Preparation and application of mass spectrometry ionization source component special for mycotoxin analysis

Abstract

The application discloses a mass spectrometry ionization source element special for mycotoxin analysis and a preparation method and application thereof. The mass spectrometry ionization source element for trace mycotoxin detection is integrated with separation and ionization and comprises a conductive substrate and an extraction layer formed on at least part of the surface of the conductive substrate, wherein the extraction layer comprises a covalent organic framework material composed of a repeating unit shown in formula I. The mass spectrometry ionization source element has a uniform extraction layer coating, a large specific surface area, a porous structure, can be used for targeted large-flux enrichment of various trace mycotoxins, has strong adsorption, and can be directly subjected to mass spectrometry detection through ionization ionization, so that the detection steps are simple, the background noise of detection is low, and the sensitivity and accuracy are high. In addition, the mass spectrometry ionization source element can be repeatedly used, and the use cost is low.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of analytical chemistry, in particular, to a separation and ionization integrated mass spectrometry ionization source element for trace mycotoxin detection and a preparation method and application thereof. BACKGROUND

[0002] Liquid chromatography tandem mass spectrometry (LC-MS / MS) is a standard detection method for mycotoxins, which can accurately detect trace mycotoxins, but the pre-treatment is complex and the chromatographic separation time is long, which cannot meet the rapid detection requirement. With the increasing attention to the timeliness, convenience and accuracy of detection, enzyme-linked immunosorbent assay, electrochemical sensor, near-infrared spectroscopy, Raman spectroscopy and other technologies for rapid on-site detection of mycotoxins have been gradually developed, but these technologies also have the disadvantages of false positives, low repeatability and poor sensitivity.

[0003] Solid substrate electrospray ionization mass spectrometry (SSESI-MS) is an atmospheric pressure ionization technology that directly uses a solid carrier for sample electrospray ionization. The prior art is actively improving the solid substrate surface structure in SSESI-MS to improve the sensitivity and selectivity of the technology, mainly by coating modification to enhance the extraction efficiency and enrichment effect of the target compound on the surface of the solid substrate. At present, the common modification materials mainly include metal organic framework materials, molecularly imprinted polymer materials, covalent organic framework materials, etc., most of which can only extract and enrich a single type of mycotoxin, and there is still a lack of methods for rapid detection of multiple mycotoxins with large throughput.

[0004] Therefore, a solid substrate mass spectrometry ionization source for rapid detection of multiple mycotoxins in complex matrices and a corresponding detection method need to be researched. SUMMARY

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

[0006] According to one aspect of the present application, a separation and ionization integrated mass spectrometry ionization source element for trace mycotoxin detection is provided. According to an embodiment of the present application, the mass spectrometry ionization source element includes: an electrically conductive substrate; and an extraction layer formed on at least a part of the surface of the electrically conductive substrate, the extraction layer including a covalent organic framework material composed of a repeating unit represented by Formula I.

[0007]

[0008] The mass spectrometry ionization source element according to the embodiment of the present application has a uniform extraction layer coating, a large specific surface area, a porous structure, can enrich a plurality of trace mycotoxins with a large flux in a targeted manner, has a large number of enriched mycotoxin types, has strong adsorption, and can be directly subjected to mass spectrometry detection through ionization ionization, without a chromatographic separation process, so that the detection process is simple, fast, and has low background noise, high sensitivity and high accuracy. In addition, the mass spectrometry ionization source element can be reused, and has low use cost.

[0009] In addition, the separation and ionization integrated mass spectrometry ionization source element for trace mycotoxin detection according to the above-mentioned embodiments of the present application can also have the following additional technical features:

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

[0011] According to the embodiment of the present application, the conductive substrate is 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 the embodiment of the present application, the thickness of the conductive substrate is 0.1-0.5 mm.

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

[0014] According to the embodiment of the present application, the contact angle of the hydrophilicity of the extraction layer is 55-65°, and preferably 60°.

[0015] According to another aspect of the present application, a method for preparing the aforementioned separation and ionization integrated mass spectrometry ionization source element for trace mycotoxin detection is provided. According to the embodiment of the present application, the method comprises: acidizing a conductive substrate to obtain an acidized conductive substrate; performing first contact of the acidized conductive substrate with an organic solution containing an amino monomer, performing ultrasonic and oscillation treatment to coat the amino monomer on the surface of the acidized conductive substrate to obtain a first reaction mixture; and performing second contact of an organic solution containing an aldehyde monomer with the first reaction mixture to perform a Schiff base reaction to obtain the separation and ionization integrated mass spectrometry ionization source element for trace mycotoxin detection.

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

[0017] According to an embodiment of the present application, 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 application, the aldehyde monomer is 2,3-dihydroxy-p-xylyleneglycol (BDD), tri-aldehyde phloroglucinol (Tp), 2,5-dimethoxy-p-xylyleneglycol (DMTP) or 2,3-dihydroxy-p-xylyleneglycol (DHA).

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

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

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

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

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

[0024] According to an embodiment of the present application, 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 application, the organic solvents of the organic solution containing the amino monomer and the organic solution containing the aldehyde monomer are both tetrahydrofuran.

[0026] According to another aspect of the present application, the present application provides a separation-ionization integrated mass spectrometry device. According to an embodiment of the present application, the device comprises: the aforementioned separation-ionization integrated mass spectrometry ion source element for trace mycotoxin detection; an open mass spectrometry detector, the open mass spectrometry detector comprising a sample inlet, the sample inlet being oppositely arranged with the tip of the mass spectrometry ion source element; and a high-voltage power supply, the high-voltage power supply being connected with the mass spectrometry ion source element.

[0027] The separation-ionization integrated mass spectrometry device according to the embodiment of the present application, the mass spectrometry ionization source element for enriching mycotoxins is directly connected with 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 elution solvent, the generated ions directly enter the mass spectrometer through the inlet of the mass spectrometer to obtain a collection signal, without a chromatographic separation process, the detection step is simple and fast, a plurality of trace mycotoxins can be detected at the same time, the detection flux is large, the background noise of detection is low, and the device is especially suitable for enrichment and detection of multiple types of mycotoxins in food samples.

[0028] According to another aspect of the present application, a method for enriching mycotoxins is provided. According to the embodiment of the present application, the method comprises: performing extraction processing on a sample to be tested to obtain a sample to be tested; and performing oscillation contact processing on the sample to be tested and the separation-ionization integrated mass spectrometry ionization source element for detecting trace mycotoxins to obtain the mass spectrometry ionization source element with the mycotoxins adsorbed on the surface. Thus, the method can specifically enrich a plurality of trace mycotoxins, has strong adsorption, and is beneficial to fully enrich mycotoxins in a complex matrix such as food. Moreover, the enrichment method of the embodiment of the present application is simple to operate, does not require a complex sample pretreatment process, and has high sample extraction efficiency. In addition, the mass spectrometry ionization source element can be reused, and the use cost is low.

[0029] According to the embodiment of the present application, the rotation speed of the oscillation contact processing is 1000-1500 revolutions, and the time is 25-35 minutes.

[0030] According to another aspect of the present application, a method for qualitatively / quantitatively detecting mycotoxins is provided. According to the embodiment of the present application, the method comprises: performing enrichment processing on mycotoxins in a sample to be tested by using the method for enriching mycotoxins to obtain the mass spectrometry ionization source element with the mycotoxins adsorbed on the surface; and performing detection on the mass spectrometry ionization source element with the mycotoxins adsorbed on the surface by using the separation-ionization integrated mass spectrometry device to qualitatively / quantitatively detect the mycotoxins. Thus, the mass spectrometry ionization source element can specifically enrich a plurality of trace mycotoxins in food, has strong adsorption, and is beneficial to fully enrich mycotoxins in a complex matrix. Moreover, the mass spectrometry ionization source element for enriching mycotoxins is directly connected with 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 elution solvent, the generated ions directly enter the mass spectrometer through the inlet of the mass spectrometer to obtain a collection signal, without a chromatographic separation process, the detection step is simple and fast, a plurality of trace mycotoxins can be detected at the same time, the detection flux is large, the background noise of detection is low, the detection sensitivity is high, and the device is especially suitable for large flux, fast, and accurate analysis of micro-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 application, the detection condition of the separation-ionization integrated mass spectrometer is: high voltage power supply voltage: -3.5 kV; ionization eluent: methanol solution containing 0.05-0.15% formic acid.

[0032] According to an embodiment of the present application, the detection condition of the mass spectrometer is: detection mode: multiple reaction monitoring (MRM); atomization gas pressure: 55 psi; auxiliary gas pressure: 50 psi; curtain gas pressure: 30 psi; ion spray voltage: 4500 V; ion source temperature: 550 ℃; residence time: 100 ms.

[0033] According to an embodiment of the present application, the volume of the ionization eluent is 4-6 mL.

[0034] According to an embodiment of the present application, 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).

[0035] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0036] The above and / or additional aspects and advantages of the present application will become apparent and be readily appreciated from the following description, including the appended drawings.

[0037] Figure 1 FIG. 6 shows a schematic diagram of scanning electron microscope results according to an embodiment of the present application, wherein a is a blank stainless steel substrate surface, b is a stainless steel substrate surface after acid treatment, c and d are the surface of a separation-ionization integrated mass spectrometer ionization source element and the cross section of the element for trace mycotoxin detection, respectively;

[0038] Figure 2 FIG. 8 shows a schematic diagram of substrate water contact angle according to an embodiment of the present application, wherein a is a blank stainless steel substrate, b is a stainless steel substrate after acid treatment, c is the surface of a separation-ionization integrated mass spectrometer ionization source element for trace mycotoxin detection;

[0039] Figure 3 FIG. 10 shows a schematic diagram of the results of the selective extraction ability of the element on three types of trace mycotoxins and interfering substances fumonisin (FBs) according to an embodiment of the present application;

[0040] Figure 4This diagram illustrates the result of reusing a mass spectrometry ionization source element according to an embodiment of the present invention.

[0041] Figure 5 An optimization of extraction conditions according to an embodiment of the present invention is shown;

[0042] Figure 6 An optimization of the parsing conditions according to an embodiment of the present invention is shown;

[0043] Figure 7 A method for preparing a separation and ionization integrated mass spectrometry ionization source element and using it for analysis and detection is shown according to an embodiment of the present invention. Detailed Implementation

[0044] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

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

[0046] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0047] According to one aspect of the present invention, an integrated mass spectrometry ionization source element for the detection of trace mycotoxins is provided. The mass spectrometry ionization source element according to embodiments of the present invention has a uniform extraction layer coating with a large specific surface area and a porous structure, enabling targeted enrichment of three types of trace mycotoxins: aflatoxin, ochratoxin, and zearalenone. It enriches a wide variety of mycotoxins, exhibits strong adsorption capacity, and allows for direct mass spectrometry detection via ionization, eliminating the need for chromatographic separation. This results in a simple and rapid detection process with low background noise, high sensitivity, and high accuracy. Furthermore, the mass spectrometry ionization source element is reusable, leading to low operating costs.

[0048] For the purpose of facilitating the understanding of the mass spectrometry ionization source element of the embodiments of the present application, the mass spectrometry ionization source element according to the embodiments of the present application is explained and described herein, which comprises:

[0049] The conductive substrate

[0050] According to the embodiments of the present application, the conductive substrate is formed of stainless steel. The inventors have found that when paper-based or wood-based solid substrate is used, the formation of electrospray after high voltage is applied thereon has high background interference and the generated mass spectrum signal is unstable due to the poor conductivity of the paper-based or wood-based solid substrate when used for open solid substrate electrospray mass spectrometry. The stainless steel substrate can directly have high voltage applied thereon due to its good conductivity, has high ionization efficiency and can further simplify the experimental operation.

[0051] According to the embodiments of the present application, the conductive substrate is isosceles triangular. Thus, the stainless steel sheet is cut into a triangular shape, which has multiple application scenarios and can not only be used as a common extraction plate but also be applied to open electrospray mass spectrometry. When the extraction element is applied to open electrospray mass spectrometry, it is fixed at the horizontal front end position of the mass spectrometry sampling port, the target substance adsorbed on the extraction element is eluted by spraying solvent, ionized at the tip and forms a Taylor cone spray, and directly enters the mass spectrometry detection. According to the embodiments of the present application, the height of the isosceles triangle is 1.5-2.5 cm and the base is 0.5-1.5 cm. Thus, the spray Taylor cone is formed at the tip of the triangle, and the angle is too small or too large, which hinders the elution solvent from forming a spray due to surface tension.

[0052] According to the embodiments of the present application, the thickness of the conductive substrate is 0.1-0.5 mm. Thus, the substrate with the thickness can make the liquid quickly wet the entire surface to form a uniform liquid film, which is conducive to generating stable and uniform spray, and can enhance the electric field strength and stability, accelerate heat transfer and evaporation speed, and thus improve the accuracy and repeatability of analysis.

[0053] The extraction layer

[0054] According to the embodiments of the present application, 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 composed of a repeating unit represented by formula I. Thus, the extraction layer has a large specific surface area, a porous structure and a granular accumulation shape, and can specifically enrich three types of trace fungal toxins, i.e., aflatoxins, ochratoxins and zearalenone, and has strong adsorption.

[0055] According to the embodiments of the present application, the thickness of the extraction layer is 10-20 μm. Thus, the thickness of the extraction layer makes the diffusion path between the functional particles or ligands on the porous matrix of the film and the liquid flow short, the mass transfer fast, the separation time of the film adsorption significantly shortened, and the separation efficiency improved.

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

[0057] According to an embodiment of the present application, 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).

[0058] According to another aspect of the present application, the present application 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 application, the prepared mass spectrometry ionization source element has uniform extraction layer coating, large specific surface area, porous structure and strong stability, can specifically enrich three types of trace mycotoxins, i.e. aflatoxin, ochratoxin and zearalenone, has strong adsorption capacity, and the preparation method has mild conditions, simple steps, good reproducibility of extraction coating and is convenient for industrialized production.

[0059] In order to facilitate the 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 the embodiment of the present application, which comprises:

[0060] S100 acidification treatment

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

[0062] S200 first contact

[0063] The acidized conductive substrate is subjected to first contact with an organic solution containing amino monomers, and is subjected to ultrasonic and oscillation treatment, so that the amino monomers are coated on the surface of the acidized conductive substrate to obtain a first reaction mixture.

[0064] According to the embodiment of the present application, the ultrasonic time is 15-25 minutes, the oscillation time is 0.5-1.5 hours, the temperature is 55-65℃, and the rotation speed is 180-220 rpm. Thus, the amino monomers are fully dissolved in the organic solvent, and the reactants are fully contacted for reaction.

[0065] S300 Schiff base reaction

[0066] The organic solution containing aldehyde group monomer is secondly contacted 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 group 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 application, 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 application, the aldehyde group monomer is 2,3-dihydroxy-p-xylylene diamaldehyde (BDD), tri-aldehyde phloroglucinol (Tp), 2,5-dimethoxy-p-xylylene diamaldehyde (DMTP) or 2,3-dihydroxy-p-xylylene diamaldehyde (DHA).

[0069] According to an embodiment of the present application, the amino monomer is 1,3,5-tris(4-aminophenoxy)benzene and the aldehyde group monomer is 2,3-dihydroxy-p-xylylene diamaldehyde. Thus, the rigid organic skeleton structure formed by the reaction of the two monomers has a specific stereo-geometric configuration and can form π-π conjugation, H-bonding, etc. with the target objects, so as to realize the targeted simultaneous adsorption of the three types of 9 target objects of aflatoxin, ochratoxin and zearalenone through the spatial recognition and intermolecular forces.

[0070] According to an embodiment of the present application, the concentration of the 1,3,5-tris(4-aminophenoxy)benzene is 7-9 mg / mL, preferably 8 mg / mL. Thus, the concentration can not only ensure the high binding rate of the amino monomer to the active sites of the stainless steel substrate, but also avoid unnecessary waste caused by excessive use of raw materials.

[0071] According to an embodiment of the present application, the concentration of the 2,3-dihydroxy-p-xylylene diamaldehyde is 12-13 mg / mL, preferably 12.5 mg / mL. Thus, the concentration can not only ensure the high reaction conversion rate of the reaction of the aldehyde group monomer and the amino monomer, so as to maximize the coverage of the synthesized extraction layer on the surface of the stainless steel substrate, but also avoid unnecessary waste caused by excessive use of raw materials.

[0072] According to an embodiment of the present application, the molar ratio of the 1,3,5-tris(4-aminophenoxy)benzene to the 2,3-dihydroxy-p-phenylenedimethanal is 1:1-2, preferably 2:3. Thus, 1 1,3,5-tris(4-aminophenoxy)benzene molecule has 3 amino groups and 1 2,3-dihydroxy-p-phenylenedimethanal has 2 aldehyde groups. In order to ensure that each reaction group can participate in the reaction and promote the reactants to be maximally converted into the target product, the molar ratio is selected to be 2:3. And under this ratio, the generated product can have a more regular and orderly arrangement in the molecular structure, reducing the occurrence of side reactions.

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

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

[0075] According to an embodiment of the present application, the organic solvents of the organic solution containing the amino monomer and the organic solution containing the aldehyde group monomer are both tetrahydrofuran. Thus, the solubility of the amino monomer and the aldehyde group monomer is high, which can make the monomers uniformly dispersed in the solution, reduce the problem of uneven reaction caused by too high or too low local concentration, and further improve the yield and quality stability of the product.

[0076] According to another aspect of the present application, the present application provides a separation and ionization integrated mass spectrometry device. According to an embodiment of the present application, the device comprises: the aforementioned separation and ionization integrated mass spectrometry ionization source element for trace mycotoxin detection; an open mass spectrometry detector, the open mass spectrometry detector comprising a sample inlet, the sample inlet being oppositely arranged with the tip of the mass spectrometry ionization source element; and a high-voltage power supply, the high-voltage power supply being connected with the mass spectrometry ionization source element.

[0077] The separation and ionization integrated mass spectrometry device according to the embodiment of the present application, the aforementioned mass spectrometry ionization substrate for enriching mycotoxins is directly connected with the high-voltage power supply. Under the action of high-voltage electricity, the target substance 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 needed, the detection step is simple and fast, and three types of trace mycotoxins, i.e., aflatoxins, ochratoxins, and zearalenone, can be detected at the same time. The detection flux is large, and the background noise of the detection is low. The device is especially suitable for the enrichment and detection of multiple types of mycotoxins in food samples.

[0078] According to another aspect of the present application, the present application provides a method for enriching mycotoxins. According to an embodiment of the present application, the method comprises: extracting a sample to be tested to obtain a test liquid; and oscillating the test liquid with the aforementioned separation-ionization integrated mass spectrometry ionization source element for detecting trace mycotoxins to obtain a mass spectrometry ionization source element with the mycotoxins adsorbed on the surface. Thus, the method can specifically enrich the three types of trace mycotoxins, i.e., aflatoxins, ochratoxins, and zearalenone, and has strong adsorption capacity, which is conducive to fully enriching mycotoxins in complex matrices, such as food. In addition, the enrichment method of the embodiment of the present application is simple to operate and does not require a complex sample pretreatment process, and has high sample extraction efficiency. Furthermore, the mass spectrometry ionization source element can be reused, and the use cost is low.

[0079] According to an embodiment of the present application, the rotation speed of the oscillating contact process is 1000-1500 revolutions, and the time is 25-35 minutes. Thus, the mass spectrometry ionization source element can fully adsorb the mycotoxins in the test liquid.

[0080] According to another aspect of the present application, the present application provides a method for qualitatively / quantitatively detecting mycotoxins. According to Figure 7 , according to an embodiment of the present application, the method comprises: enriching mycotoxins in a sample to be tested by using the aforementioned method for enriching mycotoxins to obtain a mass spectrometry ionization source element with the mycotoxins adsorbed on the surface; and detecting the mass spectrometry ionization source element with the mycotoxins adsorbed on the surface by using the aforementioned separation-ionization integrated mass spectrometry device to qualitatively / quantitatively detect the mycotoxins. Thus, the aforementioned mass spectrometry ionization source element can specifically enrich the three types of trace mycotoxins, i.e., aflatoxins, ochratoxins, and zearalenone, in food, and has strong adsorption capacity, which is conducive to fully enriching mycotoxins in complex matrices. In addition, the mass spectrometry ionization source element for enriching mycotoxins is directly connected to a high-voltage power supply, and under the action of high voltage, the target substance on the mass spectrometry ionization source element is ionized under the action of an elution solvent, the generated ions directly enter the mass spectrometer through the inlet of the mass spectrometry detector to obtain a collection signal, and there is no need for a chromatographic separation process. Thus, the detection process is simple and fast, and the method can simultaneously detect the three types of trace mycotoxins, i.e., aflatoxins, ochratoxins, and zearalenone, has a large detection flux, a low background noise, and a high detection sensitivity, and is especially suitable for large-flux, rapid, and accurate analysis of micro-trace mycotoxins. Furthermore, the mass spectrometry ionization source element can be reused, and the detection cost is low.

[0081] According to the embodiment of the present application, the detection condition of the separation-ionization integrated mass spectrometer is: high voltage power supply voltage: -3.5 kV; ionization elution solvent: methanol solution containing 0.05-0.15% formic acid. Thus, when the voltage is -3.5 kV, the signal response is stronger; when the elution solvent is methanol containing 0.05-0.15% formic acid, the signal response is higher, and thus, the selection of the above voltage and elution solvent is conducive to improving the detection signal of the test substance, and the sensitivity and accuracy of the detection are higher.

[0082] According to the embodiment of the present application, the detection condition of the mass spectrometer is: detection mode: multiple reaction monitoring (MRM); atomization gas pressure: 55 psi; auxiliary gas pressure: 50 psi; curtain gas pressure: 30 psi; ion spray voltage: 4500 V; ion source temperature: 550℃; residence time: 100 ms. Thus, under the above conditions, the mycotoxin is detected, the target substance response value is high, the background noise of the detection is low, the detection sensitivity and accuracy are high, and it is especially suitable for rapid and accurate analysis of micro trace mycotoxin.

[0083] According to the embodiment of the present application, the volume of the ionization elution solvent is 4-6 mL. Thus, the volume of the ionization elution solvent is conducive to the full elution of the target substance, and the waste of the solvent is avoided.

[0084] According to the embodiment of the present application, 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).

[0085] The present application will be described below with reference to specific examples, and it should be noted that these examples are merely illustrative and should not be construed as limiting the present application.

[0086] The solutions of the present application will be explained below with reference to examples. Those skilled in the art will understand that the following examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If the specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be purchased, for example, from Sigma Company.

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

[0088] Table 1

[0089]

[0090] Example 1

[0091] By using the method of the embodiment of the present application, a trace mycotoxin separation-ionization integrated mass spectrometry ionization source element with stainless steel as the conductive substrate and TAPOB-BDD covalent organic framework as the extraction layer is prepared, and the preparation process is as follows:

[0092] (a) The stainless steel substrate is 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 treated with 2 mol / L sulfuric acid for 3 hours under ultrasonic treatment, then washed repeatedly with ultrapure water until neutral, and continues to be ultrasonically treated in ultrapure water for 3 hours, quickly dried by nitrogen blowing, and stored in acetonitrile for standby, to obtain an acidified stainless steel substrate.

[0094] (c) TAPOB (80 mg, 0.2 mmol) is weighed into a 200 mL conical flask, followed by the addition of 10 mL THF, and shaken uniformly. The acidified stainless steel substrate in step (b) is added, and after ultrasonic treatment for 20 minutes, it is transferred to a shaking table for reaction at 60°C under a shaking frequency of 200 rpm for 1 hour.

[0095] (d) BDD (50 mg, 0.30 mmol) is weighed into 4 mL THF, and the mixed solution is added to the reaction system of step (c), and 500 μL acetic acid is added dropwise. The reaction system is placed on a shaking table, and continues to be shaken at 60°C under a shaking frequency of 200 rpm for 3 hours, to obtain a trace mycotoxin separation-ionization integrated mass spectrometry ionization source element.

[0096] (e) After the reaction is completed, the element is washed with ethanol and acetonitrile alternately for 3 times, and soaked in acetonitrile for storage, for standby.

[0097] Example 2

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

[0099] 1. The morphology of the surface of the blank stainless steel substrate, the surface of the acid-treated stainless steel substrate, the surface of the mass spectrometry ionization source element prepared in Example 1, and the cross section of the element are characterized by scanning electron microscopy (ZEISS GeminiSEM 300) with an acceleration voltage of 3 kV. The experimental results are shown in Figure 1 , the surface morphology of the blank stainless steel substrate is smooth Figure 1 (a), while the surface of the stainless steel substrate treated with sulfuric acid presents a rough morphology Figure 1b), which is due to the corrosion of the surface of the stainless steel substrate by sulfuric acid, the removal of the surface stainless steel coating, and further oxidation after removal. This provides more binding sites for the modification of amino monomers, which is conducive to the synthesis of the covalent organic framework extraction layer. The granular and dense accumulation structure is visible on the surface of the synthesized ion source element Figure 1 c), which illustrates the successful modification of the covalent organic framework extraction layer. These granular structures have multiple functional groups on their surfaces, which can provide sites for selective adsorption of target substances. The cross-section of the element is observed Figure 1 d), the thickness of the extraction layer on the surface of the element is about 15 μm, further confirming 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 ion source element of Example 1 were analyzed by a contact angle measuring instrument (SZ-CAMC32). The contact angle of the blank stainless steel substrate is 98° Figure 2 a), which indicates that it may be hydrophobic due to the presence of an antioxidant oil layer. After acid treatment, the surface roughness of the stainless steel substrate increases, and accordingly its contact angle decreases to 90° Figure 2 b). Subsequently, after modification of the covalent organic framework extraction layer, the contact angle of the element decreases significantly to 60° Figure 2 c), indicating that the element has good hydrophilicity. This improvement in hydrophilicity promotes the adsorption of mycotoxins in aqueous solution, which is conducive to the extraction and adsorption process.

[0101] Example 3

[0102] In this example, the trace mycotoxin separation-ion integrated mass spectrometry ion source element of Example 1 was used to simultaneously enrich aflatoxins (aflatoxin B1 AFB1, aflatoxin B2 AFB2, aflatoxin G1 AFG1, aflatoxin G2 AFG2), ochratoxin (OTA), and zearalenone (zearalenone ZEN, zearalanone ZEA, zearalenol ZEL, zearalanol ZAL) at the same concentration in a blank solvent (10 μg / L), and the selectivity difference of the trace mycotoxin separation-ion integrated mass spectrometry ion element for target compounds and interferents was compared (aflatoxins, ochratoxins, and zearalenones often exist simultaneously in moldy grains, but the content of fumonisin is relatively high, with a limit of mg / kg, while the content of aflatoxins, ochratoxins, and zearalenones is relatively low, with a limit of μg / kg. The presence of fumonisin interferes with the accurate quantification of trace target compounds). The results are as follows Figure 3As shown, the selectivity of the mass spectrometry ionization source element of Example 1 to the nine mycotoxins of Class 3 is about 4.6-7.5 times that of fumonisin, indicating that the element of Example 1 has selectivity to the nine mycotoxins of Class 3.

[0103] Example 4

[0104] In this embodiment, the trace mycotoxin separation-ionization integrated mass spectrometry ionization source element prepared in Example 1 is used to selectively enrich the nine mycotoxins of Class 3 in a corn sample, and the element is used as an open electrospray ionization mass spectrometry ion source for direct detection analysis, as follows:

[0105] (1) Extraction method

[0106] A commercially available instant corn sample is crushed, 2.0 g is weighed and placed in a 50 mL centrifuge tube, 10 mL of extraction solvent (acetonitrile: water: methanol, v:v:v = 80:19:1) is added, and after ultrasonic extraction for 30 minutes, 10000 rpm 4°C centrifugation is performed for 15 minutes. 5 mL of supernatant is taken, dried by nitrogen blowing, and then redissolved in 10 mL of 5% acetonitrile aqueous solution. The mass spectrometry ionization source element of Example 1 is added, and the extraction is shaken at 1500 rpm for 30 minutes. The element loaded with the target compound is dried and then subjected to open electrospray ionization mass spectrometry detection.

[0107] (2) Detection method

[0108] The element loaded with the target compound is placed on a specially designed three-dimensional moving platform, the tip of the element is adjusted to be 10 mm away from the mass spectrometry inlet, and is slightly tilted downward to help overcome the surface tension and achieve ionization. 20 μL of methanol solution (containing 0.1% formic acid) is added to the surface of the element, 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, which enters the mass spectrometry analysis. The mass spectrometry conditions include: detection mode: multiple reaction monitoring (MRM); atomization gas pressure (GS1): 55 psi; auxiliary gas pressure (GS2): 50 psi; gas curtain pressure: 30 psi; ion spray voltage (IS): 4500 V, ion source temperature: 550°C; monitored ion pairs and collision energies are shown in Table 2. The peak area of the analyte quantitative ion is used for quantification.

[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 reproducibility of the element are shown in Table 3. The quantification limit of the method is much lower than the maximum residue limit of GB 2761-2017 "National Food Safety Standard Maximum Residue Limits of Mycotoxins in Food", meeting the requirements of residue analysis, with good inter-day and intra-day precision, and good reproducibility between batches of elements.

[0114] The target was added to the blank corn sample at concentrations of 0.5, 5 and 25 μg / kg to investigate the recovery of the method. The average recovery of the nine mycotoxins was 83.62-96.97%, meeting the analysis requirements of daily detection.

[0115] Table 3

[0116]

[0117] Example 5

[0118] In this embodiment, the trace mycotoxin separation-ionization integrated mass spectrometry ionization source element of Example 1 was reused, and the experimental method of Example 4 was used to extract and detect the sample to investigate the reusability of the element. The results are shown in Figure 4 In the 8 experiments, the recovery of the 3 types of 9 mycotoxins remained stable, proving that the trace mycotoxin separation-ionization integrated mass spectrometry ionization source element of the embodiment 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 sample, with the difference being that the extraction time, extraction solvent volume, spray solvent type, formic acid concentration in the spray solvent, and spray voltage were changed respectively to investigate the influence of different extraction conditions and elution, spray conditions on the recovery. Specifically, the influence 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 of the 3 types of 9 mycotoxins was investigated. All experiments were set in triplicate, and the average value was taken as the result. The error bar after plotting represents the standard deviation between the parallel data.

[0121] The experimental results are shown in Figure 5 , 6 Specifically, as shown in Figure 5As shown in a, the adsorption amount of the element to the target analyte continuously increased in the adsorption time of 0-40 min, and then reached saturation, indicating that the adsorption kinetics reached equilibrium. Therefore, the preferred extraction time was determined to be 30 min. As shown in b, when the volume of the extraction solution increased, the response of the target compound increased, and the response reached stability after 5 mL. Therefore, 5 mL was the most economical and environmentally friendly extraction volume under the premise of ensuring the extraction effect. Figure 5 As shown in b, when the volume of the extraction solution increased, the response of the target compound increased, and the response reached stability after 5 mL. Therefore, 5 mL was the most economical and environmentally friendly extraction volume under the premise of ensuring the extraction effect. Figure 6 As shown in a, when ethanol and acetonitrile were on the COF solid matrix, their surface tension was very large. This high surface tension made it difficult for the ionization source tip droplet to form smoothly, and the target accumulated on the surface of the solid matrix, and the amount entering the mass spectrometer decreased. The molecular viscosity of ethyl acetate was relatively high, and this characteristic significantly prolonged the time required for ionization. In contrast, methanol had excellent elution and ionization performance, and therefore became the most ideal elution solvent. Figure 6 As shown in b, the addition of formic acid in the spray solvent could significantly improve the ionization efficiency. This is because formic acid can provide the necessary protons in the ionization process, thereby enhancing the mass spectrometry signal. The concentration of formic acid in the methanol solution was optimized, and the results showed that a 0.1% formic acid solution was the most ideal for improving the mass spectrometry response. Therefore, a 0.1% formic acid-methanol solution was selected as the preferred spray solvent. Figure 6 As shown in c, when the voltage was set to be low (less than 2.5 kV), the driving force for the elution of the target was obviously insufficient, which seriously hindered the effective signal collection of the mass spectrometer. If the voltage was too high, the solvent would move rapidly on the solid substrate, causing the target to diffuse, and the effective electrospray process was inhibited, which was also not conducive to the signal collection of the mass spectrometer. The results showed that when the voltage was in the range of 3.1-3.7 kV, the mass spectrometry response increased with the increase of the voltage, and when the voltage exceeded 3.5 kV, the mass spectrometry signal was significantly weakened. Therefore, 3.5 kV was determined to be the preferred spray voltage.

[0122] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0123] Although the embodiments of the present application have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, replacements and variations can be made to these embodiments without departing from the principles and purposes of the present application, and the scope of the present application is defined by the claims and their equivalents.

Claims

1. A separation, ionization integrated mass spectrometry ionization source element for trace mycotoxin detection, characterized by, comprise: a conductive substrate; and an extraction layer formed on at least part of the surface of the conductive substrate, the extraction layer comprising a covalent organic framework material composed of a repeating unit represented by Formula I; ； The method for preparing the separation and ionization integrated mass spectrometry ionization source element for trace mycotoxin detection comprises: acidifying the conductive substrate to obtain an acidified conductive substrate; contacting the acidified conductive substrate with an organic solution containing an amino monomer, performing ultrasonic and oscillation treatment to coat the amino monomer on the surface of the acidified conductive substrate to obtain a first reaction mixture; and contacting an organic solution containing an aldehyde monomer with the first reaction mixture to perform a Schiff base reaction to obtain the separation and ionization integrated mass spectrometry ionization source element for trace mycotoxin detection; the amino monomer is 1,3,5-tris(4-aminophenoxy)benzene; the aldehyde monomer is 2,3-dihydroxy-p-xylylformaldehyde, the concentration of the 1,3,5-tris(4-aminophenoxy)benzene is 7-9 mg / mL; the concentration of the 2,3-dihydroxy-p-xylylformaldehyde is 12-13 mg / mL; the molar ratio of the 1,3,5-tris(4-aminophenoxy)benzene to the 2,3-dihydroxy-p-xylylformaldehyde is 2:3; the catalyst for the Schiff base reaction is acetic acid, 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, the organic solvents of the organic solution containing the amino monomer and the organic solution containing the aldehyde monomer are both tetrahydrofuran; 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).

2. The mass spectrometry ionization source element of claim 1, wherein, the conductive substrate is formed of stainless steel, optionally, the conductive substrate is in the shape of an isosceles triangle with a height of 1.5-2.5 cm and a base of 0.5-1.5 cm, optionally, the thickness of the conductive substrate is 0.1-0.5 mm, optionally, the thickness of the extraction layer is 10-20 μm.

3. The mass spectrometry ionization source element of claim 1, wherein, the contact angle of the extraction layer is 55-65°.

4. The mass spectrometry ionization source element of claim 1, wherein, the concentration of the 1,3,5-tris(4-aminophenoxy)benzene is 8 mg / mL; and the concentration of the 2,3-dihydroxy-p-xylylformaldehyde is 12.5 mg / mL.

5. A separation-ionization integrated mass spectrometer device, characterized by comprising: comprise: the separation and ionization integrated mass spectrometry ionization source element for trace mycotoxin detection according to any one of claims 1-4; an open mass spectrometry detector comprising a sample inlet arranged opposite to the tip of the mass spectrometry ionization source element; and a high-voltage power supply connected to the mass spectrometry ionization source element.

6. A method of enriching mycotoxins, characterized by, comprise: extracting a sample to be tested to obtain a sample solution; and The to-be-tested liquid is subjected to shock contact treatment with the separation-ionization integrated mass spectrometry ionization source element for trace fungal toxin detection according to any one of claims 1-4, so as to obtain the mass spectrometry ionization source element with the fungal toxin adsorbed on the surface, The rotation speed of the shock contact treatment is 1000-1500 revolutions, and the time is 25-35 minutes.

7. A method for the qualitative / quantitative detection of mycotoxins, characterized in that, Comprise: The fungal toxin in the to-be-tested sample is subjected to enrichment treatment by using the method for fungal toxin enrichment according to claim 6, so as to obtain the mass spectrometry ionization source element with the fungal toxin adsorbed on the surface; And The mass spectrometry ionization source element with the fungal toxin adsorbed on the surface is detected by using the separation-ionization integrated mass spectrometry device according to claim 5, so as to perform qualitative / quantitative detection on the fungal toxin, The detection conditions of the separation-ionization integrated mass spectrometry device are: High-voltage power supply voltage: -3.5 kV; Ionization elution solvent: methanol solution containing 0.05-0.15% formic acid; The detection conditions of the mass spectrometry detector are: Detection mode: multiple reaction monitoring (MRM); Atomization gas pressure: 55 psi; Auxiliary gas pressure: 50 psi; Air curtain gas pressure: 30 psi; Ion spray voltage: 4500 V; Ion source temperature: 550 ℃; Residence time: 100 ms, The volume of the ionization elution solvent is 4-6 mL, The fungal toxin 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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