Method for detecting triglyceride positional isomers

Abstract

The application discloses a method for detecting triglyceride positional isomers, which comprises the following steps: providing a triglyceride sample to be detected; mixing the triglyceride sample with a salt compound to obtain a to-be-detected liquid; detecting the to-be-detected liquid by using a high-resolution mass spectrometer so as to obtain a primary mass spectrum result and a secondary mass spectrum result; and obtaining the structure type of each branched chain of the triglyceride sample based on the primary mass spectrum result and the secondary mass spectrum result, wherein the triglyceride sample contains three fatty acid chains in sequence, namely sn1, sn2 and sn3, and the structure type of the triglyceride sample comprises AAA type, AAB type, ABA type and ABC type according to the structure difference of the sn1, sn2 and sn3. The method obtains the primary and secondary mass spectrum fragment modes of rich information by using the high-resolution mass spectrometer, the fine cleavage rule of the TGA positional isomer, and a comprehensive characterization strategy of the sn positional isomer of the TGA is constructed.

Description

TECHNICAL FIELD

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

[0002] The development of lipidomics requires standards with defined structure and purity, which are applied to the development of lipid analysis methods and the quality control of analysis results. Triglycerides (TAGs) are an important class of lipids with physiological functions, which are generated by esterification of three fatty acids (FAs) and one molecule of glycerol. According to the positions of FAs on the glycerol backbone, they are named as sn-1, sn-2 and sn-3. Different arrangements of fatty acyl chains connected to the glycerol backbone result in a large number of positional fatty acyl chain isomers (sn position isomers). The positional difference of fatty acyl chains has important influences 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, while palmitic acid at the sn-1 and sn-3 positions is easily hydrolyzed by intestinal lipases into free palmitic acid, which reacts with calcium, magnesium and other minerals to reduce the absorption and utilization efficiency of fat by the human body.

[0003] Therefore, the identification of triglyceride sn-position isomers is to be studied for lipid structure characterization. SUMMARY

[0004] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, one object of the present application is to provide a method for detecting triglyceride positional isomers, in which electron activation dissociation (EAD) is applied to positively charged lipid ions with different energy electron beams to make the spectrum show a rich information fragment pattern, thereby realizing the identification of the position of fatty acyl groups in the lipid structure.

[0005] According to an aspect of the present application, a method for detecting triglyceride positional isomers is provided. According to an embodiment of the present application, the method comprises: providing a triglyceride sample to be detected; mixing the triglyceride sample to be detected with a salt compound to obtain a sample to be detected; detecting the sample to be detected by using a high-resolution mass spectrometer to obtain a primary mass spectrum result and a secondary mass spectrum result; and obtaining the structure type of each branch of the triglyceride sample based on the primary mass spectrum result and the secondary mass spectrum result, wherein the triglyceride sample contains three fatty acid chains in the order of sn1, sn2 and sn3, and the structure type of the triglyceride sample includes AAA type, AAB type, ABA type and ABC type according to the structure differences of the sn1, sn2 and sn3.

[0006] According to the method for detecting triglyceride positional isomers in an embodiment of the present invention, a high-resolution mass spectrometer is used to obtain information-rich primary and secondary mass spectral fragments and fine fragmentation patterns of TGA positional isomers, thereby constructing a comprehensive TGA sn positional isomer characterization strategy, improving the efficiency and accuracy of fatty acid acyl chain structure characterization, providing support for in-depth exploration of lipid structure, and thus achieving rapid identification of fatty acyl positions in lipid structures.

[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 as follows: 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 of the triglyceride sample, wherein M represents triglyceride, N represents a cation bound to 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 is compared with the lipid compound database to obtain the possible composition of fatty acids; based on the possible composition of the fatty acids, 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.

[0011] According to an embodiment of the present invention, in the secondary mass spectrometry results, in the fragment ion [M+N-2R] + There are 3 fragment ions within 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 triglyceride sample differs by 2Da, the triglyceride sample structure type is the ABA type or the AAA type.

[0012] According to an embodiment of the present invention, in the secondary mass spectrometry results, the fragment ion [M+N-2R] + There are 5 fragment ions in the mass-to-charge ratio range [M+N-2R] + , in addition to 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 triglyceride sample structure type is the ABC type or the AAB type.

[0013] According to an embodiment of the present invention, in the secondary mass spectrometry results, in the fragment ion [M+N-2R] + There are 4 fragment ions [M+N-2R] in the mass-to-charge ratio range + , in addition to 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 2 Da, and the triglyceride sample structure type 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 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.

[0017] According to an embodiment of the present invention, 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 [AA] + The fragment ion of the ion is [M+N-Rsn2] + , [AB] + The fragment ion of ion is [M+NRsn1 / sn3 ] + ; the [AA] of the AAB type + The fragment ion of the ion is [M+N-Rsn3] + , the [AB] + The fragment ion of the ion is [M+N-R sn1 / sn2 ] + .

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

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

[0020] Wherein, [AB] + is the [AB] + Mass spectrum peak of the fragment of the ABA type or AAB type triglyceride;

[0021] [AA] + is the [AA] + Mass spectrum peak of the fragment of the ABA type or AAB type triglyceride.

[0022] According to an embodiment of the present application, there are three fragment ions [M+N-R] + in the secondary mass spectrum result, then the structure type of the triglyceride sample is the ABC type.

[0023] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter in the description of embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0024] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the description of the embodiments, taken in conjunction with the following drawings in which:

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

[0026] Figure 2 shows the secondary mass spectrum comparison of POP16:0 / 18:1 / 16:0, OPP18:1 / 16:0 / 16:0 in EAD cleavage mode according to one embodiment of the present application;

[0027] Figure 3A secondary mass spectrum contrast chart of POS 16:0 / 18:1 / 18:0, PS O16:0 / 18:0 / 18:1, OPS 18:1 / 16:0 / 18:0 in EAD cleavage mode according to one embodiment of the present application is shown.

[0028] Figure 4 A flow chart of identification of TAG fatty acyl chain position isomers according to one embodiment of the present application is shown. DETAILED DESCRIPTION

[0029] Embodiments of the present application are described in detail below with reference to several drawings. The embodiments of the present application described below are examples for explaining the present application and should not be understood as limiting the present application.

[0030] In the description of the present application, the terms "longitudinal", "transverse", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", and the like indicate the orientation or positional relationship shown in the drawings, and are only for the purpose of facilitating the description of the present application and not requiring the present application to be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application.

[0031] It should be noted that the terms "first", "second" are only for the purpose of description and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features defined with "first", "second" can explicitly or implicitly include one or more of the features. Further, in the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0032] According to one aspect of the present application, the present application provides a method for detecting triglyceride position isomers. According to the method for detecting triglyceride position isomers according to an embodiment of the present application, the first and second mass spectrum fragments with rich information are obtained by a high-resolution mass spectrometer, the fine cleavage rule of TGA position isomers is constructed, a comprehensive sn position isomer characterization strategy of TGA is constructed, the efficiency and accuracy of fatty acyl chain structure characterization are improved, and support is provided for in-depth exploration of lipid structure, so as to realize rapid identification of fatty acyl position in lipid structure.

[0033] According to the embodiment of the present application, the high-resolution mass spectrometer adopts an electron excited dissociation (EAD) ionization mode to apply an electron beam of different energy to the positively charged lipid ions, so that the spectrum shows a fragment pattern with rich information, thereby realizing the identification of the fatty acyl position in the lipid structure; the EAD technology can not only observe the fatty acyl chain information obtained by different cleavage modes of position isomers, but also calculate the fatty acid chain composition and position information through neutral loss and fragment ion ratio method, and the two identification methods are verified with each other to provide a complete characterization strategy. More importantly, certain EAD specific product ions play an important role in the analysis of the structure of triglycerides, thereby enhancing the confidence of the triglyceride structure analysis.

[0034] In order to facilitate the understanding of the method for detecting the triglyceride position isomer, according to the embodiment of the present application, the method is explained as follows, which comprises the following steps:

[0035] S100 providing a sample

[0036] According to the embodiment of the present application, a triglyceride sample to be detected is provided. The sample is a purified triglyceride sample.

[0037] S200 ionization of the sample

[0038] According to the embodiment of the present application, the triglyceride sample is mixed with a salt compound to obtain a sample to be detected. TAG is a non-polar compound and is not easy to ionize, so some salt (such as sodium or ammonium cation) must be added to form an adduct for all analysis work.

[0039] According to the embodiment of the present application, the triglyceride sample is mixed with sodium salt. The sodium TAG is a more easily cleaved target parent ion than the ammoniated TAG, because the sodium parent ion is easy to lose double chains. In contrast, the ammoniated TAG tends to cleave at the NH4 + group, resulting in no fragment loss inside the fatty acid chain. Therefore, it is preferred to add sodium acetate to form a sodium TAG ion.

[0040] S300 mass spectrometry detection

[0041] According to the embodiment of the present application, the sample to be detected is detected by using a high-resolution mass spectrometer, so as to obtain a primary mass spectrum result and a secondary mass spectrum result.

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

[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] + , obtaining the structural type of each branch of the triglyceride sample, 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 a 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. + .

[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 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 is 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, and the R group molecular composition of the possible TAG of this molecular weight can be obtained. 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 results, in the fragment ion [M+N-2R] + There are 3 fragment ions within 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 fragment ion [M+N-2R] differs from the mass-to-charge ratio of the fragment ion [M+N-R] by 2 Da, then the structure type of the triglyceride sample is the ABA type or the AAA type. Whether the structure type is the ABA type or the AAA type can be further determined by the following two methods:

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

[0050] According to an embodiment of the present application, based on the mass-to-charge ratio of the fragment ion [M+N-2R] + , it is determined whether the structure type of the triglyceride sample is the ABC type or the AAB type. That is, the mass-to-charge ratio of the fragment ion [M+N-2R] + is compared with the molecular weight of the possible R group obtained by analysis, the composition of R is obtained, and it is determined whether the structure type is the ABC type or the AAB type.

[0051] Method two (I value method):

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

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

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

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

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

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

[0058] According to an embodiment of the present invention, in the secondary mass spectrometry results, the fragment ion [M+N-2R] + There are 5 fragment ions in the mass-to-charge ratio range [M+N-2R] + , in addition to 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 2 Da, 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. 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 triglyceride sample structure type is the ABC type. This method can be used alone to determine ABC type molecules, 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 results, in the fragment ion [M+N-2R] + There are 4 fragment ions [M+N-2R] in the mass-to-charge ratio range + , in addition to a single fragment ion [M+N-2R] + , the remaining 3 fragment ions [M+N-2R] + The mass-to-charge ratio of the triglyceride sample is 2Da different from that of 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 determine whether the triglyceride sample is ABA type or AAA type. + Determine the ABC method for confirmation.

[0061] For the difficult-to-distinguish ABA type and AAB type TAG, further confirmation can be made by the following method:

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

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

[0064] wherein I = abundance of [AB] + abundance of [AA] + (1)

[0065] wherein [AB] + is the mass spectrum peak of the [AB] + fragment of the ABA type or AAB type triglyceride;

[0066] [AA] + is the mass spectrum peak of the [AA] + fragment of the ABA type or AAB type triglyceride.

[0067] The present application is described below with reference to specific embodiments, it should be noted that these embodiments are merely illustrative, and cannot be understood as limiting the present application.

[0068] The schemes of the present application will be explained below with reference to examples. Those skilled in the art will understand that the examples below are only for illustration of the present application and should not be regarded as limiting the scope of the present application. The specific techniques or conditions not mentioned in the examples are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The reagents or instruments not mentioned by the manufacturer are all conventional products that can be purchased, for example, from Sigma Company.

[0069] Example 1

[0070] The method of the present application is used to verify the structure type of the triglyceride standard, which is as follows:

[0071] 1. Experimental section

[0072] 1.1 Instruments and reagents

[0073] Instruments: Zeno-TOF 7600 high-resolution mass spectrometer and SCIEX 3.0 data processing software (AB Sciex Company, USA) Milli-Q ultrapure water system (Millipore Company, USA); Harvard Apparatus syringe pump Pump 11 Elite

[0074] Reagents: n-hexane, isopropyl alcohol (chromatographically pure, Thermo Fisher Company, USA); sodium acetate (chromatographically pure, Sigma-Aldrich Company, USA);

[0075] Lipid standards: 17 kinds of triglyceride standards were purchased from ZZSTANDARD. In this example, the lipidomic nomenclature proposed by Marshall et al. is used: only one sn-position is defined, and the triglyceride is abbreviated in a pair of parentheses, and the isomerism at the sn position is divided by a slash, for example, OPO (18:1 / 16:0 / 18:1) represents 1,3-dioleic acid-2-palmitic acid triglyceride, i.e. ABA type TAG; OOP (18:1 / 18:1 / 16:0) represents 1,2-dioleic acid-3-palmitic acid triglyceride, i.e. AAB type TAG. For details, see the table below;

[0076] Table 1. Basic information of 17 positional isomer compounds

[0077]

[0078]

[0079] 1.2 Preparation of standard solution:

[0080] Preparation of standard stock solution: The triglyceride standards were accurately weighed and dissolved in n-hexane to prepare a 0.5 mg / mL stock solution, which was stored at -20 °C until use.

[0081] Preparation of standard working solution: 1 mL of the standard stock solution was diluted with isopropanol: water (9: 1 v / v) containing 0.5 mM sodium acetate to prepare a 0.05 mg / mL working solution, which was stored at -20 °C until use.

[0082] 1.3 Mass spectrometry method:

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

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

[0085]

[0086]

[0087] 1.4 Data analysis

[0088] The raw data were processed using SCIEX OS software 3.3 with a browser and analysis module. All the 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 and forms a [M+Na-R1] in the mass range of 550-650Da + Characteristic peaks and molecular ion loss of diacyl chains generate three [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 mass range of 550-650 Da, the parent ion [M+Na] + Loss of one C18:0(S) fatty acyl group to form one ion fragment [M+Na-RxCOOH] + m / z = 629.54, this fragment ion can be C18:0(S) fatty acyl group loss at any position of -sn1, -sn2, -sn3. Therefore, AAA type TAGs only have one obvious characteristic ion in the mass range of 550-650 Da, and the fatty acyl chain can be determined according to the mass of the lost fragment; in the mass range of 300-350 Da, three characteristic peaks are observed: 1) the parent ion loses fatty acyl groups at sn1 and sn3 positions at the same time, producing a characteristic ion [M+Na-R1COOH-R3COOCH3] m / z = 333.27; 2) the parent ion loses fatty acyl groups at sn1 and sn2 positions at the same time (or breaks sn3 and sn2 positions at the same time), but there are two ways to break the adjacent fatty acyl groups Figure 1 ), one is to break the C-O bond of the glycerol backbone, producing a characteristic fragment [M+Na-R2COOH-R 3 / 1 COOH] m / z = 347.29, which contains a methylene group (-CH2-); the other is to break the C-C bond of the glycerol backbone at -sn1 / -sn3 and -sn2 positions, producing a characteristic fragment [M+Na-R2CO-R 3 / 1 COOCH3] m / z = 349.27, in which an oxygen atom (-O-) replaces (-CH2-), so the mass-to-charge ratio of the two characteristic fragment ions only differs by 2 Da. AAA type TAGs have three obvious characteristic ions in the mass range of 300-350 Da. OOO (18:1 / 18:1 / 18:1), LLL (18:2 / 18:2 / 18:2), and LnLnLn (18:3 / 18:3 / 18:3) have the same characteristic fragmentation rules, and the characteristic fragments are shown in Table 3, and the mass spectrum is shown in Figure 1 .

[0098] 2.2 Characterization of ABA and AAB position isomer TAGs

[0099] From the EAD mass spectra of the two groups of ABA and AAB type TAGs, three groups of characteristic peaks can be observed: the molecular ion peak [M+Na] + , two [M+Na-R1]+ characteristic peaks in the mass range of 550-650 Da formed by the loss of a single acyl chain from the molecular ion, and three or five [M+Na-R1-R2]+ characteristic ion peaks in the mass range of 300-350 Da produced by the loss of a double acyl chain from the molecular ion, and the typical characteristic fragment ions are shown in Table 3. Taking the mass spectra of POP 16:0 / 18:1 / 16:0 and OPP as examples 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) or C16:0 (P) at any of the -sn1, -sn2, and -sn3 positions 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 550-650 Da, 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] + Therefore, the I value of AAB-type TGA should be less than 2.0, and the I value of ABA-type TGA 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 type triglycerides is in the range of 3.8 to 3.9, and that of AAB type 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] + ions, 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] ratio of OPO, SOS and POP basically meets this rule; under this assumption, the [AB] + / [AA] + of AAB type TGA should be 1.5, and the I values of PLL, OLL and OPP are 1.448, 1.474 and 1.625 respectively, which also basically meet this rule. The [AB] + / / [AA] + of OOP is 2.008, which is speculated to be due to the presence of OPO impurities in the raw material, resulting in an increase in the abundance of [AB] + . Of course, the above assumption is only based on a small amount of experiments, and the inventors will further accumulate relevant data to perfect this assumption. 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 ratio of the fragment ion abundance of glyceride ion [AB] + / [AA] + , AAB and ABA type position isomerization TGA can be identified, which is one of the very important identification bases.

[0102] Table 2. Relative abundance ratio of glyceride fragment ions under EAD cleavage mode

[0103]

[0104] I* represents the mass spectrometry response value in 6 time periods (0-1 min, 1-2 min, 2-3 min, 3-4 min, 4-5 min, 5-6 min) within 6 min; AAB / AAB type TAGs I* represents [AB] + :[AA] + , ABC type TAGs I* represents [AB] + :[BC] + :[AC] + .

[0105] 3) In the mass range of 300-350 Da, the characteristic peaks of AAB and ABA type position isomerization TGA S are observed to be significantly different. Similarly, taking POP (16:0 / 18:1 / 16:0) as an example, first, the parent ion loses sn1 and sn3 fatty acyl groups [P] + , producing [M+Na-P-P] +The characteristic ion m / z = 333.26 can be directly identified as the fatty acyl chain at the -sn2 position, thus characterizing the position isomer of ABA type TAGs; secondly, the parent ion loses the fatty acyl groups at the -sn1 (or -sn3) and -sn2 positions, the fatty acyl chains at the -sn1 and -sn3 positions are the same, but there are two ways of breaking the fatty acyl chain at the -sn2 position Figure 2 ), which is the same as the AAA type TAGs, thus, the ABA type TAGs 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 also loses the fatty acyl groups at the sn1 and sn3 positions to produce [M+Na-P-O] + The characteristic ion m / z = 305.29; however, because the fatty acyl chains at the -sn1 and -sn3 positions are different, the parent ion produces two characteristic fragments m / z = 347.25 and m / z = 345.28 with a mass-to-charge ratio difference of 2 Da when it loses the fatty acyl groups at the -sn1 and -sn2 positions, and produces two characteristic fragments m / z = 319.26 and m / z = 321.24 with a mass-to-charge ratio difference of 2 Da when it loses the fatty acyl groups at the -sn3 and -sn2 positions, thus, the AAB type TAGs have five obvious characteristic ions in the mass range of 300-350 Da, of which two pairs of characteristic fragment ions have a mass-to-charge ratio difference of only 2 Da. The other two ABA and three AAB type TAGs have the same characteristic fragmentation rules.

[0106] 3.3 Characterization of ABC type TAGs

[0107] From the observation of the EAD mass spectrum fragments of the six ABC type TAGs, three groups of characteristic peaks can also be observed: the molecular ion peak [M+Na] + , the molecular ion loses a single acyl chain to form three [M+Na-R1] + characteristic peaks in the mass range of 550-650 Da, and the molecular ion loses a double acyl chain to produce four or five [M+Na-R1-R2] + characteristic ion peaks, and the typical characteristic fragment ions are shown in Table 3. From the observation of the EAD mass spectrum fragments of the six ABC type TAGs, three groups of characteristic peaks can also be observed: the molecular ion peak [M+Na] Figure 3 It can be seen that 1) the molecular ion peaks of the three position isomers POS 16:0 / 18:1 / 18:0, PSO 16:0 / 18:0 / 18:1, and OPS 18:1 / 16:0 / 18:0 are all [M+Na] + m / z = 883.74; 2) in the range of 550-650 Da, the molecular ion [M+Na] + of the three isomers loses a fatty acyl chain at any of the -sn1, -sn2, and -sn3 positions to 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] + The 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 fragmentation and I value trends were also observed in the spectra of the two isomers PSO16:0 / 18:0 / 18:1 and OPS18:1 / 16:0 / 18:0. The results in Table 2 show 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 acid produces fragments [M+Na-R2COOH] + The abundance is lower than that of the 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, which also basically met the law. Therefore, by judging the ratio of the abundance of diglyceride fragment ions, the -sn2 fatty acyl chain could be identified, thereby characterizing the positional isomer of ABC-type TGA.

[0109] 3) In the mass range of 300-350 Da, ABC-type positional isomer TGA S There are 4-5 characteristic peaks. Similarly, taking POS as an example, the parent ion loses the sn1 and sn3 fatty acyl groups at the same time, generating an [M+Na-P-S] + The characteristic ion m / z = 331.26 can directly identify the -sn2 fatty acyl chain, thereby characterizing the positional isomer of ABC-type TGA according to the fragment mass number; secondly, the parent ion loses the -sn1 (or -sn3) and sn2 fatty acyl groups at the same time, which also generates two characteristic fragment ions m / z = 349.27, 347.29 ([P] loss) with a mass-to-charge ratio difference of only 2 Da, and since the -sn1 and -sn3 fatty acyl chains of ABC-type TGA are different, two other characteristic fragment ions m / z = 321.24, 319.26 ([S] loss) with a mass-to-charge ratio difference of only 2 Da are also generated. However, for OPS 18:1 / 16:0 / 18:0, since the mass number difference between the -sn1 and -sn3 fatty acyl chains is 2 Da, it indicates that the carbon chain length of -sn1 or -sn3 is the same, only with a difference of 1 double bond, so both can generate a fragment peak with a mass-to-charge ratio of m / z = 347.25, so OPS appears a total of 4 fragment peaks in the mass range of 300-350 Da.

[0110] 2.4 Identification strategy of TAG fatty acid acyl chain positional isomer

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

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

[0113] Second, the position of fatty acyl chains is characterized by characteristic fragment ions in the range of 550-650 Da mass-to-charge ratio according to the EAD secondary mass spectrum (MS / MS) of TGA. 1) If one fragment ion of losing a single acyl chain [M+Na-R]+ appears, it is determined as AAA type, and the type of fatty acyl chain is determined according to the mass number of loss; 2) If two fragment ions of losing a single acyl chain [M+Na-R]+ appear, it is initially determined as ABA or AAB type, and then identified according to the ratio I of [AB] + / [AA] + When I>2, it is ABA type, and when I<2, it is AAB type, and then the position of three fatty acyl chains is identified according to two characteristic fragment ions and the mass number of loss fragments; 3) If three fragment ions of losing a single acyl chain [M+Na-R]+ appear, it is initially determined as ABC type, and then identified according to the ratio ≈2 / 2 / 1 of [AB] + / [BC] + / [AC] + According to the mass-to-charge ratio of the lower abundance fragment, the fatty acyl chain at the -sn2 position is identified, and thus the position of the fatty acyl chain of TGA is determined;

[0114] Third, the position of fatty acyl chains is characterized by characteristic fragment ions in the range of 300-350 Da mass-to-charge ratio according to the EAD secondary mass spectrum (MS / MS) of TGA. 1) If three fragment ions of losing a diacyl chain [M+Na-2R] + appear, characterized by one single fragment ion and two fragment ions with a mass-to-charge ratio difference of only 2 Da, it is initially determined as AAA type or ABA type. Then the type and position of fatty acyl chains are identified according to the mass-to-charge ratio of three characteristic fragment ions; 2) If five fragment ions of losing a single acyl chain [M+Na-2R] + appear, characterized by one single fragment ion and two pairs of fragment ions with a mass-to-charge ratio difference of only 2 Da (a total of four), it is initially determined as ABC or AAB type, and the mass number difference of fatty acyl chains at the -sn1 and -sn3 positions is greater than 2 Da. Then the type and position of fatty acyl chains are identified according to the mass-to-charge ratio of five characteristic fragment ions; 3) If four fragment ions of losing a single acyl chain [M+Na-R] +The fragment ions of 1 single fragment ion, 3 fragment ions with mass-to-charge ratio difference of 2 Da in turn, can be preliminarily determined as AAB type or ABC type, and the mass number difference of the fatty acyl chains at the-sn1 position and the-sn3 position is 2 Da, indicating that the fatty acyl chains at the-sn1 position or the-sn3 position have the same carbon chain length and only differ by 1 double bond. In summary, according to the number, mass-to-charge ratio and abundance ratio of the fragment ions observed in the two mass-to-charge ratio ranges of 300-350 Da and 550-650 Da on the secondary mass spectrum, the composition and position of the fatty acids are determined, and 2 sets of data are verified with each other, greatly improving the confidence of the fatty acid acyl chain structure characterization.

[0115] In the description of the present specification, the description referring to the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0116] Although the embodiments of the present application have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, replacements and variations can be made to the embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the claims and their equivalents.

Claims

1. A method for detecting triglyceride positional 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; Detecting the test liquid using a high-resolution mass spectrometer to obtain primary mass spectrometry results and secondary mass spectrometry results; as well as Based on the primary mass spectrometry results and the secondary mass spectrometry results, obtaining the structural type of each branch of the triglyceride sample, 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; The detection conditions of the high-resolution mass spectrometer are: Working solution flow rate: 30 uL / 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; Based on the molecular ion peak [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 of the triglyceride sample, wherein M represents triglyceride, N represents a cation bound to the ionized triglyceride, and R represents the fatty acid acyl chain of the triglyceride sample.

2. The method according to claim 1, characterized in that Based on the molecular 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 acids, the fragment ions [M+NR] were determined. + The mass-to-charge ratio range and the fragment ions [M+N-2R] + mass-to-charge ratio range.

3. The method according to claim 2, characterized in that In the secondary mass spectrometry results, the fragment ion [M+N-2R] + There are 3 fragment ions within 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 differs by 2Da, and the triglyceride sample structure type is the ABA type or the AAA type. In the secondary mass spectrometry results, the fragment ion [M+N-2R] + There are 5 fragment ions in the mass-to-charge ratio range [M+N-2R] + , in addition to 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 differs by 2Da, and the triglyceride sample structure type is the ABC type or the AAB type. In the secondary mass spectrometry results, the fragment ion [M+N-2R] + There are 4 fragment ions [M+N-2R] in the mass-to-charge ratio range + , in addition to 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 2 Da, and the triglyceride sample structure type is the ABC type or the AAB type.

4. The method according to claim 3, 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; or 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.

5. The method according to claim 2 or 3, characterized in that In the secondary mass spectrometry results, 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.

6. The method according to claim 2 or 3, characterized in that In the secondary mass spectrometry results, 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 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 ] + .

7. The method according to claim 6, characterized in that 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. Where I = [AB] + Abundance ÷ [AA] + Abundance (1) Among them, [AB] + [AB] of the ABA or AAB triglyceride + Mass spectrometric peaks of fragments; [AA] + [AA] of the ABA or AAB triglyceride + Mass spectrometric peaks of fragments.

8. The method according to claim 2 or 3, characterized in that There are three fragment ions [M+NR] in the secondary mass spectrometry results. + , then the triglyceride sample structure type is the ABC type.

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