Preparation and application of mass spectrum ionization source element special for mycotoxin analysis

Abstract

The invention discloses a mass spectrum ionization source element special for fungaltoxin analysis and a preparation method and application thereof. The separation and ionization integrated mass spectrum ionization source element for trace fungaltoxin detection comprises a conductive substrate; the extraction layer is formed on at least part of the surface of the conductive substrate, and the extraction layer comprises a covalent organic framework material formed by repeating units shown in the formula I. According to the mass spectrum ionization source element, the extraction layer is uniform in coating and large in specific surface area, has a porous structure, can specifically enrich multiple trace fungaltoxins in a high-flux manner, is high in adsorption capacity, can directly perform mass spectrum detection through ionization and ionization, and is simple in detection steps and high in detection efficiency. And the detected background noise is low, and the sensitivity and the accuracy are high. In addition, the mass spectrum ionization source element can be reused and is low in use cost.

Description

Technical Field

[0001] The present invention relates to the field of analytical chemistry, and in particular to a separation and ionization integrated mass spectrometry ionization source element for detecting trace mycotoxins, and a preparation method and application thereof. Background Art

[0002] Liquid chromatography tandem mass spectrometry (LC-MS / MS) is a standard method for the detection of mycotoxins, which can accurately detect trace amounts of mycotoxins, but the pretreatment is complex and the chromatographic separation time is long, which cannot meet the needs of rapid detection. As the timeliness, convenience and accuracy of detection are increasingly valued, enzyme-linked immunosorbent assay, electrochemical sensors, near-infrared spectroscopy, Raman spectroscopy and other technologies that can achieve on-site rapid detection of mycotoxins have gradually been developed, but these technologies also have the disadvantages of false positives, low repeatability and poor sensitivity.

[0003] Solid substrate electrospray mass spectrometry (SSESI-MS) is a normal pressure ionization technique that uses a solid support to directly perform sample electrospray ionization. Existing technologies are actively improving the surface structure of the solid substrate in SSESI-MS to enhance the sensitivity and selectivity of the technology, mainly by coating modification to enhance the extraction efficiency and enrichment effect of target compounds on the surface of the solid substrate. At present, common modification materials mainly include metal organic framework materials, molecular imprinting polymer materials, covalent organic framework materials, etc. Most of them can only extract and enrich a single type of fungal toxin, and there is still a lack of methods for rapid detection of multiple fungal toxins with high throughput.

[0004] Therefore, solid-based mass spectrometry ionization sources and corresponding detection methods for rapid detection of multiple fungal toxins in complex matrices need to be studied. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, one object of the present invention is to provide a separation and ionization integrated mass spectrometry ionization source element for the detection of trace mycotoxins, wherein the extraction layer of the element has a large specific surface area and a porous structure, has a broad-spectrum and efficient enrichment effect on mycotoxins, and is particularly suitable for the extraction, enrichment and detection of trace mycotoxins in complex matrices.

[0006] According to one aspect of the present invention, the present invention provides a separation and ionization integrated mass spectrometry ionization source element for detecting trace mycotoxins. According to an embodiment of the present invention, the mass spectrometry ionization source element comprises: a conductive substrate; and an extraction layer, the extraction layer is formed on at least a portion of the surface of the conductive substrate, and the extraction layer comprises a covalent organic framework material composed of a repeating unit shown in Formula I.

[0007]

[0008] According to the mass spectrometry ionization source element of the embodiment of the present invention, the extraction layer coating is uniform, the specific surface area is large, and the porous structure is provided, so that a variety of trace mycotoxins can be enriched in a targeted manner with a large flux, a variety of mycotoxins can be enriched, and the adsorption capacity is strong. Moreover, the mass spectrometry ionization source element can directly perform mass spectrometry detection through ionization ionization without the need for a chromatographic separation process, and the detection steps are simple and fast, and the detection background noise is low, and the sensitivity and accuracy are high. In addition, the mass spectrometry ionization source element can be reused, and the use cost is low.

[0009] In addition, the separation and ionization integrated mass spectrometry ionization source element for trace mycotoxin detection according to the above embodiment of the present invention may also have the following additional technical features:

[0010] According to an embodiment of the present invention, the conductive substrate is formed of stainless steel.

[0011] According to an embodiment of the present invention, the conductive substrate is in the shape of an isosceles triangle, the height of the isosceles triangle is 1.5-2.5 cm, and the base is 0.5-1.5 cm.

[0012] According to an embodiment of the present invention, the thickness of the conductive substrate is 0.1-0.5 mm.

[0013] According to an embodiment of the present invention, the thickness of the extraction layer is 10-20 μm.

[0014] According to an embodiment of the present invention, the hydrophilic contact angle of the extraction layer is 55-65°, preferably 60°.

[0015] According to another aspect of the present invention, the present invention provides a method for preparing the aforementioned separation and ionization integrated mass spectrometry ionization source element for detecting trace mycotoxins. According to an embodiment of the present invention, the method includes: subjecting a conductive substrate to an acidification treatment to obtain an acidified conductive substrate; subjecting the acidified conductive substrate to a first contact with an organic solution containing an amino monomer, subjecting the acidified conductive substrate to an ultrasonic and oscillating treatment to allow the amino monomer to cover the surface of the acidified conductive substrate, and obtaining a first reaction mixture; and subjecting an organic solution containing an aldehyde monomer to a second contact with the first reaction mixture, subjecting the organic solution to a Schiff base reaction, and obtaining the separation and ionization integrated mass spectrometry ionization source element for detecting trace mycotoxins.

[0016] According to the preparation method of the embodiment of the present invention, the extraction layer coating of the prepared mass spectrometry ionization source element is uniform, has a large specific surface area, has a porous structure, and has strong stability. It can enrich a variety of trace fungal toxins in a targeted and large-flux manner and has strong adsorption capacity. In addition, the preparation method has mild conditions, simple steps, good reproducibility of the extraction coating, and is convenient for industrial production.

[0017] According to an embodiment of the present invention, the amino monomer is 1,3,5-tris(4-aminophenoxy)benzene (TAPOB), benzidine (BD), p-diaminoazobenzene (DAAB) or 1,3,5-tris(4-aminophenyl)triazine (TAPT).

[0018] According to an embodiment of the present invention, the aldehyde monomer is 2,3-dihydroxyterephthalaldehyde (BDD), trialdehyde phloroglucinol (Tp), 2,5-dimethoxyterephthalaldehyde (DMTP) or 2,3-dihydroxyterephthalaldehyde (DHA).

[0019] According to an embodiment of the present invention, the amino monomer is 1,3,5-tris(4-aminophenoxy)benzene; and the aldehyde monomer is 2,3-dihydroxyterephthalaldehyde.

[0020] According to an embodiment of the present invention, the concentration of 1,3,5-tris(4-aminophenoxy)benzene is 7-9 mg / mL, preferably 8 mg / mL.

[0021] According to an embodiment of the present invention, the concentration of the 2,3-dihydroxyterephthalaldehyde is 12-13 mg / mL, preferably 12.5 mg / mL.

[0022] According to an embodiment of the present invention, the molar ratio of 1,3,5-tris(4-aminophenoxy)benzene to 2,3-dihydroxyterephthalaldehyde is 1:1-2, preferably 2:3.

[0023] According to an embodiment of the present invention, the catalyst for the Schiff base reaction is acetic acid.

[0024] According to an embodiment of the present invention, the volume ratio of the first reaction mixture, the organic solution containing the aldehyde monomer and the acetic acid is 19-21:7-9:1.

[0025] According to an embodiment of the present invention, the organic solvents of the organic solution containing amino monomers and the organic solution containing aldehyde monomers are both tetrahydrofuran.

[0026] According to another aspect of the present invention, the present invention provides a separation and ionization integrated mass spectrometer. According to an embodiment of the present invention, the device comprises: the aforementioned separation and ionization integrated mass spectrometer ionization source element for detecting trace mycotoxins; an open mass spectrometer detector, the open mass spectrometer detector comprising an injection port, the injection port is arranged opposite to the tip of the mass spectrometer ionization source element; and a high-voltage power supply, the high-voltage power supply is connected to the mass spectrometer ionization source element.

[0027] According to the separation and ionization integrated mass spectrometry device of the embodiment of the present invention, the aforementioned mass spectrometry ionization substrate for enriching mycotoxins is directly connected to the high-voltage power supply. Under the action of high voltage electricity, the target on the mass spectrometry ionization source element is ionized under the action of the elution solvent, and the generated ions directly enter the mass spectrometer through the mass spectrometer inlet to obtain a collection signal. No chromatographic separation process is required, the detection steps are simple and the speed is fast. It can detect multiple trace mycotoxins at the same time, the detection throughput is large, and the detection background noise is low. It is particularly suitable for the enrichment and detection of multiple categories of mycotoxins in food samples.

[0028] According to another aspect of the present invention, the present invention provides a method for enriching mycotoxins. According to an embodiment of the present invention, the method comprises: extracting and treating the sample to be tested so as to obtain a liquid to be tested; and subjecting the liquid to be tested to an oscillating contact treatment with the aforementioned separation and ionization integrated mass spectrometry ionization source element for trace mycotoxin detection so as to obtain a mass spectrometry ionization source element with the mycotoxin adsorbed on the surface. Thus, the method can specifically enrich a variety of trace mycotoxins, has a strong adsorption force, and is conducive to fully enriching complex matrices, such as mycotoxins in food. Moreover, the enrichment method of the embodiment of the present invention is simple to operate, does not require a complex sample pretreatment process, and has a high sample extraction efficiency. In addition, the mass spectrometry ionization source element can be reused and has a low cost of use.

[0029] According to an embodiment of the present invention, the rotation speed of the oscillating contact treatment is 1000-1500 revolutions per minute, and the time is 25-35 minutes.

[0030] According to another aspect of the present invention, the present invention provides a method for qualitative / quantitative detection of fungal toxins. According to an embodiment of the present invention, the method comprises: enriching the fungal toxins in the sample to be tested using the aforementioned method for enriching the fungal toxins, so as to obtain a mass spectrometry ionization source element with the fungal toxins adsorbed on the surface; and detecting the mass spectrometry ionization source element with the fungal toxins adsorbed on the surface using the aforementioned separation and ionization integrated mass spectrometry device, so as to qualitatively / quantitatively detect the fungal toxins. Thus, the aforementioned mass spectrometry ionization source element can be targeted to enrich a variety of trace mycotoxins in food, has strong adsorption, is conducive to fully enriching mycotoxins in complex matrices, and the mass spectrometry ionization source element for enriching mycotoxins is directly connected to a high-voltage power supply. Under the action of high voltage electricity, the target on the mass spectrometry ionization source element is ionized under the action of an elution solvent, and the generated ions directly enter the mass spectrometer through the entrance of the mass spectrometry detector to obtain a collection signal, without the need for a chromatographic separation process, the detection step is simple, the speed is fast, and a variety of trace mycotoxins can be detected simultaneously, the detection flux is large, the background noise of the detection is low, and the detection sensitivity is high, which is particularly suitable for high-throughput, rapid, and precise analysis of trace mycotoxins. In addition, the mass spectrometry ionization source element can be reused, and the detection cost is low.

[0031] According to an embodiment of the present invention, the detection conditions of the separation and ionization integrated mass spectrometer are as follows: high voltage power supply voltage: -3.5 kV; ionization elution solvent: methanol solution containing 0.05-0.15% formic acid.

[0032] According to an embodiment of the present invention, the detection conditions of the mass spectrometer detector are: detection mode: multiple reaction monitoring (MRM); nebulizing gas pressure: 55psi; auxiliary gas pressure: 50psi; curtain gas pressure: 30psi; ion spray voltage: 4500V; ion source temperature: 550°C; dwell time: 100ms.

[0033] According to an embodiment of the present invention, the volume of the ionization elution solvent is 4-6 mL.

[0034] According to an embodiment of the present invention, the fungal toxin is selected from aflatoxin B 1 (AFB 1 ), Aflatoxin B 2 (AFB 2 ), Aflatoxin G 1 (AFG 1 ), Aflatoxin G 2 (AFG 2 ), at least one of zearalenone (ZEN), zearalenone (ZEA), zearalenol (ZEL), zearalanol (ZAL) and ochratoxin (OTA).

[0035] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0037] Figure 1 A schematic diagram of scanning electron microscopy results according to an embodiment of the present invention is shown, wherein a is the surface of a blank stainless steel substrate, b is the surface of a stainless steel substrate after acid treatment, and c and d are the surface and cross section of a separation-ionization integrated mass spectrometry ionization source element for trace mycotoxin detection, respectively;

[0038] Figure 2 A schematic diagram of substrate water contact angle according to an embodiment of the present invention is shown, wherein a is a blank stainless steel substrate, b is a stainless steel substrate after acid treatment, and c is a separation-ionization integrated mass spectrometry ionization source element for trace mycotoxin detection;

[0039] Figure 3A schematic diagram showing the results of the selective extraction capability of the element according to one embodiment of the present invention for three types of trace mycotoxins and interfering fumonisins (FBs);

[0040] Figure 4 A schematic diagram showing the results of reusing a mass spectrometry ionization source element according to an embodiment of the present invention;

[0041] Figure 5 shows the optimization of extraction conditions according to one embodiment of the present invention;

[0042] Figure 6 shows the optimization of parsing conditions according to one embodiment of the present invention;

[0043] Figure 7 A method for preparing a separation and ionization integrated mass spectrometer ionization source element and using it for analysis and detection according to one embodiment of the present invention is shown. DETAILED DESCRIPTION

[0044] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0045] In the description of the present invention, the terms "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and do not require that the present invention must be constructed and operated in a specific orientation. Therefore, they should not be understood as limitations on the present invention.

[0046] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0047] According to one aspect of the present invention, the present invention provides a separation and ionization integrated mass spectrometry ionization source element for the detection of trace mycotoxins. According to the mass spectrometry ionization source element of the embodiment of the present invention, the extraction layer coating is uniform, the specific surface area is large, and it has a porous structure. It can be targeted to enrich the three types of trace mycotoxins, aflatoxin, ochratoxin, and zearalenone. It can enrich a variety of mycotoxins and has strong adsorption. Moreover, the mass spectrometry ionization source element can directly perform mass spectrometry detection through ionization ionization without the need for a chromatographic separation process. The detection steps are simple and fast, and the detection background noise is low, and the sensitivity and accuracy are high. In addition, the mass spectrometry ionization source element can be reused and has low cost of use.

[0048] In order to facilitate understanding of the mass spectrometry ionization source element according to the embodiment of the present invention, the mass spectrometry ionization source element is explained here according to the embodiment of the present invention. The mass spectrometry ionization source element includes:

[0049] Conductive substrate

[0050] According to an embodiment of the present invention, the conductive substrate is formed of stainless steel. The inventors found that when a paper base or a wood base is used as a solid substrate, when used for open solid substrate electrospray mass spectrometry, due to the poor conductivity of the paper base or the wood base, the electrospray formed after applying high voltage thereon has high background interference, and the generated mass spectrometry signal is unstable. However, due to the good conductivity of the stainless steel substrate, high voltage can be directly applied to the substrate, the ionization efficiency is high, and the experimental operation can be further simplified.

[0051] According to an embodiment of the present invention, the conductive substrate is in the shape of an isosceles triangle. Thus, the stainless steel sheet is cut into a triangle, so that it has multiple application scenarios. It can not only be used as an ordinary extraction plate, but also be convenient for application in open electrospray mass spectrometry detection. When the extraction element is applied to open electrospray mass spectrometry detection, it is fixed at the horizontal front end position of the mass spectrometry injection port. By applying high voltage electricity, the spray solvent can elute the target adsorbed on the extraction element, ionize at the tip and form a Taylor cone spray, which directly enters the mass spectrometry detection. According to an embodiment of the present invention, the height of the isosceles triangle is 1.5-2.5 cm, and the base is 0.5-1.5 cm. Thus, a spray Taylor cone is formed at the tip of the triangle. If the angle is too small or too large, the elution solvent will be hindered from spraying due to surface tension.

[0052] According to an embodiment of the present invention, the thickness of the conductive substrate is 0.1-0.5 mm. Thus, a substrate of this thickness can allow the liquid to more quickly infiltrate the entire surface, forming a uniform liquid film, which is conducive to generating a stable and uniform spray, and can enhance the electric field strength and stability, accelerate heat transfer and evaporation speed, thereby improving the accuracy and repeatability of the analysis.

[0053] Extraction layer

[0054] According to an embodiment of the present invention, the extraction layer is formed on at least part of the surface of the conductive substrate, and the extraction layer includes a covalent organic framework material composed of repeating units shown in Formula I. As a result, the extraction layer has a large specific surface area, a porous structure, and is in the form of granular accumulation, and can specifically enrich three types of trace mycotoxins, namely, aflatoxin, ochratoxin, and zearalenone, and has a strong adsorption capacity.

[0055] According to an embodiment of the present invention, the thickness of the extraction layer is 10-20 μm. Therefore, the thickness of the extraction layer shortens the diffusion path between the functional particles or ligands on the membrane porous matrix and the liquid flow, accelerates mass transfer, significantly shortens the separation time of membrane adsorption, and improves separation efficiency.

[0056] According to an embodiment of the present invention, the hydrophilic contact angle of the extraction layer is 55-65°, preferably 60°. Thus, the adsorption layer has good hydrophilicity, which is conducive to the adsorption of the target in the aqueous solution.

[0057] According to an embodiment of the present invention, the fungal toxin is selected from aflatoxin B 1 (AFB 1 ), Aflatoxin B 2 (AFB 2 ), Aflatoxin G 1 (AFG 1 ), Aflatoxin G 2 (AFG 2 ), at least one of zearalenone (ZEN), zearalenone (ZEA), zearalenol (ZEL), zearalanol (ZAL) and ochratoxin (OTA).

[0058] According to another aspect of the present invention, the present invention provides a method for preparing the aforementioned separation and ionization integrated mass spectrometry ionization source element for trace mycotoxin detection. According to the preparation method of the embodiment of the present invention, the extraction layer coating of the prepared mass spectrometry ionization source element is uniform, has a large specific surface area, has a porous structure, and has strong stability. It can be targeted to enrich the three types of trace mycotoxins, aflatoxin, ochratoxin, and zearalenone, and has strong adsorption capacity. In addition, the preparation method has mild conditions, simple steps, good reproducibility of the extraction coating, and is convenient for industrial production.

[0059] In order to facilitate understanding of the method for preparing the aforementioned separation and ionization integrated mass spectrometry ionization source element for trace mycotoxin detection, reference is made to Figure 7 The method is explained here. According to an embodiment of the present invention, the method includes:

[0060] S100 acid treatment

[0061] The conductive substrate is subjected to an acidification treatment to obtain an acidified conductive substrate, thereby eliminating the interference of stains or other ions on the surface of the stainless steel, so that the subsequent reaction has a better synthesis effect.

[0062] S200 First Contact

[0063] The acidified conductive substrate is first contacted with an organic solution containing an amino monomer, and is subjected to ultrasonic and oscillation treatment so that the amino monomer covers the surface of the acidified conductive substrate to obtain a first reaction mixture.

[0064] According to an embodiment of the present invention, the ultrasonication time is 15-25 minutes, the shaking time is 0.5-1.5 hours, the temperature is 55-65° C., and the rotation speed is 180-220 rpm. This is conducive to the full dissolution of the amino monomer in the organic solvent, and the reactants are fully contacted to react.

[0065] S300 Schiff base reaction

[0066] The organic solution containing the aldehyde monomer is brought into second contact with the first reaction mixture to perform a Schiff base reaction, so as to obtain the separation and ionization integrated mass spectrometry ionization source element for trace mycotoxin detection. Thus, through the Schiff base reaction, the aldehyde monomer is polymerized with the amino monomer on the conductive substrate to form an organic polymer (COP).

[0067] According to an embodiment of the present invention, the amino monomer is 1,3,5-tris(4-aminophenoxy)benzene (TAPOB), benzidine (BD), p-diaminoazobenzene (DAAB) or 1,3,5-tris(4-aminophenyl)triazine (TAPT).

[0068] According to an embodiment of the present invention, the aldehyde monomer is 2,3-dihydroxyterephthalaldehyde (BDD), trialdehyde phloroglucinol (Tp), 2,5-dimethoxyterephthalaldehyde (DMTP) or 2,3-dihydroxyterephthalaldehyde (DHA).

[0069] According to an embodiment of the present invention, the amino monomer is 1,3,5-tris(4-aminophenoxy)benzene; the aldehyde monomer is 2,3-dihydroxyterephthalaldehyde. Thus, the rigid organic skeleton structure formed by the reaction of the two monomers has a specific three-dimensional geometric configuration, and can form π-π conjugation, H bonds, etc. with the target, and can achieve targeted simultaneous adsorption of 9 types of targets including aflatoxin, ochratoxin, and zearalenone through spatial recognition and intermolecular forces.

[0070] According to an embodiment of the present invention, the concentration of 1,3,5-tris(4-aminophenoxy)benzene is 7-9 mg / mL, preferably 8 mg / mL. Thus, this concentration can ensure a high binding rate between the amino monomer and the active sites of the stainless steel substrate, and avoid unnecessary waste due to excessive use of raw materials.

[0071] According to an embodiment of the present invention, the concentration of 2,3-dihydroxyterephthalaldehyde is 12-13 mg / mL, preferably 12.5 mg / mL. Thus, this concentration can ensure a high reaction conversion rate of the aldehyde monomer and the amino monomer, so that the synthesized extraction layer covers the surface of the stainless steel substrate to the greatest extent, and can also avoid unnecessary waste caused by excessive use of raw materials.

[0072] According to an embodiment of the present invention, the molar ratio of 1,3,5-tris(4-aminophenoxy)benzene to 2,3-dihydroxyterephthalaldehyde is 1:1-2, preferably 2:3. Thus, one 1,3,5-tris(4-aminophenoxy)benzene molecule has 3 amino groups, and one 2,3-dihydroxyterephthalaldehyde has 2 aldehyde groups. To ensure that each reactive group can participate in the reaction and promote the maximum conversion of the reactants into the target product, the molar ratio should be selected to be 2:3. And under this ratio, the generated product can present a more regular and orderly arrangement in the molecular structure, reducing the occurrence of side reactions.

[0073] According to an embodiment of the present invention, the catalyst of the Schiff base reaction is acetic acid, thereby achieving good catalytic effect and high reaction efficiency.

[0074] According to an embodiment of the present invention, the volume ratio of the first reaction mixture, the organic solution containing the aldehyde monomer and the acetic acid is 19-21: 7-9: 1. Thus, this volume ratio range enables the components in the reaction system to be fully contacted and the catalyst acetic acid to have a suitable concentration, thereby ensuring that the reaction proceeds at a relatively ideal rate.

[0075] According to an embodiment of the present invention, the organic solvents of the organic solution containing amino monomers and the organic solution containing aldehyde monomers are both tetrahydrofuran. As a result, the solubility of amino monomers and aldehyde monomers is high, and the monomers can be evenly dispersed in the solution, reducing the problem of uneven reaction caused by excessively high or low local concentrations, thereby improving the yield and quality stability of the product.

[0076] According to another aspect of the present invention, the present invention provides a separation and ionization integrated mass spectrometer. According to an embodiment of the present invention, the device comprises: the aforementioned separation and ionization integrated mass spectrometer ionization source element for detecting trace mycotoxins; an open mass spectrometer detector, the open mass spectrometer detector comprising an injection port, the injection port is arranged opposite to the tip of the mass spectrometer ionization source element; and a high-voltage power supply, the high-voltage power supply is connected to the mass spectrometer ionization source element.

[0077] According to the separation and ionization integrated mass spectrometry device of the embodiment of the present invention, the aforementioned mass spectrometry ionization substrate for enriching fungal toxins is directly connected to the high-voltage power supply. Under the action of high voltage electricity, the target on the mass spectrometry ionization source element is ionized under the action of the elution solvent, and the generated ions directly enter the mass spectrometer through the mass spectrometer inlet to obtain a collection signal. No chromatographic separation process is required, the detection steps are simple and the speed is fast. It can simultaneously detect three types of trace fungal toxins, namely aflatoxin, ochratoxin and zearalenone, has a large detection throughput and low detection background noise, and is particularly suitable for the enrichment and detection of multiple types of fungal toxins in food samples.

[0078] According to another aspect of the present invention, the present invention provides a method for enriching mycotoxins. According to an embodiment of the present invention, the method comprises: extracting and treating the sample to be tested so as to obtain a liquid to be tested; and subjecting the liquid to be tested to an oscillating contact treatment with the aforementioned separation and ionization integrated mass spectrometry ionization source element for trace mycotoxin detection so as to obtain a mass spectrometry ionization source element with the mycotoxin adsorbed on the surface. Thus, the method can specifically enrich the three types of trace mycotoxins, namely aflatoxin, ochratoxin, and zearalenone, and has a strong adsorption force, which is conducive to fully enriching complex matrices, such as mycotoxins in food. Moreover, the enrichment method of the embodiment of the present invention is simple to operate, does not require a complex sample pretreatment process, and has a high sample extraction efficiency. In addition, the mass spectrometry ionization source element can be reused and has a low cost of use.

[0079] According to an embodiment of the present invention, the rotation speed of the oscillating contact treatment is 1000-1500 revolutions and the time is 25-35 minutes, which is conducive to the mass spectrometry ionization source element fully absorbing the fungal toxins in the test solution.

[0080] According to another aspect of the present invention, the present invention provides a method for qualitative / quantitative detection of fungal toxins. Figure 7According to an embodiment of the present invention, the method includes: using the aforementioned method for enriching fungal toxins to enrich the fungal toxins in the sample to be tested, so as to obtain a mass spectrometry ionization source element with the fungal toxins adsorbed on the surface; and using the aforementioned separation and ionization integrated mass spectrometry device to detect the mass spectrometry ionization source element with the fungal toxins adsorbed on the surface, so as to perform qualitative / quantitative detection of the fungal toxins. Thus, the aforementioned mass spectrometry ionization source element can be targeted to enrich 9 types of trace mycotoxins in 3 categories in food, has strong adsorption force, is conducive to fully enriching mycotoxins in complex matrices, and the mass spectrometry ionization source element for enriching mycotoxins is directly connected to a high-voltage power supply. Under the action of high voltage electricity, the target on the mass spectrometry ionization source element is ionized under the action of the elution solvent, and the generated ions directly enter the mass spectrometer through the entrance of the mass spectrometry detector to obtain a collection signal, without the need for a chromatographic separation process, the detection step is simple and fast, and the 3 types of trace mycotoxins, aflatoxin, ochratoxin, and zearalenone, can be detected simultaneously, with a large detection flux, low background noise, and high detection sensitivity, which is particularly suitable for high-throughput, rapid, and precise analysis of trace mycotoxins. In addition, the mass spectrometry ionization source element can be reused, and the detection cost is low.

[0081] According to an embodiment of the present invention, the detection conditions of the separation and ionization integrated mass spectrometer are: high voltage power supply voltage: -3.5kV; ionization elution solvent: methanol solution containing 0.05-0.15% formic acid. Therefore, when the voltage is -3.5kV, the signal response is stronger; the signal response is higher when methanol containing 0.05-0.15% formic acid is used as the elution solvent. Furthermore, the selection of the above voltage and elution solvent is conducive to improving the detection signal of the object to be detected, making the detection sensitivity and accuracy higher.

[0082] According to an embodiment of the present invention, the detection conditions of the mass spectrometer are: detection mode: multiple reaction monitoring (MRM); nebulizer gas pressure: 55psi; auxiliary gas pressure: 50psi; curtain gas pressure: 30psi; ion spray voltage: 4500V; ion source temperature: 550°C; dwell time: 100ms. Therefore, under the above conditions, the detection of mycotoxins has a high target response value, low background noise, high detection sensitivity and accuracy, and is particularly suitable for rapid and accurate analysis of trace mycotoxins.

[0083] According to an embodiment of the present invention, the volume of the ionization elution solvent is 4-6 mL. Therefore, this volume of ionization elution solvent is not only conducive to the full elution of the target, but also avoids the waste of solvent.

[0084] According to an embodiment of the present invention, the fungal toxin is selected from aflatoxin B 1 (AFB 1 ), Aflatoxin B 2 (AFB2 ), Aflatoxin G 1 (AFG 1 ), Aflatoxin G 2 (AFG 2 ), at least one of zearalenone (ZEN), zearalenone (ZEA), zearalenol (ZEL), zearalanol (ZAL) and ochratoxin (OTA).

[0085] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and should not be construed as limiting the present invention.

[0086] Below in conjunction with embodiment, the scheme of the present invention will be explained.It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.Unrepresented specific techniques or conditions in the embodiment, according to the technology or conditions described in the document in this area or according to the product specification sheet.Reagents used or instruments that do not indicate manufacturers are conventional products that can be obtained commercially, for example, can be purchased from Sigma company.

[0087] The materials and reagents used in the examples of the present invention are shown in Table 1.

[0088] Table 1

[0089]

[0090] Example 1

[0091] Using the method of the embodiment of the present invention, a trace fungal toxin separation-ionization integrated mass spectrometry ionization source element is prepared with stainless steel as a conductive substrate and TAPOB-BDD covalent organic framework as an extraction layer. The preparation process is as follows:

[0092] (a) The stainless steel substrate was cut into an isosceles triangle with a waist length of 2 cm, a base length of 1 cm, and a thickness of 0.3 mm.

[0093] (b) The stainless steel sheet in step (a) is ultrasonically treated with 2 mol / L sulfuric acid for 3 hours, then repeatedly washed with ultrapure water until neutral, ultrasonicated in ultrapure water for another 3 hours, quickly dried with nitrogen blown, and stored in acetonitrile for later use, thereby obtaining an acidified stainless steel substrate.

[0094] (c) Weigh TAPOB (80 mg, 0.2 mmol) into a 200 mL conical flask, then add 10 mL THF and shake evenly. Add the stainless steel substrate acidified in step (b), sonicate for 20 minutes, then transfer to a shaker and react at 60°C and 200 rpm for 1 hour.

[0095] (d) Weigh BDD (50 mg, 0.30 mmol) and dissolve it in 4 mL THF, add the mixed solution to the reaction system of step (c), and drop 500 μL acetic acid. The reaction system is placed on a shaker and shaken at 60° C. and 200 rpm for 3 hours to obtain a trace fungal toxin separation-ionization integrated mass spectrometry ionization source element.

[0096] (e) After the reaction is completed, the component is washed alternately with ethanol and acetonitrile for three times, and then immersed in acetonitrile for storage.

[0097] Example 2

[0098] In this example, the element prepared in Example 1 was characterized by scanning electron microscopy and contact angle to prove the successful preparation and physical and chemical properties of the element, as follows:

[0099] 1. The morphology of the blank stainless steel substrate surface, the acid-treated stainless steel substrate surface, the mass spectrometry ionization source element surface and the element cross section prepared in Example 1 were characterized by scanning electron microscopy (ZEISS GeminiSEM 300) at an acceleration voltage of 3 kV. Figure 1 As shown, the surface morphology of the blank stainless steel substrate is flat and smooth ( Figure 1 a), while the surface of the stainless steel substrate treated with sulfuric acid presents a rough and uneven appearance ( Figure 1 b), this is because sulfuric acid corrodes the surface of the stainless steel substrate, and after removing the stainless steel coating on the surface, it is further oxidized. This provides more binding sites for the modification of the amino monomer, which is beneficial to the synthesis of the covalent organic framework extraction layer. A granular dense stacking structure can be seen on the surface of the synthesized ionization source element ( Figure 1 c), indicating the successful modification of the covalent organic framework extraction layer. These granular structures have a variety of functional groups on their surfaces, which can provide sites for selective adsorption of target substances. The cross section of the element was observed ( Figure 1 d), the thickness of the extraction layer on the surface of the component is about 15 μm, which further confirms that the covalent organic framework extraction layer has been successfully modified on the surface of the stainless steel substrate.

[0100] 2. The water contact angles of the blank stainless steel substrate, the acid-treated stainless steel substrate, and the mass spectrometry ionization source element of Example 1 were analyzed by a contact angle meter (SZ-CAMC32). The contact angle of the blank stainless steel substrate was 98° ( Figure 2 a), which indicates that it may be hydrophobic due to the presence of an anti-oxidative oil layer. After acidification, the surface roughness of the stainless steel substrate increased, and its contact angle decreased to 90° ( Figure 2 b). Subsequently, after modification with the covalent organic framework extraction layer, the contact angle of the element dropped significantly to 60° ( Figure 2c), indicating that the element has good hydrophilicity. This improvement in hydrophilicity promotes the adsorption of fungal toxins in aqueous solution and is beneficial to the extraction and adsorption process.

[0101] Example 3

[0102] In this example, the trace mycotoxin separation-ionization integrated mass spectrometry ionization source element of Example 1 was used to analyze the aflatoxins (aflatoxin B1 AFB) added to the blank solvent at the same concentration. 1 、Aflatoxin B2 AFB 2 , Aflatoxin G1 AFG 1 , Aflatoxin G2 AFG 2 ), ochratoxin (OTA), zearalenone (zearalenone ZEN, zearalenone ZEA, zearalenol ZEL, zearalenol ZAL), and fumonisin (fumonisin B1FB 1 、Fumonisin B2 FB 2 ), and enrichment treatment (concentration of 10μg / L) was performed at the same time to compare the selectivity differences of the trace mycotoxin separation-ionization integrated mass spectrometry ionization element for target compounds and interferences (aflatoxin, ochratoxin, zearalenone and fumonisin often exist in moldy grains at the same time, but the content of fumonisin is relatively high, and the limit is generally at the mg / kg level. The content of aflatoxin, ochratoxin and zearalenone is relatively low, and the limit is generally at the μg / kg level. The presence of fumonisin interferes with the precise quantification of trace targets). The results are as follows Figure 3 As shown, the selectivity of the mass spectrometry ionization source element of Example 1 to 9 fungal toxins of 3 categories is about 4.6-7.5 times that of fumonisins, indicating that the element of Example 1 is selective to 9 fungal toxins of 3 categories.

[0103] Example 4

[0104] In this example, the trace mycotoxin separation-ionization integrated mass spectrometry ionization source element prepared in Example 1 was used to selectively enrich 9 types of mycotoxins in corn samples, and the element was used as an open electrospray ionization mass spectrometry ion source for direct detection and analysis, as follows:

[0105] (1) Extraction method

[0106] Take the commercially available instant corn sample and crush it, weigh 2.0g and place it in a 50mL centrifuge tube, add 10mL of extraction solvent (acetonitrile: water: methanol, v:v:v = 80:19:1), ultrasonically extract for 30 minutes, and centrifuge at 10000rpm 4℃ for 15 minutes. Take out 5mL of supernatant, blow dry with nitrogen, and then dissolve in 10mL of 5% acetonitrile aqueous solution. Add the mass spectrometry ionization source element of Example 1, and shake and extract at 1500rpm for 30 minutes. Take out the element loaded with the target compound, dry it, and perform open electrospray ionization mass spectrometry detection.

[0107] (2) Detection method

[0108] The element loaded with the target compound is placed on a special three-dimensional mobile platform, and the tip of the element is adjusted to be 10 mm away from the mass spectrometer injection port and slightly tilted downward to help overcome surface tension to achieve ionization. 20 μL of methanol solution (containing 0.1% formic acid) is dripped onto the surface of the element, and a high voltage of +3.5 kV is applied to the tail of the element to generate analyte ions at the tip and form a spray for mass spectrometry analysis. The mass spectrometry conditions include: detection mode: multiple reaction monitoring (MRM); nebulizer gas pressure (GS1): 55 psi; auxiliary gas pressure (GS2): 50 psi; curtain gas pressure: 30 psi; ion spray voltage (IS): 4500 V, ion source temperature: 550 ° C; monitoring ion pairs and collision energy are shown in Table 2. Quantification is performed using the peak area of ​​the analyte quantitative ion.

[0109] Table 2

[0110]

[0111] *Quantitative ion

[0112] (3) Experimental results

[0113] The detection limit, quantification limit and precision of the method, linear regression equation, linear range, determination coefficient, and batch-to-batch reproducibility of the components are shown in Table 3. The quantification limit of this method is much lower than the maximum residue limit of GB 2761-2017 National Food Safety Standard Limits of Mycotoxins in Food, meeting the requirements of residue analysis, with good intra-day and inter-day precision, and good reproducibility between component batches.

[0114] The target substances were added to blank corn samples at concentrations of 0.5, 5 and 25 μg / kg, respectively, to investigate the recovery rate of the method. The average recovery rates of the nine mycotoxins were 83.62-96.97%, which met the analytical requirements of daily testing.

[0115] Table 3

[0116]

[0117] Example 5

[0118] In this example, the trace fungal toxin separation-ionization integrated mass spectrometry ionization source element of Example 1 was reused, and the spiked sample was extracted and analyzed using the experimental method of Example 4 to examine the reusability of the element. Figure 4 As shown, in the 8 experiments conducted, the recovery rates of 9 types of fungal toxins in 3 categories remained stable, proving that the trace fungal toxin separation-ionization integrated mass spectrometry ionization source element of the embodiment of the present invention can be reused more than 8 times.

[0119] Example 6

[0120] In this embodiment, the trace mycotoxin separation-ionization integrated mass spectrometry ionization source element of Example 1 was used to extract and detect the spiked sample. The difference was that the extraction time, extraction solvent volume, spray solvent type, formic acid concentration in the spray solvent, spray voltage and other parameters were changed respectively, and the effects of different extraction conditions and elution and spray conditions on the recovery rate were investigated respectively. Specifically, the effects of extraction time (10-50 minutes), extraction solvent volume (1-10 mL), spray solvent type (ethanol, methanol, acetonitrile, ethyl acetate), formic acid concentration in the spray solvent (0.1-0.5%), and spray voltage (3.1-3.7 kV) on the recovery rates of 9 types of mycotoxins in 3 categories were investigated respectively. All experiments were set up in triplicate, and the results were averaged. The error bars after plotting represent the standard deviation between the parallel data.

[0121] The experimental results are as follows Figure 5 , 6 As shown, specifically, Figure 5 As shown in a, during the adsorption time of 0 to 40 min, the adsorption amount of the target analyte by the element continued to increase, and then reached saturation, indicating that the adsorption kinetics reached equilibrium. Therefore, the optimal extraction time was determined to be 30 min. Figure 5 As shown in b, when the volume of the extraction solution increases, the response of the target compound increases, and the response reaches a stable level after 5 mL. Therefore, 5 mL is a relatively economical and environmentally friendly extraction volume under the premise of ensuring the extraction effect. Figure 6 As shown in a, when ethanol and acetonitrile are on the COF solid matrix, their surface tension is very large. This high surface tension makes it difficult for droplets to form smoothly at the tip of the ionization source, and the target continues to accumulate on the surface of the solid matrix, reducing the number of targets entering the mass spectrometer. The molecular viscosity of ethyl acetate is relatively high, and this characteristic significantly prolongs the time required for the ionization process. In contrast, methanol has excellent elution and ionization properties, making it the most ideal elution solvent. Figure 6As shown in Figure 2b, adding formic acid to the spray solvent can significantly improve the ionization efficiency. This is because formic acid can provide the protons necessary for the ionization process, thereby enhancing the mass spectrometry signal. The formic acid concentration in the methanol solution was optimized, and the results showed that 0.1% formic acid solution was the most ideal for improving the mass spectrometry response. Therefore, 0.1% formic acid-methanol solution was selected as the preferred spray solvent. Figure 6 As shown in Figure c, when the voltage is set low (less than 2.5kV), the driving force for elution of the target is obviously insufficient, which seriously hinders the effective signal collection of the mass spectrometer. If the voltage is too high, the solvent will move quickly on the solid substrate, resulting in diffusion of the target, and the effective electrospray process will be suppressed, which is also not conducive to mass spectrometry signal collection. The results show that when the voltage is in the range of 3.1-3.7kV, the mass spectrometry response will increase with the increase of voltage, and when the voltage exceeds 3.5kV, the mass spectrometry signal will be significantly weakened. Therefore, 3.5kV is determined to be the preferred spray voltage.

[0122] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0123] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A separation and ionization integrated mass spectrometry ionization source element for detecting trace mycotoxins, characterized in that: include: Conductive substrate; as well as An extraction layer is formed on at least a portion of the surface of the conductive substrate, and the extraction layer comprises a covalent organic framework material composed of repeating units shown in Formula I.

2. The mass spectrometry ionization source element according to claim 1, characterized in that: The conductive substrate is formed of stainless steel, Optionally, the conductive substrate is in the form of an isosceles triangle, the height of the isosceles triangle is 1.5-2.5 cm, the base is 0.5-1.5 cm, Optionally, the thickness of the conductive substrate is 0.1-0.5 mm, Optionally, the extraction layer has a thickness of 10-20 μm.

3. The mass spectrometry ionization source element according to claim 1, characterized in that: The hydrophilic contact angle of the extraction layer is 55-65°, preferably 60°.

4. A method for preparing the separation and ionization integrated mass spectrometry ionization source element for detecting trace mycotoxins according to any one of claims 1 to 3, characterized in that: include: Acidifying the conductive substrate to obtain an acidified conductive substrate; The acidified conductive substrate is first contacted with an organic solution containing an amino monomer, and subjected to ultrasonic and oscillation treatment so that the amino monomer covers the surface of the acidified conductive substrate to obtain a first reaction mixture; and The organic solution containing the aldehyde monomer is brought into second contact with the first reaction mixture to perform a Schiff base reaction, so as to obtain the separation and ionization integrated mass spectrometry ionization source element for detecting trace fungal toxins.

5. The method according to claim 4, characterized in that The amino monomer is 1,3,5-tris(4-aminophenoxy)benzene (TAPOB), benzidine (BD), p-diaminoazobenzene (DAAB) or 1,3,5-tris(4-aminophenyl)triazine (TAPT); Optionally, the aldehyde monomer is 2,3-dihydroxyterephthalaldehyde (BDD), trialdehyde phloroglucinol (Tp), 2,5-dimethoxyterephthalaldehyde (DMTP) or 2,3-dihydroxyterephthalaldehyde (DHA).

6. The method according to claim 5, characterized in that The amino monomer is 1,3,5-tris(4-aminophenoxy)benzene; the aldehyde monomer is 2,3-dihydroxyterephthalaldehyde, Optionally, the concentration of 1,3,5-tris(4-aminophenoxy)benzene is 7-9 mg / mL, preferably 8 mg / mL; Optionally, the concentration of the 2,3-dihydroxyterephthalaldehyde is 12-13 mg / mL, preferably 12.5 mg / mL; Optionally, the molar ratio of 1,3,5-tris(4-aminophenoxy)benzene to 2,3-dihydroxyterephthalaldehyde is 1:1-2, preferably 2:

3.

7. The method according to claim 4, characterized in that The catalyst for the Schiff base reaction is acetic acid, Optionally, the volume ratio of the first reaction mixture, the organic solution containing the aldehyde monomer and the acetic acid is 19-21:7-9:1, Optionally, the organic solvents of the organic solution containing amino monomers and the organic solution containing aldehyde monomers are both tetrahydrofuran.

8. A separation and ionization integrated mass spectrometer, characterized in that: include: The separation and ionization integrated mass spectrometry ionization source element for detecting trace mycotoxins according to any one of claims 1 to 3; An open mass spectrometer detector, the open mass spectrometer detector comprising an injection port, the injection port being arranged opposite to the tip of the mass spectrometer ionization source element; and A high voltage power supply is connected to the mass spectrometer ionization source element.

9. A method for enriching mycotoxins, characterized in that: include: Extracting and processing the sample to be tested to obtain a test solution; as well as The test liquid is subjected to an oscillating contact treatment with the separation and ionization integrated mass spectrometry ionization source element for detecting trace mycotoxins according to any one of claims 1 to 3, so as to obtain a mass spectrometry ionization source element with the mycotoxin adsorbed on the surface, Optionally, the rotation speed of the oscillating contact treatment is 1000-1500 rpm and the time is 25-35 minutes.

10. A method for qualitative / quantitative detection of fungal toxins, characterized in that: include: The method for enriching mycotoxins according to claim 9 is used to enrich the mycotoxins in the sample to be tested, so as to obtain a mass spectrometry ionization source element with the mycotoxins adsorbed on the surface; as well as The separation and ionization integrated mass spectrometer according to claim 8 is used to detect the mass spectrometer ionization source element on the surface adsorbing the fungal toxin, so as to perform qualitative / quantitative detection of the fungal toxin. Optionally, the detection conditions of the separation and ionization integrated mass spectrometry device are: High voltage power supply voltage: -3.5kV; Ionization elution solvent: methanol solution containing 0.05-0.15% formic acid; Optionally, the detection conditions of the mass spectrometer detector are: Detection method: multiple reaction monitoring (MRM); Atomizing gas pressure: 55psi; Auxiliary gas pressure: 50psi; Air curtain air pressure: 30psi; Ion spray voltage: 4500V; Ion source temperature: 550°C; Dwell time: 100ms, Optionally, the volume of the ionization elution solvent is 4-6 mL, Optionally, the mycotoxin is at least one selected from aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), zearalenone (ZEN), zearalenone (ZEA), zearalenol (ZEL), zearalanol (ZAL) and ochratoxin (OTA).

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