Vegetable oil oxidized triglyceride high coverage analysis method based on list dependence mode

Abstract

The invention provides a vegetable oil oxidized triglyceride high coverage analysis method based on a list dependence mode, and belongs to the technical field of detection. The method comprises the following steps: S1, predicting the molecular formula of potential oxidized triglyceride in grease by adopting an enumeration method based on the fatty acyl composition of plant oil triglyceride and the oxidation product formation rule of the plant oil triglyceride; s2, constructing a vegetable oil non-targeted detection priority ion list and an exclusion ion list; S3, determining oxidized triglyceride in the vegetable oil; and S4, matching the mass-to-charge ratio of the first-stage mass spectrum, screening suspected oxidized triglyceride molecules, analyzing the composition of fatty acyl, the type of oxidation functional groups and the oxidation position through the characteristic fragments of the second-stage mass spectrum, and determining the fine structure of the oxidized triglyceride molecules. By adopting the vegetable oil oxidized triglyceride high-coverage analysis method based on the list dependence mode, high-coverage and high-sensitivity analysis of oxidized triglyceride in vegetable oil can be realized.

Description

Technical Field

[0001] The present invention relates to the field of detection technology, and in particular to a high-coverage analysis method for oxidized triglycerides in vegetable oil based on a list-dependent pattern. Background Art

[0002] Oxidized triglycerides are one of the core products of oil oxidation. The type of oxidized functional groups (e.g., hydroperoxy, epoxy, hydroxyl, carbonyl, etc.) and the sites of oxidation (e.g., in-situ and ortho-positions of double bonds) directly reflect the degree of oil oxidation and the differences in oxidation pathways. Deciphering the fine structure of oxidized triglycerides is crucial for tracking the dynamic progression of oil oxidation and elucidating its mechanisms. Non-targeted analytical methods based on liquid chromatography-mass spectrometry can perform indiscriminate screening of oil oxidation products and are widely used for the identification and screening of oil oxides.

[0003] The non-targeted analysis method based on liquid chromatography-mass spectrometry is based on the MS of the compound. 2 The fragment information in the spectrum is used to identify the molecular structure. Data-dependent mode is a widely used MS 2 Spectral acquisition mode, however, traditional data-dependent mode acquires compound MS based on ion intensity 2 Spectral information, that is, the parent ion with ion intensity greater than a certain threshold will be preferentially selected to collect its secondary mass spectrometry 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. The main substance in vegetable oil is triglyceride, and the target compound oxidized triglyceride has low abundance (especially in the early stage of oil oxidation), and the two have similar polarity, making it difficult to effectively separate them by chromatography and prone to co-elution. Due to the ion shielding effect of mass spectrometry, that is, high-abundance triglycerides are preferentially fragmented, resulting in MS of low-abundance oxidized triglycerides. 2 Low spectral acquisition rate and missing diagnostic ions result in low identification coverage and accuracy in the traditional non-targeted method for the determination of oxidized triglycerides in vegetable oils.

[0004] The data-dependent acquisition mode based on list priority prioritizes the parent ions in the ion list to acquire their MS 2 Spectra, which can effectively improve the MS of target compounds 2 The information acquisition rate is increased, thereby improving detection coverage and sensitivity. Furthermore, by setting an exclusion list of ions, interfering substances in complex samples can be effectively screened. However, list-based detection methods require the foreknowledge of the quasi-molecular ion information of the analyte to form the list. This strategy is quite challenging for the diverse and structurally unknown oxidized triglycerides in vegetable oils. Summary of the Invention

[0005] The purpose of the present invention is to provide a high coverage analysis method for oxidized triglycerides in vegetable oils based on a list-dependent model, which can achieve high coverage and high sensitivity analysis of oxidized triglycerides in vegetable oils.

[0006] To achieve the above object, the present invention provides a high coverage analysis method for vegetable oil oxidized triglycerides based on a list-dependent model, comprising the following steps:

[0007] Step S1: Based on the fatty acyl composition of vegetable oil triglycerides and the formation pattern of their oxidation products, an enumeration method is used to predict the molecular formula of potential oxidized triglycerides in the oil;

[0008] Step S2: Constructing a priority ion list and an exclusion ion list for non-targeted detection of vegetable oils:

[0009] Step S3, measuring the oxidized triglycerides in the vegetable oil;

[0010] Step S4: matching the mass-to-charge ratio of the primary mass spectrometry to screen suspected oxidized triglyceride molecules, analyzing the fatty acyl composition, oxidized functional group type and oxidation position through the secondary mass spectrometry characteristic fragments, and determining the fine structure of the oxidized triglyceride molecules.

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

[0012] Preferably, in step S1, the oxidized form includes hydroperoxides, epoxides, hydroxyl compounds and carbonyl compounds.

[0013] Preferably, in step S2, based on the molecular formula of the vegetable oil oxidized triglyceride obtained in step S1, its NH4 + with Na + In the adduct mode, ions with the same mass-to-charge ratio are combined to form a priority ion list for non-targeted detection of vegetable oils;

[0014] Based on the molecular formula of vegetable oil triglyceride obtained in step S1, calculate its NH4 + with Na + In the adduct mode, ions with the same mass-to-charge ratio are combined to form an excluded ion list.

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

[0016] Chromatographic conditions: A C18 column was used, mobile phase A was a mixture of water and methanol in a volume ratio of 95:5, and mobile phase B was a mixture of isopropanol and methanol in a volume ratio of 80:20, with 5 mmol of ammonium acetate and 0.1 mmol of sodium acetate added, respectively;

[0017] Gradient elution program: initially 85% mobile phase B for 5 min, linearly increased to 90% mobile phase B from 5 min to 10 min and maintained for 15 min, linearly increased to 95% mobile phase B from 15 min to 25 min and maintained for 28 min, linearly increased to 100% mobile phase B from 28 min to 35 min and maintained for 53 min, and restored to 85% mobile phase B equilibrium;

[0018] Mass spectrometry conditions: electrospray ionization positive mode, full scan range m / z 800 to 1200, list-priority data-dependent acquisition mode with triggered secondary mass spectrometry fragmentation in the m / z range of 100 to 1200.

[0019] Preferably, the chromatographic conditions are as follows: the inner diameter of the chromatographic column is 4.6 mm, the length is 250 mm, the filler particle size 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 spectrometry conditions, the desolvation line temperature ranges from 150° C. to 250° C., the nebulizing 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 spectrometry characteristic fragment analysis includes:

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

[0023] Determination of oxidized functional groups and positions: Based on the characteristic neutral loss fragments, the loss of water molecules and 88 mass units correspond to the 13-position hydroperoxy characteristics, and the loss of 140 mass units corresponds to the 9-position hydroperoxy characteristics, which are used to determine the peroxide group; the loss of 83 or 100 mass units corresponds to the 12- and 13-position epoxy characteristics, and the loss of 124 or 140 mass units corresponds to the 9- and 10-position epoxy characteristics, which are used to determine the epoxy group.

[0024] Therefore, the present invention adopts the above-mentioned high coverage analysis method of vegetable oil oxidized triglycerides based on the list-dependent model, and the beneficial technical effects are as follows: this method does not require complex pre-treatment and can achieve high coverage and high sensitivity detection of low-abundance oxidized triglycerides in vegetable oil. Compared with traditional non-targeted methods, oxidized triglyceride MS 2 The spectral acquisition rate was greatly improved, and the spectral information was significantly improved, which increased the detection rate of oxidized triglycerides in vegetable oils. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0026] Figure 2 Oxidized triglycerides in Na + Schematic diagram of fragment formation in adduct mode;

[0027] Figure 3 To illustrate the structural identification process of oxidized triglycerides, we use the precursor ion of m / z 912.7661 as an example. Figure 3 A in the figure is the extracted ion chromatogram (EIC) of m / z 912.7661. Figure 3 B in the MS is m / z 912.7661 at 38.2 min 2 Spectrum, Figure 3 The C in the MS is m / z 912.7661 at 38.3 min. 2 Spectrum, Figure 3 D in the MS is m / z 912.7661 at 38.6 min 2 Spectrum;

[0028] Figure 4 The precursor ion distribution diagrams of potential oxidized triglycerides obtained by the method proposed by the present invention and the traditional non-targeted scanning method, wherein: Figure 4 A in the present invention is the method, Figure 4 B in the figure is the traditional non-targeted scanning method;

[0029] Figure 5 The MS of m / z 927.6694 obtained by the method proposed in the present invention and the traditional non-targeted method respectively 2 Spectrum, where Figure 5 A in the present invention is the method, Figure 5 B in FIG is a traditional non-targeted scanning method. DETAILED DESCRIPTION

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

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

[0032] The equipment and materials used in the examples are as follows: an LCMS-9030 liquid phase mass spectrometer (Shimadzu, Japan) and a C18 chromatographic column (250 × 4.6 mm × 5 μm, Agilent); the mobile phases are methanol and isopropanol, and the detection object is walnut oil heated in an oven at 60°C for 15 h.

[0033] Example 1

[0034] (1) Construction of a database of molecular formulas of potential oxidized triglycerides 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, hydroperoxides (HOO-), epoxides (ep-), hydroxyl compounds (HO-), and carbonyl compounds (oxo-), were arranged and combined. Since the molecular formula was predicted, the configurational isomers were not considered. A total of 1295 potential oxidized triglycerides were predicted.

[0035] Construction of walnut oil triglyceride exclusion ion list and oxidized triglyceride priority ion list: 48 walnut oil triglycerides with the same molecular formula were merged to obtain 30 different triglyceride molecular formulas, and NH4 was matched for each molecular formula. + and Na + In addition mode, the corresponding quasi-molecular ion mass-to-charge ratio is obtained to form an excluded ion list (Table 1). In the list-priority data-dependent scanning mode, the mass spectrometer detector can shield the ions in the excluded ion list to reduce the collection of interfering ion information; the same molecular formula of the predicted potential oxidized triglycerides is merged and matched with NH4 + and Na + In addition mode, the corresponding quasi-molecular ion mass-to-charge ratio is obtained to form a priority ion list (Table 2 and Table 3). In the list priority data dependent scanning mode, the mass spectrometer detector can preferentially collect the MS of the parent ion in the list. 2 spectra, increasing the acquisition rate of molecular structure information of low-abundance oxidized triglycerides.

[0036] Table 1 List of excluded ions

[0037]

[0038]

[0039] Table 2NH4 + Priority ion list in adduct mode

[0040] Serial number Molecular formula Priority ions Serial number Molecular formula Priority ions 1 <![CDATA[C 53 H 94 O7]]> 860.7347 12 <![CDATA[C 57 H 90 O7]]> 904.7034 2 <![CDATA[C 53 H 96 O7]]> 862.7504 13 <![CDATA[C 57 H 92 O7]]> 906.7191 3 <![CDATA[C 53 H 98 O7]]> 864.766 14 <![CDATA[C 57 H 94 O7]]> 908.7347 4 <![CDATA[C 53 H 100 O7]]> 866.7817 15 <![CDATA[C 57 H 96 O7]]> 910.7504 5 <![CDATA[C 55 H 92 O7]]> 882.7191 16 <![CDATA[C 57 H 98 O7]]> 912.766 6 <![CDATA[C 55 H 94 O7]]> 884.7347 17 <![CDATA[C 57 H 100 O7]]> 914.7817 7 <![CDATA[C 55 H 96 O7]]> 886.7504 18 <![CDATA[C 57 H 102 O7]]> 916.7974 8 <![CDATA[C 55 H 98 O7]]> 888.766 19 <![CDATA[C 57 H 104 O7]]> 918.813 9 <![CDATA[C 55 H 100 O7]]> 890.7817 20 <![CDATA[C 57 H 106 O7]]> 920.8287 10 <![CDATA[C 55 H 102 O7]]> 892.7974 21 <![CDATA[C 57 H 108 O7]]> 922.8443 11 <![CDATA[C 55 H 104 O7]]> 894.813

[0041] Table 3Na + Priority ion list in adduct 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 isopropyl alcohol to make a 1 mg / mL solution, which is diluted to 0.1 mg / mL during measurement.

[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 °C; the injection volume was 1 μL; the flow rate was 0.5 mL / min; the mobile phase A was a mixed solution of water and methanol (v:v = 95:5), and the mobile phase B was a mixed solution of isopropanol and methanol (v:v = 80:20), with 5 mmol of ammonium acetate and 0.1 mmol of sodium acetate added for NH4 + and Na + Oxidized triglycerides were determined in the adduct mode; the gradient elution conditions were: starting with 85% mobile phase B, maintaining for 5 min, linearly increasing to 90% mobile phase B from 5 min to 10 min and maintaining for 15 min, then linearly increasing to 95% mobile phase B from 15 min to 25 min and maintaining for 28 min, linearly increasing to 100% mobile phase B from 28 min to 35 min and maintaining for 53 min, then restoring the initial concentration of 85% mobile phase B, and performing the next injection at equilibrium.

[0047] (5) Mass spectrometry detection conditions: Ionization was performed using an electrospray ion source in positive ion mode, a desolvation line temperature of 250°C, a nebulizer gas flow rate of 5 L / min, and a drying gas flow rate of 10 L / min. 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, the peak was extracted using the mass spectrometry software Labsolution, and the exact mass-to-charge ratio of the extracted molecular formula was matched with the predicted potential oxidized triglycerides to screen out the suspected oxidized triglyceride molecules; further, the ion MS was analyzed to determine the peak. 2 Characteristic fragments in the spectrum are used to annotate the fine structures of the oxidized triglyceride molecules, such as the fatty acyl composition, oxidized functional group type, and oxidized position ( Figure 1 、 Figure 2 , oxidized triglycerides in NH4 + and Na + Schematic diagram of fragment formation in adduct ion mode; Figure 3The molecular structure identification process of oxidized triglycerides was described using an ion with a mass-to-charge ratio of 912.7661 as an example. In this example, a list-priority model was used to successfully identify 109 oxidized triglyceride molecules in walnut oil. These molecules included six major categories of oxidative modification products: monoepoxy (36 species), monohydroperoxy (42 species), hydroperoxy-epoxy complex (16 species), dihydroperoxy (8 species), diepoxy (5 species), and hydroxy-epoxy (2 species). See Table 4 for details.

[0049]

[0050]

[0051]

[0052]

[0053]

[0054]

[0055]

[0056] (7) Compared with the traditional non-targeted method, the oxidized triglyceride MS of the method proposed in this invention is 2 Spectrum acquisition rate increased significantly: in traditional DDA mode ( Figure 4 ), potentially oxidized triglycerides accounted for only 7.3% of the precursor ions triggering fragmentation, with the remaining fragmentation events being background ions (e.g., solvent adducts and column bleed products). In contrast, in data collected using list-priority data-dependent mode, potentially oxidized triglycerides accounted for 90.7% of the fragmentation.

[0057] The method proposed by the present invention not only improves the MS of vegetable oil oxidized triglycerides 2 The spectrum information acquisition rate is improved significantly, and the spectrum quality is also significantly improved. Figure 5 As shown, for the oxidized triglycerides with low abundance in walnut oil, the traditional method or the MS 2 The spectrum has little fingerprint information about the molecular structure, or cannot trigger the fragmentation process of low-abundance oxidized triglyceride parent ions, resulting in MS 2 The spectrum is missing, but the method proposed in this invention can collect high-quality MS of low-abundance oxidized triglycerides in walnut oil. 2 Spectra are used for structural identification and to improve detection coverage, sensitivity and accuracy.

[0058] It is worth noting that the contents not elaborated in detail in the present invention are all prior art and are well known to those skilled in the art.

[0059] Therefore, the present invention adopts the above-mentioned high coverage analysis method of oxidized triglycerides in vegetable oil based on the list-dependent model, which can achieve high coverage and high sensitivity analysis of oxidized triglycerides in vegetable oil.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements 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 oil based on a list-dependent model, characterized in that: The following steps are involved: Step S1: Based on the fatty acyl composition of vegetable oil triglycerides and the formation pattern of their oxidation products, an enumeration method is used to predict the molecular formula of potential oxidized triglycerides in the oil; Step S2, constructing a priority ion list and an exclusion ion list for non-targeted detection of vegetable oil; Step S3, measuring the oxidized triglycerides in the vegetable oil; Step S4: matching the mass-to-charge ratio of the primary mass spectrometer to screen suspected oxidized triglyceride molecules, analyzing the fatty acyl composition, oxidized functional group type and oxidation position through the secondary mass spectrometer characteristic fragments, and determining the molecular structure of the oxidized triglyceride.

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

3. The high coverage analysis method for vegetable oil oxidized triglycerides based on list-dependent mode according to claim 1, characterized in that: In step S1, the oxidized forms include hydroperoxides, epoxides, hydroxyl compounds and carbonyl compounds.

4. The high coverage analysis method for vegetable oil oxidized triglycerides based on list-dependent mode according to claim 1, characterized in that: In step S2, based on the molecular formula of the vegetable oil oxidized triglyceride obtained in step S1, its NH4 + with Na + In the adduct mode, ions with the same mass-to-charge ratio are combined to form a priority ion list for non-targeted detection of vegetable oils; Based on the molecular formula of vegetable oil triglyceride obtained in step S1, calculate its NH4 + with Na + In the adduct mode, ions with the same mass-to-charge ratio are combined to form an excluded ion list.

5. The high coverage analysis method for vegetable oil oxidized triglycerides based on list-dependent mode according to claim 1, characterized in that: In step S3, the oxidized triglycerides in the vegetable oil are measured, comprising the following steps: Chromatographic conditions: A C18 column was used, mobile phase A was a mixture of water and methanol in a volume ratio of 95:5, and mobile phase B was a mixture of isopropanol and methanol in a volume ratio of 80:20, with 5 mmol of ammonium acetate and 0.1 mmol of sodium acetate added, respectively; Gradient elution program: initially 85% mobile phase B for 5 min, linearly increased to 90% mobile phase B from 5 min to 10 min and maintained for 15 min, linearly increased to 95% mobile phase B from 15 min to 25 min and maintained for 28 min, linearly increased to 100% mobile phase B from 28 min to 35 min and maintained for 53 min, and restored to 85% mobile phase B equilibrium; Mass spectrometry conditions: electrospray ionization positive mode, full scan range m / z 800 to 1200, list-priority data-dependent acquisition mode with triggered secondary mass spectrometry fragmentation in the m / z range of 100 to 1200.

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

7. The high coverage analysis method for vegetable oil oxidized triglycerides based on list-dependent mode according to claim 4, characterized in that: In the mass spectrometry conditions, the desolvation line temperature ranged from 150°C to 250°C, the nebulizing gas flow rate ranged from 2 L / min to 7 L / min, and the drying gas flow rate ranged from 3 L / min to 10 L / min.

8. The high coverage analysis method for vegetable oil oxidized triglycerides based on list-dependent mode according to claim 4, characterized in that: Secondary mass spectrometry characteristic fragment analysis includes: Fatty acyl group determination: based on the diacylglycerol fragment ions formed by the loss of oxidized or unoxidized acyl groups; Determination of oxidized functional groups and positions: Based on the characteristic neutral loss fragments, the loss of water molecules and 88 mass units correspond to the 13-position hydroperoxy characteristics, and the loss of 140 mass units corresponds to the 9-position hydroperoxy characteristics, which are used to determine the peroxide group; the loss of 83 or 100 mass units corresponds to the 12- and 13-position epoxy characteristics, and the loss of 124 or 140 mass units corresponds to the 9- and 10-position epoxy characteristics, which are used to determine the epoxy group.

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