High-resolution mass spectrometry for the detection of hidden mycotoxins in grains and their products

Abstract

The present invention discloses a high-resolution mass spectrometry detection method for concealed mycotoxins in grains and their products, which belongs to the field of instrument detection technology and includes the following steps: S1. Sample extraction: using acetonitrile aqueous solution as an extracting solution, placing magnetic iron oxide nanoparticle-nanocage composite material and ceramic homogenizer into the sample, freezing, centrifuging and then blowing to volume with nitrogen; S2. Purification: transferring the extract obtained in S1 to a purification tube, vortex centrifuging and filtering after micro-solid phase extraction; S3. Detecting the filtrate by liquid chromatography-quadrupole-time of flight mass spectrometry. The problem that mycotoxins are difficult to detect in the prior art due to the large difference in the physicochemical properties and the lack of high-resolution mass spectrometry technology for detecting mycotoxins and concealed mycotoxins is solved, and at the same time, the problem that traditional pre-treatment methods and detection technologies are difficult to apply to the screening and confirmation of dozens of common mycotoxins and concealed toxins is solved. It has the advantages of a large regulatory scope and high detection efficiency.

Description

Technical Field

[0001] The present invention relates to the field of instrument detection technology, and in particular to a high-resolution mass spectrometry detection method for concealed mycotoxins in grains and their products. Background Art

[0002] Mycotoxin contamination is one of the important factors affecting global food safety. Mycotoxins are secondary metabolites produced by fungi, which have carcinogenic, teratogenic, mutagenic, immunotoxic and estrogenic effects on humans and animals. At present, mycotoxins have been listed as the most important chronic dietary risk factor, and their risk level is higher than that of plant toxins, pesticide residues and organic synthetic pollutants. In addition, under natural conditions, mycotoxin molecules can combine with highly polar groups such as glucose, malonic acid or glutathione to generate hidden mycotoxins. Hidden mycotoxins often exist in grains and their products at the same time as their prototypes. After entering the bodies of humans and animals, hidden toxins can release toxic toxin prototype molecules through metabolic processes such as hydrolysis and oxidation-reduction, posing serious hidden dangers to food safety and the health of humans and animals.

[0003] In recent years, the rapid screening and detection of multiple compounds simultaneously has become one of the research hotspots in the field of residue analysis. At present, the most commonly used methods for detecting mycotoxins and hidden toxins are liquid chromatography-mass spectrometry (LC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS / MS), which have the advantages of high separation, high selectivity, and high sensitivity, and are ideal detection methods for trace analytes in trace samples or complex matrix samples. However, the mass spectra of LC-MS and LC-MS / MS are mostly low-resolution mass spectra, which are difficult to fully elucidate the structural fragmentation information of the compounds, and may produce false positive phenomena. They have certain limitations when dealing with multiple target screening objects in complex matrix samples. High-resolution mass spectrometry (HRMS) can make up for the above limitations. More and more food safety experts realize that the "screening + confirmation" analysis mode using HRMS has significant advantages in reducing regulatory loopholes, improving efficiency, and reducing costs. In recent years, the rapid development of HRMS has indeed provided strong technical support for the "screening + confirmation" analysis model. The liquid chromatography-high-resolution mass spectrometry analysis technology based on HRMS has received more and more attention and application in trace multi-component detection. It can determine the precise molecular mass of compounds and their fragment ions at ultra-high resolution, has strong anti-interference ability, and is suitable for high-throughput screening and confirmation analysis of target compounds. At the same time, due to the large differences in the physical and chemical properties of mycotoxins, traditional pretreatment methods are difficult to apply. For example, traditional mycotoxin purification mostly relies on multifunctional extraction columns for extracting toxins, which have many purification steps, few coverage components, and low detection efficiency. It is very necessary to establish a sample pretreatment method that matches HRMS detection.

[0004] Therefore, it is urgent to establish a method that can screen and confirm dozens of common mycotoxins and hidden toxin components in complex matrices through a single analysis. By building a screening database of 40 mycotoxins and hidden mycotoxins, combined with improved rapid pre-treatment methods, we can achieve improvements in regulatory scope, detection technology, and timeliness. Summary of the invention

[0005] To this end, the present invention provides a high-resolution mass spectrometry detection method for concealed mycotoxins in grains and their products, so as to solve the problem in the prior art that traditional pretreatment methods and detection technologies are difficult to apply to the screening and confirmation of dozens of common mycotoxins and concealed toxin components due to the large differences in the physicochemical properties of mycotoxins and the lack of technology for high-resolution mass spectrometry detection of mycotoxins and concealed mycotoxins.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The high-resolution mass spectrometry method for detecting hidden mycotoxins in grains and their products provided by the present invention comprises the following steps:

[0008] S1. Sample extraction: using acetonitrile aqueous solution as the extracting solution, placing magnetic iron oxide nanoparticles-nanocage composite materials and ceramic homogenizers into the sample, freezing, centrifuging, and then blowing with nitrogen to constant volume;

[0009] S2. Purification: transfer the extract obtained in S1 into a purification tube, vortex centrifuge, perform micro-solid phase extraction, and then filter;

[0010] S3. performing liquid chromatography-quadrupole-time of flight mass spectrometry detection on the filtrate;

[0011] S4. Draw a matrix matching standard curve: prepare a blank extract according to the extraction and purification steps, and prepare a matrix standard working solution;

[0012] S5. Establish a high-resolution mass spectrometry database.

[0013] Furthermore, the specific steps of extracting the S1 sample are:

[0014] (1) Accurately weigh 5 g of grain sample into a 50 mL centrifuge tube, then add magnetic iron oxide nanoparticle-nanocage composite material, ceramic homogenizer, 20 mL of 79% acetonitrile aqueous solution and CHO4P, vortex for 3 min, centrifuge at 9500 rpm for 10 min, and aspirate the supernatant into a clean 50 mL centrifuge tube;

[0015] (2) Place the centrifuge tube in a -30℃ freezer for 1 hour, add MgSO4, NaCl, Na3C6H5O7, Na2C6H6O7·3H2O and C6H 14, vortex oscillation for 3 minutes, centrifuge at 9500r / min for 10 minutes, transfer all the supernatant to a 15mL centrifuge tube, blow nitrogen at 40℃ to less than 5mL, make the volume with acetonitrile to 5mL, and mix well.

[0016] Furthermore, the preparation method of the magnetic iron oxide nanoparticle-nanocage composite material is:

[0017] (1) ultrasonically dispersing magnetic iron oxide nanoparticles in a nitric acid solution, stirring evenly and standing for 20-40 minutes, washing the magnetic iron oxide nanoparticles with deionized water for 3-6 times, and then ultrasonically dispersing them in deionized water to obtain an intermediate solution, placing the intermediate solution in a sealed environment, keeping it warm at 180-200° C. for 15-20 hours, and then washing and drying the reactant to obtain iron oxide magnetic nanoparticles;

[0018] (2) Weigh 2.0 g of iron oxide magnetic nanoparticles and 1.25 g of copper acetate, heat them, and fully calcine them in a tubular furnace. A mixed gas of Ar / H2 with a volume percentage concentration of 95% / 5% is passed through the tubular furnace. The temperature is set to first increase from 25°C to 500-900°C at a rate of 5°C / min and keep warm for 2 hours. After the reaction is completed, the temperature is cooled to room temperature at a rate of 10°C / min to obtain a magnetic iron oxide nanoparticle-nanocage composite material.

[0019] Furthermore, the specific steps of the S2 purification are:

[0020] (1) Transfer the extract obtained in S1 to an activated 15 mL Bond Elut EMR-Lipid dSPE cleanup tube, vortex mix for 2 min, and quickly centrifuge at 9500 rpm for 10 min at 10°C to obtain the supernatant;

[0021] (2) adding the supernatant to a micro solid phase extraction column, adding an ethanol solvent mixed with sodium chloride as an eluent for elution, collecting the eluate, centrifuging the eluate, taking the intermediate liquid, passing it through a 0.22 μm organic filter membrane, and providing it for detection by a liquid chromatography-quadrupole-time of flight mass spectrometer.

[0022] Furthermore, the parameters of the liquid chromatography-quadrupole-time of flight mass spectrometry detection in S3 include:

[0023] a) Chromatographic column: AcquityUPCLHSST3;

[0024] b) Mobile phase A: 0.1% formic acid in water, mobile phase B: 50% methanol / acetonitrile solution containing 0.1% formic acid in water;

[0025] The elution method is gradient elution, and the gradient elution program is:

[0026] 0-3min, 95% mobile phase A, 5% mobile phase B;

[0027] From 3 to 9 minutes, mobile phase A was linearly reduced to 1%, and mobile phase B was linearly increased to 99% and maintained for 6 minutes;

[0028] From 15 to 15.1 min, mobile phase A was linearly increased to 95%, and mobile phase B was linearly decreased to 5% and maintained for 4.9 min.

[0029] Furthermore, the mass spectrometry conditions include:

[0030] HESI ionization mode was adopted; spray voltage was 5500V; capillary temperature was 325℃; heater temperature was 550℃; Gas was 155psi; Gas was 260psi; Curtain Gas was 35unit; CAD Gas was 7unit; scanning mode was: SWATH / TOF-MS / TOF-MS / MS; positive ion acquisition mode; TOF-MS parameters: acquisition range was 150-785m / z; DP voltage was 80V; collision capacity was CE5V; accumulation time was 0.16s; TOF-MS / MS parameters: acquisition range was 50-785m / z; accumulation time was 0.06s; charge number was 1.

[0031] Furthermore, the method of establishing a high-resolution mass spectrometry database in S5 is:

[0032] Under chromatographic separation conditions, 40 common mycotoxins and hidden mycotoxins were analyzed to obtain the mass numbers of calculated compound sum ions as mass spectrometry theoretical information; standard spectra and characteristic fragments were collected as primary and secondary mass spectrometry information; search and rapid comparison methods were established; after obtaining sample data, the collected mass spectrometry information was quickly searched for matching with hundreds of compounds collected in the database to compare samples and conduct mass spectrometry confirmation.

[0033] Furthermore, the activation process of the 15 mL Bond Elut EMR-Lipid dSPE purification tube in S2 is as follows: before use, the purification tube is placed in a container, 3 mL of ethyl acetate and 5 mL of deionized water are added, and the purification tube is washed with acetate buffer and vortexed for 30 seconds for activation.

[0034] Furthermore, 4 g of MgSO4, 1 g of NaCl, 1 g of Na3C6H5O7, 0.5 g of Na2C6H6O7·3H2O and 0.5 g of C6H 14 The volume ratio of the 79% acetonitrile aqueous solution is 79:20:1 for acetonitrile / water / acetic acid.

[0035] Furthermore, the sample grains in S1 are specifically soybeans, corn, rice or wheat, and are weighed to an accuracy of 0.01 g.

[0036] The present invention has the following advantages:

[0037] 1. This application uses high-performance liquid chromatography-quadrupole / time-of-flight mass spectrometry to determine 40 fungal toxins in grains, and can complete high-throughput screening and confirmation of 40 fungal toxins within 20 minutes. High-resolution mass spectrometry eliminates matrix interference in sample analysis, and combined with improved pretreatment and cleanup methods, it greatly improves the qualitative accuracy and quantitative limit of the method.

[0038] 2. In the pre-treatment process of the present application, a magnetic iron oxide nanoparticle-nanocage composite material is added, which can use a magnetic field to quickly separate the target toxin from a complex sample matrix, thereby achieving efficient and rapid separation and purification; shortening the sample pre-treatment time and improving the efficiency of analysis; the magnetic iron oxide nanoparticle-nanocage composite material has a large specific surface area and adsorption sites, which can enhance the adsorption performance of the sample, effectively enrich trace target toxins, protect the target toxins from damage and loss, improve the sensitivity and accuracy of detection, reduce analytical errors and reduce detection limits, thereby better meeting the restrictions of regulations and standards.

[0039] 3. In this application, the dSPE purification tube is combined with micro solid phase extraction to effectively remove interfering substances in the sample, improve the recovery rate and purity of the target, and thus obtain more accurate analysis results. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0041] The structures, proportions, sizes, etc. illustrated in this specification are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size shall still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0042] Figure 1(a)-Figure 1(g)It is an extracted ion current chromatogram of 10ug / L matrix standard solution of 40 kinds of mycotoxins in the rice matrix provided in Example 2 of the present invention, specifically including Figure 1 (a), Figure 1 (b), Figure 1 (c), Figure 1 (d), Figure 1 (e), Figure 1 (f) and Figure 1 (g);

[0043] Figure 2 This is a comparative analysis chart of the recovery rates of typical mycotoxins when using different extraction solvents provided in Example 3 of the present invention;

[0044] In the picture: 1.acetonitrile / water (50: 50, v / v); 2. acetonitrile / water / aceticacid (50: 49: 1, v / v / v); 3. acetonitrile / water (70: 30, v / v); 4.acetonitrile / water / aceticacid (70: 29: 1, v / v / v); 5. acetonitrile / water (84: 16, v / v); 6. acetonitrile / water / acetic acid(84:15:1,v / v / v);

[0045] Figure 3 It is a full scan chromatogram of the sample solution of untreated soybean provided in Example 6 of the present invention, after PSA / C18 dSPE purification and EMR-Lipid dSPE treatment, wherein A is soybean--QuEChERS extract before dSPE, B is soybean--QuEChERS extract obtained by C18 / PSA purification, and C is soybean--QuEChERS extract obtained by EMR-Lipid purification;

[0046] Figure 4 The four matrix effect quantity diagrams of 40 species provided in Example 6 of the present invention;

[0047] Figure 5 The full scan chromatogram and TOF-MS and TOF-MS / MS mass spectra of aflatoxin B1 provided in Example 7 of the present invention. DETAILED DESCRIPTION

[0048] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] The high-resolution mass spectrometry method for detecting hidden mycotoxins in grains and their products provided by the present invention comprises the following steps:

[0050] S1. Sample extraction: using acetonitrile aqueous solution as the extraction solution, placing magnetic iron oxide nanoparticle-nanocage composite material and a ceramic homogenizer into the sample, freezing, centrifuging and then blowing with nitrogen to make it constant volume;

[0051] S2. Purification: transfer the extract obtained in S1 into a purification tube, vortex centrifuge, perform micro-solid phase extraction, and then filter;

[0052] S3. performing liquid chromatography-quadrupole-time of flight mass spectrometry detection on the filtrate;

[0053] S4. Draw a matrix matching standard curve: prepare a blank extract according to the extraction and purification steps, and prepare a matrix standard working solution;

[0054] S5. Establish a high-resolution mass spectrometry database.

[0055] The specific steps of S1 sample extraction are:

[0056] (1) Accurately weigh 5 g of grain sample into a 50 mL centrifuge tube, then add magnetic iron oxide nanoparticle-nanocage composite material, ceramic homogenizer, 20 mL of 79% acetonitrile aqueous solution and CHO4P, vortex for 3 min, centrifuge at 9500 rpm for 10 min, and aspirate the supernatant into a clean 50 mL centrifuge tube;

[0057] (2) Place the centrifuge tube in a -30℃ freezer for 1 hour, add MgSO4, NaCl, Na3C6H5O7, Na2C6H6O7·3H2O and C6H 14 , vortex oscillation for 3 minutes, centrifuge at 9500r / min for 10 minutes, transfer all the supernatant to a 15mL centrifuge tube, blow nitrogen at 40℃ to less than 5mL, make the volume with acetonitrile to 5mL, and mix well.

[0058] The preparation method of the magnetic iron oxide nanoparticle-nanocage composite material is as follows:

[0059] (1) ultrasonically dispersing magnetic iron oxide nanoparticles in a nitric acid solution, stirring evenly and standing for 20-40 minutes, washing the magnetic iron oxide nanoparticles with deionized water for 3-6 times, and then ultrasonically dispersing them in deionized water to obtain an intermediate solution, placing the intermediate solution in a sealed environment, keeping it warm at 180-200° C. for 15-20 hours, and then washing and drying the reactant to obtain iron oxide magnetic nanoparticles;

[0060] (2) Weigh 2.0 g of iron oxide magnetic nanoparticles and 1.25 g of copper acetate, heat them, and fully calcine them in a tubular furnace. A mixed gas of Ar / H2 with a volume percentage concentration of 95% / 5% is passed through the tubular furnace. The temperature is set to first increase from 25°C to 500-900°C at a rate of 5°C / min and keep warm for 2 hours. After the reaction is completed, the temperature is cooled to room temperature at a rate of 10°C / min to obtain a magnetic iron oxide nanoparticle-nanocage composite material.

[0061] The specific steps of S2 purification are:

[0062] (1) Transfer the extract obtained in S1 to an activated 15 mL Bond Elut EMR-Lipid dSPE cleanup tube, vortex mix for 2 min, and quickly centrifuge at 9500 rpm for 10 min at 10°C to obtain the supernatant;

[0063] (2) adding the supernatant to a micro solid phase extraction column, adding an ethanol solvent mixed with sodium chloride as an eluent for elution, collecting the eluate, centrifuging the eluate, taking the intermediate liquid, passing it through a 0.22 μm organic filter membrane, and providing it for detection by a liquid chromatography-quadrupole-time of flight mass spectrometer.

[0064] The parameters for liquid chromatography-quadrupole-time of flight mass spectrometry detection in S3 include:

[0065] a) Chromatographic column: AcquityUPCLHSST3;

[0066] b) Mobile phase A: 0.1% formic acid in water, mobile phase B: 50% methanol / acetonitrile solution containing 0.1% formic acid in water;

[0067] The elution method is gradient elution, and the gradient elution program is:

[0068] 0-3min, 95% mobile phase A, 5% mobile phase B;

[0069] From 3 to 9 minutes, mobile phase A was linearly reduced to 1%, and mobile phase B was linearly increased to 99% and maintained for 6 minutes;

[0070] From 15 to 15.1 min, mobile phase A was linearly increased to 95%, and mobile phase B was linearly decreased to 5% and maintained for 4.9 min.

[0071] Mass spectrometry conditions included:

[0072] HESI ionization mode was adopted; spray voltage was 5500V; capillary temperature was 325℃; heater temperature was 550℃; Gas was 155psi; Gas was 260psi; curtain gas was 35unit; CADGas was 7unit; scanning mode was SWATH / TOF-MS / TOF-MS / MS; positive ion acquisition mode; TOF-MS parameters were as follows: acquisition range was 150-785m / z; DP voltage was 80V; collision capacity was CE5V; accumulation time was 0.16s; TOF-MS / MS parameters were as follows: acquisition range was 50-785m / z; accumulation time was 0.06s; charge number was 1.

[0073] S5 The method for establishing a high-resolution mass spectrometry database is:

[0074] Under chromatographic separation conditions, 40 common mycotoxins and hidden mycotoxins were analyzed to obtain the mass numbers of calculated compound sum ions as mass spectrometry theoretical information; standard spectra and characteristic fragments were collected as primary and secondary mass spectrometry information; search and rapid comparison methods were established; after obtaining sample data, the collected mass spectrometry information was quickly searched for matching with hundreds of compounds collected in the database to compare samples and conduct mass spectrometry confirmation.

[0075] The activation process of the 15mL Bond Elut EMR-Lipid dSPE cleanup tube in S2 is as follows: before use, place the cleanup tube in a container, add 3mL of ethyl acetate and 5mL of deionized water, wash the cleanup tube with pH 4-5 acetate buffer, and vortex for 30s to activate.

[0076] Add 4g MgSO4, 1g NaCl, 1g Na3C6H5O7, 0.5g Na2C6H6O7·3H2O and C6H 14 0.5 g; 79% acetonitrile aqueous solution has a volume ratio of acetonitrile / water / acetic acid of 79:20:1.

[0077] The sample grains in S1 are specifically soybeans, corn, rice, wheat, etc., and are weighed to an accuracy of 0.01g.

[0078] Materials and Methods

[0079] 1. Instruments and Equipment

[0080] Liquid chromatography-quadrupole time-of-flight mass spectrometer (X500R QTOF, ABSCIEX, USA); HITACHI TF-123 tissue chopper (HITACHI, Japan); vortex mixer (Thermo, USA); Sigma low-temperature centrifuge (maximum speed 10000r / min, Sigma, USA); Gilson pipette (adjustable range: 10-100μL, 20-200μL, 10-1000μL, 1000-5000μL, Gilson, France).

[0081] 2. Reagents and Materials

[0082] Homogenizer (Omni, USA); vortex oscillator (Heidolph, Germany); high-speed refrigerated centrifuge (Sigma, Germany); high-purity water generator (Millipore, USA); 0.22 μm organic filter membrane.

[0083] Methanol, acetonitrile, n-hexane, formic acid, and ammonium acetate were all chromatographically pure (Merck, Germany); anhydrous MgSO4, NaCl, sodium citrate, and disodium citrate trihydrate were all analytically pure; water was Milli-Q ultrapure water; 15mL Bond Elut EMR-Lipid dSPE purification tube (Agilent, USA). The purity of the 40 mycotoxin standard mixed standard was ≥97% (Alta, Tianjin), and the internal standard solution was 13C17-15-A-DON, with a concentration of 10.0 mg / L (stored at -18°C away from light). The matrix blank sample solution was used to dilute and prepare the mixed standard working solution, and the concentrations of the standard curve series solutions were 0.005, 0.010, 0.025, 0.050, and 0.100 mg / L, respectively, and the internal standard concentration was 0.050 mg / L.

[0084] Samples: soybeans, corn, rice or wheat, all of which are commercially available samples.

[0085] 3. Instrument parameters

[0086] 3.1 Chromatographic conditions:

[0087] a) Chromatographic column: Acquity UPCL HSST3, 1.8 μm, 150 mm × 2.1 mm (id) (Waters, USA);

[0088] b) Mobile phase A: 0.1% formic acid in water, B: 50% methanol / acetonitrile solution (containing 0.1% formic acid in water), mobile phase gradient is shown in Table 1;

[0089] c) Flow rate: 300 μL / min;

[0090] d) Injection volume: 10L;

[0091] e) Column temperature: 25°C.

[0092] The elution method is gradient elution. The gradient elution program is shown in Table 1:

[0093] Table 1 Liquid chromatography gradient elution program

[0094]

[0095] 3.2 Mass spectrometry conditions

[0096] Ion source and gas parameters: HESI ionization mode; spray voltage 5500V; capillary temperature 325℃; heater temperature 550℃; Gas 155psi; Gas 260psi; Curtain Gas 35unit; CAD Gas 7unit; scanning mode: SWATH / TOF-MS / TOF-MS / MS; positive ion acquisition mode. TOF-MS parameters: acquisition range 150-785m / z; DP voltage: 80V; collision capacity CE5V; accumulation time: 0.16s. TOF-MS / MS parameters: acquisition range 50-785m / z; accumulation time: 0.06s; charge number 1; mass spectrometry scanning parameters are shown in Table 2.

[0097] Table 2: TOF-MS / MS mass spectrometry scanning parameters

[0098]

[0099] Example 1

[0100] This embodiment provides a high-resolution mass spectrometry method for detecting hidden mycotoxins in grains and their products, comprising the following steps:

[0101] The specific steps of S1 sample extraction are:

[0102] (1) Accurately weigh 5 g of grain sample into a 50 mL centrifuge tube, then add 7 mg of magnetic iron oxide nanoparticle-nanocage composite material, 1 ceramic homogenizer, 20 mL of 79% acetonitrile aqueous solution and CHO4P, vortex for 3 min, centrifuge at 9500 rpm for 10 min, and aspirate the supernatant into a clean 50 mL centrifuge tube;

[0103] (2) Place the centrifuge tube in a -30℃ freezer for 1 hour, add MgSO4 2g, NaCl 3g, Na3C6H5O7 0.8g, Na2C6H6O7·3H2O 0.7g and C6H 141.5mL, vortex oscillation for 3min, centrifuge at 9500r / min for 10min, transfer all the supernatant to a 15mL centrifuge tube, blow nitrogen at 40℃ until it is less than 5mL, make the volume with acetonitrile to 5mL, and mix well to obtain the extract.

[0104] Wherein, the preparation method of the magnetic iron oxide nanoparticle-nanocage composite material is:

[0105] (1) ultrasonically dispersing 0.15 g of magnetic iron oxide nanoparticles with a diameter of 40 nm in 30 mL of a 2% nitric acid solution, stirring evenly and standing for 20-40 min, washing the magnetic iron oxide nanoparticles with deionized water for 3-6 times, and then ultrasonically dispersing them in 50 mL of deionized water for 40 min to obtain an intermediate solution, placing the intermediate solution in a sealed environment, and keeping it at 180-200° C. for 15-20 h, and then washing and drying the resulting reactant to obtain iron oxide magnetic nanoparticles;

[0106] (2) Weigh 2.0 g of iron oxide magnetic nanoparticles and 1.25 g of copper acetate, heat them, and fully calcine them in a tubular furnace. A mixed gas of Ar / H2 with a volume percentage concentration of 95% / 5% is passed through the tubular furnace. The temperature is set to first increase from 25°C to 500-900°C at a rate of 5°C / min and keep warm for 2 hours. After the reaction is completed, the temperature is cooled to room temperature at a rate of 10°C / min to obtain a magnetic iron oxide nanoparticle-nanocage composite material.

[0107] The specific steps of S2 purification are:

[0108] (1) Transfer the extract obtained in S1 to an activated 15 mL Bond Elut EMR-Lipid dSPE cleanup tube, vortex mix for 2 min, and quickly centrifuge at 9500 rpm for 10 min at 10°C to obtain the supernatant;

[0109] (2) adding all the supernatant to a micro solid phase extraction column, adding 95% ethanol solvent mixed with sodium chloride as an eluent for elution, collecting the eluate, centrifuging the eluate, taking the intermediate liquid, passing it through a 0.22 μm organic filter membrane, and providing it for detection by liquid chromatography-quadrupole-time of flight mass spectrometry.

[0110] The parameters for liquid chromatography-quadrupole-time of flight mass spectrometry detection in S3 include:

[0111] a) Chromatographic column: AcquityUPCLHSST3;

[0112] b) Mobile phase A: 0.1% formic acid in water, mobile phase B: 50% methanol / acetonitrile solution containing 0.1% formic acid in water;

[0113] The elution method is gradient elution, and the gradient elution program is:

[0114] 0-3min, 95% mobile phase A, 5% mobile phase B;

[0115] From 3 to 9 minutes, mobile phase A was linearly reduced to 1%, and mobile phase B was linearly increased to 99% and maintained for 6 minutes;

[0116] From 15 to 15.1 min, mobile phase A was linearly increased to 95%, and mobile phase B was linearly decreased to 5% and maintained for 4.9 min.

[0117] Mass spectrometry conditions included:

[0118] HESI ionization mode was adopted; spray voltage was 5500V; capillary temperature was 325℃; heater temperature was 550℃; Gas was 155psi; Gas was 260psi; Curtain Gas was 35unit; CAD Gas was 7unit; scanning mode was: SWATH / TOF-MS / TOF-MS / MS; positive ion acquisition mode; TOF-MS parameters: acquisition range was 150-785m / z; DP voltage was 80V; collision capacity was CE5V; accumulation time was 0.16s; TOF-MS / MS parameters: acquisition range was 50-785m / z; accumulation time was 0.06s; charge number was 1.

[0119] S4. Draw a matrix matching standard curve: prepare a blank extract according to the extraction and purification steps, and prepare a matrix standard working solution;

[0120] S5 The method for establishing a high-resolution mass spectrometry database is:

[0121] Under chromatographic separation conditions, 40 common mycotoxins and hidden mycotoxins were analyzed to obtain the mass numbers of calculated compound sum ions as mass spectrometry theoretical information; standard spectra and characteristic fragments were collected as primary and secondary mass spectrometry information; search and rapid comparison methods were established; after obtaining sample data, the collected mass spectrometry information was quickly searched for matching with hundreds of compounds collected in the database to compare samples and conduct mass spectrometry confirmation.

[0122] Example 2

[0123] Optimization of chromatographic separation conditions

[0124] (1) Selection of chromatographic column

[0125] Since most of the 40 target mycotoxins in this application are weakly polar compounds, and some are moderately polar, the Acquity UPCL HSS T3 column with reversed-phase bonded polar groups was selected, which can retain both weakly polar compounds and water-soluble, highly polar organic compounds. The results showed that all 40 compounds were well separated and retained.

[0126] (2) Selection and optimization of mobile phase

[0127] In this example, the chromatographic separation effects of the above 40 substances in acetonitrile-water, methanol-water and 50% acetonitrile / methanol-water mobile phase systems [0.1% formic acid, 0.005 mol / L ammonium acetate (containing 0.01% (v / v) formic acid, pH about 4.5) and 0.005 mol / L ammonium acetate (containing 0.1% (v / v) formic acid, pH about 3.2) were added to the aqueous phase and the organic phase, respectively] were compared.

[0128] There are differences in the separation of target compounds under different mobile phase conditions, and the mass spectrometry response strengths are different. In the methanol mobile phase system, most compounds have a strong response and relatively good separation, but the peak shape of individual compounds is slightly poor. Under acetonitrile mobile phase conditions, the peak shape of many compounds is better, but the separation of isomers is poor. The advantage of a 50% acetonitrile / methanol mixed mobile phase is that isomers are better separated, and both peak shape and separation can be taken into account. This may be due to the elution ability and viscosity differences of acetonitrile and methanol. The results show that the optimal chromatographic peak shape and mass spectrometry response were obtained when a mixed solution of 0.1% formic acid water-50% methanol / acetonitrile solution (containing 0.1% formic acid water) was used as the mobile phase. Figure 1(a) to Figure 1(g) It can be seen that the 40 fungal toxins are well retained and distributed in the chromatographic column, and the chromatographic peaks are evenly distributed in each time period of the mobile phase gradient, indicating that the separation effect is optimal.

[0129] Example 3

[0130] Since the polarity of the 40 kinds of mycotoxins in this application is quite different, a magnetic iron oxide nanoparticle-nanocage composite material is added during the pre-treatment process. The nanoscale size of the magnetic iron oxide nanoparticle-nanocage composite material is fully in contact with other components in the sample to improve the adsorption effect. Secondly, the magnetic iron oxide nanoparticles have magnetic responsiveness and can be easily separated and manipulated by magnetic force, which simplifies the sample processing process. In addition, the magnetic iron oxide nanoparticle-nanocage composite material also has a porous structure to provide a larger specific surface area, further enhancing its adsorption capacity, and then quickly separating the target toxin from the complex sample matrix.

[0131] When selecting the extraction solvent, the properties of the solvent, the properties of the fungal toxins and the characteristics of the sample matrix should be considered. Commonly used extraction reagents include acetonitrile, methanol, acetic acid, ethyl acetate and deionized water. The extraction effect of methanol-water solution as the extraction reagent is not as good as that of acetonitrile-water solution. Acetonitrile has good solubility and strong permeability, and is suitable for extracting a relatively wide range of fungal toxins with polarity. Acetonitrile can promote the dissolution of fungal toxins from tissues and improve the extraction efficiency through proper acidification. Acetonitrile water can increase the extraction efficiency of polar compounds. In this example, the extraction efficiency of 40 fungal toxins was investigated using 6 different extraction solvents (1. acetonitrile / water (50:50, v / v); 2. acetonitrile / water / formic acid (50:49:1, v / v / v); 3. acetonitrile / water (70:30, v / v); 4. acetonitrile / water / formic acid (79:20:1, v / v / v); 5. acetonitrile / water (84:16, v / v); 6. acetonitrile / water / formic acid (84:15:1, v / v / v)), and the recovery rate of the target substance with a spike level of 0.100 mg / kg in soybean matrix was used as the investigation index.

[0132] The results show that if Figure 2 As shown in the figure, the larger the proportion of acetonitrile, the more suitable it is for the extraction of most toxins (the extraction reagents with acetonitrile volume ratio of 79% and 74% are better than the acetonitrile extraction reagent with volume ratio of 49%); most toxins can be extracted well under acidic conditions, except that the extraction recovery rates of Fumonisin B1, FumonisinB2, Fumonisin B3, Deoxynivalenol, HT-2, Toxin, Alternariol monomethyl ether, Diacetoxyscirpenol, Penicillic Acid, and Alternariol are slightly lower, and the recovery rates of the remaining 30 toxins are all over 60%.

[0133] This extraction method improves the pretreatment method by adding magnetic iron oxide nanoparticle-nanocage composite materials and optimizing and examining and selecting 6 different extraction solvents to achieve efficient and rapid separation and purification.

[0134] Example 4

[0135] Optimization of purification adsorbents

[0136] Immunoaffinity columns are commonly used to analyze a variety of mycotoxins, including aflatoxins. However, these columns are expensive and require unique and diverse workflows that are not convenient for food laboratories. The fast, easy, economical, efficient, robust, and safe QuEChERS method uses a simple three-step process (extraction, cleanup, and analysis) for the sample. Therefore, this method is very promising for the pretreatment of a variety of different analytes and matrices, including aflatoxins, in cereals and dairy products. QuEChERS cleanup using C18 and PSA has some limitations when analyzing samples such as meat and milk that contain a lot of lipids. This limitation comes from non-selective interactions with the target analytes and the fact that the major lipid species are almost impossible to remove. Any remaining lipids will accumulate in the analytical flow path, resulting in increased maintenance, chromatographic anomalies, and greatly reduced data accuracy and precision.

[0137] Enhanced Matrix Removal (EMR-Lipid) is a novel adsorbent that combines unique size exclusion and hydrophobic interactions between the adsorbent and long aliphatic functional groups to selectively remove major lipids from samples without retaining other unwanted analytes. This adsorbent can be used in dSPE cleanup via QuEChERS and protein precipitation workflows, helping to achieve simple and efficient cleanup.

[0138] The sample matrix of this application is a grain sample, which is rich in lipids. If the pre-treatment purification is not good, the lipids will accumulate in the instrument and chromatographic column, thereby shortening the life of the instrument and chromatographic column, and also causing the analytical sensitivity of the analyte to decrease due to ion suppression. Figure 3 As shown, the sample extract in this application was cleaned up by EMR-LipiddSPE, which achieved excellent matrix removal.

[0139] Example 5

[0140] Sample amount optimization

[0141] The purification capacity of a certain amount of QuEChERS adsorption filler for samples is fixed. By optimizing the sample amount, not only can the purification process be made more efficient, but it also plays an important role in reducing matrix interference and improving recovery. In theory, at the same added concentration level, increasing the concentration multiple of the extract is beneficial to improving the sensitivity of the method. However, in actual sample analysis, the concentration operation not only concentrates the interfering components in the matrix while concentrating the components to be tested, but even fails to achieve the effect of improving sensitivity due to the superposition of matrix effects. Embodiment 5g soybean samples of screening concentration levels are purified by combining dSPE purification tubes and micro solid phase extraction, and the final volume is adjusted to 10mL of extract. The purified solution (50% methanol water) is difficult to concentrate, but the instrument detection sensitivity can meet the quantitative requirements, so 5g sample amount is used as the sample dosage of this application.

[0142] Example 6

[0143] Elimination of sample matrix effects

[0144] The matrix effect of mass spectrometry is mainly caused by the co-elution components of the analyte affecting the ionization efficiency of the electrospray interface, which manifests as ion enhancement or suppression. This application investigates the matrix effect of soybeans, rice, corn, and wheat. Standard samples are added after pure reagents and different matrix extracts, and the signal peak areas of the target in pure reagents and matrix solutions are compared. The relative intensity of the matrix response (ME) is (peak area of ​​matrix standard sample / peak area of ​​reagent standard sample) 100%. The results show (see Table 3 and Figure 4 ), most of the items have matrix effects in the above four matrices, among which α-zearalanol, α-zearalenol, gliotoxin, diacetoxyscirpenol, neosolaniol, resiniferatoxin (RSF-Resiniferatoxin), and penicillin A (PenitremA) show matrix enhancement effects, and other items show matrix inhibition effects, which may be related to the properties of the substances. Although the optimized purification method can effectively reduce the interfering components in the sample extract, in order to eliminate the interference of matrix effects to the greatest extent, this application uses the matrix matching method for quantification. The method uses the extract of the blank sample as the dilution solution of the standard solution, so that the standard solution and the sample solution have as similar ionization environments as possible, thereby reducing the influence of the matrix effect and ensuring the sensitivity and reproducibility of the method.

[0145] Table 3: Matrix effects of 40 compounds in soybean, rice, corn and wheat

[0146]

[0147] Example 7 Optimization of mass spectrometry conditions

[0148] (1) Resolution selection

[0149] High-resolution mass spectrometry can achieve baseline separation of target compounds and isobaric interfering substances in the matrix at an appropriate resolution, thereby achieving more accurate qualitative capabilities. When the primary mass spectrometry has a resolution of >20,000 and a mass spectrometry accuracy of ≤5ppm, complete baseline separation of the target compound and the interfering substance can be achieved, effectively removing matrix interference, and the extracted ion chromatogram is clear and complete, allowing accurate qualitative and quantitative analysis. The primary mass spectrometry and secondary mass spectrometry of this application both use a resolution of ≥35,000, and the resulting spectra have both the accurate mass number of the parent ion and the full scan information of the secondary mass spectrometry, which fully meets the qualitative and quantitative requirements. Figure 5 The SWATH scanning method was used to obtain the full scan chromatogram, TOF-MS mass spectrum and TOF-MS / MS mass spectrum of aflatoxin B1.

[0150] (2) Selection of parent ion charging method

[0151] The ion source was an electrospray ionization source, and the data were collected in positive mode. The parent ion charging mode of the 38 compounds was [M+H] + , the charging mode of the two compounds is [M+NH4] + , one compound is charged in the form of [M+Na] + (See Table 4).

[0152] (3) Level 1 Accurate Mass Database

[0153] Samples were injected under optimized chromatographic-mass spectrometry conditions, and a primary mass spectrometry full scan was performed on the 40 compounds to obtain information such as retention time, parent ion mass-to-charge ratio, mass deviation, and ionization form of the target. The name, retention time, molecular formula, and accurate relative molecular mass of the compound were entered into the software to establish a primary accurate mass database of the 40 compounds (see Table 4).

[0154] Table 4: Retention time, limit of quantification and mass spectrum information of 40 compounds (soybean matrix)

[0155]

[0156] (4) Secondary mass spectrometry library

[0157] If the response value of the target object in the retention time window exceeds the threshold, it will automatically trigger the acquisition and superposition of secondary fragment spectra at different collision energies (20, 35, 50 eV), thereby establishing a secondary information library of 40 fungal toxins, including parent ions, retention times, superimposed secondary mass spectra at different collision energies, and other information.

[0158] Example 8

[0159] According to the latest version of the EU's "Guidance Document on Quality Control and Method Validation for Pesticide Residue Analysis in Food and Feed" (SANTE / 11813 / 2017), when using high-resolution mass spectrometry for confirmation, at least one high-resolution parent ion and one characteristic fragment ion are required, and the mass deviation is ≤5ppm. This application is confirmed by the matching degree of one high-resolution parent ion and the secondary spectrum, and the mass deviation of the parent ion and the fragment ion is ≤5ppm. Taking aflatoxin B1 as an example (see Figure 5), the full scan chromatogram, TOF-MS and TOF-MS / MS mass spectra of aflatoxin B1, after deducting background interference, the parent ion and retention time deviation are ≤±0.2min, which indicates that the confirmation is passed. Otherwise, it is necessary to further confirm it through factors such as secondary spectrum matching (≥70%) and isotope precise relative mass number matching.

[0160] Example 9

[0161] Methodological validation

[0162] (1) Precision and accuracy of screening methods

[0163] Standard solutions (0.100 mg / L) of 40 compounds were added to blank matrix (soybean, corn, rice, wheat matrix) samples for determination. In the soybean matrix spiked sample solution, the deviation of the primary accurate mass number of the 40 compounds was ≤4.57×10 -6 , the matching degree of the secondary full scan mass spectra was above 73.3%, and the retention time deviations were all within ±0.2min (see Table 4).

[0164] (2) Linear relationship and quantitative limit of quantitative method

[0165] This method achieves synchronous quantitative analysis by extracting the precise relative molecular mass of the primary mass spectrometer on the basis of rapid screening. The number of collection points for each compound can be more than 20 to ensure the accuracy of the quantitative results. Standard curves were drawn with various blank matrix standard solutions, and the concentration points were 0.005 mg / L, 0.010 mg / L, 0.025 mg / L, 0.050 mg / L and 0.100 mg / L. The MS1 peak area ratio Y of each target compound and the internal standard (13C17-3Ac-DON concentration 0.050 mg / L) was used as the ordinate, and the concentration ratio X was used as the abscissa. Each target compound had a good linear relationship in the range of 0.005 mg / L-0.100 mg / L, and the correlation coefficient (R 2 ) are all greater than 0.98, as shown in Table 5:

[0166] Table 5 Reference linear equations and correlation coefficients for 40 compounds

[0167]

[0168] The standard solution was added to the matrix blank sample, and the method quantification limit was determined with S / N ≥ 10.

[0169] The results showed that among the target substances, 4 substances had a quantification limit of 0.005 mg / kg, 32 substances had a quantification limit of 0.010 mg / kg, 3 substances had a quantification limit of 0.025 mg / kg, and 1 substance had a quantification limit of 0.050 mg / kg.

[0170] (3) Recovery and precision

[0171] Four food matrices (soybean, corn, rice, wheat matrix) were selected to investigate the accuracy of this method. In the above blank matrix, the concentration of 0.01 (aflatoxin 0.005), 0.05 and 0.1 mg / kg was added and recovered. The concentration was determined under the conditions of steps S1 and S2. Six parallel samples were made for each added concentration level to ensure that the signal-to-noise ratio (S / N) of all substances at the screening concentration met the regulatory requirements. The recovery rate of some substances was low, but the signal was strong enough. The average recovery rate of 40 compounds at three levels in soybean, corn, rice and wheat ranged from 82.8-120%, 94.1-113%, 107-121%, and 100-125%, respectively, and the relative standard deviation (RSD) ranged from 3.7-24.6%, 1.9-24.1%, 0.8-17.9%, and 2.2-15.2% (n=6).

[0172] Example 10

[0173] Actual sample measurement

[0174] 21 soybean, 27 wheat and 41 corn samples were tested on the market. Deoxynivalenol was detected in 27 batches of wheat flour, with a detection rate of 100% and a content ranging from 0.034-0.537 mg / kg; deoxynivalenol was detected in 26 batches of corn flour, with a detection rate of 63.6% and a content ranging from 0.022-0.223 mg / kg. No hidden mycotoxins were screened out.

[0175] This application uses high-performance liquid chromatography-quadrupole / time-of-flight mass spectrometry to determine 40 fungal toxins in grains, and high-throughput screening and confirmation of 40 fungal toxins can be completed within 20 minutes. High-resolution mass spectrometry eliminates matrix interference in sample analysis, and combined with improved pretreatment methods, it greatly improves the qualitative accuracy and quantitative limit of the method. Based on accurate relative molecular mass, secondary mass spectrometry and relative retention time, a screening database for 40 fungal toxins was constructed, and a rapid screening and confirmation method for 40 fungal toxins was established.

[0176] Although the present invention has been described in detail above by general description and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made to the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all belong to the scope of protection claimed by the present invention.

Claims

1. High-resolution mass spectrometry detection method for hidden mycotoxins in grains and their products. It is characterized in that The following steps are involved: S1. Sample extraction: using acetonitrile aqueous solution as the extracting solution, placing magnetic iron oxide nanoparticles-nanocage composite materials and ceramic homogenizers into the sample, freezing, centrifuging, and then blowing with nitrogen to constant volume; S2. Purification: transfer the extract obtained in S1 into a purification tube, vortex centrifuge and perform micro-solid phase extraction followed by filtration; S3. performing liquid chromatography-quadrupole-time of flight mass spectrometry detection on the filtrate; S4. Draw a matrix matching standard curve: prepare a blank extract according to the extraction and purification steps, and prepare a matrix standard working solution; S5. Establish a high-resolution mass spectrometry database; The specific steps of S1 sample extraction are: (1) Accurately weigh 5 g of grain sample into a 50 mL centrifuge tube, then add magnetic iron oxide nanoparticle-nanocage composite material, ceramic homogenizer, 20 mL of 79% acetonitrile aqueous solution and CHO 4 P, vortex for 3 min, centrifuge at 9500 rpm for 10 min, and pipette the supernatant into a clean 50 mL centrifuge tube; (2) Place the centrifuge tube in a -30℃ freezer for 1 hour and add MgSO 4 , NaCl, Na 3 C 6 H 5 O 7 、Na 2 C 6 H 6 O 7 ·3H 2 O and C 6 H 14 , vortex oscillation for 3 minutes, centrifuge at 9500r / min for 10 minutes, transfer all the supernatant to a 15mL centrifuge tube, blow nitrogen at 40℃ until it is less than 5mL, make the volume with acetonitrile to 5mL, and mix well; The preparation method of the magnetic iron oxide nanoparticle-nanocage composite material is as follows: (1) ultrasonically dispersing magnetic iron oxide nanoparticles in a nitric acid solution, stirring evenly and standing for 20-40 minutes, washing the magnetic iron oxide nanoparticles with deionized water for 3-6 times, and then ultrasonically dispersing them in deionized water to obtain an intermediate solution, placing the intermediate solution in a sealed environment, and keeping it at 180-200° C. for 15-20 hours, and then washing and drying the reactant to obtain iron oxide magnetic nanoparticles; (2) Weigh 2.0 g of iron oxide magnetic nanoparticles and 1.25 g of copper acetate and heat them to fully calcine them in a tube furnace. The tube furnace is filled with Ar / H with a volume percentage concentration of 95% / 5%. 2 The temperature of the mixed gas is set to be raised from 25°C to 500-900°C at a rate of 5°C / min and kept at this temperature for 2 hours. After the reaction is completed, the temperature is lowered to room temperature at a rate of 10°C / min to obtain a magnetic iron oxide nanoparticle-nanocage composite material; The specific steps of the S2 purification are: (1) Transfer the extract obtained in S1 to an activated 15 mL Bond Elut EMR-Lipid dSPE cleanup tube, vortex mix for 2 min, and quickly centrifuge at 9500 rpm for 10 min at 10°C to obtain the supernatant. (2) adding the supernatant to a micro solid phase extraction column, adding an ethanol solvent mixed with sodium chloride as an eluent for elution, collecting the eluate, centrifuging the eluate, taking the intermediate liquid, passing it through a 0.22 μm organic filter membrane, and providing it for detection by liquid chromatography-quadrupole-time of flight mass spectrometry; The parameters of the liquid chromatography-quadrupole-time of flight mass spectrometry detection in S3 include: a) Chromatographic column: Acquity UPLC HSS T3; b) Mobile phase A: 0.1% formic acid in water, mobile phase B: 50% methanol / acetonitrile solution containing 0.1% formic acid in water; The elution method is gradient elution, and the gradient elution program is: 0-3min, 95% mobile phase A, 5% mobile phase B; From 3 to 9 minutes, mobile phase A was linearly reduced to 1%, and mobile phase B was linearly increased to 99% and maintained for 6 minutes; From 15 to 15.1 min, mobile phase A increased linearly to 95%, and mobile phase B decreased linearly to 5% and maintained for 4.9 min.

2. The high-resolution mass spectrometry method for detecting hidden mycotoxins in grains and their products according to claim 1, It is characterized in that Mass spectrometry conditions included: HESI ionization mode was adopted; spray voltage was 5500V; capillary temperature was 325℃; heater temperature was 550℃; Gas was 155psi; Gas2 was 60psi; curtain gas was 35unit; CADGas was 7unit; scanning mode was SWATH / TOF-MS / TOF-MS / MS; positive ion acquisition mode; TOF-MS parameters were as follows: acquisition range was 150-785m / z; DP voltage was 80V; collision capacity was CE5V; accumulation time was 0.16s; TOF-MS / MS parameters were as follows: acquisition range was 50-785m / z; accumulation time was 0.06s; charge number was 1.

3. The high-resolution mass spectrometry method for detecting hidden mycotoxins in grains and their products according to claim 1, It is characterized in that The method of S5 establishing a high-resolution mass spectrometry database is: Under chromatographic separation conditions, 40 common mycotoxins and hidden mycotoxins were analyzed to obtain the mass numbers of calculated compound sum ions as mass spectrometry theoretical information; standard spectra and characteristic fragments were collected as primary and secondary mass spectrometry information; search and rapid comparison methods were established; after obtaining sample data, the collected mass spectrometry information was quickly searched for matching with hundreds of compounds collected in the database to compare samples and conduct mass spectrometry confirmation.

4. The high-resolution mass spectrometry method for detecting hidden mycotoxins in grains and their products according to claim 1, It is characterized in that The activation process of the 15 mL Bond Elut EMR-Lipid dSPE purification tube in S2 is as follows: before use, the purification tube is placed in a container, 3 mL of ethyl acetate and 5 mL of deionized water are added, and the purification tube is washed with acetate buffer and vortexed for 30 seconds for activation.

5. The high-resolution mass spectrometry method for detecting hidden mycotoxins in grains and their products according to claim 1, It is characterized in that The S1 is added with MgSO 4 is 4g, NaCl is 1g, Na 3 C 6 H 5 O 7 1g, Na 2 C 6 H 6 O 7 ·3H 2 O is 0.5 g and C 6 H 14 The volume ratio of the 79% acetonitrile aqueous solution is 79:20:1 for acetonitrile / water / acetic acid.

6. The high-resolution mass spectrometry method for detecting hidden mycotoxins in grains and their products according to claim 1, It is characterized in that The sample grains in S1 are specifically soybeans, corn, rice or wheat, and are weighed to an accuracy of 0.01 g.

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