Oxidized triglyceride mass spectrum analysis method based on thermal oxidation reaction mechanism and mass spectrum fragmentation rule prediction spectrum library

Abstract

The invention relates to the technical field of food chemistry, and particularly discloses an oxidized triglyceride mass spectrum analysis method based on a thermal oxidation reaction mechanism and a mass spectrum fragmentation rule prediction spectrum library, which comprises the following steps: P1, constructing possible oxidized acyl chains based on the types of unsaturated fatty acids in common vegetable oil and oxidation mechanisms of different double bond sites of molecules of the unsaturated fatty acids, predicting the molecular structure of the oxidized triglyceride according to the acyl chain composition and the Cartesian product, and calculating the molecular formula, molecular mass and adduct ion mass of the oxidized triglyceride; p2, calculating mass spectrum collision induced dissociation core fragments according to general laws of different types of oxidized triglyceride, and constructing primary and secondary prediction spectrum libraries of oxidized triglyceride; p3, collecting sample data based on a non-targeted liquid chromatography-mass spectrometry method; p4, annotating and identifying oxidized triglyceride molecules in the vegetable oil sample; according to the method, high-throughput detection of oxidized triglyceride molecules in vegetable oil can be realized without complicated pretreatment and enrichment methods.

Description

Technical Field

[0001] This invention relates to the field of food chemistry technology, and in particular to a mass spectrometry analysis method for oxidized triglycerides based on the thermal oxidation reaction mechanism and mass spectrometry fragmentation rules to predict the spectral library. Background Technology

[0002] Edible oils undergo thermal oxidation during heat processing / cooking, producing complex oxidation products. Oxidized triglycerides are primary oxidation products and important oxidation intermediates in this process. Analysis of oxidized triglycerides can be used to monitor the degree of oxidation and safety of edible oils. Oxidized triglycerides are cytotoxic and can easily affect human health when ingested with oils; therefore, the analysis of oxidized triglycerides in edible oils has received significant attention from researchers.

[0003] Because all three fatty acyl chains of triglycerides can be oxidized, the types and structures of oxidized triglycerides produced by thermal oxidation are extremely complex. Oxidized triglycerides mainly include hydroxyl groups, peroxidized hydroxyl groups, epoxy groups, ketone groups, and groups containing multiple oxidized groups. Currently, the main method for detecting oxidized triglycerides is liquid chromatography-mass spectrometry (LC-MS). The separation capabilities of chromatography and the structural resolution capabilities of mass spectrometry enable researchers to perform precise analysis of oxidized triglycerides in different oil samples. However, oxidized triglycerides are unstable, commercially available standards are very limited, and there is no comprehensive secondary mass spectrometry database for oxidized triglycerides, making the analysis and identification of oxidized triglycerides in edible oils a major challenge. Summary of the Invention

[0004] To address the problems mentioned in the background art, the present invention aims to provide a mass spectrometry analysis method for oxidized triglycerides based on the thermal oxidation reaction mechanism and the mass spectrometry fragmentation rule prediction library.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows: A mass spectrometry analysis method for oxidized triglycerides based on thermal oxidation reaction mechanism and mass spectrometry fragmentation rules predicting the spectral library includes the following steps: P1. Based on the general rules and oxidation mechanisms of thermal oxidation of unsaturated fatty acids: 1) The α-carbon at the side of the unsaturated double bond readily undergoes a hydrogen extraction reaction, combining with oxygen to form peroxidized hydroxyl fatty acids; the peroxidized hydroxyl group undergoes a dehydration reaction to produce ketone fatty acids; the peroxidized hydroxyl group provides a hydroxyl group, and the α-carbon of the double bond provides a hydrogen atom, dehydrating to form epoxy-based fatty acids; 2) Fatty acids containing oxidized groups undergo β-cleavage to produce short-chain alkanes and break fatty acid structures. Based on the above reaction mechanisms and the Cartesian product combination of oxidized triglycerides, the molecular structure of oxidized triglycerides is predicted, and the molecular formula, molecular mass, and adduct ion mass of oxidized triglycerides are calculated.

[0006] P2. Based on the general rules of different types of oxidized triglycerides, calculate the collision-induced dissociation core fragments in mass spectrometry and construct the primary and secondary prediction spectral libraries of oxidized triglycerides.

[0007] P3. Perform non-targeted chromatographic-mass spectrometry analysis on oxidized vegetable oil samples.

[0008] P4. Using the predicted molecular mass of oxidized triglycerides, the mass of the adduct, and the predicted secondary spectral library, the oxidized triglycerides in the sample are matched and the secondary spectral similarity is calculated to achieve structural annotation of oxidized triglyceride molecules.

[0009] Furthermore, in step P1, the fatty acid acyl chain oxidation mechanism includes free radical chain reaction, peroxidation, epoxidation, β-cleavage, dehydration reaction, etc., and the types of unsaturated fatty acyl chains included are: palmitoleic acid with 16 carbon atoms, 1 double bond, and double bond position ω-7; oleic acid with 18 carbon atoms, 1 double bond, and double bond position ω-9; linoleic acid with 18 carbon atoms, 2 double bonds, and double bond position ω-6; and eicosenoic acid with 20 carbon atoms, 1 double bond, and double bond position ω-9.

[0010] Furthermore, in step P2, the spectral library predicted based on the fragmentation pattern of oxidized triglycerides induced by mass spectrometry collision-induced dissociation includes characteristic fragments: diglyceride fragments [M-R1COOH] formed by the neutral loss of acyl chains in oxidized triglycerides. + [M-R2COOH] + [M-R3COOH] + Acyl chain fragments [R1COOH-H2O] + [R2COOH-H2O] + [R3COOH-H2O] + ; adduct deamination fragment [M+NH4-17] + .

[0011] Furthermore, the determination of oxidized triglycerides in the vegetable oil sample in step P3 includes the following steps: Chromatographic elution conditions: A C18 column was used. Mobile phase A was a 1:1:1 mixture of water, methanol, and acetonitrile (v / v), and mobile phase B was a 5:1 mixture of isopropanol and acetonitrile (v / v), with 5 mM ammonium acetate added to each. The chromatographic elution program was as follows: Initial mobile phase B was 20% B, held for 0.5 min, then from 0.5 min to 1.5 min, mobile phase B linearly increased to 40% B; from 1.5 min to 2 min, mobile phase B increased to 60% B; from 2 min to 10 min, mobile phase B linearly increased to 95%, held for 2 min, and then from 13 min, mobile phase B decreased to 20% B.

[0012] The chromatographic conditions used were a C18 column with a packing material of 2.6 μm, a length of 100 mm, an inner diameter of 2.1 mm, a flow rate of 0.4 mL / min, a column temperature of 60 degrees Celsius, and an injection volume of 3 μL.

[0013] In the mass spectrometry conditions, electrospray mass spectrometry in positive ion mode was used, with a mass-to-charge ratio mass scan range of 200-1200. MS2 was acquired in a dependent acquisition mode, with 15 candidate high-abundance precursor ions selected for each scan interval to trigger secondary spectrum acquisition.

[0014] Furthermore, in step P4, the spectral matching and identification method for oxidized triglycerides is as follows: The collected raw data undergoes primary molecular weight matching, followed by secondary spectral matching. Higher spectral similarity results in higher accuracy. To improve accuracy, the spectral identification rule for oxidized triglycerides is: [M+NH4] oxidized triglyceride precursor ion + The oxidized triglyceride loses ammonia molecules first, followed by water molecules; after losing the acyl chain, the diglyceride fragments exhibit oxygen-containing and dehydrated characteristics; in the low-mass range ( mz (100-300) will have obvious fatty acyl chains and oxidized fatty acyl chain fragments.

[0015] The beneficial effects of this invention are as follows: The analytical method provided by this invention does not require complex pretreatment and enrichment methods, and can achieve high-throughput detection of oxidized triglyceride molecules in vegetable oils. It can construct a predictive spectral library of oxidized triglycerides through oxidation reaction and mass spectrometry fragmentation patterns without the need for standard reference materials, thereby improving the coverage and reliability of oxidized triglyceride analysis and reducing the difficulty of structural analysis. Attached Figure Description

[0016] Figure 1 A schematic diagram of the oxidation reaction mechanism of oxidized triglycerides (taking 18:1 as an example); Figure 2 A schematic diagram of the mass spectrometry fragmentation mechanism of oxidized triglycerides; Figure 3 This is a schematic diagram of annotation and secondary spectrum matching for oxidized triglycerides. Detailed Implementation

[0017] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art are within the protection scope of the present invention.

[0018] Example The construction and annotation of a secondary mass spectrometry library for predicting oxidized triglycerides in rapeseed oil are as follows: (1) Take 10 mL of rapeseed oil in a borosilicate glass tube and heat it at 180℃ for 90 min. After heating, take about 10 mg of rapeseed oil, dissolve it in chromatographic grade isopropanol, and then analyze it by mass spectrometry.

[0019] (2) The fatty acids contained in rapeseed oil include: palmitic acid (16:0), palmitoleic acid (16:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), eicosanoic acid (20:0), eicosenoic acid (20:1), docosanoic acid (22:0), etc., which include products produced by oxidation reaction (hydroxyl, epoxy, peroxyhydroxyl, etc.). There are a total of 196 possible fatty acid precursors and oxidation products, as shown in Table 1.

[0020] Table 1 Major fatty acid precursors and oxidation products (3) Construction of the second-order spectra of oxidized triglycerides based on reaction mechanism: Oxidation products of 196 fatty acid precursors were combined using Cartesian product methods and weight removal to obtain 1,274,196 oxidized triglyceride products with different acyl chain combinations. Based on the mass spectrometric fragmentation mechanism of oxidized triglycerides, the secondary fragments of each oxidized triglyceride molecule were predicted and calculated: diglyceride fragments [M-R1COOH] formed by the neutral loss of acyl chains in oxidized triglycerides. + [M-R2COOH] + [M-R3COOH] + Acyl chain fragments [R1COOH-H2O] + [R2COOH-H2O] + [R3COOH-H2O] + ; adduct deamination fragment [M+NH4-17] + Based on the fragmentation pattern and the ease of bond breaking, the abundance of oxidized triglycerides was predicted, and a total of 1,274,196 predicted secondary mass spectra of oxidized triglycerides were obtained. Some of the spectra are shown in Table 2. The 1,274,196 predicted secondary spectra were compiled into MSP format.

[0021] Table 2. Predicted secondary mass spectrometry information of oxidized triglycerides (OxTGs) (partial). (4) Chromatographic elution conditions for rapeseed oil: A C18 column (Pyromex 2.1 × 100 mm, 2.6 μm) was used, and the measurement temperature was 60℃. Mobile phase A was a 1:1:1 mixture of water, methanol, and acetonitrile (v / v), and mobile phase B was a 5:1 mixture of isopropanol and acetonitrile (v / v), with 5 mM ammonium acetate added to each. The chromatographic elution program was as follows: initial mobile phase B was 20% B, held for 0.5 min; from 0.5 min to 1.5 min, mobile phase B linearly increased to 40% B; from 1.5 min to 2 min, mobile phase B increased to 60% B; from 2 min to 10 min, mobile phase B linearly increased to 95% B, held for 2 min, and from 13 min, mobile phase B decreased to 20% B. After needle equilibration, the next sampling was performed.

[0022] (5) Mass spectrometry detection conditions for rapeseed oil: Ionization was performed using electrospray mass spectrometry in positive ion mode, with an ion source temperature of 550℃, a declustering voltage of 80 eV, and a collision energy of 12 eV. The mass-to-charge ratio mass scan range was 200-1200. MS2 was performed in acquisition-dependent mode, with 15 candidate high-abundance precursor ions selected for each scan interval to trigger secondary spectrum acquisition.

[0023] (6) Identification of oxidized triglycerides in rapeseed oil: Peak extraction was performed using the open-source mass spectrometry data analysis software MS Dial 5, and the peaks were imported into a predicted spectral library for identification and annotation of oxidized triglycerides. The first-order mass tolerance was 0.01 Da, and the second-order mass tolerance was 0.025 Da; the added ions selected were [M+H]+ and [M+NH4]+. Figure 1 The oxidation reaction mechanism of triglycerides is illustrated using a triglyceride that theoretically contains an 18:1 ratio of unsaturated fatty acids as an example. Figure 2 Taking epoxidized triglyceride TG 18:1_18:1_18:1(O) as an example, we can illustrate the mass spectrometry fragmentation pattern and main fragment ions of epoxidized triglyceride. Figure 3 Taking a mass-to-charge ratio of 918.8096 as an example, this study illustrates the identification and matching of oxidized triglycerides from rapeseed oil. This example employs an oxidized triglyceride analysis method based on the thermal oxidation reaction mechanism and mass spectrometry fragmentation rules to predict the spectral library, identifying 51 triglyceride precursors and oxidized triglycerides from rapeseed oil: 30 triglyceride precursors and 21 oxidized triglycerides (including six categories: cleavage products, hydroxyl groups, ketone groups, epoxy groups, peroxide hydroxyl groups, and complex polykaryotic groups), as detailed in Table 3.

[0024] Table 3. List of Oxyglycerols (OxTGs) in Heated Rapeseed Oil Identified Based on Predicted Secondary Spectral Library Compared with traditional methods, the method described in this invention solves the problem of the lack of corresponding secondary mass spectrometry annotation libraries for oxidized triglycerides. By constructing a predicted secondary spectral library containing 1,274,196 non-repeating oxidation products through oxidation reaction mechanisms and mass spectrometry fragmentation patterns, it improves the identification coverage of oxidized triglycerides and enhances the annotation capability of complex oxidation products. The method used in this invention can be used to identify complex triglyceride precursors and oxidized triglycerides in rapeseed oil, achieving high-coverage, high-throughput analysis of oxidized triglycerides in vegetable oils.

[0025] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A mass spectrometry analysis method for oxidized triglycerides based on thermal oxidation reaction mechanism and mass spectrometry fragmentation rules to predict the spectral library, characterized in that, Includes the following steps: P1. Based on the types of unsaturated fatty acids in common vegetable oils and their oxidation mechanisms at different double bond sites, construct possible oxidized acyl chains. Predict the molecular structure of oxidized triglycerides based on the combination of the acyl chain composition and the Cartesian product of the oxidized triglycerides. Calculate the molecular formula, molecular weight, and adduct ion mass ([M+NH4)) of the oxidized triglycerides. + ); P2. Based on the general rules of different types of oxidized triglycerides, calculate the collision-induced dissociation core fragments in mass spectrometry and construct the primary and secondary prediction spectral libraries of oxidized triglycerides. P3. Sample data acquisition based on non-targeted liquid chromatography-mass spectrometry; P4. Annotate and identify oxidized triglyceride molecules in vegetable oil samples: Use the predicted molecular mass of oxidized triglycerides, the mass of adducts, and the secondary predicted spectral library in step 2) to match and calculate the secondary spectral similarity of oxidized triglycerides in the samples, so as to achieve structural annotation of oxidized triglyceride molecules.

2. The method for mass spectrometry analysis of oxidized triglycerides based on thermal oxidation reaction mechanism and mass spectrometry fragmentation rule prediction library as described in claim 1, characterized in that, In step P1, the fatty acid acyl chain oxidation mechanism includes free radical chain reaction, peroxidation, epoxidation, β-cleavage, and dehydration reaction. The unsaturated fatty acyl chain types include: palmitoleic acid with 16 carbon atoms, 1 double bond, and double bond position ω-7; oleic acid with 18 carbon atoms, 1 double bond, and double bond position ω-9; linoleic acid with 18 carbon atoms, 2 double bonds, and double bond position ω-6; and eicosenoic acid with 20 carbon atoms, 1 double bond, and double bond position ω-9.

3. The method for mass spectrometry analysis of oxidized triglycerides based on thermal oxidation reaction mechanism and mass spectrometry fragmentation rule prediction library as described in claim 1, characterized in that, In step P2, the predicted spectral library based on the fragmentation pattern of oxidized triglycerides induced by mass spectrometry collision-induced dissociation includes the following characteristic fragments: diglyceride fragments [M-R1COOH] formed by the neutral loss of acyl chains in oxidized triglycerides. + [M-R2COOH] + [M-R3COOH] + Acyl chain fragments [R1COOH-H2O] + [R2COOH-H2O] + [R3COOH-H2O] + ; adduct deamination fragment [M+NH4-17] + .

4. The mass spectrometry analysis method for oxidized triglycerides based on thermal oxidation reaction mechanism and mass spectrometry fragmentation rule prediction library as described in claim 1, characterized in that, In step P3, the chromatographic and physical conditions are as follows: Chromatographic elution conditions: A C18 column was used. Mobile phase A was a mixture of water, methanol and acetonitrile in a volume ratio of 1:1:

1. Mobile phase B was a mixture of isopropanol and acetonitrile in a volume ratio of 5:

1. 5 mM ammonium acetate was added to each phase. Chromatographic elution program: Initial mobile phase B is 20% B, hold for 0.5 min, 0.5 min - 1.5 min, mobile phase B linearly increases to 40% B; 1.5 min - 2 min, mobile phase B increases to 60% B; 2 min - 10 min, mobile phase increases linearly to 95%, hold for 2 min, 13 min, mobile phase B decreases to 20% B; In the mass spectrometry conditions, electrospray mass spectrometry in positive ion mode was used, with a mass-to-charge ratio mass scan range of 200-1200. MS2 was acquired in a dependent acquisition mode, with 15 candidate high-abundance precursor ions selected for each scan interval to trigger secondary spectrum acquisition.

5. The mass spectrometry analysis method for oxidized triglycerides based on thermal oxidation reaction mechanism and mass spectrometry fragmentation rule prediction library as described in claim 4, characterized in that, In the chromatographic analysis, a C18 column was used with a packing material of 2.6 μm, a length of 100 mm, an inner diameter of 2.1 mm, a flow rate of 0.4 mL / min, a column temperature of 60 degrees Celsius, and an injection volume of 3 μL.

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