA sn positional isomer characterization strategy is constructed, the efficiency and accuracy of fatty acid acyl chain structure characterization are improved, support is provided for in-depth exploration of lipid structure, and rapid identification of fatty acyl positions in lipid structures is achieved.

[0007] In addition, the method for detecting triglyceride positional 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; ion source type: TurboVTM ion source; electrospray ionization: ESI(+); electron kinetic energy of EAD: 10eV; electron beam current: 7000nA; electrospray voltage: +5500V; ion source temperature: 300°C; nebulizing gas: 30psi; heating gas: 30psi; curtain gas: 30psi.

[0009] 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] + , obtaining the structural type of each branch chain of the triglyceride sample, wherein M represents triglyceride, N represents a cation combined with the ionized triglyceride, and R represents the fatty acid acyl chain of the triglyceride sample.

[0010] According to an embodiment of the present invention, based on the ion peak [M+N] + The mass-to-charge ratio of the fragment ions [M+NR] is determined by comparing it with the lipid compound database to obtain the possible composition of the fatty acids. + The mass-to-charge ratio range and the fragment ions [M+N-2R] + mass-to-charge ratio range.

[0011] According to an embodiment of the present invention, in the secondary mass spectrometry result, in the fragment ion [M+N-2R] + There are 3 fragment ions in the mass-to-charge ratio range [M+N-2R] + , and there are two fragment ions [M+N-2R] + If the mass-to-charge ratio of the samples differs by 2Da, the structure type of the triglyceride sample is the ABA type or the AAA type.

[0012] According to an embodiment of the present invention, in the secondary mass spectrometry result, the fragment ion [M+N-2R] + There are 5 fragment ions in the mass-to-charge ratio range [M+N-2R] + , except for a single fragment ion [M+N-2R] + , the remaining four fragment ions [M+N-2R] + Divided into 2 groups, the fragment ions in each group [M+N-2R] + If the mass-to-charge ratio of the triglyceride sample differs by 2Da, the structure type of the triglyceride sample is the ABC type or the AAB type.

[0013] According to an embodiment of the present invention, in the secondary mass spectrometry result, in the fragment ion [M+N-2R] + There are 4 fragment ions in the mass-to-charge ratio range [M+N-2R] + , except for a single fragment ion [M+N-2R] + , the remaining 3 fragment ions [M+N-2R] + The mass-to-charge ratios of the triglyceride samples differ by 2Da respectively, and the structure type of the triglyceride sample is the ABC type or the AAB type.

[0014] According to an embodiment of the present invention, based on the fragment ion [M+N-2R] + The mass-to-charge ratio is used to determine whether the triglyceride sample structure type is the ABA type or the AAA type.

[0015] According to an embodiment of the present invention, based on the fragment ion [M+N-2R] + The mass-to-charge ratio is used to determine whether the triglyceride sample structure type is the ABC type or the AAB type.

[0016] According to an embodiment of the present invention, in the secondary mass spectrometry result, in the fragment ion [M+NR] + In the mass-to-charge ratio range, there is only one fragment ion [M+NR] + , then the triglyceride sample structure type is the AAA type.

[0017] According to an embodiment of the present invention, in the secondary mass spectrometry result, in the fragment ion [M+NR] + In the mass-to-charge ratio range, there are two fragment ions [M+NR] + , then the triglyceride sample structure type is the ABA type or AAB type, wherein the ABA type [AA] + The fragment ion of the ion is [M+N-Rsn2] + , [AB] + The fragment ion of the ion is [M+NRsn1 / sn3 ] + ; The AAB type [AA] + The fragment ion of the ion is [M+N-Rsn3] + , [AB] + The fragment ion of the ion is [M+NR sn1 / sn2 ] + .

[0018] According to an embodiment of the present invention, the I value is calculated based on formula (1). When the I value is <2.0, the triglyceride sample structure type is the AAB type; when the I value is >2.0, the triglyceride sample structure type is the ABA type.

[0019] Where I = [AB] + Abundance ÷ [AA] + Abundance (1)

[0020] Among them, [AB] + [AB] of the ABA or AAB triglyceride + Mass spectrum peaks of fragments;

[0021] [AA] + [AA] of the ABA or AAB triglyceride + Mass spectrum peaks of fragments.

[0022] According to an embodiment of the present invention, there are three fragment ions [M+NR] in the secondary mass spectrometry result. + , then the triglyceride sample structure type is the ABC type.

[0023] 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

[0024] 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:

[0025] Figure 1 shows a secondary mass spectrum of SSS18:0 / 18:0 / 18:0 in EAD fragmentation mode according to one embodiment of the present invention;

[0026] Figure 2 A secondary mass spectrometry comparison diagram of POP16:0 / 18:1 / 16:0 and OPP18:1 / 16:0 / 16:0 in EAD pyrolysis mode according to one embodiment of the present invention is shown;

[0027] Figure 3A comparison of secondary mass spectra of POS16:0 / 18:1 / 18:0, PSO16:0 / 18:0 / 18:1, and OPS18:1 / 16:0 / 18:0 in EAD fragmentation mode according to one embodiment of the present invention is shown;

[0028] Figure 4 A schematic diagram of the identification process of TAG fatty acid acyl chain position isomers according to one embodiment of the present invention is shown. DETAILED DESCRIPTION

[0029] 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.

[0030] In the description of the present invention, the terms "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and do not require that the present invention must be constructed and operated in a specific orientation. Therefore, they should not be understood as limitations on the present invention.

[0031] 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.

[0032] According to one aspect of the present invention, the present invention provides a method for detecting triglyceride positional isomers. According to the method for detecting triglyceride positional isomers according to an embodiment of the present invention, primary and secondary mass spectrometric fragments with rich information are obtained by a high-resolution mass spectrometer, and the fine cracking rules of TGA positional isomers are constructed to construct a comprehensive TGA sn positional isomer characterization strategy, improve the efficiency and accuracy of fatty acid acyl chain structure characterization, provide support for in-depth exploration of lipid structure, and thus realize rapid identification of fatty acyl positions in lipid structures.

[0033] According to an embodiment of the present invention, a high-resolution mass spectrometer uses an electron-excited dissociation (EAD) ionization mode to apply electron beams of different energies to positively charged lipid ions, so that the spectrum shows a fragmentation pattern rich in information, thereby realizing the identification of fatty acyl positions in lipid structures; EAD technology can not only observe the positional isomers through different cleavage patterns to obtain fatty acyl chain information, but also calculate the composition and position of fatty acid chains through neutral loss and fragment ion ratio method. The two identification methods verify each other to provide a complete characterization strategy. More importantly, some EAD-specific product ions play an important role in triglyceride structure analysis, enhancing the confidence of triglyceride structure analysis.

[0034] In order to facilitate understanding of the method for detecting triglyceride positional isomers, the method is explained according to an embodiment of the present invention. The method comprises:

[0035] S100 Samples Available

[0036] According to an embodiment of the present invention, a triglyceride sample to be tested is provided, which is a purified triglyceride sample.

[0037] S200 Sample Ionization

[0038] 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.

[0039] 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.

[0040] S300 Mass Spectrometry

[0041] 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.

[0042] According to an embodiment of the present invention, the detection conditions of the high-resolution mass spectrometer are: working solution flow rate: 30uL / min; ion source type: TurboVTM ion source; electrospray ionization: ESI(+); electron kinetic energy of EAD: 10eV; electron beam current: 7000nA; electrospray voltage: +5500V; ion source temperature: 300°C; nebulizing gas: 30psi; heating gas: 30psi; curtain gas: 30psi.

[0043] 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] + , the structural type of each branch of the triglyceride sample is obtained, 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, a fatty acid acyl chain is broken, that is, one R group is reduced, and a fragment ion [M+NR] is produced. + , the two fatty acid acyl chains are broken, that is, two R groups are reduced, and the fragment ion [M+N-2R] is produced. + .

[0044] S400 Configuration Analysis

[0045] According to an embodiment of the present invention, based on the primary mass spectrometry results and the secondary mass spectrometry results, the structural type of each branch chain of the triglyceride sample is obtained, wherein the three fatty acid chains contained in the triglyceride sample are sn1, sn2 and sn3, respectively. According to the structural differences of sn1, sn2 and sn3, the structural types of the triglyceride sample include AAA type, AAB type, ABA type and ABC type.

[0046] 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.

[0047] After determining the possible molecular composition and fragment ions of TAG [M+NR] + and [M+N-2R] + After the mass-to-charge ratio range was determined, the structure of TAG was further analyzed based on the secondary spectrum:

[0048] According to an embodiment of the present invention, in the secondary mass spectrometry result, in the fragment ion [M+N-2R] + There are 3 fragment ions in the mass-to-charge ratio range [M+N-2R] + , and there are two fragment ions [M+N-2R]+ If the mass-to-charge ratio of the sample is different by 2Da, the structure type of the triglyceride sample is the ABA type or the AAA type. The following two methods can be used to further confirm whether it is the ABA type or the AAA type:

[0049] Method 1 (mass-to-charge ratio comparison method):

[0050] According to an embodiment of the present invention, based on the fragment ion [M+N-2R] + The mass-to-charge ratio of the triglyceride sample is used to determine whether the triglyceride sample structure type is the ABC type or the AAB type. That is, the fragment ion [M+N-2R] + The mass-to-charge ratio is compared with the molecular weight of the possible R group obtained by our analysis to obtain the composition of R, thus determining whether the structural type is the ABC type or the AAB type.

[0051] Method 2 (I value method):

[0052] According to an embodiment of the present invention, in the secondary mass spectrometry result, in the fragment ion [M+NR] + In the mass-to-charge ratio range, there is only one fragment ion [M+NR] + , then the structure type of the triglyceride sample is the AAA type. This method can be used alone to determine the AAA type molecule, or it can be combined with the above method to confirm whether the TAG molecule structure is ABA type.

[0053] According to an embodiment of the present invention, in the secondary mass spectrometry result, in the fragment ion [M+NR] + In the mass-to-charge ratio range, there are two fragment ions [M+NR] + , then the triglyceride sample structure type is the ABA type or AAB type, wherein the ABA type [AA] + The fragment ion of the ion is [M+N-Rsn2] + , [AB] + The fragment ion of the ion is [M+NR sn1 / sn3 ] + ; The AAB type [AA] + The fragment ion of the ion is [M+N-Rsn3] + , [AB] + The fragment ion of the ion is [M+NR sn1 / sn2 ] + .

[0054] According to an embodiment of the present invention, the I value is calculated based on formula (1). When the I value is <2.0, the triglyceride sample structure type is the AAB type; when the I value is >2.0, the triglyceride sample structure type is the ABA type.

[0055] Where I = [AB] + Abundance ÷ [AA] + Abundance (1)

[0056] Among them, [AB] + [AB] of the ABA or AAB triglyceride + Mass spectrum peaks of fragments;

[0057] [AA] + [AA] of the ABA or AAB triglyceride + Mass spectrum peaks of fragments.

[0058] According to an embodiment of the present invention, in the secondary mass spectrometry result, the fragment ion [M+N-2R] + There are 5 fragment ions in the mass-to-charge ratio range [M+N-2R] + , except for a single fragment ion [M+N-2R] + , the remaining four fragment ions [M+N-2R] + Divided into 2 groups, the fragment ions in each group [M+N-2R] + If the mass-to-charge ratio of the sample is different by 2Da, the structure type of the triglyceride sample is the ABC type or the AAB type. The above mass-to-charge ratio comparison method can be used to further confirm whether it is ABA type or AAA type. It can also be confirmed by the following method:

[0059] According to an embodiment of the present invention, there are three fragment ions [M+NR] in the secondary mass spectrometry result. + , then the structure type of the triglyceride sample is the ABC type. This method can be used alone to determine the ABC type molecule, or it can be combined with the above method to confirm whether the TAG molecular structure is ABC type.

[0060] According to an embodiment of the present invention, in the secondary mass spectrometry result, in the fragment ion [M+N-2R] + There are 4 fragment ions in the mass-to-charge ratio range [M+N-2R] + , except for a single fragment ion [M+N-2R] + , the remaining 3 fragment ions [M+N-2R] + The mass-to-charge ratios of the triglyceride samples differ by 2Da, and the triglyceride sample structure type is the ABC type or the AAB type. The above mass-to-charge ratio comparison method can be used to further confirm whether it is ABA type or AAA type. The above fragment ion [M+NR] can also be used to further confirm whether it is ABA type or AAA type. + Determine the ABC method for confirmation.

[0061] For ABA-type and AAB-type TAGs that are difficult to distinguish, further confirmation can be performed by the following methods:

[0062] According to an embodiment of the present invention, in the secondary mass spectrometry result, in the fragment ion [M+NR] + In the mass-to-charge ratio range, there are two fragment ions [M+NR] + , then the triglyceride sample structure type is the ABA type or AAB type, wherein the ABA type [AA] + The fragment ion of the ion is [M+N-Rsn2] + , [AB] + The fragment ion of the ion is [M+NR sn1 / sn3 ] + ; The AAB type [AA] + The fragment ion of the ion is [M+N-Rsn3] + , [AB] + The fragment ion of the ion is [M+NR sn1 / sn2 ] + .

[0063] According to an embodiment of the present invention, the I value is calculated based on formula (1). When the I value is <2.0, the triglyceride sample structure type is the AAB type; when the I value is >2.0, the triglyceride sample structure type is the ABA type.

[0064] Where I = [AB] + Abundance ÷ [AA] + Abundance (1)

[0065] Among them, [AB] + [AB] of the ABA or AAB triglyceride + Mass spectrum peaks of fragments;

[0066] [AA] + [AA] of the ABA or AAB triglyceride + Mass spectrum peaks of fragments.

[0067] 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.

[0068] 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.

[0069] Example 1

[0070] The structural type of the triglyceride standard was verified using the method of the embodiment of the present invention, as follows:

[0071] 1. Experimental part

[0072] 1.1 Instruments and reagents

[0073] Instruments: Zeno-TOF7600 high-resolution mass spectrometer and SCIEX3.0 data processing software (ABSciex, USA); Milli-Q ultrapure water system (Millipore, USA); Harvard Apparatus syringe pump Pump11Elite

[0074] Reagents: n-hexane, isopropanol (chromatographic grade, ThermoFisher, USA); sodium acetate (chromatographic grade, Sigma-Aldrich, USA);

[0075] Lipid standards: 17 triglyceride standards were purchased from ZZSTANDARD. This example uses the lipidomics terminology proposed by Marshall et al.: only one sn-position is defined, the abbreviation of triglyceride is indicated in a pair of brackets, and the isomerism at the sn-position is divided by a slash, for example, OPO (18:1 / 16:0 / 18:1) is represented by 1,3-dioleoyl-2-palmitoyl triglyceride, i.e., ABA-type TAG; OOP (18:1 / 18:1 / 16:0) is represented by 1,2-dioleoyl-3-palmitoyl triglyceride, i.e., AAB-type TAG. See the table below for details;

[0076] Table 1. Basic information of 17 positional isomers

[0077]

[0078]

[0079] 1.2 Preparation of standard solution:

[0080] 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;

[0081] 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.

[0082] 1.3 Mass spectrometry method:

[0083] The working solution was directly injected into the TurboVTM ion source (SCIEX) at a flow rate of 30uL / min by syringe pump injection, and the precursor ions were generated by electrospray ionization ESI(+). The acquisition time was 6min, and the source parameters used are listed in Table 2; in this work, the electron kinetic energy (KE) of EAD was set to 10eV and the electron beam current was 7000nA.

[0084] Table 2. Ion source parameters used in EAD experiments

[0085]

[0086]

[0087] 1.4 Data Analysis

[0088] Raw data were processed using SCIEXOS software 3.3 with a browser and analysis module. All vertical line graphs were processed using Origin 2022 (Origin Genetics Lab, USA).

[0089] 2. Results and Discussion

[0090] In the embodiment, 17 TGAs were screened, including 4 AAA type, 4 AAB type, 3 ABA type and 6 ABC type positional isomers. AAA type triglycerides are SSS (18:0 / 18:0 / 18:0), OOO (18:1 / 18:1 / 18:1), LLL (18:2 / 18:2 / 18:2), LnLnLn (18:3 / 18:3 / 18:3); 4 AAB type triglycerides are OPP (18:1 / 16:0 / 16:0), OOP (18:1 / 18:1 / 16:0), PLL (16:0 / 18:2 / 18:2), OLL (18:1 / 18:2 / 18:2); 3 ABA type triglycerides are POP (16:0 / 18:1 / 16:0), OPO (18:1 / 16:0 / 18:1), SOS (18:0 / 18:1 / 18:0); the two groups of BAC / ABC / ACB type triglyceride positional isomers are POS (16:0 / 18:1 / 18:0), PSO (16:0 / 18:0 / 18:1), OPS (18:1 / 16:0 / 18:0), and OPL (18:1 / 16:0 / 18:2), OLP (18:1 / 18:2 / 16:0), POL (16:0 / 18:1 / 18:2).

[0091] In this example, sodium acetate was added to form sodium TAG ions. The diagnostic characteristic peaks of the positional isomers of 17 TAGs are shown in Table 3.

[0092] Table 3. Diagnostic characteristic peaks of 17 TAGs positional isomers in EAD collision mode

[0093]

[0094]

[0095] 2.1 Characterization of AAA-type TAGs

[0096] From the EAD mass spectra of the four AAA-type TAGs, three groups of characteristic peaks can be observed: molecular ion peak [M+Na] + , the molecular ion loses a single acyl chain to form a [M+Na-R1] in the mass range of 550-650Da + Characteristic peaks, molecular ion loss of diacyl chain produces 3 [M+Na-R1-R2] in the mass range of 300-350Da + The characteristic ion peaks and typical characteristic fragment ions are shown in Table 3.

[0097] Take the mass spectrum of SSS (18:0 / 18:0 / 18:0) as an example ( Figure 1 ), observed [M+Na] +Peak m / z = 913.82; in the range of 550-650Da, parent ion [M+Na] + Loss of a C18:0(S) fatty acyl group forms an ion fragment [M+Na-RxCOOH] + m / z = 629.54, this fragment ion can be the loss of C18:0 (S) fatty acyl group at any position of -sn1, -sn2, -sn3. Therefore, AAA-type TAGs only have one obvious characteristic ion in the mass range of 550-650Da, and the fatty acid acyl chain can be determined based on the mass of the lost fragment; in the mass range of 300-350Da, three characteristic peaks are observed: 1) the parent ion loses the fatty acyl groups at the sn1 and sn3 positions at the same time, generating the characteristic ion [M+Na-R1COOH-R3COOCH3] m / z = 333.27; 2) the parent ion loses the fatty acyl groups at the sn1 and sn2 positions at the same time (or the sn3 and sn2 positions are broken at the same time), but there are two ways for the adjacent fatty acyl groups to break ( Figure 1 ), one is that the CO bond of the glycerol skeleton is broken to produce [M+Na-R2COOH-R 3 / 1 COOH]m / z=347.29, this characteristic fragment contains methylene (-CH2-); secondly, the CC bonds at -sn1 / -sn3 and -sn2 of the glycerol backbone are broken, producing [M+Na-R2CO-R 3 / 1 COOCH3]m / z=349.27In this characteristic fragment, the oxygen atom (-O-) replaces (-CH2-), so the mass-to-charge ratio of the two characteristic fragment ions differs by only 2 Da. Three obvious characteristic ions appear in the mass range of 300-350Da for AAA-type TAGs. OOO (18:1 / 18:1 / 18:1), LLL (18:2 / 18:2 / 18:2), LnLnLn (18:3 / 18:3 / 18:3) have the same characteristic fragmentation rules. The characteristic fragments are shown in Table 3, and the mass spectra are shown in Figure 1 .

[0098] 2.2 Characterization of ABA and AAB positional isomers of TAGs

[0099] From the EAD mass spectra of two groups of ABA and AAB type TAGs, three groups of characteristic peaks can be observed: molecular ion peak [M+Na] + , the molecular ion loses the single acyl chain to form two [M+Na-R1]+ characteristic peaks in the mass range of 550-650Da, and the molecular ion loses the double acyl chain to produce 3 or 5 [M+Na-R1-R2]+ characteristic ion peaks in the mass range of 300-350Da. The typical characteristic fragment ions are shown in Table 3. Take the mass spectra of POP16:0 / 18:1 / 16:0 and OPP as an example ( Figure 2), 1) The same parent ion [M+Na] was observed for both isomers + Peak m / z = 855.72; 2) In the range of 550-650Da, the parent ions of this pair of isomers [M+Na] + Loss of a fatty acid acyl chain C18:1 (O) fatty acyl or C16:0 (P) fatty acyl at any position of -sn1, -sn2, or -sn3 can produce [PP] + Fragment m / z = 573.2 and [PO] + Fragment m / z = 599.5. Therefore, ABA and AAB type TAGs have the same two characteristic ions in the mass range of 550-650Da, which can be distinguished from AAA type (only one characteristic fragment), but this pair of characteristic ions cannot directly distinguish ABA from AAB type TAGs.

[0100] To this end, the [AB] + With [AA] + Abundance ratio of characteristic fragments (I). Without considering steric hindrance, theoretically they can all produce 1 [AA] + Ions, 2 [AB] + ions, so the I value should be 2.0, but due to steric hindrance, the loss of sn-2 fatty acids produces fragments [M+Na-R2COOH] + The abundance is lower than the fragment ions [M+Na-R 1 / 3 COOH] + Abundance, therefore, the I value of AAB type TGA should be less than 2.0, and the I value of ABA type should be greater than 2.0. This example analyzes the I values ​​of three ABA type TGAs (POP (16:0 / 18:1 / 16:0), OPO (18:1 / 16:0 / 18:1), SOS (18:0 / 18:1 / 18:0)) and four AAB TGAs (OPP (18:1 / 16:0 / 16:0), OOP (18:1 / 18:1 / 16:0), PLL (16:0 / 18:2 / 18:2), OLL 18:1 / 18:2 / 18:2)).

[0101] From the results in Table 2, we can see that the I value of ABA triglycerides is in the range of 3.8 to 3.9, and that of AAB triglycerides is in the range of 1.4 to 2.0. + / [AA] + ratio, it can be assumed that under steric hindrance, 1 mol of ABA parent ion will produce 1 mol [AB] + Ion, 1 mol [BA] +ions and 1 / 2 mol of AA + ions, so the I value of this type of TAG should be 4.0, and the [AB]+ / [AA] ratios of OPO, SOS, and POP basically conform to this rule; under this assumption, the [AB] + / [AA] + It should be 1.5. The I values ​​of PLL, OLL and OPP are 1.448, 1.474 and 1.625 respectively, which basically conforms to this rule. + / [AA] + The ratio is 2.008, which suggests that there may be OPO impurities in the raw materials, resulting in [AB] + Of course, the above hypothesis is only an inference based on a small number of experiments. The inventor will further accumulate relevant data to improve the hypothesis. However, it is certain that the I value of AAB type TAG is <2.0, while the I value of ABA type TAG is >2.0. Therefore, by judging the diacylglycerol ion [AB] + / [AA] + The ratio of fragment ion abundance can be used to identify AAB and ABA type positional isomers in TGA, which is one of the most important identification bases.

[0102] Table 2. Relative abundance ratio of diacylglycerol fragment ions in EAD fragmentation mode

[0103]

[0104] I* is expressed as the mass spectrometer response value comparison of 6 time periods (0-1min, 1-2min, 2-3min, 3-4min, 4-5min, 5-6min) within 6min; I* of AAB / AAB type TAGs is expressed as [AB] + :[AA] + , ABC-type TAGs I* is represented by [AB] + :[BC] + :[AC] + .

[0105] 3) TGA of AAB and ABA positional isomers in the mass range of 300-350Da S Similarly, taking POP (16:0 / 18:1 / 16:0) as an example, first, the parent ion loses both the sn1 and sn3 fatty acyl groups [P] + , producing [M+Na-PP] +Characteristic ion m / z = 333.26. According to the mass number of the fragment, the fatty acyl chain at the -sn2 position can be directly identified, thereby characterizing the positional isomers of ABA-type TGA. Secondly, the parent ion loses the fatty acyl groups at the -sn1 (or -sn3) and sn2 positions at the same time. Although the fatty acyl chains at the -sn1 and -sn3 positions are the same, there are also two ways to break with the -sn2 fatty acyl group ( Figure 2 ), which is the same as AAA-type TAGs. Therefore, ABA-type TAG will also produce two characteristic fragment ions with a mass-to-charge ratio difference of only 2 Da. However, for OPP (18:1 / 16:0 / 16:0), the parent ion will also lose both the sn1 and sn3 fatty acyl groups, producing [M+Na-PO] + Characteristic ion m / z = 305.29; however, due to the different fatty acyl chains at -sn1 and -sn3, when the parent ion loses the fatty acyl groups at -sn1 and sn2 simultaneously, two characteristic fragments m / z = 347.25 and m / z = 345.28 with a mass-to-charge ratio difference of 2Da are generated, and when the parent ion loses the fatty acyl groups at -sn3 and -sn2 simultaneously, two characteristic fragments m / z = 319.26 and m / z = 321.24 with a mass-to-charge ratio difference of 2Da are generated. Therefore, AAB type TAGs have 5 obvious characteristic ions in the mass range of 300-350Da, among which there are 2 pairs of characteristic fragment ions with a mass-to-charge ratio difference of only 2Da. The other 2 ABA and 3 AAB type TAGs have the same characteristic fragmentation rules.

[0106] 3.3 Characterization of ABC-type TAGs

[0107] From the observation of EAD mass spectrometry fragments of 6 ABC-type TAGs, three groups of characteristic peaks can also be observed: molecular ion peak [M+Na] + , the molecular ion loses a single acyl chain to form three [M+Na-R1] in the mass range of 550-650Da + Characteristic peaks, molecular ion loss of diacyl chains in the mass range of 300-350Da produce 4 or 5 [M+Na-R1-R2] + Characteristic ion peaks and typical characteristic fragment ions are shown in Table 3. Figure 3 It can be seen that 1) the molecular ion peaks of the three position isomers of POS16:0 / 18:1 / 18:0, PSO16:0 / 18:0 / 18:1, and OPS18:1 / 16:0 / 18:0 are all [M+Na] + m / z=883.74; 2) In the range of 550-650Da, the molecular ions of these three isomers [M+Na] + Loss of any fatty acid acyl chain at -sn1, -sn2, or -sn3 can produce [PO] +Fragment m / z = 599.99, [PS] + Fragment m / z = 601.99 and [SO] + m / z = 627.99. Therefore, after the three ABC isomers lose one fatty acyl chain, three [M+Na-R1] + Characteristic peaks can characterize the TGA fatty acid composition based on the mass number of the lost fatty acyl chain, but the connection position with the glycerol hydroxyl group cannot be determined.

[0108] To this end, the [AB] + , [BC] + and [AC] + The abundance ratio of the three characteristic fragments (I). Taking POS (16:0 / 18:1 / 18:0) as an example, Figure 3 It can be seen that [BC] + Fragment ion m / z = 627.57, [AB] + Fragment ions m / z = 599.55 and [AC] + The abundance ratio of the fragment ion m / z = 601.56 is approximately 1:1:0.5 ([AB] + :[BC] + :[AC] + ), indicating that due to steric hindrance, the sn-2 oleic acid C18:1 is more difficult to break than the sn-1 and sn-3 fatty acids. The same cleavage and I value trends were also observed in the spectra of the two isomers of PSO16:0 / 18:0 / 18:1 and OPS18:1 / 16:0 / 18:0. From the results in Table 2, it can be seen that the I value of ABC triglycerides is in the range of 1:(0.91~1.06):(0.53~0.60). Without considering steric hindrance, they can theoretically produce 1 [AB] + Ion, 1 [BC] + Ion, 1 [AC] + ions, so the I value should be 1:1:1, but due to steric hindrance, the loss of sn-2 fatty acids produces fragments [M+Na-R2COOH] + The abundance is lower than the abundance of fragment ions generated by the loss of sn-1 or sn-3 [M+Na-R 1 / 3 COOH] + , so the I value of ABC type TGA is about 1:1:0.5. The [AB] of POL, PLO and OPL in the table + :[BC] + :[AC] +The ratios were 1:1.06:0.5, 1:0.99:0.60, and 1:1.06:0.58, respectively, which were also basically in line with this rule. Therefore, by determining the ratio of the abundance of diacylglycerol fragment ions, the -sn2 fatty acyl chain can be identified, thereby characterizing the positional isomers of ABC-type TGA.

[0109] 3) ABC-type positional isomerism TGA in the mass range of 300-350Da S With 4-5 significant characteristic peaks. Similarly, taking POS as an example, the parent ion loses both the sn1 and sn3 fatty acyl groups, producing a [M+Na-PS] + The characteristic ion m / z = 331.26, based on the fragment mass number, the fatty acyl chain at the -sn2 position can be directly identified, thereby characterizing the positional isomers of ABC-type TGA; secondly, the parent ion loses both the -sn1 (or -sn3) and sn2 fatty acyl groups at the same time, which will also produce two characteristic fragment ions m / z = 349.27 and 347.29 (loss of [P]) with a mass-to-charge ratio difference of only 2 Da. At the same time, due to the different types of fatty acyl chains at the -sn1 and -sn3 positions of ABC-type TGA, another two characteristic fragment ions m / z = 321.24 and 319.26 (loss of [S]) with a mass-to-charge ratio difference of only 2 Da will be produced. However, for OPS18:1 / 16:0 / 18:0, since the mass number difference between the fatty acyl chains at the -sn1 position and the -sn3 position is 2Da, it means that the carbon chain lengths at the -sn1 or -sn3 positions are the same and only differ by one double bond, so both can produce fragment peaks with a mass-to-charge ratio of m / z = 347.25. Therefore, OPS has a total of 4 fragment peaks in the mass range of 300-350Da.

[0110] 2.4 Flowchart for identification of positional isomers of TAG fatty acid acyl chains (strategy)

[0111] This example establishes a new strategy for identifying TAG fatty acid acyl chain position isomers based on EAD technology to provide more accurate and reliable characterization results. This example also summarizes the secondary characteristic mass spectrometry data of triglycerides reported in the literature, see Appendix Table 1, and combines the data of this example to establish a new strategy for identifying TAG fatty acid acyl chain position isomers, such as Figure 4 shown.

[0112] First, based on the mass-to-charge ratio of the molecular ion peak [M+Na]+, the possible composition of fatty acids is obtained by comparing with Lipidmaps or other open source databases;

[0113] Second, characterize the position of the fatty acyl chain based on the characteristic fragment ions in the mass-to-charge ratio range of 550-650Da in the EAD secondary mass spectrum (MS / MS) of TGA. 1) If there is one fragment ion with a missing single acyl chain [M+Na-R]+, it can be determined as AAA type, and the type of fatty acyl chain can be determined based on the missing mass number; 2) If there are two fragment ions with a missing single acyl chain [M+Na-R]+, it can be preliminarily determined as ABA or AAB type, and then based on [AB] + / [AA] + The ratio I is used for identification. When I>2, it is ABA type, and when I<2, it is AAB type. Then, the positions of the three fatty acyl chains are identified based on the two characteristic fragments and the mass numbers of their missing fragments. 3) If there are three fragment ions with missing single acyl chains [M+Na-R]+, it can be preliminarily determined to be ABC type. Then, based on [AB] + / [BC] + / [AC] + The ratio is ≈2 / 2 / 1. Based on the mass-to-charge ratio of the less abundant fragments, the fatty acyl chain at the -sn2 position can be identified, thereby determining the position of the fatty acyl chain by TGA;

[0114] Third, the position of the fatty acyl chains was characterized based on the characteristic fragment ions in the mass-to-charge ratio range of 300-350 Da in the EAD secondary mass spectrum (MS / MS) of TGA. 1) If there are three missing diacyl chains [M+Na-2R] + The fragment ions are characterized by 1 single fragment ion and 2 fragment ions with a mass-to-charge ratio difference of only 2Da, which can be preliminarily determined to be AAA type or ABA type. Then, the type and position of the fatty acyl chain can be identified based on the mass-to-charge ratio of the 3 characteristic fragments; 2) If there are 5 missing single acyl chains [M+Na-2R] + The fragment ions are characterized by 1 single fragment ion and 2 pairs of fragment ions with a mass-to-charge ratio difference of only 2Da (4 in total). Based on this, it can be preliminarily determined to be ABC or AAB type, and the mass difference between the fatty acyl chains at -sn1 and -sn3 is greater than 2Da. Then the type and position of the fatty acyl chain are identified based on the mass-to-charge ratio of the 5 characteristic fragments; 3) If 4 missing single acyl chains [M+Na-R] +The fragment ions are characterized by a single fragment ion mass-to-charge ratio and three fragment ions with mass-to-charge ratios that differ by 2Da in sequence. It can be preliminarily determined to be AAB type or ABC type, and the difference in the mass number of the fatty acyl chain at -sn1 and -sn3 is 2Da, indicating that the carbon chain length of the fatty acid acyl chain at -sn1 or -sn3 is the same and only differs by one double bond. In summary, the composition and position of fatty acids are determined based on the number of fragment ions, mass-to-charge ratios, and abundance ratios observed in the mass-to-charge ratio range of 300-350Da and 550-650Da on the secondary mass spectrum. The two sets of data verify each other, greatly improving the confidence in the characterization of the fatty acid acyl chain structure.

[0115] 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.

[0116] 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 positional isomers of triglycerides, 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; as well as Based on the primary mass spectrometry results and the secondary mass spectrometry results, the structural types of each branch chain of the triglyceride sample are obtained, wherein the three fatty acid chains contained in the triglyceride sample are sn1, sn2 and sn3, respectively. According to the structural differences of sn1, sn2 and sn3, the structural types of the triglyceride sample include AAA type, AAB type, ABA type and ABC type.

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; Ion source type: TurboVTM ion source; Electrospray ionization: ESI(+); Electron kinetic energy of EAD: 10 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: 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] + , obtaining the structural type of each branch chain of the triglyceride sample, wherein M represents triglyceride, N represents a cation combined with the ionized triglyceride, and R represents the fatty acid acyl chain of the triglyceride sample.

4. The method according to claim 1, characterized in that: Based on the ion peak [M+N] + The mass-to-charge ratio is compared with the lipid compound database to obtain the possible composition of fatty acids; Based on the possible composition of the fatty acid, the fragment ions [M+NR] were determined respectively. + The mass-to-charge ratio range and the fragment ions [M+N-2R] + mass-to-charge ratio range.

5. The method according to claim 4, characterized in that: In the secondary mass spectrometry results, in the fragment ion [M+N-2R] + There are 3 fragment ions in the mass-to-charge ratio range [M+N-2R] + , and there are two fragment ions [M+N-2R] + The mass-to-charge ratio of the triglyceride sample is different by 2Da, then the triglyceride sample structure type is the ABA type or the AAA type, Optionally, in the secondary mass spectrometry result, the fragment ion [M+N-2R] + There are 5 fragment ions in the mass-to-charge ratio range [M+N-2R] + , except for a single fragment ion [M+N-2R] + , the remaining four fragment ions [M+N-2R] + Divided into 2 groups, the fragment ions in each group [M+N-2R] + The mass-to-charge ratio of the triglyceride sample is different by 2Da, then the triglyceride sample structure type is the ABC type or the AAB type, Optionally, in the secondary mass spectrometry result, in the fragment ion [M+N-2R] + There are 4 fragment ions in the mass-to-charge ratio range [M+N-2R] + , except for a single fragment ion [M+N-2R] + , the remaining 3 fragment ions [M+N-2R] + The mass-to-charge ratios of the triglyceride samples differ by 2Da respectively, and the structure type of the triglyceride sample is the ABC type or the AAB type.

6. The method according to claim 5, characterized in that Based on the fragment ion [M+N-2R] + The mass-to-charge ratio of the triglyceride sample is used to determine whether the triglyceride sample structure type is the ABA type or the AAA type; Optionally, based on the fragment ion [M+N-2R] + The mass-to-charge ratio is used to determine whether the triglyceride sample structure type is the ABC type or the AAB type.

7. The method according to claim 4 or 5, characterized in that: In the secondary mass spectrometry results, in the fragment ion [M+NR] + In the mass-to-charge ratio range, there is only one fragment ion [M+NR] + , then the triglyceride sample structure type is the AAA type.

8. The method according to claim 4 or 5, characterized in that: In the secondary mass spectrometry results, in the fragment ion [M+NR] + In the mass-to-charge ratio range, there are two fragment ions [M+NR] + , then the triglyceride sample structure type is the ABA type or AAB type, wherein, The ABA type [AA] + The fragment ion of the ion is [M+N-Rsn2] + , [AB] + The fragment ion of the ion is [M+NR sn1 / sn3 ] + ; The AAB type [AA] + The fragment ion of the ion is [M+N-Rsn3] + , [AB] + The fragment ion of ion is [M+NR sn1 / sn2 ] + .

9. The method according to claim 8, characterized in that The I value is calculated based on formula (1). When the I value is less than 2.0, the triglyceride sample structure type is the AAB type; when the I value is greater than 2.0, the triglyceride sample structure type is the ABA type. Where I = [AB] + Abundance ÷ [AA] + Abundance (1) Among them, [AB] + [AB] of the ABA or AAB triglyceride + Mass spectrum peaks of fragments; [AA] + [AA] of the ABA or AAB triglyceride + Mass spectrum peaks of fragments.

10. The method according to claim 4 or 5, characterized in that: There are three fragment ions in the secondary mass spectrometry results [M+NR] + , then the triglyceride sample structure type is the ABC type.

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