Method for detecting diglyceride compounds based on chemical derivatization

Abstract

The invention belongs to the technical field of detection and analysis, and particularly relates to a method for detecting diglyceride compounds based on chemical derivatization. The invention discloses application of a chemical derivative shown in a structural formula 1 in mass spectrometry or liquid chromatography-mass spectrometry of diglyceride substance derivatization treatment, one end of the chemical derivative is a carboxyl group, the other end of the chemical derivative is a compound of NR2, the molecular structural formula 1 is NR2-Ph-COOH or NR2-Ph-CH2-COOH, R is selected from H,-CH3,-CH2CH3 and-CH2CH2CH3, and R is selected from H,-CH3,-CH2CH3 and-CH2CH2CH3. The-CH (CH3) 2 carbon chain length is less than 3, and Ph is a benzene ring. The method can be used for derivatization treatment before detection of the diglyceride compounds, the mass spectrum response abundance is improved, and detection analysis of the diglyceride compounds is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of detection and analysis, and specifically relates to a method for detecting diglyceride compounds based on chemical derivatization. Background Art

[0002] Diacylglcerol (DAG) is a type of structural lipid in which a fatty acid in a triglyceride is replaced by a hydroxyl group. It is widely present in edible vegetable oils, with a content of no more than 10%. Diacylglcerol is mainly composed of two isomers: 1,3-diacylglycerol (1,3-DAG) and 1,2-diacylglycerol (1,2-DAG). The chemical properties of 1,3-diacylglycerol are more stable, and the ratio of the two isomers in natural vegetable oils is about 7:3. The safety of diacylglycerol has been extensively scientifically evaluated worldwide. The U.S. Food and Drug Administration (FDA) has listed it as a generally recognized as safe ingredient (GARS). my country listed diacylglycerol oil as a new resource food in 2009, and revised the product quality standard requirements for diacylglycerol oil in 2021, using soybean oil, rapeseed oil, etc. as raw materials, lipase preparations, water, glycerin, etc. as main auxiliary materials, and the diacylglycerol content should be greater than or equal to 40%. Diacylcerol oil is mainly synthesized by oil chemical method or enzymatic modification, and then made by distillation separation, decolorization, deodorization and other processes. It is a product of hydrolysis of one bond of triglyceride compound or esterification condensation reaction of one molecule of glycerol and two molecules of fatty acid. Diacylcerol compound has a different metabolic pathway from triglyceride (TAG). TAG is hydrolyzed into 2-monoglyceride, which is synthesized into triglyceride by pancreatic lipase in small intestinal mucosal cells and accumulates in human liver and adipose tissue, while 1,3-diacylcerol is hydrolyzed into 1-monoglyceride. Due to the lack of esterification synthase that directly acts on 1-monoglyceride in small intestinal mucosal cells, it cannot be converted into triglyceride and accumulates in the body, and is finally converted into energy through β-oxidation. Diacylcerol is a low-calorie alternative lipid to natural TAG. Intake of equal mass of diacylglycerol and TAG can provide the human body with considerable energy to maintain normal life activities, but the calorie of diacylglycerol is 30% lower than that of TAG. Diacylcerol has significant differences in metabolic pathways from TAG and can regulate the expression of genes related to lipase oxidation. It has a wide range of physiological activities and nutritional functions, can reduce body fat, lower blood lipids, inhibit body weight, and reduce the incidence of chronic syndromes such as diabetes, fatty liver, and cardiovascular diseases. Therefore, studying the composition and content distribution of diacylglycerol in edible vegetable oils is of great significance to evaluating the important role of diacylglycerol in human health.

[0003] At present, the main methods for detecting diglycerides are thin layer chromatography, nuclear magnetic resonance, near infrared spectroscopy, high performance liquid chromatography, etc. Thin layer chromatography uses silica gel plates to separate diglycerides from actual samples based on the difference in polarity of the analyte, which is suitable for qualitative analysis. Nuclear magnetic resonance is a spectral technology based on the absorption of radio frequency radiation by atomic nuclei. By comparing the characteristic indicators of camellia oil diglyceride, a quantitative relationship between its characteristic indicators and content is established, thereby realizing the monitoring of diglyceride components. Near infrared spectroscopy is a simple, rapid and non-destructive detection technology. By detecting the infrared absorption spectrum of diglyceride, a model is established to analyze the diglyceride component and total amount in vegetable oil. High performance liquid chromatography-ultraviolet spectroscopy first atomizes the test liquid to form an aerosol, evaporates the solvent in a heated drift tube, and then analyzes the content of diglyceride. This technology has stable repeatability and high accuracy. In recent years, ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) has been widely used in the quantitative detection of small molecule compounds due to its advantages such as excellent separation performance of liquid chromatography and high selectivity and high sensitivity of mass spectrometry.

[0004] Previous studies have shown that chemical derivatization is often used to increase the volatility of gas chromatography analytes, or to enhance the signal of liquid chromatography analysis, and to introduce chromophores to facilitate ultraviolet or fluorescence detection. With the rapid development of liquid chromatography-mass spectrometry technology, the commonly used electrospray ionization source (ESI) is suitable for the detection of most compounds. However, some compounds are still difficult to separate and detect. The main reasons are: (1) the lack of ionized groups in the molecular structure makes it difficult to achieve efficient ionization; (2) the chemical properties are active and have unstable functional groups; (3) the ionization efficiency of different substances of the same type varies greatly, and mass spectrometry analysis interferes with each other; (4) the compound content is low, the pretreatment steps are cumbersome, and the sensitivity is poor, making it difficult to perform quantitative analysis using conventional liquid chromatography-mass spectrometry detection technology. For mass spectrometry analysis of such substances, in addition to relying on the development and breakthrough of interface technology, introducing easily ionized groups through chemical derivatization to change their physical and chemical properties is an efficient sensitivity enhancement technology. Derivatization reaction is a specific chemical reaction between the reactive groups in the target compound and the derivatization reagent, which converts the target compound into a substance with a similar chemical structure. The modified groups in the derivatization reagent can enhance the specificity of the target compound and has been applied to the precise quantitative analysis of small molecule compounds such as free fatty acids, phenolic acids, and sterols.

[0005] In view of the bottleneck problems of low ionization efficiency and poor separation in liquid chromatography-mass spectrometry detection of diacylglycerol, it is of great significance to develop a chemical derivatization analysis method for detecting diacylglycerol. Summary of the invention

[0006] The purpose of the present invention is to provide a chemical derivatization agent for detecting diglycerides and a method for detecting diglycerides based on the chemical derivatization agent in order to solve the bottleneck problem of diglyceride detection technology in the prior art. The chemical derivatization agent can be used for the detection and analysis of diglycerides, solving the problems of poor mass spectrometry response, low chromatographic separation and low sensitivity of diglycerides.

[0007] In order to achieve the above-mentioned invention object, the present invention adopts the following technical scheme:

[0008] The chemical derivatization agent described in structural formula 1 is used in the derivatization treatment of diglyceride substances for mass spectrometry analysis or liquid chromatography-mass spectrometry analysis. The chemical derivatization agent has a carboxyl group at one end and a NR2 compound at the other end. Structural formula 1 is: NR2-Ph-COOH or NR2-Ph-CH2-COOH, wherein the R group is selected from H, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2 and other alkane substituent groups with a carbon chain length of less than 3, and Ph is a benzene ring.

[0009] According to the above scheme, the chemical derivatizing agent is 4-diethylaminobenzoic acid, 4-dimethylaminophenylacetic acid, 4-aminobenzoic acid, 4-dimethylaminobenzoic acid or 3-dimethylaminobenzoic acid.

[0010] According to the above scheme, the chemical derivatization reagent is preferably 3-dimethylaminobenzoic acid or 4-dimethylaminobenzoic acid. More preferably, 3-dimethylaminobenzoic acid is preferred. 3-Dimethylaminobenzoic acid or 4-dimethylaminobenzoic acid not only introduces an easily ionized tertiary amine group, but also increases the hydrophobic group benzene ring, so that the mass spectrometry sensitivity of 1,3-diaceryl compounds derived from N,N-dimethylbenzoic acid is improved, the liquid phase separation is improved, and the separation time is shortened. Compared with the underivatized 1,3-diaceryl, the quantitative or qualitative parent ion of the diglyceryl after chemical derivatization is [M+H] + or [M+NH4] + , the ions are easy to carry charge and have high cleavage efficiency. Based on the analysis of the cleavage rules of N,N-dimethylbenzoic acid diglyceride derivative ions, the cleavage fragments of N,N-dimethylbenzoic acid groups can be used as qualitative and / or quantitative ions, that is, the substituted aminobenzoic acid group fragment ions of the products produced during the cleavage of the chemical derivatizer. When the chemical derivatizer is 3-dimethylaminobenzoic acid or 4-dimethylaminobenzoic acid, it is usually the substituted aminobenzoyl ion [C9H 10 NO]· or substituted aminophenyl ion [C8H 13 N]·, substituted aminophenyl acid glyceride derivatives qualitative and / or quantitative ion fragments substituted aminobenzoyl ion [C9H 10 NO]· or substituted aminophenyl ion [C8H 13N]· was significantly higher than that of the underivatized diglycerides and aromatic acid derivatives.

[0011] According to the above scheme, the mass spectrometry analysis or liquid chromatography-mass spectrometry analysis uses a positive ion mode, and the quantitative or qualitative parent ion is [M+H] + or [M+NH4] + The daughter ions are the fragment ions generated during the cleavage of diglyceride derivatives derived from chemical derivatization agents. For example, when the chemical derivatization agent is 3-dimethylaminobenzoic acid or 4-dimethylaminobenzoic acid, the corresponding daughter ions are [C9H 10 NO] or [C8H 13 N]·.

[0012] The chemical derivatizing agent of the present invention has strong esterification activity under mild derivatization conditions, and can be used for derivatization treatment of diglyceride compounds before liquid chromatography mass spectrometry detection (the hydroxyl group of diglyceride and the carboxyl group in the chemical derivatizing agent are coupled and esterified, a molecule of water is removed and an ester bond is formed), and the abundance of mass spectrometry response of specific ion fragmentation can be improved. The diglyceride derivative is easy to carry a charge in the positive ion mode, and the mass spectrometry response signal is significantly higher than that in the negative ion mode. Based on the diglyceride derivative compound [M+H] derivatized by the derivatizing agent + or [M+NH4] + The parent ion is the fragment ion of the product produced during the cracking process of the diglyceride derivative, and the quantitative and / or directional analysis and detection of the diglyceride compound is realized, and the operation is simple.

[0013] On this basis, the present invention further provides a method for detecting diglyceride substances in a sample, wherein the sample is chemically derivatized with a chemical derivatizing agent of the present invention, such as converting diglyceride substances in a vegetable oil sample into N,N-substituted benzoic acid diglyceride compounds in an equal molar ratio, and then detected by UHPLC-MS / MS to select N,N-substituted benzoic acid diglyceride compounds [M+H] + or [M+NH4] + As the parent ion, its specific ion is used as the quantitative ion to analyze the diglyceride content. The specific ion refers to the fragment ion of 3-dimethylaminobenzoic acid produced when the diglyceride derived from 3-dimethylaminobenzoic acid is fragmented by mass spectrometry. It has the advantages of simple operation, high sensitivity, large detection flux, and applicability to a variety of matrix types, and realizes the simultaneous quantitative detection of diglyceride analytes in complex oil samples.

[0014] The present invention further provides a method for detecting diacylglycerol substances in a vegetable oil sample, the main steps of which include: (1) pretreatment of the vegetable oil sample, (2) chemical derivatization treatment of the sample using the chemical derivatization agent described in the above structural formula 1, and (3) analysis of diacylglycerol substances based on mass spectrometry or liquid chromatography-mass spectrometry after the chemical derivatization treatment.

[0015] According to the above scheme, the chemical derivatization to the hydroxyl group of diglycerol is coupled with the chemical derivatization agent described in structural formula 1 for esterification, the chemical derivatization conditions are the conditions for coupling esterification of small molecule compounds, the derivatized chemical functional group is the hydroxyl group of diglycerol, the catalyst for coupling esterification can be 4-dimethylaminopyridine (DMAP), and the esterification coupling agent can be 1-ethyl-(3-diaminopropyl)-3-ethylcarbodiimide (EDC).

[0016] According to the above scheme, the reaction solvent of the above coupling esterification is acetonitrile, methanol, acetone, ethyl acetate, isopropanol or dichloromethane, and the reaction temperature is 70-80°C.

[0017] According to the above scheme, the above detection and analysis process is to select N, N-substituted benzoic acid diglyceride compounds [M+H] + or [M+NH4] + The specific ion used as the parent ion is used as the quantitative ion to analyze the diacylglycerol content. The specific ion refers to the fragment ion of 3-dimethylaminobenzoic acid generated when the diacylglycerol derived from 3-dimethylaminobenzoic acid is fragmented in the mass spectrometer.

[0018] According to the above scheme, the vegetable oil samples are vegetable oils such as rapeseed oil, soybean oil, peanut oil, corn oil, linseed oil, olive oil, and diglyceride oils made from the above vegetable oils as raw materials.

[0019] According to the above scheme, the diglyceride compounds include 1,3-diglyceride and 1,2-diglyceride; the 1,3-diglyceride includes 1-palmitoyl-3-stearic acid diglyceride, 1-palmitoyl-3-oleic acid diglyceride, 1-palmitoyl-3-linoleic acid diglyceride, 1-palmitoyl-3-linolenic acid diglyceride, 1-stearic acid-3-oleic acid diglyceride, 1-linoleic acid-3-linolenic acid diglyceride, 1,3-dipalmitoyl-3-stearic acid diglyceride 1,3-distearic acid diglyceride, 1,3-dioleic acid diglyceride, 1,3-dilinoleic acid diglyceride, 1,3-dilinolenic acid diglyceride, 1,3-undecenoic acid diglyceride, 1,3-dodecenoic acid diglyceride, 1,3-tridecanoic acid diglyceride, 1,3-tetradecanoic acid diglyceride, 1,3-tetradecenoic acid diglyceride, 1,3-pentadecanoic acid diglyceride, 1,3-heptadecanoic acid diglyceride , 1,3-heptadecenoic acid diglyceride, 1,3-eicosanoic acid diglyceride, 1,3-heneicosanoic acid diglyceride, 1-myristic acid-3-lauric acid diglyceride, 1-myristic acid-3-docosanoic acid diglyceride, 1-myristic acid-3-oleic acid diglyceride, 1-palmitic acid-3-eicosanoic acid diglyceride, 1-stearic acid-3-eicosanoic acid diglyceride, 1-oleic acid-3-eicosanoic acid diglyceride; the 1,2- The diglycerides include 1-myristic acid-2-lauric acid diglyceride, 1-palmitic acid-2-lauric acid diglyceride, 1,2-palmitic acid diglyceride, 1-palmitic acid-2-stearic acid diglyceride, 1-oleic acid-2-palmitic acid diglyceride, 1,2-dioleic acid diglyceride, 1-oleic acid-2-palmitic acid diglyceride, 1-stearic acid-2-lauric acid diglyceride, 1,2-stearic acid diglyceride, and 1,2-heptadecanoic acid diglyceride.

[0020] According to the above scheme, step (3) is a method for quantitatively detecting diglycerides by UHPLC-MS / MS. When the present invention uses UHPLC-MS / MS analysis, the specific analysis conditions can be: the mobile phase is 0.1% formic acid acetonitrile and 0.1% formic acid solution, gradient program elution, the chromatographic separation column is C18 (100×2.1mm, 3.0μm), flow rate: 250μL / min; mass spectrometry analysis conditions: triple tandem quadrupole mass spectrometer, ESI ion source, positive ion mode, quantitative parent ion is [M+H] + or [M+NH4] + The daughter ions are fragment ions produced during the cleavage of diacylglycerol derivatives derived from chemical derivatization agents.

[0021] Beneficial effects of the present invention:

[0022] The present invention uses an aminophenyl acid compound as a chemical derivatization reagent with strong esterification activity under mild derivatization conditions, and is used for the derivatization of diglyceride substances. After derivatization treatment, the diglyceride substances in the sample are converted into N,N-substituted phenyl acid diglyceride compounds, which can be used for the detection of diglyceride substances, and have the advantages of high detection sensitivity, large detection throughput, and being applicable to a variety of matrix types.

[0023] The esterification reaction between the aminophenyl acid compound and the hydroxyl group of diglyceride in the present invention has strong specificity, mild conditions, high derivatization efficiency and high yield, which is beneficial to reducing the matrix interference caused by lipid components in the sample to mass spectrometry detection.

[0024] The present invention adopts chemical derivatization to enhance the sensitivity, improve the mass spectrometer response, and improve the separation, and establishes a diglyceride detection and analysis method. The diglyceride UHPLC-MS / MS detection method established in this way can be applied to the qualitative and / or quantitative detection of diglycerides in complex oil matrix samples, which is of great significance for clarifying the composition and content distribution of diglycerides in edible vegetable oils and accurately evaluating the nutritional function of diglycerides.

[0025] The detection and analysis method of diglyceride compounds in vegetable oil samples provided by the present invention converts diglyceride substances into N, N-substituted phenyl acid diglyceride compounds in equal molar ratios through chemical derivatization, which can achieve baseline separation and no interference from impurity peaks. It can not only improve the mass spectrometry ionization efficiency, realize the detection of diglyceride compounds, and improve the detection sensitivity of diglyceride compounds, but also improve the detection flux of diglyceride compounds. In combination with UHPLC-MS / MS technology, the content of diglyceride compounds in samples can be quickly and accurately quantitatively analyzed, including vegetable oils such as rapeseed oil, soybean oil, peanut oil, corn oil, linseed oil, olive oil, and diglyceride oils made from the above vegetable oils as raw materials, with the advantages of simple operation, high sensitivity, large detection flux, and applicable to many types of oil matrixes. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the mass spectrometry fragmentation rule of diglyceride derivatives.

[0027] Figure 2 It is the molecular structure of diglyceride.

[0028] Figure 3 This is the multiple reaction monitoring (MRM) chromatogram of 1,3-dioleylglycerol derived from 4-diethylaminobenzoic acid.

[0029] Figure 4 It is the MRM chromatogram of 1-myristic acid-3-oleic acid diglyceride and 1-palmitic acid-3-oleic acid diglyceride derived from 4-dimethylaminophenylacetic acid.

[0030] Figure 5 It is the MRM chromatogram of 1-palmitoyl-3-stearic acid diglyceride, 1,3-dioleic acid diglyceride, 1-myristic acid-3-oleic acid diglyceride, and 1-palmitoyl-3-oleic acid diglyceride derived from 4-aminobenzoic acid.

[0031] Figure 6 It is the MRM chromatogram of 1-palmitoyl-3-stearic acid diglyceride, 1,3-dioleic acid diglyceride, 1-myristic acid-3-oleic acid diglyceride, 1-palmitoyl-3-oleic acid diglyceride, 1-palmitoyl-3-linolenic acid diglyceride, and 1-linoleic acid-3-linolenic acid diglyceride derived from 3-dimethylaminobenzoic acid.

[0032] Figure 7 It is the MRM chromatogram of 1-palmitoyl-3-stearic acid diglyceride, 1,3-dioleic acid diglyceride, 1-myristic acid-3-oleic acid diglyceride, 1-palmitoyl-3-oleic acid diglyceride, 1-palmitoyl-3-linolenic acid diglyceride, and 1-linoleic acid-3-linolenic acid diglyceride derived from 4-dimethylaminobenzoic acid.

[0033] Figure 8 The MRM chromatograms of the 3-dimethylaminobenzoic acid derivative of 1,3-dioleylglycerol and the underivatized 1,3-dioleylglycerol are shown in Figure 1. Among them, a is the MRM chromatogram before derivatization, and b is the MRM chromatogram after derivatization with the 3-dimethylaminobenzoic acid derivative.

[0034] Fig. 9 The MRM chromatograms of the 3-dimethylaminobenzoic acid derivative of 1-oleoyl-3-eicosanoyl diglycerol and the underivatized 1-oleoyl-3-eicosanoyl diglycerol are shown in Figure 1. Among them, a is the MRM chromatogram before derivatization, and b is the MRM chromatogram after derivatization with the 3-dimethylaminobenzoic acid derivative.

[0035] Fig.10 It is the MRM chromatogram of diacylglycerol derivatives. DETAILED DESCRIPTION

[0036] The principles and features of the present invention are described below in conjunction with the accompanying drawings and specific embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0037] The present invention provides the use of a chemical derivatizing agent as described in structural formula 1 in the derivatization treatment of diglyceride substances for liquid chromatography-mass spectrometry analysis. The chemical derivatizing agent has a carboxyl group at one end and a NR2 compound at the other end. The molecular structural formula 1 is: NR2-Ph-COOH or NR2-Ph-CH2-COOH, wherein the R group is selected from H, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2 and other alkane substituent groups with a carbon chain length of less than 3, and Ph is a benzene ring.

[0038] In some specific embodiments, the chemical derivatizing agent is 4-diethylaminobenzoic acid, 4-dimethylaminophenylacetic acid, 4-aminobenzoic acid, 4-dimethylaminobenzoic acid or 3-dimethylaminobenzoic acid.

[0039] The present invention also provides a method for detecting diacylglycerol substances in a vegetable oil sample, the main steps of which include: (1) pretreatment of the vegetable oil sample, (2) chemical derivatization treatment of the sample using the chemical derivatization agent described in the above structural formula 1, and (3) analysis of diacylglycerol substances based on mass spectrometry or liquid chromatography-mass spectrometry after the chemical derivatization treatment.

[0040] Furthermore, the chemical derivatization is a hydroxyl group of diglycerol ester and a chemical derivatization agent described in formula 1 for coupling esterification, the chemical derivatization conditions are the coupling esterification conditions of small molecule compounds, the derived chemical functional group is the hydroxyl group of diglycerol ester, in some embodiments, the coupling esterification catalyst is 4-dimethylaminopyridine (DMAP), and the esterification coupling agent can be 1-ethyl-(3-diaminopropyl)-3-ethylcarbodiimide (EDC).

[0041] The reaction solvent of the above coupling esterification is acetonitrile, methanol, acetone, ethyl acetate, isopropanol, dichloromethane, etc., and the reaction temperature is 70-80°C.

[0042] The above-mentioned diglyceride compounds include 1,3-diglyceride and 1,2-diglyceride, and the structural formula is shown as follows: Figure 2As shown; the 1,3-diglycerol includes 1-palmitoyl-3-stearic acid diglyceride, 1-palmitoyl-3-oleic acid diglyceride, 1-palmitoyl-3-linoleic acid diglyceride, 1-palmitoyl-3-linolenic acid diglyceride, 1-stearic acid-3-oleic acid diglyceride, 1-linoleic acid-3-linolenic acid diglyceride, 1,3-dipalmitoyl-3-stearic acid diglyceride, 1,3-dioleic acid diglyceride, 1,3-dilinolenic acid diglyceride, 1,3-undecenoic acid diglyceride, 1,3-dodecenoic acid diglyceride, 1,3-tridecanoic acid diglyceride, 1,3-tetradecanoic acid diglyceride, 1,3-tetradecenoic acid diglyceride, 1,3-pentadecanoic acid diglyceride, 1,3-hept ... 3-eicosanoic acid diglyceride, 1,3-heneicosanoic acid diglyceride, 1-myristic acid-3-lauric acid diglyceride, 1-myristic acid-3-behenic acid diglyceride, 1-myristic acid-3-oleic acid diglyceride, 1-palmitic acid-3-eicosanoic acid diglyceride, 1-stearic acid-3-eicosanoic acid diglyceride, 1-oleic acid-3-eicosanoic acid diglyceride; the 1,2-glyceride includes 1- Myristic acid-2-lauric acid diglyceride, 1-palmitic acid-2-lauric acid diglyceride, 1,2-palmitic acid diglyceride, 1-palmitic acid-2-stearic acid diglyceride, 1-oleic acid-2-palmitic acid diglyceride, 1,2-dioleic acid diglyceride, 1-oleic acid-2-palmitic acid diglyceride, 1-stearic acid-2-lauric acid diglyceride, 1,2-stearic acid diglyceride, 1,2-heptadecanoic acid diglyceride.

[0043] In some embodiments, the liquid chromatography mass spectrometry analysis uses a positive ion mode, and the quantitative or qualitative precursor ion is [M+H] + or [M+NH4] + The daughter ions are the fragment ions generated during the cleavage of diglyceride derivatives derived from chemical derivatization agents. For example, when the chemical derivatization agent is 3-dimethylaminobenzoic acid or 4-dimethylaminobenzoic acid, the corresponding daughter ions are [C9H 10 NO] or [C8H 13 N]·.

[0044] In a specific embodiment, the main steps of the method for quantitatively detecting diacylglycerol compounds based on chemical derivatization UHPLC-MS / MS of the present invention include: (1) pretreatment of vegetable oil samples, (2) chemical derivatization treatment, and (3) quantitative analysis of diacylglycerol compounds. The specific operation of the vegetable oil sample pretreatment step 1 is as follows: weigh 0.50g of sample, add 5mL methyl tert-butyl ether / methanol / water (v:v:v, 10:3:2.5) and 1.5mL methanol, ultrasonically mix, add 1.25mL ultrapure water, ultrasonically mix, centrifuge at 4500rpm for 5min, take 100μL of the upper clear liquid, blow it to near dryness with nitrogen, and place it in a 10mL reaction tube for derivatization treatment.

[0045] Step 2 Chemical derivatization is the esterification of the hydroxyl group of diglyceride with substituted amino acid benzoic acid: 0.5 mL of 3-dimethylaminobenzoic acid solution (concentration 4 mg mL -1 ), 0.5 mL 4-dimethylaminopyridine solution (concentration 2 mg mL -1 ), 0.5 mL 1-ethyl-(3-dimethylaminopropyl) solution (concentration 5 mg mL -1 ) was added to the reaction tube and heated to 75°C for 5 h. 1 mL of the sample was blown almost dry with liquid nitrogen, re-dissolved in 0.5 mL of acetonitrile, and passed through a 0.22 μm organic membrane for UHPLC-MS / MS analysis.

[0046] Step 3 Analysis of the instrumental analysis parameters of UHPLC-MS / MS for diglyceride in the sample: the chromatographic column is ThermoHypersil GOLD (100×2.1 mm, 3.0 μm), the flow rate is 0.25 mL / min, the injection volume is 1 μL, the column temperature is 40°C, the nebulizer gas flow rate is 3 L / min, the interface temperature is 300°C, and the DL temperature is 250°C; the heating block temperature is 400°C; the mobile phases A and B of the ultra-high performance liquid chromatography (UHPLC) are 0.1% formic acid acetonitrile solution and 0.1% formic acid solution, respectively, and the gradient elution program is shown in Table 1.

[0047] Table 1 UHPLC-MS / MS chromatographic gradient elution program

[0048]

[0049] Mass spectrometry analysis conditions: triple quadrupole mass spectrometer; ESI ion source; positive ion mode; collision energy of the optimized 1,3-dioleyl diglyceride derivative (m / z 786.20→123.15Da): -46eV; ion pair before derivatization of 1-oleyl-3-eicosanoyl diglyceride (m / z 799.15→148.15Da): -46eV; ion pair of underivatized 1-oleyl-3-eicosanoyl diglyceride (m / z 669.10→339.30Da): -52eV.

[0050] The diglycerides in the vegetable oil samples were converted into N,N-substituted benzoic acid diglycerides or N,N-substituted phenylacetic acid diglycerides in equal molar ratios by chemical derivatization. The MS detection selected N,N-substituted benzoic acid diglycerides or N,N-substituted phenylacetic acid diglycerides [M+H] + or [M+NH4] + As the parent ion, its specific ion is used as the quantitative ion to analyze the diglyceride content. The specific ion is the fragment ion of N,N-substituted benzoic acid generated during mass spectrometry fragmentation of N,N-substituted benzoic acid diglyceride compounds or N,N-substituted phenylacetic acid diglyceride compounds.

[0051] Different derivatization reagents have different derivatization activities, and the diacylglycerol derivatives after derivatization also have different mass spectrometry response abundances.

[0052] The following are typical examples of chemical derivatization of different N,N-substituted benzoic acids or N,N-substituted phenylacetic acids:

[0053] 4-Diethylaminobenzoic acid was used to derivatize 1,3-dioleylglycerol and detected by UHPLC-MS / MS ( Figure 3 ).

[0054] 1-Myristic acid-3-oleic acid diglyceride and 1-palmitic acid-3-oleic acid diglyceride were derivatized with 4-dimethylaminophenylacetic acid and detected by UHPLC-MS / MS ( Figure 4 ).

[0055] 4-Aminobenzoic acid was used as a chemical derivatizing agent to derivatize 1-palmitoyl-3-stearic acid diglyceride, 1,3-dioleic acid diglyceride, 1-myristic acid-3-oleic acid diglyceride and 1-palmitoyl-3-oleic acid diglyceride, and the results were detected by UHPLC-MS / MS. Figure 5 ).

[0056] 3-Dimethylaminobenzoic acid and 4-dimethylaminobenzoic acid can derive various typical diglyceride compounds, realizing the simultaneous quantitative detection of diglyceride analytes in complex oil samples. The present invention lists six typical diglyceride compounds ( Figure 6 and 7 ), including 1-palmitoyl-3-stearic acid diglyceride, 1,3-dioleic acid diglyceride, 1-myristic acid-3-oleic acid diglyceride, 1-palmitoyl-3-oleic acid diglyceride, 1-palmitoyl-3-linolenic acid diglyceride, and 1-linoleic acid-3-linolenic acid diglyceride.

[0057] Compared with 4-dimethylaminobenzoic acid, the meta-carboxyl group of 3-dimethylaminobenzoic acid has strong acidity, which is conducive to further improving the derivatization efficiency, mass spectrometry ionization efficiency and signal response, and further improving the chromatographic separation.

[0058] Direct detection of diglyceride compounds is difficult to ionize due to the high polarity of diglyceride, resulting in failure to detect or low detection sensitivity. Taking 1,3-dioleyl diglyceride and 1-oleyl-3-eicosanoyl diglyceride as examples, the detection limit of 1,3-dioleyl diglyceride before derivatization is 5 mg kg -1 After derivatization with the chemical derivatizing agent 3-dimethylaminobenzoic acid of the present invention, the detection limit is 4 μg kg -1 The chromatographic results are as follows: baseline separation, no impurity interference, and a significant increase in sensitivity by 3 orders of magnitude. The chromatographic results in multiple reaction ion monitoring mode (MRM) are as follows: Figure 8 As shown; for 1-oleoyl-3-eicosanoylglycerol diester, the background interference is large and the sensitivity is low before derivatization. After derivatization with the chemical derivatizing agent 3-dimethylaminobenzoic acid of the present invention, the MRM chromatographic results are as follows Fig. 9 As shown, the detection limit was 4 μg kg -1 , the chromatographic peak shape is symmetrical and there are no interfering peaks.

[0059] In summary, the present invention provides a method for detecting diglyceride compounds in vegetable oil samples, and establishes a chemical derivatization method based on a benzoic acid compound or a phenylacetic acid compound derivatizing agent, which has the advantages of simple operation, high sensitivity, applicability to a variety of matrix types, and large detection flux. Therefore, the present invention solves the technical problems of low ionization efficiency and poor separation in the prior art and has broad industrial application prospects.

[0060] The present invention embodiment lists 30 kinds of diglyceride compounds ( Fig.10), including 1-palmitoyl-3-stearic acid diglyceride, 1-palmitoyl-3-oleic acid diglyceride, 1-palmitoyl-3-linoleic acid diglyceride, 1-palmitoyl-3-linolenic acid diglyceride, 1-stearic acid-3-oleic acid diglyceride, 1-linoleic acid-3-linolenic acid diglyceride, 1,3-stearic acid diglyceride, 1,3-oleic acid diglyceride, 1,3-linoleic acid diglyceride, 1,3-linolenic acid diglyceride, 1-myristic acid-3-lauric acid diglyceride, 1-myristic acid-3-behenic acid diglyceride, 1-myristic acid-3-oleic acid diglyceride, 1-palmitoyl-3-eicosanoic acid diglyceride, 1-stearic acid-3-eicosanoic acid diglyceride, 1,3-palmitoyl-3-oleic acid diglyceride 27 1,3-diglycerides including palmitic acid diglyceride, 1,3-undecenoic acid diglyceride, 1,3-dodecenoic acid diglyceride, 1,3-tridecanoic acid diglyceride, 1,3-myristic acid diglyceride, 1,3-tetradecenoic acid diglyceride, 1,3-pentadecanoic acid diglyceride, 1,3-heptadecanoic acid diglyceride, 1,3-heptadecanoic acid diglyceride, 1,3-eicosanoic acid diglyceride, 1,3-heneicosanoic acid diglyceride, 1-oleo-3-eicosanoic acid diglyceride and 3 1,2-diglycerides including 1-myristic acid-2-lauric acid diglyceride, 1-palmitic acid-2-lauric acid diglyceride, 1-oleic acid-2-palmitic acid diglyceride. N,N-substituted benzoic acid diglyceride compounds [M+H] were selected in the test. + or [M+NH4] + As the precursor ion, the specific ion replaces the aminobenzoyl ion [C9H 10 NO]· or substituted aminophenyl ion [C8H 13 The content of diglycerides was analyzed by quantitative ion analysis. In addition, the quantitative ion fragment of substituted aminophenyl glyceride derivatives, substituted aminobenzoyl ion [C9H 10 NO]· or substituted aminophenyl ion [C8H 13 The intensity of N]· was significantly higher than that of the underivatized diacylglycerol and the aromatic acid-derivatized diacylglycerol.

[0061] The above embodiments will help those skilled in the art to further understand the present invention, but do not constitute any limitation to the present invention. Any modification, equivalent substitution and improvement made within the spirit and principle of the present invention shall be included in the protection scope of the present invention. The above description is only a preferred specific implementation of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field shall make equivalent substitutions or changes according to the technical solution and inventive concept of the present invention within the technical scope disclosed by the present invention, which shall be included in the protection scope of the present invention.

Claims

1. Use of the chemical derivatizing agent described in structural formula 1 in the derivatization treatment of diglyceride substances for mass spectrometry analysis or liquid chromatography-mass spectrometry analysis, characterized in that: The chemical derivatizing agent has a carboxyl group at one end and a NR2 compound at the other end, and the structural formula 1 is: NR2-Ph-COOH or NR2-Ph-CH2-COOH, wherein the R group is selected from H, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2, an alkane substituent group with a carbon chain length of less than 3, and Ph is a benzene ring.

2. The use according to claim 1, characterized in that: The chemical derivatizing agent is 4-diethylaminobenzoic acid, 4-dimethylaminophenylacetic acid, 4-aminobenzoic acid, 4-dimethylaminobenzoic acid or 3-dimethylaminobenzoic acid.

3. The use according to claim 1, characterized in that: The chemical derivatization reagent is 3-dimethylaminobenzoic acid or 4-dimethylaminobenzoic acid.

4. The use according to claim 1, characterized in that: The mass spectrometry or liquid chromatography-mass spectrometry uses a positive ion mode, and the quantitative or qualitative parent ion is [M+H] + or [M+NH4] + The daughter ions are fragment ions produced during the cleavage of diacylglycerol derivatives derived from chemical derivatization agents.

5. A method for detecting diglycerides in a vegetable oil sample, characterized in that: The main steps include: (1) pretreatment of vegetable oil samples, (2) chemical derivatization of the samples using a chemical derivatization agent as described in structural formula 1 in claim 1, and (3) analysis of diglyceride substances based on mass spectrometry or liquid chromatography-mass spectrometry after the chemical derivatization.

6. The detection method according to claim 5, characterized in that: The chemical derivatization is a coupling esterification of the hydroxyl group of diglyceride with the chemical derivatization agent described in structural formula 1. The chemical derivatization conditions are the coupling esterification conditions of small molecule compounds, and the derived chemical functional group is the hydroxyl group of diglyceride.

7. The detection method according to claim 6, characterized in that: The reaction solvent of the coupling esterification is acetonitrile, methanol, acetone, ethyl acetate, isopropanol or dichloromethane, and the reaction temperature is 70-80°C.

8. The detection method according to claim 5, characterized in that: The vegetable oil samples are rapeseed oil, soybean oil, peanut oil, corn oil, linseed oil, olive oil, vegetable oil and diglyceride oil made from the above vegetable oils as raw materials.

9. The detection method according to claim 5, characterized in that: The diglyceride compounds include 1,3-diglyceride and 1,2-diglyceride; the 1,3-diglyceride includes 1-palmitoyl-3-stearic acid diglyceride, 1-palmitoyl-3-oleic acid diglyceride, 1-palmitoyl-3-linoleic acid diglyceride, 1-palmitoyl-3-linolenic acid diglyceride, 1-stearic acid-3-oleic acid diglyceride, 1-linoleic acid-3-linolenic acid diglyceride, 1,3-dipalmitoyl-3-stearic acid diglyceride Palmitic acid diglyceride, 1,3-distearic acid diglyceride, 1,3-dioleic acid diglyceride, 1,3-dilinoleic acid diglyceride, 1,3-dilinolenic acid diglyceride, 1,3-undecenoic acid diglyceride, 1,3-dodecenoic acid diglyceride, 1,3-tridecanoic acid diglyceride, 1,3-tetradecanoic acid diglyceride, 1,3-tetradecenoic acid diglyceride, 1,3-pentadecanoic acid diglyceride, 1,3-decenoic acid diglyceride Heptadecanoic acid diglyceride, 1,3-heptadecanoic acid diglyceride, 1,3-eicosanoic acid diglyceride, 1,3-heneicosanoic acid diglyceride, 1-myristic acid-3-lauric acid diglyceride, 1-myristic acid-3-behenic acid diglyceride, 1-myristic acid-3-oleic acid diglyceride, 1-palmitoyl-3-eicosanoic acid diglyceride, 1-stearic acid-3-eicosanoic acid diglyceride, 1-oleic acid-3-diglyceride Decanoic acid diglyceride; the 1,2-diglyceride includes 1-palmitoyl-2-lauric acid diglyceride, 1,2-dioleic acid diglyceride, 1-stearic acid-2-lauric acid diglyceride, 1-oleic acid-2-palmitoyl acid diglyceride, 1-palmitoyl-2-stearic acid diglyceride, 1,2-palmitic acid diglyceride, 1,2-stearic acid diglyceride, 1,2-heptadecanoyl acid diglyceride, and 1,2-linoleic acid diglyceride.

10. The detection method according to claim 5, characterized in that: The detection and analysis method of diglyceride was UHPLC-MS / MS, and the analysis conditions were as follows: the mobile phase was 0.1% formic acid acetonitrile and 0.1% formic acid solution, gradient elution, the chromatographic separation column was C18, and the flow rate was 250 μL / min; the mass spectrometry analysis conditions were triple tandem quadrupole mass spectrometer, ESI ion source, positive ion mode, and the quantitative parent ion was [M+H] + or [M+NH4] + The daughter ions are fragment ions produced during the cleavage of diacylglycerol derivatives derived from chemical derivatization agents.

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