Omega-3 lipid detection method based on inorganic matrix enhanced MALDI mass spectrum

Abstract

The invention relates to an omega-3 lipid detection method based on an inorganic matrix enhanced MALDI mass spectrum. The invention discloses an omega-3 supplement identification technology of an inorganic matrix enhanced MALDI mass spectrum, which can efficiently and accurately carry out comprehensive analysis on lipid types, double bond positions and oxidation degrees in a complex lipid sample. The technology comprises the following steps: selecting an omega-3 supplement, mixing the omega-3 supplement, a cycloaddition reagent and an inorganic matrix, carrying out MALDI laser-driven online cycloaddition reaction, and analyzing the lipid double-bond isomer by mass spectrum data. The selected 25nm hydrophilic anatase titanium dioxide obviously improves the sensitivity of lipid double bond identification, the vacuum environment of the MALDI mass spectrum and shorter laser irradiation time (30s) effectively protect lipid from additional oxidation, and the original oxidation information of the lipid is retained. The method successfully identifies 30 main omega-3 lipids in four omega-3 supplements, and reveals significant differences of lipid types, omega-3 purity and lipid oxidation degrees. The method has a wide application prospect in the aspect of high-efficiency and high-throughput screening of other complex lipid information samples such as omega-3 supplements and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of detection methods, and particularly relates to a method for identifying fatty acids, triglycerides and phospholipid C-C isomers through an online cycloaddition reaction of MALDI mass spectrometry based on a highly ionizable inorganic matrix, and an application of the identification method in high-throughput screening of lipid information in ω-3 oil samples. Background Art

[0002] The intake of polyunsaturated lipids represented by ω-3 fatty acids can participate in regulating the human lipid metabolism pathway and has the effects of antioxidation and inflammation regulation. Such polyunsaturated lipids play a role in promoting health and reducing the risks of cardiovascular diseases, non-alcoholic fatty liver diseases and neurodegenerative diseases. ω-3 lipids are mainly derived from the fats or viscera of marine animals, and these fish oils are concentrated and sold in the form of ethyl esters or acylglycerols. Algae, fungi and single-cell oils have recently become popular as new renewable sources of ω-3 supplements. Krill and the plant seed oils of some special plants (such as perilla seeds) have also been successfully marketed as ω-3 supplements. However, the existing forms (such as fatty acids, phospholipids, triglycerides) and contents of lipids in ω-3 supplements from different sources are different, which directly affects the bioavailability of ω-3 supplements, and due to the doping of isomeric ω-6 lipids, their purity is also affected. In addition, polyunsaturated lipids are inevitably oxidized during the production process, and such oxidized lipids will have a negative impact on health after being ingested. Therefore, it is necessary to evaluate the form, purity and oxidation degree of ω-3 lipids in ω-3 supplements.

[0003] National and international standards (GB 5009.227-2016 and CXS 329-2017) state that the oxidation state of ω-3 supplements is determined by analyzing peroxide value (PV), p-anisidine value (PAV) and total oxidation value (TOTOX, TOTOX = 2×PV + PAV). The PV of the final product must be lower than 5 meq / kg, the PAV must be lower than 20, and the TOTOX must be lower than 26 to be acceptable. However, the instability of hydroperoxides and the non-selectivity of the anisidine reaction to aldehydes have prompted people to develop new methods to improve the evaluation effect of lipid quality.

[0004] Literatures (Srigley, C.T.; Rader, J.I. J. Agric. Food Chem. 2014, 62(29), 7268–7278 and Suh, J.H.; Niu, Y.S.; Hung, W.-L.; Ho, C.-T.; Wang, Y. Talanta 2017, 168, 31–42.) reported methods for evaluating the lipid content and composition in marine animal-based ω-3 supplements using gas chromatography and liquid chromatography tandem mass spectrometry. However, the separation step of chromatography makes the detection time of chromatography-mass spectrometry coupling methods often reach 30 - 50 min, and more precise chromatographic columns are required to distinguish lipid isomers. Literatures (Damerau, A.; Ahonen, E.; Kortesniemi, M.; Puganen, A.; Tarvainen, M.; Linderborg, K.M. Food Chemistry 2020, 330, 127194 and Killeen, D.P.; Marshall, S.N.; Burgess, E.J.; Gordon, K.C.; Perry, N.B. J. Agric. Food Chem. 2017, 65(17), 3551–3558.) reported the use of nuclear magnetic resonance spectroscopy and Raman spectroscopy to detect the content and oxidation degree of polyunsaturated lipids in ω-3 supplements. However, the detection throughput of such single detections is limited and it is difficult to meet the demand of the rapidly developing ω-3 supplement market for quickly and accurately obtaining information on the purity, lipid form, and oxidation degree of ω-3 lipids in a large number of samples.

[0005] Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), as a high-throughput mass spectrometry detection method, can rapidly screen a large number of samples in a short time with high throughput. However, due to the lack of a chromatographic separation step, conventional MALDI MS is difficult to distinguish lipid isomers. The photo-driven [2 + 2] cycloaddition reaction has the characteristics of simple operation, fast derivatization reaction rate, and high specificity for C=C double bonds, and can identify the position of the C=C double bond in unsaturated lipids through the derivatization fragments generated by the cycloaddition product in the secondary mass spectrometry. However, the off-line derivatization method used in this method will inevitably cause lipid oxidation, interfering with the accuracy of lipid information evaluation, so it is an important obstacle to the application of this method. Summary of the Invention

[0006] In order to overcome the problems of insufficient detection throughput, long analysis time, high instrument requirements, and insufficient accuracy in existing methods, the present invention aims to provide an efficient, high-throughput, simple detection device, and highly accurate MALDI MS online reaction analysis method for evaluating the lipid form, purity, and oxidation degree of ω-3 supplements. ω-3 lipids refer to lipids in which the first unsaturated double bond on the side chain is located between the 3rd and 4th carbon atoms starting from the methyl end, mainly including ω-3 ethyl esters, fatty acids, triglycerides, diglycerides, re-esterified triglycerides, re-esterified diglycerides, or phospholipids.

[0007] A method for detecting ω-3 lipids based on inorganic matrix enhanced MALDI mass spectrometry provided by the present invention is characterized by comprising the following steps:

[0008] Step 1, mixing a sample containing ω-3 lipids with an inorganic matrix and a cycloaddition reagent, spotting on a sample target of a MALDI mass spectrometer, and air-drying;

[0009] Step 2, feeding the spotted target plate into MALDI MS to perform an online cycloaddition reaction driven by MALDI laser, and ionizing and performing mass spectrometry analysis on the sample containing ω-3 lipids by using a MALDI mass spectrometer, wherein the working parameters of the MALDI mass spectrometer are a laser frequency of 5 Hz to 200 Hz and a laser power of 10 - 100% of the maximum power of the instrument;

[0010] Step 3, the reaction mode adopted for the MALDI mass spectrometry analysis is a 355 nm laser pulse equipped by the MALDI mass spectrometer, and the categories and structures of ω-3 lipids are identified according to the mass spectrometry data;

[0011] The inorganic matrix is anatase and rutile titanium dioxide, cerium oxide, zinc oxide, copper oxide, graphite phase carbon nitride, iron(III) oxide, tin dioxide, magnesium oxide, aluminum oxide, iron titanate, barium titanate, lead titanate, and the particle size is 5 - 150 nm;

[0012] The cycloaddition reagent is benzo[g]isoquinoline-5,10-dione, 2-benzoylpyridine, 3-benzoylpyridine, or 4-benzoylpyridine.

[0013] In one embodiment, the cycloaddition reagent is 3-benzoylpyridine.

[0014] In one embodiment, the inorganic matrix is 25 nm anatase titanium dioxide.

[0015] In one embodiment, the sample containing ω-3 lipids includes fish oil, seal oil, krill oil, perilla oil, or olive oil.

[0016] In one embodiment, the concentration range of the inorganic matrix is from 0.75 mg / mL -1 to 20 mg / mL -1 , the concentration of the cycloaddition reagent is 1 - 100 mM, and the concentration of hydrochloric acid is 0.1 - 100 mM.

[0017] In one embodiment, the concentration of the sample containing ω-3 lipids is 0.01 mM - 100 mM.

[0018] In one embodiment, the reaction equivalent ratio of the sample containing ω-3 lipids to the cycloaddition reagent is 0.5:1 to 16:1.

[0019] In one embodiment, the volume ratio of the sample containing ω-3 lipids : inorganic matrix : cycloaddition reagent is 1:2:1.

[0020] In one embodiment, the spotting volume is 0.5 μL - 3 μL, and after spotting, it is air-dried at 21°C for 0.5 - 10 min.

[0021] In one embodiment, Step 2 is specifically as follows: Open the mass spectrometry software. After the instrument is evacuated, adjust the method to the RP, RN, or LIFT mode, and adjust the working parameters of the MALDI mass spectrometer to a laser frequency of 5 Hz to 200 Hz and a laser power of 10 - 100% of the maximum power; click the start button, manually move the laser spot to the entire area of the sample spot to ensure complete reaction, and each sample spot stays for 5 - 30 s to ensure sufficient on-line cycloaddition reaction.

[0022] In one embodiment, for the lipid content quantification method: In the mass spectrometry results, take the ratio R = T / S of the signal intensity T of the mass-to-charge ratio of the mass spectrometry target signal molecule fragment and the signal intensity S of the mass-to-charge ratio of the internal standard fragment within the same time as the quantification basis, obtain a linear equation using the detection results of the standard lipid sample, and calculate the concentration of the target lipid double bond isomer in the sample to be measured.

[0023] In one embodiment, for the relative quantification method of lipid isomers: In the mass spectrometry results, take the signal intensities T1 and T2 of the mass-to-charge ratios corresponding to the product fragments of two lipid isomers as the quantification basis, and obtain the molar ratio of the lipid double bond isomers using R2 = T1 / T2.

[0024] In one embodiment, an internal standard is added to the sample for quantification by internal standard method.

[0025] In one embodiment, the internal standard includes crystal violet or methyl violet, and the concentration is 0.05 - 0.5 μmol / L.

[0026] In one embodiment, in the obtained mass spectrometry, the mass-to-charge ratio of the mass spectrometry signal molecule or its fragment is the signal of the sample to be measured, and the mass-to-charge ratio of the internal standard substance or its fragment is the internal standard signal.

[0027] In one embodiment, step 3 is specifically: using fatty acid FA 18:3(Δ9,12,15) as the mass spectrometry signal molecule and crystal violet as the internal standard, a series of fatty acid standard solutions with concentrations of c1, c2, …, c n are prepared, spotted on the MALDI target plate, the laser frequencies of the first-order and second-order mass spectrometry are 20 Hz and 60 Hz respectively, the laser powers are 50% and 60% of the maximum power respectively, and the laser gain is set to 7X; in the obtained mass spectrometry, the possible ionization forms of the product of the lipid double bond and the cycloaddition reagent are [M+CR+H] + , [M+CR+Na] + , [M+CR+K] + , where M is the relative molecular mass of the target lipid isomer and CR is the relative molecular mass of the cycloaddition reagent, and the mass-to-charge ratio generated by the internal standard crystal violet is m / z = 372.2; the data analysis of the mass-to-charge ratio of the mass spectrometry signal molecule or its fragment specifically includes:

[0028] 3-1) In the obtained mass spectrometry, find the mass-to-charge ratio of the mass spectrometry fragment of the target lipid isomer, and obtain the signal intensity (Tc1, Tc2,..., Tc n ) of the mass-to-charge ratio of the mass spectrometry signal molecule fragment of FA 18:3(Δ9,12,15) in one detection and the signal intensity (Sc1, Sc2,..., Sc n ) of the mass-to-charge ratio of the mass spectrometry signal molecule fragment of the internal standard in the same detection; calculate the signal intensity ratio Rc of Tc to Sc: Rc1 = Tc1 / Sc1, Rc2 = Tc2 / Sc2,..., Rc n = Tc n / Sc n。 Similarly, obtain the mass-to-charge ratio signal intensity ratios Rx1, Rx2,..., Rx m of the lipid isomer mass spectrometry and the internal standard signal molecule fragment in the sample to be measured;

[0029] 3-2) Using c1, c2, …, c n as the abscissa values and Rc1, Rc2,..., Rc n as the ordinate values, plot a graph, perform linear fitting on the data points in the graph, and obtain a linear equation;

[0030] 3-3) Substitute Rx1, Rx2,..., Rx m into the linear equation and calculate the concentration of the corresponding lipid isomer in the sample to be measured.

[0031] In one embodiment, the sample detection order is as follows: the standard products are detected in ascending concentration order, and then the samples are detected.

[0032] In one embodiment, taking the fatty acid isomers FA 18:3(Δ9,12,15) and FA 18:3(Δ6,9,12) as examples, a series of fatty acid isomer mixtures with molar ratios of c1, c2, …, c n are prepared, spotted on the MALDI target plate. The laser frequencies of the first - stage and second - stage mass spectrometry are 20Hz and 60Hz respectively, the laser powers are 50% and 60% of the maximum power respectively, and the laser gain is set to 7X. In the obtained mass spectrometry diagram, the possible ionization forms of the product of the lipid double - bond isomer and the cycloaddition reagent are [M + CR + H]+, [M + CR + Na]+, [M + CR + K]+, where M is the relative molecular mass of the target lipid isomer and CR is the relative molecular mass of the cycloaddition reagent. The specific analysis of the mass - to - charge ratio data of the mass spectrometry signal molecules or their fragments includes:

[0033] 4 - 1) In the obtained mass spectrometry diagram, find the mass - to - charge ratios of the mass spectrometry fragments of the target lipid isomers respectively, and obtain the signal intensities (Tc1, Tc2,..., Tc n ) of the mass - to - charge ratios of the mass spectrometry signal molecule fragments of FA18:3(Δ9,12,15) in one detection and the signal intensities (Sc1, Sc2,..., Sc n ) of the mass - to - charge ratios of the mass spectrometry signal molecule fragments of FA 18:3(Δ6,9,12) in the same detection; calculate the signal intensity ratio Rc of Tc to Sc: Rc1 = Tc1 / Sc1, Rc2 = Tc2 / Sc2,..., Rc n = Tc n / Sc n。 Calculate the mass - to - charge ratio signal intensity ratios Rx1, Rx2,..., Rx m of the mass spectrometry signal molecule fragments of the lipid isomer to be detected;

[0034] 4 - 2) Using c1, c2, …, c n as the abscissa values and Rc1, Rc2,..., Rc n as the ordinate values, plot a graph, perform linear fitting on the data points in the graph to obtain a linear equation;

[0035] 4 - 3) Substitute Rx1, Rx2,..., Rx m into the linear equation to calculate the molar ratio of the corresponding lipid isomer in the sample to be detected.

[0036] The second object of the present invention is to provide a ω-3 lipid structure identification system constructed based on the above method, including: a sample processing unit for mixing a sample containing ω-3 lipids with an inorganic matrix and a cycloaddition reagent;

[0037] a MALDI mass spectrometer connected to the sample processing unit for ionizing and performing mass spectrometry analysis on the mixed sample;

[0038] a data processing unit for receiving the mass spectrometry data of the MALDI mass spectrometer and identifying the category of ω-3 lipids, the relative content of ω-3 / ω-6 lipids, and the oxidation degree of ω-3 supplements.

[0039] The implementation steps of the MALDI online analysis method provided by the present invention for evaluating the lipid morphology, purity, and oxidation degree in a sample containing ω-3 lipids include the preparation of the actual sample, the preparation of the mixed solution of the sample, inorganic matrix, and cycloaddition reagent, the adjustment of spotting on the target and instrument parameters, the online reaction of MALDI MS, data processing, and other steps.

[0040] The online mass spectrometry isomer analysis method provided by the present invention has the characteristics of high efficiency and high throughput. The inorganic substances used as the MALDI matrix can improve the ionization efficiency of the product and realize the online reaction during the ionization process. In addition, the online reaction utilizes the vacuum environment of MALDI itself for the cycloaddition reaction, reducing exogenous lipid oxidation by-products and ensuring the authenticity of the detection results. The online mass spectrometry isomer analysis method in the present invention can be extended to the simultaneous detection of multiple lipid isomers, ensuring the accurate evaluation of lipid morphology, ω-3 lipid purity, and lipid oxidation degree in ω-3 supplements by the online lipid isomer detection method in the present invention. BRIEF DESCRIPTION OF THE DRAWINGS [[ID=,15]]

[0041] Figure 1 It is a schematic flow chart of the MALDI online lipid isomer analysis method of the present invention.

[0042] Figure 2 It is the optimization result of the cycloaddition reagent of the online lipid isomer analysis method of the present invention; 3-benzoylpyridine (3-BzPy), 2-benzoylpyridine (2-BzPy), 4-benzoylpyridine (4-BzPy), benzo[g]isoquinoline-5,10-dione (BIQD).

[0043] Figure 3 It is the optimization result of the inorganic nanomaterial matrix of the online lipid isomer analysis method of the present invention.

[0044] Figure 4 It is the optimization result of the morphology and size of the titanium dioxide matrix of the online lipid isomer analysis method of the present invention. <,

[0045] Figure 5 It is the mass spectrum obtained for the identification of the carbon-carbon double bonds of fatty acid FA 18:3(Δ9,12,15) in Example 2, and the mass spectrometry signal intensity is used to plot the relationship curve.

[0046] Figure 6 It is the mass spectrum for the identification of the carbon-carbon double bonds of triglyceride TG 18:2(Δ9,12) / 18:2(Δ9,12) / 18:3(Δ9,12,15) in Example 2, and the mass spectrometry signal intensity is used to plot the relationship curve.

[0047] Figure 7 It is the mass spectrum obtained for the identification of the carbon-carbon double bonds of phosphatidylethanolamine PE 16:0_18:1(Δ9) in Example 2, and the mass spectrometry signal intensity is used to plot the relationship curve.

[0048] Figure 8 It is the linear relationship diagram of the molar ratio of two lipid isomers FA 18:3(Δ9,12,15) and FA 18:3(Δ6,9,12) to the intensity of lipid double bond fragments in Example 3.

[0049] Figure 9 It is the distribution diagram of ω-3 lipid types and relative contents in four ω-3 supplements in Example 4.

[0050] Figure 10 It is the ω-3 lipid purity diagram of four ω-3 supplements in Example 5.

[0051] Figure 11 It is to compare the oxidation degree of lipids in four ω-3 supplements between offline reaction and online reaction in Comparative Example 1. Detailed implementation mode

[0052] The following further elaborates the technical solution of the present invention through examples in conjunction with the accompanying drawings, but the protection scope of this application is not limited by the specific conditions of these examples.

[0053] Example 1:

[0054] To implement the on-line cycloaddition method, screening of cycloaddition reagents and inorganic matrices was first carried out. Fatty acid FA 18:3(Δ9,12,15) was used as a lipid standard, and benzo[g]isoquinoline-5,10-dione, 2-benzoylpyridine, 3-benzoylpyridine, and 4-benzoylpyridine were candidate cycloaddition reagents. These reagents all showed absorption bands near 355 nm in the ultraviolet-visible absorption spectrum, indicating the possibility of generating biradicals under MALDI laser activation. The specific steps are as follows:

[0055] (1) Online MALDI lipid isomer identification was performed using a Bruker autoflex maX MALDI TOF / TOF mass spectrometer equipped with a 355 nm SmartBeam II laser.

[0056] (2) Fatty acid FA 18:3(Δ9,12,15) was dissolved and diluted with methanol. 25 nm hydrophilic anatase titanium dioxide was dispersed with ethanol and ultrasonicated to make it uniformly dispersed. During the online detection process, lipid standards (0.08 - 18 mmol L -1 ), benzo[g]isoquinoline-5,10-dione, 2-benzoylpyridine, 3-benzoylpyridine, 4-benzoylpyridine (100 mmol L -1 ), and titanium dioxide dispersion (7.5 mg mL -1 ) were mixed at a volume ratio of 1:1:2, then spotted on a MALDI target plate and dried (21 °C, ~30 seconds), and the prepared samples were waiting for detection.

[0057] MALDI-TOF MS conditions: Ion source 1 (18.94 kV), ion source (16.58 kV), lens voltage (8.41 kV), reflector 1 (20.90 kV), reflector 2 (9.63 kV), pulsed ion extraction (120 ns), detector gain (7×; 2117 V), and smart beam parameter settings (4_large). For MS and MS / MS modes, the laser power was optimized to 50% and 60% of the maximum laser power respectively, and the laser frequency was optimized to 20 Hz and 60 Hz respectively.

[0058] (3) Acquisition was performed in MS mode to obtain the molecular ion peaks of the on-line cycloaddition reaction of the target fatty acid with the cycloaddition reagent [M+H] + m / z 488.3 (benzo[g]isoquinoline-5,10-dione cycloaddition product), m / z 462.2 (benzoylpyridine cycloaddition product). In MS / MS mode, the product precursor ions detected were collected for the second-order mass spectrum, and the second-order mass spectrum-derived ion signal peaks m / z 236.1 (benzo[g]isoquinoline-5,10-dione cycloaddition product-derived ion), m / z 210.2 (benzoylpyridine cycloaddition product-derived ion) were marked and the intensities of the derived ion signal peaks were compared.

[0059] The ability of the cycloaddition product to produce lipid-indicating double bond position-derived ions was studied using MS / MS. As Figure 2 shown, 3-benzoylpyridine produced the highest double bond characteristic ions compared with 2-benzoylpyridine and 4-benzoylpyridine. Therefore, 3-benzoylpyridine was selected as the best on-line cycloaddition reaction reagent.

[0060] Fourteen inorganic substances (mainly metals and metal oxides that absorb 355 nm) were used as the matrix to test the on-line cycloaddition reaction. The specific steps are as follows:

[0061] (1) An on-line MALDI lipid isomer identification was carried out using a Bruker autoflex maX MALDI TOF / TOF mass spectrometer equipped with a 355 nm SmartBeam II laser.

[0062] (2) Fatty acid FA 18:3 (Δ9,12,15) was dissolved and diluted with methanol. The inorganic matrix and the organic small molecule matrix were dispersed with methanol, ethanol and water, and ultrasonicated to make them evenly dispersed. During the on-line detection process, lipid standards (0.08 - 18 mmol L -1 ), 3-benzoylpyridine (100 mmol L -1 ), and the matrix dispersion (7.5 - 15 mg mL -1 ) were mixed at a volume ratio of 1:1:2, and then spotted on the MALDI target plate and dried to prepare the sample for detection.

[0063] MALDI-TOF MS conditions: Ion source 1 (18.94 kV), ion source (16.58 kV), lens voltage (8.41 kV), reflector 1 (20.90 kV), reflector 2 (9.63 kV), pulsed ion extraction (120 ns), detector gain (7×; 2117 V), and smart beam parameter settings (4_large). For the MS and MS / MS modes, the laser power was optimized to 50% and 60% of the maximum laser power respectively, and the laser frequency was optimized to 20 Hz and 60 Hz respectively.

[0064] (3) Acquisition was carried out in the MS mode to obtain the [M+H] + molecular ion peak m / z 462.2 of the on-line cycloaddition reaction of the target fatty acid with 3-benzoylpyridine, and the intensity of the molecular ion signal peak was compared.

[0065] Compared with common small molecule organic matrices such as α-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB), titanium dioxide, gold nanoparticles and iron(III) oxide achieved higher product signal-to-noise ratios. Among them, titanium dioxide had the highest signal-to-noise ratio, which was about 10.25 times higher than that of the common CHCA and DHB matrices ( Figure 3 ). The effects of titanium dioxide with different crystal structures and sizes on the on-line cycloaddition reaction were further studied ( Figure 4) Among different sizes, 25-nm anatase titanium dioxide is the most effective matrix for the on-line cycloaddition reaction, and unmodified hydrophilic titanium dioxide shows a higher enhancement effect than lipophilic titanium dioxide. Finally, 25-nm hydrophilic anatase is selected as the MALDI matrix to enhance the on-line cycloaddition reaction.

[0066] Example 2:

[0067] The on-line cycloaddition method of the present invention is used to determine the positions of carbon-carbon double bonds of fatty acid FA 18:3(Δ9,12,15), triglyceride TG 18:2(Δ9,12) / 18:2(Δ9,12) / 18:3(Δ9,12,15), and phosphatidylethanolamine PE 16:0_18:1(Δ9).

[0068] The specific steps are as follows:

[0069] (1) An on-line MALDI lipid isomer identification is performed using a Bruker autoflex maX MALDI TOF / TOF mass spectrometer equipped with a 355-nm SmartBeam II laser.

[0070] (2) Fatty acid FA 18:3(Δ9,12,15), triglyceride TG 18:2(Δ9,12) / 18:2(Δ9,12) / 18:3(Δ9,12,15), and phosphatidylethanolamine PE 16:0_18:1(Δ9) standards are dissolved and diluted with methanol. 25-nm hydrophilic anatase titanium dioxide is dispersed in ethanol (EtOH) to 7.5 mg mL -1 , and ultrasonic treatment is used to make it uniformly dispersed. During the on-line detection process, lipid standards (0.08 - 18 mmol L -1 ), 3-BzPy (80 mmol L -1 ), and titanium dioxide dispersion (7.5 mg mL -1 ) are mixed at a volume ratio of 1:1:2, and then spotted on a MALDI target plate and dried (21 °C, ~30 seconds). The prepared samples are waiting for detection.

[0071] MALDI-TOF MS conditions: Ion source 1 (18.94 kV), ion source (16.58 kV), lens voltage (8.41 kV), reflector 1 (20.90 kV), reflector 2 (9.63 kV), pulsed ion extraction (120 ns), detector gain (7×; 2117 V), and SmartBeam parameter settings (4_large). For the MS and MS / MS modes, the laser power is optimized to 50% and 60% of the maximum laser power respectively, and the laser frequency is optimized to 20 Hz and 60 Hz respectively.

[0072] (3) Collect in MS mode to obtain the [M+H] of the on-line cycloaddition reaction of the target fatty acid and 3-BzPy + The molecular ion peak is m / z 462.2. In MS / MS mode, collect the secondary mass spectrum of the detected product parent ion m / z 462.2 to obtain Figure 5 , and mark the secondary mass spectrometry signal peak as the derived fragment peak indicating the position of the carbon-carbon double bond.

[0073] (4) Collect in MS mode to obtain the [M+H] of the on-line cycloaddition reaction of the target triglyceride and 3-BzPy + The molecular ion peak is m / z 1060.9. In MS / MS mode, collect the secondary mass spectrum of the detected product parent ion m / z 1060.9 to obtain Figure 6 , and mark the secondary mass spectrometry signal peak as the derived fragment peak indicating the position of the carbon-carbon double bond.

[0074] (5) Collect in MS mode to obtain the [M+K] of the on-line cycloaddition reaction of the target phosphatidylethanolamine and 3-BzPy + The molecular ion peak is m / z 939.6. In MS / MS mode, collect the secondary mass spectrum of the detected product parent ion m / z 939.6 to obtain Figure 7 , and mark the secondary mass spectrometry signal peak as the derived fragment peak indicating the position of the carbon-carbon double bond.

[0075] Example 3:

[0076] Use the on-line cycloaddition method of the present invention to determine the molar ratio of fatty acid isomers FA 18:3(Δ9,12,15) and FA 18:3(Δ6,9,12).

[0077] The specific steps are as follows:

[0078] (1) Use a Bruker autoflex maX MALDI TOF / TOF mass spectrometer equipped with a 355nm SmartBeam II laser for on-line MALDI lipid isomer identification.

[0079] (2) Use methanol to dissolve and dilute the fatty acid isomer standards FA 18:3(Δ9,12,15) and FA 18:3(Δ6,9,12). Disperse 25nm hydrophilic anatase titanium dioxide into 7.5mg mL with ethanol (EtOH) -1 , and ultrasonically disperse it evenly. During the on-line detection process, two fatty acid isomer standards with different concentrations (0.08 - 18mmol L -1 ), 3-BzPy (80mmol L -1 ), and titanium dioxide dispersion (7.5mg mL -1) and HCl (100 mmol L -1 ) were mixed at a volume ratio of 1:1:2:1, maintaining the total fatty acid concentration at 10 mmol L -1 . Then, it was spotted on a MALDI target plate and dried (21 °C, ~30 seconds). The prepared sample was waiting for detection.

[0080] MALDI-TOF MS conditions: Ion source 1 (18.94 kV), ion source (16.58 kV), lens voltage (8.41 kV), reflector 1 (20.90 kV), reflector 2 (9.63 kV), pulsed ion extraction (120 ns), detector gain (7×; 2117 V), and intelligent beam parameter setting (4_large). For MS and MS / MS modes, the laser power was optimized to 50% and 60% of the maximum laser power respectively, and the laser frequency was optimized to 20 Hz and 60 Hz respectively.

[0081] (3) Acquisition was carried out in MS mode to obtain the [M+H] molecular ion peak m / z 462.2 of the on-line cycloaddition reaction of the two target fatty acid isomers with 3-BzPy. + The secondary mass spectrum of the detected product parent ion m / z 462.2 was acquired in MS / MS mode, and the derivative ion fragment peaks m / z 210.2 and m / z 252.2 indicating the carbon-carbon double bonds at the Δ15 and Δ12 positions were found among the derivative fragments.

[0082] (4) The signal intensities of the derivative ion fragment peaks m / z 210.2 and m / z 252.2 were detected, and the signal intensity ratio Rc, Rx of m / z 210.2 and m / z 252.2 was calculated for quantitative calculation. Between the molar ratio r = 1 - 19, the signal intensity ratio showed a linear relationship with the molar ratio, Rc = 1.120r + 0.1509 (R<� 2 = 0.997)( Figure 8 ).

[0083] Example 4:

[0084] The online cycloaddition method of the present invention was used to determine the types and relative contents of ω-3 lipids in four ω-3 supplements.

[0085] The specific steps are as follows:

[0086] (1) Each ω-3 supplement was separately transferred to a 2 mL brown glass bottle, and the air in the bottle was replaced with nitrogen. Subsequently, 100 μL of the ω-3 supplement was dissolved in 1000 μL of the solvent (methanol:chloroform = 8:2).

[0087] (2) The diluted sample was subjected to precursor ion scanning using a QTRAP@4500 MS (AB SCIEX, USA) to obtain possible unsaturated lipids. The instrument parameters were set as follows: curtain gas 10 Psi, ion source gas 1 18 Psi, ion source gas 2 0 Psi, spray voltage 5.5 kV, interface heating temperature 0 °C, collision gas low, declustering voltage 135 V, entrance voltage 10 V, collision energy 44 V, collision cell exit voltage 10 V.

[0088] (3) Identification and relative content analysis of ω-3 lipid species were performed using a Bruker autoflex maX MALDI TOF / TOF mass spectrometer equipped with a 355 nm SmartBeam II laser. The sample was diluted with methanol to 16 mg mL -1 , and 200 nmol L -1 of crystal violet was added as an internal standard. The sample, 3-benzoyl pyridine, titanium dioxide dispersion, and internal standard were mixed in a volume ratio of 1:1:2. 2.5 μL of the mixed solution was spotted on the target, air-dried, and then subjected to MALDI TOF MS analysis.

[0089] (4) The high-resolution m / z values of possible ω-3 unsaturated lipids were searched in the full-scan MS (referring to the LIPIDMAPS database), and further confirmed by combining the characteristic fragment ions of lipid species in MS / MS. The content ratio of each component was calculated through the signal response of different lipid species and its intensity-content correction factor (fatty acid: triglyceride: phospholipid = 1:0.4711:0.0686) ( Figure 9 ).

[0090] Example 5:

[0091] The online cycloaddition method of the present invention was used to determine the purity of ω-3 lipids in four ω-3 supplements.

[0092] The specific steps are as follows:

[0093] (1) Each ω-3 supplement was separately transferred to a 2 mL brown glass bottle, and the air in the bottle was replaced with nitrogen. Subsequently, 100 μL of the ω-3 supplement was dissolved in 1000 μL of the solvent (methanol: chloroform = 8:2).

[0094] (2) The sample was hydrolyzed in 2 mol L -1 NaOH (EtOH:H2O = 6:1) at 60 °C for 1 hour. After the reaction, the pH was adjusted to 6 with 3 molL -1 HCl.

[0095] (3) The analysis of the purity of ω-3 lipids was carried out using a Bruker autoflex maX MALDI TOF / TOF mass spectrometer equipped with a 355 nm SmartBeam II laser. The sample, 3-benzoylpyridine, titanium dioxide dispersion, and internal standard were mixed at a volume ratio of 1:1:2. 2.5 μL of the mixed solution was spotted on the target and dried before MALDI TOF MS analysis.

[0096] (4) Acquisition was carried out in the MS mode to obtain the [M+H] molecular ion peak at m / z 462.2 of the fatty acid isomers after hydrolysis of the ω-3 supplement and the on-line cycloaddition reaction with 3-benzoylpyridine. The product parent ion at m / z 462.2 detected was collected for the secondary mass spectrum in the MS / MS mode, and the derivative ion fragment peaks at m / z 210.2 and m / z 252.2 indicating the carbon-carbon double bonds at the Δ15 and Δ12 positions were found among the derivative fragments. + The signal intensities of the derivative ion fragment peaks at m / z 210.2 and m / z 252.2 were detected, and the signal intensity ratio Rc of m / z 210.2 and m / z 252.2 was calculated and substituted into the linear equation of the signal intensity ratio and the molar ratio, Rc = 1.120r + 0.1509. Finally, normalization was carried out to obtain the ω-3 lipid purity of the four samples (

[0097] ) Figure 10 )

[0098] Comparative Example 1:

[0099] The online lipid isomer method of the present invention was used to compare the oxidation degrees of the lipids of four ω-3 supplements in the off-line reaction and the on-line reaction.

[0100] The specific steps are as follows:

[0101] (1) Each ω-3 supplement was separately transferred to a 2 mL brown glass bottle, and the air in the bottle was replaced with nitrogen. Subsequently, 100 μL of the ω-3 supplement was dissolved in 1000 μL of the solvent (methanol:chloroform = 8:2).

[0102] (2) A Bruker autoflex maX MALDI TOF / TOF mass spectrometer equipped with a 355 nm SmartBeam II laser was used for the identification and oxidation degree analysis of oxidized lipids.

[0103] (4) 84 mg mL -1 of the sample was mixed with methanol (underdivatized sample) or 100 mmol L -13-Benzoyl pyridine (offline-derivatized sample) was mixed (1:1, v / v), and the offline-derivatized sample was irradiated with a 365 nm ultraviolet lamp for 15 minutes for an offline reaction. The non-derivatized sample (lipids only), the offline reaction sample (lipids and 3-benzoyl pyridine from the offline reaction), and the online reaction sample (lipids and 3-benzoyl pyridine) were simultaneously detected using MALDI-TOF MS method.

[0104] (5) Search for the high-resolution m / z values of possible oxidized lipids in the full-scan MS, focusing on the oxidized forms of the identified unsaturated lipids, such as epoxidation (+O), peroxidation (+2O), and ozonation (+3O). The degree of oxidation was evaluated using the percentage of the signal of each oxidized form of the lipid relative to the lipid signal ( Figure 11 ).

[0105] Compared with the offline derivatization reaction, online derivatization can provide almost the same information on the degree of lipid oxidation as the non-derivatized sample. This is because the rapid, online formation of cycloaddition products enhanced by the titanium dioxide matrix does not interfere with the generation of additional lipid oxidation by-products, and the MALDI TOF MS vacuum environment protects the lipid oxidation information. In contrast, the offline derivatization reaction requires long-term ultraviolet irradiation, which leads to the generation of more additional oxidation by-products and destroys the original lipid oxidation information of the lipid sample.

Claims

1. A detection method for ω-3 type lipids based on inorganic matrix enhanced MALDI mass spectrometry, characterized in that, It includes the following steps: Step 1: Mix the sample containing ω-3 lipids with an inorganic matrix and a cycloaddition reagent, spot it on the sample target of the MALDI mass spectrometer and air-dry it. Step 2: Feed the spotted target plate into the MALDI MS to perform an on-line cycloaddition reaction driven by MALDI laser, and use the MALDI mass spectrometer to ionize and perform mass spectrometry analysis on the sample containing ω-3 lipids. The working parameters of the MALDI mass spectrometer are a laser frequency of 5 Hz to 200 Hz and a laser power of 10-100% of the maximum power of the instrument. Step 3: The reaction mode used in the MALDI mass spectrometry analysis is the 355 nm laser pulse equipped with the MALDI mass spectrometer, and the category and structure of ω-3 lipids are identified based on the mass spectrometry data. The inorganic matrix is anatase or rutile titanium dioxide, cerium oxide, zinc oxide, copper oxide, graphitic carbon nitride, iron(III) oxide, tin dioxide, magnesium oxide, aluminum oxide, iron titanate, barium titanate or lead titanate, and the particle size is 5-150 nm. The cycloaddition reagent is benzo[g]isoquinoline-5,10-dione, 2-benzoylpyridine, 3-benzoylpyridine or 4-benzoylpyridine.

2. The detection method of ω-3 type lipids based on inorganic matrix enhanced MALDI mass spectrometry according to claim 1, characterized in that, The sample containing ω-3 lipids includes fish oil, seal oil, krill oil, perilla oil or olive oil.

3. The detection method of ω-3 type lipids based on inorganic matrix enhanced MALDI mass spectrometry according to claim 1, wherein, The concentration range of the inorganic matrix is from 0.75 mg / mL -1 to 20 mg / mL -1 , the concentration of the cycloaddition reagent is 1 - 100 mM, and the concentration of hydrochloric acid is 0.1 - 100 mM.

4. The detection method of ω-3 type lipids based on inorganic matrix enhanced MALDI mass spectrometry according to claim 1, wherein The reaction equivalent ratio of the sample containing ω-3 lipids to the cycloaddition reagent is 0.5:1 to 16:

1.

5. The detection method of ω-3 type lipids based on inorganic matrix enhanced MALDI mass spectrometry according to claim 1, wherein, The cycloaddition reagent is 3-benzoylpyridine; the inorganic matrix is 25 nm anatase titanium dioxide.

6. The detection method of ω-3 type lipids based on inorganic matrix enhanced MALDI mass spectrometry according to claim 1, wherein, The volume ratio of the sample containing ω-3 lipids: inorganic matrix: cycloaddition reagent is 1:2:

1.

7. The detection method of ω-3 type lipids based on inorganic matrix enhanced MALDI mass spectrometry according to claim 1, characterized in that, Step 2 is specifically as follows: Open the mass spectrometry software, after the instrument is evacuated, adjust the method to the RP, RN or LIFT mode, adjust the working parameters of the MALDI mass spectrometer to a laser frequency of 5 Hz to 200 Hz and a laser power of 10-100% of the maximum power; click the start button, manually move the laser spot to the entire area of the sample point to ensure complete reaction, and each sample point stays for 5-30 s to ensure sufficient on-line cycloaddition reaction.

8. The detection method of ω-3 type lipids based on inorganic matrix enhanced MALDI mass spectrometry according to claim 1, wherein, Lipid content quantification method: In the mass spectrometry results, take the ratio R = T / S of the signal intensity T of the mass-to-charge ratio of the mass spectrometry target signal molecule fragment and the signal intensity S of the mass-to-charge ratio of the internal standard fragment within the same time as the quantification basis, obtain a linear equation using the detection results of the standard lipid sample, and calculate the concentration of the target lipid double bond isomer in the sample to be measured.

9. The detection method of ω-3 type lipids based on inorganic matrix enhanced MALDI mass spectrometry according to claim 1, wherein, Lipid isomer relative quantification method: In the mass spectrometry results, take the signal intensities T1 and T2 of the mass-to-charge ratios corresponding to the product fragments of two lipid isomers as the quantification basis, and obtain the molar ratio of the lipid double bond isomers using R2 = T1 / T2.

10. An ω-3 lipid structure identification system constructed based on the method according to any one of claims 1 to 9, characterized in that, It includes: A sample processing unit for mixing the sample containing ω-3 lipids with an inorganic matrix and a cycloaddition reagent. A MALDI mass spectrometer connected to the sample processing unit for ionizing and performing mass spectrometry analysis on the mixed sample. A data processing unit for receiving mass spectrometry data of a MALDI mass spectrometer and identifying the class of ω-3 lipids, the relative content of ω-3 / ω-6 lipids, and the degree of oxidation of the ω-3 lipid sample.

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