High coverage analysis method of vegetable oil oxidized triglycerides based on list-dependent mode

Abstract

The application provides a plant oil oxidized triglyceride high-coverage analysis method based on a list-dependent mode, and belongs to the technical field of detection. The method comprises the following steps: S1, based on the fatty acyl group composition of plant oil triglyceride and the formation rule of oxidation products, an enumeration method is used to predict potential oxidized triglyceride molecular formulas in oil; S2, a plant oil non-target detection priority ion list and an exclusion ion list are constructed; S3, oxidized triglycerides in plant oil are determined; and S4, suspected oxidized triglyceride molecules are screened by matching the mass-to-charge ratio of primary mass spectrometry, the fatty acyl group composition, the oxidation functional group type and the oxidation position are analyzed by secondary mass spectrometry characteristic fragments, and the fine structure of the oxidized triglyceride molecules is determined. The plant oil oxidized triglyceride high-coverage analysis method based on the list-dependent mode can realize high-coverage and high-sensitivity analysis of oxidized triglycerides in plant oil.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection, in particular to a plant oil oxidized triglyceride high-coverage analysis method based on a list-dependent mode. BACKGROUND

[0002] Oxidized triglyceride is one of the core products of oil oxidation, and the types of its oxidation functional groups (such as hydroperoxy, epoxy, hydroxyl, carbonyl, etc.) and oxidation sites (such as double bond in situ and ortho region) directly reflect the degree of oil oxidation and the difference in oxidation path. Analyzing the fine structure of oxidized triglyceride is a key basis for tracing the dynamic process of oil oxidation and elucidating the mechanism of oil oxidation. Non-targeted analysis methods based on liquid chromatography-mass spectrometry technology can screen oil oxidation products without discrimination and are widely used for identification and screening of oil oxidation products.

[0003] Non-targeted analysis methods based on liquid chromatography-mass spectrometry technology identify the molecular structure according to the fragment information in the MS 2 spectrum of the compound. Data-dependent mode is a widely used MS 2 spectrum acquisition mode. However, the traditional data-dependent mode acquires the MS 2 spectrum information of the compound based on ion intensity, that is, the parent ions with ion intensity greater than a certain threshold are preferentially selected to collect their secondary mass spectrum information. This mode is beneficial to compounds with high abundance and strong ion response in the sample, but it is easy to ignore compounds with low abundance and weak ion response. In plant oil, the main substance is triglyceride, the target compound oxidized triglyceride has low abundance (especially in the early stage of oil oxidation), and the polarity of the two is similar, so they are difficult to separate by chromatography and are prone to co-elution. Due to the ion shielding effect of mass spectrometry, that is, high-abundance triglycerides are preferentially fragmented, resulting in low-abundance oxidized triglycerides with low MS 2 spectrum acquisition rate and missing diagnostic ions, causing the traditional non-targeted method to have low identification coverage and accuracy in measuring oxidized triglycerides in plant oil.

[0004] The list-dependent data-dependent acquisition mode preferentially selects parent ions containing the ion list to acquire their MS 2 spectrum, which can effectively improve the MS 2 information acquisition rate of the target compound, and further improve the coverage and sensitivity of detection. At the same time, by setting an exclusion ion list, interference substances in complex samples can be effectively shielded. However, the list-dependent detection method needs to know the accurate molecular ion information of the measured substance to form a list, and for the oxidized triglycerides in plant oil, which are diverse in structure and unknown, using this strategy has considerable challenges. SUMMARY

[0005] The application aims to provide a high-coverage analysis method for oxidized triglycerides in vegetable oil based on a list-dependent mode, which can realize high-coverage and high-sensitivity analysis of oxidized triglycerides in vegetable oil.

[0006] To achieve the above-mentioned purpose, the application provides a high-coverage analysis method for oxidized triglycerides in vegetable oil based on a list-dependent mode, which comprises the following steps:

[0007] In step S1, based on the fatty acyl composition of triglycerides in vegetable oil and the formation rule of oxidized products thereof, an enumeration method is used to predict potential oxidized triglyceride molecular formulas in oil and fat.

[0008] In step S2, a non-targeted detection priority ion list and an exclusion ion list of vegetable oil are constructed.

[0009] In step S3, oxidized triglycerides in vegetable oil are determined.

[0010] In step S4, suspected oxidized triglyceride molecules are screened by matching the mass-to-charge ratio of the first mass spectrum, and the fatty acyl composition, oxidized functional group type and oxidation position are analyzed by the characteristic fragments of the second mass spectrum to determine the fine structure of the oxidized triglyceride molecules.

[0011] Preferably, in step S1, the fatty acids include palmitic acid with 16 carbon atoms and 0 double bonds, palmitoleic acid with 16 carbon atoms and 1 double bond, stearic acid with 18 carbon atoms and 0 double bonds, oleic acid with 18 carbon atoms and 1 double bond, linoleic acid with 18 carbon atoms and 2 double bonds, linolenic acid with 18 carbon atoms and 3 double bonds, arachidic acid with 20 carbon atoms and 0 double bonds, and arachidonic acid with 20 carbon atoms and 4 double bonds.

[0012] Preferably, in step S1, the oxidation forms include hydroperoxides, epoxides, hydroxyl compounds and carbonyl compounds.

[0013] Preferably, in step S2, based on the oxidized triglyceride molecular formulas obtained in step S1, the NH4 + and Na + mass-to-charge ratios under the adduct mode are calculated, ions with the same mass-to-charge ratio are combined, and a non-targeted detection priority ion list of vegetable oil is formed.

[0014] Based on the triglyceride molecular formulas of vegetable oil obtained in step S1, the NH4 + and Na + mass-to-charge ratios under the adduct mode are calculated, ions with the same mass-to-charge ratio are combined, and an exclusion ion list is formed.

[0015] Preferably, in step S3, the determination of oxidized triglycerides in vegetable oil comprises the following steps:

[0016] Chromatographic conditions: C18 column, mobile phase A: 95:5 water and methanol, mobile phase B: 80:20 isopropanol and methanol, respectively, with 5 mmol ammonium acetate and 0.1 mmol sodium acetate;

[0017] Gradient elution program: initial 85% mobile phase B for 5 min, linearly increased to 90% mobile phase B from 5 min to 10 min and kept until 15 min, linearly increased to 95% mobile phase B from 15 min to 25 min and kept until 28 min, linearly increased to 100% mobile phase B from 28 min to 35 min and kept until 53 min, return to 85% mobile phase B for equilibration;

[0018] Mass spectrometric conditions: electrospray ionization positive ion mode, full scan range mass to charge ratio 800 to 1200, list priority data dependent acquisition mode to trigger secondary mass spectrum fragmentation in the mass to charge ratio range of 100 to 1200.

[0019] Preferably, in the chromatographic conditions, the inner diameter of the chromatographic column is 4.6 mm, the length is 250 mm, the particle size of the filler is 5 μm, the flow rate is 0.5 mL / min, the column temperature is 30°C, and the injection volume is 1 μL.

[0020] Preferably, in the mass spectrometric conditions, the desolvation line temperature ranges from 150°C to 250°C, the nebulization gas flow rate ranges from 2 L / min to 7 L / min, and the drying gas flow rate ranges from 3 L / min to 10 L / min.

[0021] Preferably, the secondary mass spectrum characteristic fragment resolution includes:

[0022] Fatty acyl group determination: based on diacylglycerol fragment ions formed by losing oxidized or unoxidized acyl groups;

[0023] Oxidized functional group and position determination: based on characteristic neutral loss fragments, wherein: loss of a water molecule, loss of 88 mass units corresponding to a 13-hydroperoxy group characteristic, loss of 140 mass units corresponding to a 9-hydroperoxy group characteristic, for judging hydroperoxy groups; loss of 83 or 100 mass units corresponding to a 12, 13-epoxy group characteristic, loss of 124 or 140 mass units corresponding to a 9, 10-epoxy group characteristic, for judging epoxy groups.

[0024] Therefore, the present application adopts the above-mentioned plant oil oxidized triglyceride high-coverage analysis method based on the list-dependent mode, and has the following beneficial technical effects: the present method does not require complex pretreatment and can realize high-coverage and high-sensitivity detection of low-abundance oxidized triglycerides in plant oils, compared with traditional non-targeted methods, the MS 2 spectrum acquisition rate is greatly improved, and the spectrum information is significantly improved, thereby improving the detection rate of oxidized triglycerides in plant oils. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 For oxidized triglycerides in NH4 + Schematic diagram of fragment formation in the additive mode;

[0026] Figure 2 For oxidized triglycerides in Na + Schematic diagram of fragment formation in the additive mode;

[0027] Figure 3 Taking the precursor ion with m / z 912.7661 as an example, this paper illustrates the process of identifying the structure of oxidized triglycerides. Figure 3 A in the figure represents the extracted ion chromatogram (EIC) at m / z 912.7661. Figure 3 B in the equation represents the MS at 38.2 min for m / z 912.7661. 2 Spectrum Figure 3 C in the figure is m / z 912.7661 at 38.3 min MS. 2 Spectrum Figure 3 D in the figure is m / z 912.7661 at 38.6 min MS. 2 Spectrum;

[0028] Figure 4 The images show the precursor ion distributions of potential oxidized triglycerides obtained using the method proposed in this invention and a conventional non-targeted scanning method, respectively. Figure 4 In this invention, A represents the method proposed in this invention. Figure 4 In this context, B represents the traditional non-targeted scanning method;

[0029] Figure 5 MS with m / z 927.6694 obtained by the method proposed in this invention and the traditional non-targeting method, respectively. 2 Spectrum, in which, Figure 5 In this invention, A represents the method proposed in this invention. Figure 5 In this context, B represents the traditional non-targeted scanning method. Detailed Implementation

[0030] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0031] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0032] The equipment and materials used in the examples were as follows: the equipment was an LCMS-9030 liquid chromatography-mass spectrometer (Shimadzu, Japan), a C18 column (250×4.6mm×5μm, Agilent); the mobile phase consisted of methanol and isopropanol; and the analyte was walnut oil that had been heated in an oven at 60℃ for 15 hours.

[0033] Example 1

[0034] (1) Construction of a database of potential oxidized triglyceride molecular formulas in walnut oil: There are 48 types of triglycerides in walnut oil. Based on the number of active sites on the unsaturated fatty acid chains in triglycerides, four oxidizing functional groups were combined: hydroperoxide (HOO-), epoxide (ep-), hydroxyl compound (HO-), and carbonyl compound (oxo-). Since the molecular formulas were predicted, configurational isomers were not considered. A total of 1295 potential oxidized triglycerides were predicted.

[0035] Construction of ion exclusion lists and preferred ion lists for oxidized triglycerides in walnut oil: 48 walnut oil triglycerides with the same molecular formula were merged to obtain 30 different triglyceride molecular formulas. NH4+ was then matched to each molecular formula. + and Na + In the addition mode, the corresponding quasi-molecular ion mass-to-charge ratio is obtained, forming an exclusion ion list (Table 1). In the list-first data-dependent scanning mode, the mass spectrometer detector can shield ions in the exclusion ion list, reducing interference ion information acquisition. Predicted potential oxidized triglycerides with the same molecular formula are combined and matched with NH4+. + and Na + In addition mode, the corresponding quasi-molecular ion mass-to-charge ratio is obtained, forming a priority ion list (Tables 2 and 3). In list-priority data-dependent scan mode, the mass spectrometer detector can preferentially acquire MS data of the precursor ions in the list. 2 Spectrograms increase the acquisition rate of molecular structure information for low-abundance oxidized triglycerides.

[0036] Table 1 List of Excluded Ions

[0037]

[0038]

[0039] Table 2 NH4 + Preferred ion list in addition mode

[0040] No. Molecular formula Preferred ion No. Molecular formula Preferred ion 1 C 53 H 94 O7]]> 860.7347 12 C 57 H 90 O7]]> 904.7034 2 C 53 H 96 O7]]> 862.7504 13 C 57 H 92 O7]]> 906.7191 3 C 53 H 98 O7]]> 864.766 14 C 57 H 94 O7]]> 908.7347 4 C 53 H 100 O7]]> 866.7817 15 C 57 H 96 O7]]> 910.7504 5 C 55 H 92 O7]]> 882.7191 16 C 57 H 98 O7]]> 912.766 6 C 55 H 94 O7]]> 884.7347 17 C 57 H 100 O7]]> 914.7817 7 C 55 H 96 O7]]> 886.7504 18 C 57 H 102 O7]]> 916.7974 8 C 55 H 98 O7]]> 888.766 19 C 57 H 104 O7]]> 918.813 9 C 55 H 100 O7]]> 890.7817 20 C 57 H 106 O7]]> 920.8287 10 C 55 H 102 O7]]> 892.7974 21 C 57 H 108 O7]]> 922.8443 11 C 55 H 104 O7]]> 894.813

[0041] Table 3 Na + Preferred ion list in addition mode

[0042]

[0043]

[0044]

[0045] (3) Walnut sample preparation: Weigh 10 mg of oxidized walnut oil sample and place it in a glass sample bottle. Then add isopropanol to prepare a 1 mg / mL solution. Dilute to 0.1 mg / mL for determination.

[0046] (4) Chromatographic conditions for walnut oil separation: The liquid chromatography column was a C18 column (Agilent 4.6 mm id × 250 mm, 5 μm); the measurement temperature was 30℃; the injection volume was 1 μL; the flow rate was 0.5 mL / min; mobile phase A was a mixture of water and methanol (v:v = 95:5), and mobile phase B was a mixture of isopropanol and methanol (v:v = 80:20). 5 mmol of ammonium acetate and 0.1 mmol of sodium acetate were added respectively for NH4+. + and Na + Oxidized triglycerides were determined in addition mode. The gradient elution conditions were as follows: starting with 85% mobile phase B, holding for 5 min, then linearly increasing to 90% mobile phase B from 5 min to 10 min and holding for 15 min, then linearly increasing to 95% mobile phase B from 15 min to 25 min and holding for 28 min, then linearly increasing to 100% mobile phase B from 28 min to 35 min and holding for 53 min, then restoring to the initial concentration of 85% mobile phase B, and performing the next injection when in equilibrium.

[0047] (5) Mass spectrometry detection conditions: Ionization was performed using an electrospray ionization source in positive ion mode, with a desolvation line temperature of 250℃ and a nebulizer gas flow rate of 5L / min; the drying gas flow rate was 10L / min; and the mass spectrometry was performed in full scan mode (m / z 800-1200), DDA list priority mode (m / z 100-1200), and MS / MS mode.

[0048] (6) Identification of oxidized triglycerides in walnut oil: First, peak extraction was performed using the built-in mass spectrometry software Labsolution. The precise mass-to-charge ratio of the extracted molecular formula was matched with the predicted potential oxidized triglycerides to screen out suspected oxidized triglyceride molecules. Further analysis was conducted using ion MS... 2 Feature fragments in the spectrum annotate the fine structure of oxidized triglyceride molecules, including the fatty acyl group composition, type of oxidized functional group, and oxidation position. Figure 1 , Figure 2 Oxidized triglycerides were respectively in NH4 + and Na + Schematic diagram of fragment formation in adsorption ion mode; Figure 3(Taking an ion with a mass-to-charge ratio of 912.7661 as an example to illustrate the identification process of oxidized triglyceride molecules). In this embodiment, a list-first approach was used to successfully identify 109 oxidized triglyceride molecules in walnut oil, covering six major categories of oxidation-modified products, including monoepoxides (36 types), monohydroperoxides (42 types), hydroperoxide-epoxide complexes (16 types), dihydroperoxides (8 types), diepoxides (5 types), and hydroxyl-epoxides (2 types), as detailed in Table 4.

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056] (7) Compared with traditional non-targeted methods, the method proposed in this invention for the MS of oxidized triglycerides 2 The spectral acquisition rate has been greatly increased: in traditional DDA mode ( Figure 4 Potentially oxidized triglycerides accounted for only 7.3% of the triggering fragmentation precursor ions, with the remaining fragmentation events being background ions (such as solvadducts and column leaching products). However, in the data collected using the list-first data-dependent mode, potentially oxidized triglycerides accounted for 90.7% of the fragmentation events.

[0057] The method proposed in this invention not only improves the MS content of oxidized triglycerides in vegetable oils 2 The spectral information acquisition rate was increased, and the spectral quality was significantly improved, such as... Figure 5 As shown, for oxidized triglycerides with low abundance in walnut oil, traditional methods or MS analysis are used. 2 The spectra contain limited fingerprint information about the molecular structure, or fail to trigger the fragmentation process of low-abundance oxidized triglyceride precursor ions, resulting in poor MS performance. 2 The spectrum was missing, but the method proposed in this invention can collect low-abundance oxidized triglycerides in walnut oil using high-quality MS. 2 Spectra are used for structural identification, improving the coverage, sensitivity, and accuracy of detection.

[0058] It is worth noting that all contents not described in detail in this invention are existing technologies and are well known to those skilled in the art.

[0059] Therefore, the present invention employs the above-mentioned list-dependent mode-based high-coverage analysis method for oxidized triglycerides in vegetable oils, which can achieve high-coverage and high-sensitivity analysis of oxidized triglycerides in vegetable oils.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A high-coverage analysis method for oxidized triglycerides in vegetable oils based on list dependency pattern, characterized in that, Includes the following steps: Step S1: Based on the fatty acyl group composition of vegetable oil triglycerides and the formation rules of their oxidation products, the enumeration method is used to predict the molecular formula of potential oxidized triglycerides in oils. Step S2: Construct a priority ion list and an exclusion ion list for non-targeted detection of plant oils; Among them, based on the molecular formula of oxidized triglycerides of vegetable oil obtained in step S1, its NH4+ is calculated. + with Na + In the addition mode, the mass-to-charge ratio is combined to form a priority ion list for non-targeted detection of plant oils by merging ions with the same mass-to-charge ratio. Based on the molecular formula of vegetable oil triglycerides obtained in step S1, calculate their NH4+. + with Na + In the addition mode, the mass-to-charge ratio is used to combine ions with the same mass-to-charge ratio to form an exclusion list of ions. Step S3: Determine the oxidized triglycerides in vegetable oils; Step S4: Match the mass-to-charge ratio of the primary mass spectrometer to screen for suspected oxidized triglyceride molecules, and use secondary mass spectrometry to analyze the fatty acyl group composition, oxidized functional group type and oxidation position to determine the structure of the oxidized triglyceride molecule. The analysis of secondary mass spectrometry characteristic fragments includes: Fatty acyl group identification: based on diglyceride fragment ions formed by the loss of oxidized or unoxidized acyl groups; Determination of oxidized functional groups and their positions: Based on the characteristic neutral missing fragments, where: the loss of water molecules and the loss of 88 mass units correspond to the 13-position hydroperoxy group feature, and the loss of 140 mass units corresponds to the 9-position hydroperoxy group feature, used to determine the peroxide group; the loss of 83 or 100 mass units corresponds to the 12- and 13-position epoxy group features, and the loss of 124 or 140 mass units corresponds to the 9- and 10-position epoxy group features, used to determine the epoxy group.

2. The method for high-coverage analysis of oxidized triglycerides in vegetable oils based on list dependency pattern according to claim 1, characterized in that, In step S1, the fatty acids include palmitic acid with 16 carbon atoms and 0 double bonds, palmitoleic acid with 16 carbon atoms and 1 double bond, stearic acid with 18 carbon atoms and 0 double bonds, oleic acid with 18 carbon atoms and 1 double bond, linoleic acid with 18 carbon atoms and 2 double bonds, linolenic acid with 18 carbon atoms and 3 double bonds, arachidic acid with 20 carbon atoms and 0 double bonds, and arachidonic acid with 20 carbon atoms and 4 double bonds.

3. The method for high-coverage analysis of oxidized triglycerides in vegetable oils based on list dependency pattern according to claim 1, characterized in that, In step S1, the oxidation forms include hydroperoxides, epoxides, hydroxy compounds, and carbonyl compounds.

4. The method for high-coverage analysis of oxidized triglycerides in vegetable oils based on list dependency pattern according to claim 1, characterized in that, In step S3, the oxidized triglycerides in vegetable oil are determined, including the following steps: Chromatographic conditions: A C18 column was used. Mobile phase A was a mixture of water and methanol with a volume ratio of 95:5, and mobile phase B was a mixture of isopropanol and methanol with a volume ratio of 80:

20. 5 mmol of ammonium acetate and 0.1 mmol of sodium acetate were added, respectively. Gradient elution program: Initially, maintain 85% mobile phase B for 5 min; from 5 min to 10 min, linearly increase to 90% mobile phase B and maintain for 15 min; from 15 min to 25 min, linearly increase to 95% mobile phase B and maintain for 28 min; from 28 min to 35 min, linearly increase to 100% mobile phase B and maintain for 53 min; then restore 85% mobile phase B to equilibrium. Mass spectrometry conditions: Electrospray ionization positive ion mode, mass-to-charge ratio of 800 to 1200 across the full scan range, and secondary mass fragmentation triggered in the mass-to-charge ratio range of 100 to 1200 using list-first data-dependent acquisition mode.

5. The method for high-coverage analysis of oxidized triglycerides in vegetable oils based on list dependency pattern according to claim 4, characterized in that, The chromatographic conditions were as follows: column inner diameter 4.6 mm, length 250 mm, packing particle size 5 µm, flow rate 0.5 mL / min, column temperature 30 °C, and injection volume 1 μL.

6. The method for high-coverage analysis of oxidized triglycerides in vegetable oils based on list dependency pattern according to claim 4, characterized in that, In the mass spectrometry conditions, the desolvation line temperature range is 150℃ to 250℃, the nebulizer gas flow rate ranges from 2L / min to 7L / min, and the drying gas flow rate ranges from 3L / min to 10L / min.

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