Zearalenone toxin separation and ionization integrated mass spectrometry ionization device and application

Abstract

The application discloses a zearalenone toxin separation and ionization integrated mass spectrometry ionization device and application thereof, wherein the mass spectrometry ionization device comprises a sample carrier table, the sample carrier table comprises a support frame, a detachable extraction element, one end of the extraction element is connected with the support frame, the extraction element comprises a conductive layer formed by stainless steel, an extraction layer located on at least part of the surface of the conductive layer, the extraction layer is formed by a covalent organic polymer, wherein the covalent organic polymer is formed by 1, 3, 5-tri (4-aminophenyl) benzene (TAPB) and 1, 3, 5-triformylphloroglucinol (Tp) through a polymerization reaction, and a high-voltage power supply connected with the extraction element. The extraction layer of the extraction element of the device is formed by a covalent organic polymer, the extraction layer has selective adsorption capacity for zearalenone mycotoxins, and the adsorption capacity is strong.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, and more specifically, to an integrated mass spectrometry ionization device for the detection of zearalenone toxins in zearalenone and its applications. Background Technology

[0002] Zearalenone toxins are a class of toxic secondary metabolites produced by filamentous fungi. They readily contaminate agricultural products and feed, causing serious health problems in humans and animals. When animals ingest feed contaminated with zearalenone toxins, these toxins are metabolized into more toxic secondary products and transferred to animal products (such as milk and eggs), thereby increasing the risk of human exposure to zearalenone toxins.

[0003] Currently, the main methods for detecting zearalenone toxins include enzyme-linked immunosorbent assay (ELISA), sensing technologies, chromatography, and liquid chromatography-mass spectrometry (LC-MS). While ELISA offers rapid detection, it is often a semi-quantitative or qualitative analysis, resulting in high false positive rates and poor repeatability. Sensing technologies typically exhibit poor stability, repeatability, and accuracy in practical applications. Furthermore, most sensing technologies can only detect one type of zearalenone toxin. Chromatography offers advantages such as high sensitivity, strong separation capabilities, and resistance to matrix interference, but it also has limitations, such as the inability to simultaneously analyze multiple zearalenone toxins and the need for complex derivatization of the target zearalenone. In contrast, LC-MS combines the high separation efficiency of liquid chromatography with the high sensitivity of mass spectrometry, enabling accurate and rapid detection of trace amounts of zearalenone toxins in food. However, zearalenone toxins are present in low concentrations in food and readily bind to complex food matrix components. Matrix interference severely affects the sensitivity of detection, requiring effective sample pretreatment techniques for efficient separation and purification. Furthermore, detection using chromatography-mass spectrometry systems is time-consuming.

[0004] Therefore, the analytical techniques for detecting zearalenone toxins in zearalenone compounds need improvement. Summary of the Invention

[0005] This invention aims to at least solve one of the technical problems existing in the prior art. Therefore, one objective of this invention is to provide an integrated mass spectrometry ionization device for the detection of zearalenone toxins. The extraction element of this device is detachable and can be removed and placed in the sample solution for adsorption of zearalenone toxins. It is then mounted on the support frame of the mass spectrometry ionization device, where high voltage is used to achieve ionization of the zearalenone toxins, thereby realizing the extraction, separation, and ionization of the target analyte. This reduces external contamination and sample loss, avoids the use of liquid chromatography, significantly shortens the detection time, and is suitable for rapid extraction and direct mass spectrometry detection of zearalenone fungal toxins in complex samples.

[0006] It should be noted that this invention was completed based on the following work of the inventors:

[0007] Compared to conventional liquid chromatography-mass spectrometry (LC-MS), open-type mass spectrometry (OSMS) for target analytes requires simpler sample pretreatment, sometimes even eliminating the need for column separation, thus significantly reducing detection time—often taking only tens of seconds to analyze a single sample. The basic structure of an open-type mass spectrometry ion source involves introducing the eluent into the injection device, applying high voltage to the eluent or injection device to create an electrospray ionization reaction. Ionization occurs at atmospheric pressure, followed by analysis by the mass spectrometer. Currently, the most common open-type mass spectrometry ion source injection devices are primarily developed from paper spray ionization sources. Electrospray mass spectrometry using a solid substrate as the carrier can be called solid-substrate ESI-MS. Currently used solid matrices include inert materials such as toothpicks, bamboo tips, porous membranes, and glass rods. However, these solid matrices suffer from drawbacks such as difficulty in preparation, small adsorption capacity, and weak enrichment ability, making it difficult to achieve selective adsorption and detection of specific or similar structural compounds. Furthermore, using inert materials as solid substrates results in high background interference and poor resistance to matrix interference. Traditional mass spectrometry ion sources require separate solution injection pumps, nitrogen generators or nitrogen cylinders, high-temperature heating modules, and power supply modules, which are bulky, inconvenient to move, and pose safety risks such as burns from high temperatures. They are only suitable for laboratory applications and difficult to use for real-time field operation, greatly limiting their application in emergency situations and on-site detection. The inventors have creatively used a stainless steel plate coated with covalent organic polymers as the solid matrix, i.e., the extraction element, which has a strong adsorption and enrichment effect on zearalenone toxins. The extraction and enrichment are highly specific. Moreover, the eluent of this extraction element is detected by mass spectrometry through ionization, simplifying the detection steps and resulting in low background noise and high specificity.

[0008] Therefore, according to one aspect of the present invention, the present invention provides a separation and ionization integrated mass spectrometry ionization device for the detection of zearalenone toxins. According to an embodiment of the present invention, the device includes: a sample stage, the sample stage comprising: a support frame; a detachable extraction element, one end of the extraction element being connected to the support frame, the extraction element comprising: a conductive layer formed of stainless steel; an extraction layer located on at least a portion of the surface of the conductive layer, the extraction layer being formed of an organic covalent polymer, wherein the covalent organic polymer is formed by the polymerization reaction of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 1,3,5-triformylphloroglucinol (Tp); and a high-voltage power supply connected to the extraction element.

[0009] According to embodiments of the present invention, the integrated mass spectrometry ionization device for the detection of zearalenone toxins comprises an extraction element whose extraction layer is formed of a covalent organic polymer. This extraction layer exhibits selective and strong adsorption capacity for zearalenone fungal toxins. Furthermore, the extraction element is easily disassembled and used for the extraction and enrichment of target substances without requiring additional solid-phase extraction pretreatment and chromatographic separation procedures. In addition, the mass spectrometry ionization device of the present invention has a simple structure, small size, and is easy to move. It can be used for on-site sample pretreatment and direct mass spectrometry detection, avoiding the use of liquid chromatography. The detection process is simple to operate, highly accurate, and fast, enabling high-efficiency detection of large batches of samples.

[0010] In addition, the integrated mass spectrometry ionization device according to the above embodiments of the present invention may also have the following additional technical features:

[0011] According to an embodiment of the present invention, the extraction element is in the shape of an isosceles triangle, wherein the length of the legs of the isosceles triangle is 1.5-2.5 cm and the length of the base is 0.5-1.5 cm.

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

[0013] According to an embodiment of the present invention, the thickness of the extraction layer is 5-30 μm.

[0014] According to an embodiment of the present invention, a method for preparing the extraction element includes: acidifying a stainless steel sheet to obtain an acidified steel sheet; contacting the acidified steel sheet with a prepolymerization solution to perform a pre-reaction, wherein the prepolymerization solution is a tetrahydrofuran (THF) solution containing 1,3,5-tris(4-aminophenyl)benzene (TAPB); and mixing a secondary reaction solution and acetic acid with the pre-reacted prepolymerization solution to perform a polymerization reaction, so as to form an adsorption layer on the surface of the acidified steel sheet, thereby obtaining the integrated extraction device, wherein the secondary reaction solution contains 1,3,5-triformylphloroglucinol (Tp).

[0015] According to an embodiment of the present invention, the concentration of 1,3,5-tris(4-aminophenyl)benzene in the prepolymer solution is 4-6 mg / mL, preferably 5 mg / mL.

[0016] According to an embodiment of the present invention, the volume ratio of the prepolymerization solution to the secondary reaction solution and the acetic acid is 40-60:15-25:1.

[0017] According to an embodiment of the present invention, the device further includes: a conductive solid sample clamp, which is sleeved on a support frame and connected to the extraction element and the high-voltage power supply.

[0018] According to an embodiment of the present invention, the device further includes: a movable insulating ruler, the insulating ruler being connected to the support frame and located at the upper end of the extraction element, the length of the insulating ruler being adjustable, and the projection of the entire length of the insulating ruler in the horizontal direction being longer than the projection of the extraction element in the horizontal direction.

[0019] According to another aspect of the present invention, a mass spectrometer is provided. According to an embodiment of the present invention, the mass spectrometer includes: a mass spectrometer detector including an inlet; and the aforementioned integrated separation and ionization mass spectrometry ionization device for the detection of zearalenone compounds, wherein the extraction element of the mass spectrometry ionization device is disposed opposite to the inlet.

[0020] The mass spectrometer according to embodiments of the present invention utilizes the aforementioned integrated mass spectrometry ionization device for the extraction and enrichment of target substances, eliminating the need for additional solid-phase extraction pretreatment and chromatographic separation procedures. Furthermore, the extraction element is detachable and, after adsorbing the target analyte, is installed on the integrated mass spectrometry ionization device as a mass spectrometry ion source. Through the action of high-voltage electricity, the ionized target compound can directly enter the mass spectrometry detector for mass spectrometry detection, avoiding the use of liquid chromatography. This results in fast analysis speed and high accuracy. In addition, the mass spectrometer of the present invention is small in size, lightweight, and easy to move and carry, making it suitable for rapid on-site sample detection applications. It should also be noted that the integrated mass spectrometry ionization device of this mass spectrometer possesses all the technical features and effects of the aforementioned integrated mass spectrometry ionization device, which will not be elaborated upon here.

[0021] According to another aspect of the present invention, the present invention provides the use of the aforementioned mass spectrometry ionization device and the aforementioned mass spectrometer in detecting the content of zearalenone compounds. Thus, the surface of the extraction element of the mass spectrometry ionization device and the mass spectrometer is formed of a covalent organic polymer containing various functional groups (C=N; -OH; -NH2), exhibiting selective adsorption capacity for zearalenone mycotoxins, strong adsorption, high detection sensitivity and accuracy, ease of movement, and suitability for the detection of complex samples, especially suitable for the detection of zearalenone mycotoxins.

[0022] According to another aspect of the present invention, the present invention provides a method for detecting the content of zearalenone compounds in a sample to be tested. According to an embodiment of the present invention, the method includes: contacting the sample to be tested with an extraction element of a mass spectrometer to extract zearalenone compounds from the sample; mounting the extraction element on a support frame; adding a spray desorption solvent to the surface of the extraction element to desorb the extracted zearalenone compounds; and applying a high voltage to the extraction element using a high-voltage power supply, ionizing the target compound and detecting it in a mass spectrometer detector to obtain the content of zearalenone compounds in the sample to be tested. Thus, this method utilizes the aforementioned extraction element, the surface of which is formed of a covalent organic polymer containing various functional groups (C=N; -OH; -NH2), exhibiting selective adsorption capacity for zearalenone mycotoxins, strong adsorption, high sensitivity and accuracy of detection, ease of movement, and suitability for the detection of complex samples, especially for the detection of zearalenone mycotoxins.

[0023] According to an embodiment of the present invention, the extraction is carried out with shaking at 1200 rpm for 20-50 minutes, preferably 30 minutes.

[0024] According to an embodiment of the present invention, the volume of the sample to be tested is 5-25 mL, preferably 10 mL.

[0025] According to an embodiment of the present invention, the distance between the tip of the extraction element and the inlet of the mass spectrometer detector is 3-8 mm, preferably 5 mm.

[0026] According to an embodiment of the present invention, the mass ionization conditions of the integrated mass spectrometry ionization device are as follows: the spray voltage of the high-voltage power supply is -2.0 to -4.0 kV, preferably -3.5 kV.

[0027] According to an embodiment of the present invention, the spray desorption solvent is a methanol solution containing (0-0.4)% formic acid, and the volume of the desorption solvent is 10-30 μL, preferably 20 μL.

[0028] According to an embodiment of the present invention, the detection conditions of the mass spectrometer detector are as follows: detection mode: multiple reaction monitoring (MRM); nebulizer gas pressure: 55 psi; auxiliary gas pressure: 50 psi; curtain gas pressure: 20 psi; ion source temperature: 550 °C; residence time: 100 ms.

[0029] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0030] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0031] Figure 1 A schematic diagram of an integrated mass spectrometry ionization device according to an embodiment of the present invention is shown;

[0032] Figure 2 A schematic diagram of an integrated mass spectrometry ionization device according to an embodiment of the present invention is shown;

[0033] Figure 3 A schematic diagram of the structure of an extraction element according to an embodiment of the present invention is shown;

[0034] Figure 4 A schematic diagram of a mass spectrometer according to an embodiment of the present invention is shown.

[0035] Figure 5 A schematic diagram showing the mass spectrometry response signal results of different extraction solvents according to an embodiment of the present invention is displayed;

[0036] Figure 6 A schematic diagram showing extraction yield results at different extraction times according to an embodiment of the present invention is provided.

[0037] Figure 7 A schematic diagram showing extraction yield results for different extraction solvent volumes according to an embodiment of the present invention is provided.

[0038] Figure 8 A schematic diagram showing signal strength results for different spray voltages according to an embodiment of the present invention is displayed;

[0039] Figure 9 A schematic diagram showing signal intensity results for different spray solvents according to an embodiment of the present invention is displayed;

[0040] Figure 10 A schematic diagram showing the elution results of formic acid at different concentrations in methanol according to an embodiment of the present invention is presented. Detailed Implementation

[0041] 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.

[0042] 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.

[0043] 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.

[0044] According to one aspect of the present invention, an integrated mass spectrometry ionization device for the detection of zearalenone toxins is provided. In the integrated mass spectrometry ionization device for the detection of zearalenone toxins according to an embodiment of the present invention, the extraction layer of the extraction element is formed of a covalent organic polymer. This extraction layer has selective adsorption capacity for zearalenone fungal toxins, exhibiting strong adsorption capacity. Furthermore, the extraction element is easily disassembled and used for the extraction and enrichment of target substances without requiring additional solid-phase extraction pretreatment and chromatographic separation procedures. In addition, the mass spectrometry ionization device of the present invention has a simple structure, small size, and is easy to move, allowing for on-site sample pretreatment and direct mass spectrometry detection, avoiding the use of liquid chromatography, and providing rapid analysis.

[0045] According to embodiments of the present invention, the covalent organic polymer has multiple functional groups (C=N; -OH; -NH2), exhibits selective adsorption capacity for zearalenone mycotoxins, and has a strong adsorption capacity.

[0046] To facilitate understanding of the aforementioned mass spectrometry ionization device, refer to... Figure 1-3 According to an embodiment of the present invention, the mass spectrometry ionization device will be explained and described in detail as follows:

[0047] According to an embodiment of the present invention, the mass spectrometry ionization device 100 includes a sample stage 170 and a high-voltage power supply 130. According to an embodiment of the present invention, the sample stage 170 includes a support frame 110 and a detachable extraction element 120, one end of which is connected to the support frame 110. The extraction element 120 includes a conductive layer 122 and an extraction layer 121. According to an embodiment of the present invention, the conductive layer 122 is formed of stainless steel; the extraction layer 121 is located on at least a portion of the surface of the conductive layer 122 and is formed of an organic covalent polymer. According to an embodiment of the present invention, the covalent organic polymer is formed by polymerization of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 1,3,5-triformylphloroglucinol (Tp). According to an embodiment of the present invention, the high-voltage power supply 130 is connected to the extraction element 120, and the high-voltage power supply 130 applies high voltage to the extraction layer 121 to provide energy for the detection of the target analyte forming a spray and ionizing at the tip of the conductive layer 122.

[0048] According to an embodiment of the integrated mass spectrometry ionization device of the present invention, the extraction layer 121 of the extraction element 120 is formed of the covalent organic polymer, which is formed by the polymerization reaction of 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 1,3,5-tricarboxymethylphloroglucinol (Tp). This covalent organic polymer has good selective adsorption capacity for zearalenone mycotoxins, strong adsorption, and is suitable for sample spraying and ionization of complex samples, especially for the detection of zearalenone mycotoxins. Furthermore, the mass spectrometry ionization device has a simple structure, small size, and is easy to disassemble and assemble. It is suitable not only for laboratory use but also for on-site detection applications that require mobility, such as in farmers' markets.

[0049] According to an embodiment of the present invention, the extraction element is an isosceles triangular stainless steel sheet with a leg length of 1.5-2.5 cm and a base length of 0.5-1.5 cm. Thus, the conductive layer is an isosceles triangle. Under the action of high voltage, the eluent dropped onto the extraction element forms a spray Taylor cone at the apex of the triangle. If the angle is too small or too large, or the thickness is too thick, the surface tension of the eluent will hinder spray formation, thus affecting the mass spectrometry signal response. If the conductive layer is less than 0.1 mm thick, its strength is reduced due to its thinness, and deformation during extraction can lead to the peeling off of the covalent organic polymer layer, affecting the adsorption effect. The aforementioned isosceles triangle shape has a suitable angle, facilitates spray formation, and results in a strong mass spectrometry signal response.

[0050] According to an embodiment of the present invention, the thickness of the conductive layer 122 is 0.1-0.5 mm. If the conductive layer 122 is too small, for example less than 0.1 mm, the strength will be reduced due to the thinness, and deformation during the extraction process will cause the covalent organic polymer layer to peel off, affecting the adsorption effect. The conductive layer 122 of the above-mentioned thickness has suitable conductivity, and the covalent polymer is firmly attached and not easily detached.

[0051] According to an embodiment of the present invention, the thickness of the extraction layer is 5-30 μm. Therefore, a thicker layer indicates a greater number of layers between repeating units of the covalent organic polymer, and thus a greater adsorption capacity.

[0052] According to an embodiment of the present invention, a method for preparing the extraction element includes: acidifying a stainless steel sheet to obtain an acidified steel sheet; contacting the acidified steel sheet with a prepolymerization solution to perform a pre-reaction, wherein the prepolymerization solution is a tetrahydrofuran (THF) solution containing 1,3,5-tris(4-aminophenyl)benzene (TAPB); and mixing a secondary reaction solution and acetic acid with the pre-reacted prepolymerization solution to perform a polymerization reaction, so as to form an adsorption layer on the surface of the acidified steel sheet, thereby obtaining the integrated extraction device, wherein the secondary reaction solution contains 1,3,5-triformylphloroglucinol (Tp).

[0053] According to an embodiment of the present invention, the concentration of 1,3,5-tris(4-aminophenyl)benzene in the prepolymerization solution is 4-6 mg / mL, preferably 5 mg / mL. Therefore, the covalent organic polymer formed under this concentration condition has a stable structure and a relatively high yield.

[0054] According to an embodiment of the present invention, the volume ratio of the prepolymerization solution to the secondary reaction solution and the acetic acid is 40-60:15-25:1. Therefore, the catalytic effect is good and the reaction rate is fast.

[0055] According to an embodiment of the present invention, the device further includes: a conductive solid sample clamp 150, which is sleeved on a support frame and connected to the extraction element 120 and the high-voltage power supply 130.

[0056] According to an embodiment of the present invention, the device further includes: a movable insulating ruler 140, which is connected to the support frame 110 and located at the upper end of the extraction element 120. The length of the insulating ruler 140 is adjustable, and the horizontal projection of the entire length of the insulating ruler 140 is longer than the horizontal projection of the extraction element 120. That is, the length of the insulating ruler on the extraction element 120 side after extension is longer than that of the extraction element, which facilitates control of the distance between the integrated mass spectrometry ionization device and the mass spectrometer.

[0057] According to an embodiment of the present invention, the output voltage of the high-voltage power supply 130 is positive or negative, with a voltage intensity of 1-6 kV. Under this voltage intensity, the sample on the extraction element forms a Taylor cone under the action of the high-voltage electric field and undergoes electrospray ionization.

[0058] According to an embodiment of the present invention, the high-voltage power supply 130 is connected to the conductive solid clamp 150 via a copper wire. The copper wire has low resistivity, good ductility, high strength, fatigue resistance, and good stability, making it suitable for stable transmission of high-voltage electricity.

[0059] According to an embodiment of the present invention, the sample stage 170 further includes a platform holder 160, an X-axis translation guide rail, and a Y-axis translation guide rail. The support frame is disposed on the X-axis translation guide rail and the Y-axis translation guide rail, thereby realizing the movement adjustment on the horizontal plane. The platform holder 160 is located at the lower end of the support frame, thereby realizing the fixation of the support frame in a specific position.

[0060] According to another aspect of the present invention, a mass spectrometer is provided. (Reference) Figure 4According to an embodiment of the present invention, the mass spectrometer includes: a mass spectrometer detector 200 and the aforementioned integrated mass spectrometer ionization device 100 for the detection of zearalenone compounds, wherein the mass spectrometer detector 200 includes an inlet 210, and the extraction element 120 of the mass spectrometer ionization device 100 is disposed opposite to the inlet 210.

[0061] The mass spectrometer according to embodiments of the present invention utilizes the aforementioned integrated mass spectrometry ionization device for the extraction and enrichment of target substances, eliminating the need for additional solid-phase extraction pretreatment and chromatographic separation procedures. Furthermore, the extraction element is detachable and, after adsorbing the target analyte, is installed on the integrated mass spectrometry ionization device as a mass spectrometry ion source. Through the action of high-voltage electricity, the ionized target compound can directly enter the mass spectrometry detector for mass spectrometry detection, avoiding the use of liquid chromatography. This results in fast analysis speed and high accuracy. In addition, the mass spectrometer according to embodiments of the present invention is small in size, lightweight, and easy to move and carry, making it suitable for rapid on-site sample detection applications. It should also be noted that the integrated mass spectrometry ionization device of this mass spectrometer possesses all the technical features and effects of the aforementioned integrated mass spectrometry ionization device, which will not be elaborated upon here.

[0062] According to another aspect of the present invention, the present invention provides the use of the aforementioned mass spectrometry ionization device and the aforementioned mass spectrometer in detecting the content of zearalenone compounds. Thus, the surface of the extraction element of the mass spectrometry ionization device and the mass spectrometer is formed of a covalent organic polymer containing various functional groups (C=N; -OH; -NH2), exhibiting selective adsorption capacity for zearalenone mycotoxins, strong adsorption, high detection sensitivity and accuracy, ease of movement, and suitability for the detection of complex samples, especially suitable for the detection of zearalenone mycotoxins.

[0063] According to another aspect of the present invention, the present invention provides a method for detecting the content of zearalenone compounds in a sample to be tested. According to an embodiment of the present invention, the method includes: contacting the sample to be tested with an extraction element of a mass spectrometer to extract zearalenone compounds from the sample; mounting the extraction element on a support frame; adding a spray desorption solvent to the surface of the extraction element to desorb the extracted zearalenone compounds; and applying a high voltage to the extraction element using a high-voltage power supply, ionizing the target compound and detecting it in a mass spectrometer detector to obtain the content of zearalenone compounds in the sample to be tested. Thus, this method utilizes the aforementioned extraction element, the surface of which is formed of a covalent organic polymer containing various functional groups (C=N; -OH; -NH2), exhibiting selective adsorption capacity for zearalenone mycotoxins, strong adsorption, high sensitivity and accuracy of detection, ease of movement, and suitability for the detection of complex samples, especially for the detection of zearalenone mycotoxins.

[0064] According to an embodiment of the present invention, the extraction is carried out with shaking at 1200 rpm for 20-50 minutes, preferably 30 minutes. This ensures sufficient contact between the extraction element and the sample, resulting in a high extraction rate of the target compound.

[0065] According to an embodiment of the present invention, the volume of the sample to be tested is 5-25 mL, preferably 10 mL. Thus, accurate detection can be achieved with a relatively small sample volume.

[0066] According to an embodiment of the present invention, the distance between the tip of the extraction element and the inlet of the mass spectrometer detector is 3-8 mm, preferably 5 mm. This results in a higher response value of the analyte in the mass spectrometer.

[0067] According to an embodiment of the present invention, the mass spectrometry ionization conditions of the integrated mass spectrometry ionization device are as follows: the spray voltage of the high-voltage power supply is -2.0 to -4.0 kV, preferably -3.5 kV. When the voltage is too low, the analytes eluted by the eluent cannot be ionized, resulting in no signal being detected within the detection time. However, if the voltage is too high, the spray movement speed will be too fast, and the target analyte cannot be fully ionized, causing the instrument to be unable to collect all effective signals. Within the above voltage range, the signal intensity of zearalenone fungal toxins is strong, with the signal intensity being even better at -3.5 kV.

[0068] According to an embodiment of the present invention, the spray desorption solvent is a methanol solution containing (0-0.4)% formic acid, and the volume of the desorption solvent is 10-30 μL, preferably 20 μL. Therefore, the desorption effect of zearalenone mycotoxins is good, and the recovery rate is high.

[0069] According to an embodiment of the present invention, the detection conditions of the mass spectrometer detector are as follows: detection mode: multiple reaction monitoring (MRM); nebulizer gas pressure: 55 psi; auxiliary gas pressure: 50 psi; curtain gas pressure: 20 psi; ion source temperature: 550 °C; residence time: 100 ms. Therefore, the mass spectrometry detection has high accuracy and sensitivity.

[0070] According to embodiments of the present invention, the zearalenone fungal toxin is at least one selected from zearalenone (ZEA), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), α-zeenol (α-ZAL), and β-zeenol (β-ZAL).

[0071] The present invention will now be described 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.

[0072] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in the art (e.g., refer to *Principles and Applications of Mass Spectrometry Analysis Technology*, edited by the Taiwan Mass Spectrometry Society and revised by Liu Huwei et al., Science Press) or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available products, such as those purchased from Sigma.

[0073] The materials and reagents used in the embodiments of this invention are shown in Table 1.

[0074] Table 1

[0075]

[0076] Example 1

[0077] According to the method of this embodiment of the invention, using zearalenone as the compound to be extracted, a covalent organic polymer plate is prepared, as follows:

[0078] (A) Cut the stainless steel sheet into an isosceles triangle with a base of 1cm and a side of 2cm.

[0079] (B) Stainless steel sheets were ultrasonically treated with 2 mol / L sulfuric acid for 2 hours. They were then repeatedly washed with ultrapure water until neutral, rinsed with acetonitrile, and dried by nitrogen blowing.

[0080] (C) The treated stainless steel sheet was placed in an Erlenmeyer flask containing 6 mL of tetrahydrofuran solution of 1,3,5-tris(4-aminophenyl)benzene. The flask was sonicated for 10 minutes and shaken on a closed shaker for 30 minutes. 2 mL of tetrahydrofuran solution containing 30.0 mg of 1,3,5-tricarboxymethylresorcinol and 200 μL of acetic acid were added dropwise. The reaction was continued at 70 rpm and 65 °C for 2 hours. The flask was then rinsed with ultrapure water and acetonitrile, and dried with nitrogen to obtain a stainless steel sheet with a covalent organic polymer modified on its surface, which is the extraction element.

[0081] Example 2

[0082] In this embodiment, the extraction element of Example 1 was used to optimize the mass spectrometry detection parameters for zearalenone compounds in milk samples, as detailed below:

[0083] 1. Experimental Methods

[0084] (1) Take 2 mL of milk sample into a 50 mL polypropylene centrifuge tube, add a mixed solution of acetonitrile:water:acetic acid (9:0.9:0.1, V:V:V) and bring the volume to 10 mL. Vortex for 3 min and then sonicate for 20 min. Then centrifuge at 4 °C, 10000 r / min for 10 min. After centrifugation, take 5 mL of the supernatant and blow it to near dryness with nitrogen, then reconstitute it with 10 mL of deionized water for later use.

[0085] (2) Zearalenone compounds were enriched to obtain the enriched product. The specific operation was as follows: take the above test solution, add an extraction element, extract at 1200 rpm for 30 min, and then take it out and quickly wash the surface with deionized water to remove non-specific co-extractants.

[0086] (3) The extraction element, which has adsorbed the target analyte, is detected using an integrated separation and ionization mass spectrometry ionization device. The analytical conditions for this mass spectrometry ionization device include: placing the extraction element on a three-dimensional moving platform, adjusting the tip of the extraction element to be aligned with the mass spectrometer inlet, and adjusting the tip of the extraction element to be 5 mm away from the mass spectrometer inlet; then, adding 20 μL of methanol containing 0.1% formic acid to the surface of the extraction element, allowing it to stand for 10 s, then turning on the high-voltage power supply and applying a high voltage of -3.5 kV. The solution to be tested moves towards the tip of the extraction element, generating analyte ions at the tip and forming a spray, which enters the mass spectrometer for analysis. A schematic diagram of the device structure is shown below. Figure 2 As shown. Mass spectrometry conditions included: detection mode: multiple reaction monitoring (MRM); electrospray voltage (IS): 5500V; nebulizer gas pressure (GS1): 55psi; auxiliary gas pressure (GS2): 50psi; curtain gas pressure (CUR): 20psi; ion source temperature (TEM): 550℃; residence time (DT): 100ms.

[0087] 2. Experimental Results

[0088] (1) The optimized MRM mass spectrometry parameters of the five zearalenone analytes are shown in Table 2, and the peak area of ​​the quantitative ion is used as the evaluation index.

[0089] Table 2:

[0090]

[0091]

[0092] Note: * indicates quantitative ions.

[0093] Example 3

[0094] In this embodiment, the effects of extraction conditions such as sample solvent, extraction time, and sample volume on the detection results of the integrated mass spectrometry ionization device of this invention were investigated. Taking the analysis of five zearalenone compounds in a milk sample as an example, five spiked samples of the zearalenone compounds to be tested were prepared (spiking concentration: 50 μg / L), and analyzed in parallel three times using the mass spectrometry detection conditions of Example 2.

[0095] 1. Extraction solvent optimization

[0096] Extraction solvent is a crucial factor for the effective separation of target analytes. To obtain better extraction results, four different extraction solvents were investigated: acetonitrile (ACN), water (H2O), acetone (Act), and methanol (MeOH). The extraction was performed in triplicate, and the extraction yield was determined using the detection procedure described above. The results are as follows: Figure 5 As shown, the mass spectrometry response signal is highest when water is used as the extraction solvent, indicating that the extraction element can have a good adsorption effect on the target analyte under aqueous phase conditions. Therefore, water is the preferred extraction solvent.

[0097] 2. Optimization of extraction time

[0098] Extraction time is a crucial factor in the extraction process. To obtain optimal extraction time, the effects of different extraction times (10, 20, 30, 40, and 50 min) on the extraction yield were investigated. The experiments were performed in triplicate, and the extraction yield was measured using the aforementioned detection steps. The results are as follows: Figure 6As shown, within 10-30 min, the signal intensities of the five ZEAs (zearalenone compounds) increased with increasing extraction time. However, when the extraction time exceeded 30 min, the signal intensities of the analytes remained almost unchanged, indicating no significant change. This is mainly because the analytes in the sample solution and those adsorbed on the extraction element reached a dynamic equilibrium, demonstrating that the extraction process can be completed in a very short time. This indicates that the extraction element has the potential for rapid analyte extraction. Therefore, an extraction time of 30 min is preferred to obtain a larger adsorption capacity.

[0099] 3. Optimization of extraction solvent volume

[0100] The effects of different extraction solvent volumes (5, 10, 15, 20, and 25 mL) on the extraction yield were studied. The experiments were performed in triplicate, and the results are shown below. Figure 7 As shown, within the extraction solvent volume range of 5-10 mL, the signal intensity increases with increasing extraction solvent volume; however, as the extraction solvent volume continues to increase, the signal intensity change is minimal. This may be because excessive milk matrix occupies some sites on the extraction element, thus affecting the extraction yield of the analyte. Therefore, 10 mL was chosen as the optimal extraction solvent volume, achieving greater extraction of zearalenone compounds while minimizing matrix interference.

[0101] 4. Optimization of spray ionization conditions

[0102] Spray ionization conditions determine the amount of target analytes eluted from the extraction element and the detectable signal of the target analytes, playing a crucial role in open-field mass spectrometry analysis. Two experimental parameters, spray voltage and spray solvent, were investigated, and the signal intensities of five ZEAs analytes were compared to determine optimal spray ionization conditions.

[0103] A. Spray voltage optimization

[0104] Spray voltage significantly affects the analyte signal in open-type mass spectrometry. This study compares different voltages. When the voltage is too low, the analyte eluted by the eluent cannot be ionized, resulting in no detectable signal within the detection time. Conversely, when the voltage is too high, the spray velocity is too fast, preventing sufficient ionization of the target analyte and preventing the instrument from collecting all effective signals. The signal intensity of five zearalenone compounds was detected by applying different voltages, with each voltage measured three times. The results are as follows: Figure 8As shown, the signal strength of the five ZEAs increases with increasing applied voltage. When the absolute voltage increases to 3.5 kV, most analytes exhibit the highest signal strength; thereafter, the signal strength of the five analytes decreases with further increases in absolute voltage. Therefore, -3.5 kV is the preferred voltage to ensure stable and high signal strength for most compounds.

[0105] B. Spray solvent optimization

[0106] For spray solvents, it is necessary not only to maximize the elution of analytes adsorbed on the extraction element, but also to achieve higher ionization efficiency for the analytes. To obtain higher signal intensity, the effects of methanol (MeOH), ethanol (EtOH), acetonitrile (ACN), and ethyl acetate (EAC) as spray solvents on the signal intensity of ZEAs were investigated, with three parallel analyses performed. The results are as follows: Figure 9 As shown, methanol provides better elution. Subsequently, 0.1%, 0.2%, 0.3%, and 0.4% formic acid were added to methanol, and the results were as follows. Figure 10 As shown, the results indicate that methanol containing 0.1% formic acid (FA) has better elution ability for the five ZEAs. Therefore, methanol containing 0.1% formic acid is preferred as the eluent.

[0107] 5. Detection limit and linear range of mass spectrometry

[0108] Five different concentrations of zearalenone compounds were detected in a blank milk matrix, and the linearity, LODs, and LOQs of the standard curves were obtained. The results are shown in Table 3. The linear ranges for the five analytes were all within the range of 0.5–50 ng / mL, and the correlation coefficients (r) were [not specified in the original text]. 2 The LODs of the five tested zearalenone compounds were greater than 0.99, with LODs of 0.1 ng / mL and LODQs of 0.4 ng / mL.

[0109] Table 3

[0110]

[0111] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0112] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A separation-ionization integrated mass spectrometry ionization device for detection of zearalenone mycotoxin, characterized in that, include: The sample stage includes: Support frame; A detachable extraction element, one end of which is connected to the support frame, the extraction element comprising: A conductive layer, wherein the conductive layer is formed of stainless steel; An extraction layer, located on at least a portion of the surface of the conductive layer, the extraction layer being formed of a covalent organic polymer, wherein the covalent organic polymer is formed by the polymerization reaction of 1,3,5-tris(4-aminophenyl)benzene and 1,3,5-tricarboxymethylphloroglucinol; and A high-voltage power supply, which is connected to the extraction element. The method for preparing the extraction element includes: The stainless steel sheet is acidified to obtain an acidified steel sheet. The acidification conditions are as follows: the stainless steel sheet is ultrasonically treated with 2 mol / L sulfuric acid for 2 hours; then it is repeatedly washed with ultrapure water until neutral, rinsed with acetonitrile, and dried by nitrogen blowing. The acid-treated steel sheet is contacted with a prepolymerization solution for a pre-reaction, wherein the prepolymerization solution is a 6 mL tetrahydrofuran solution containing 1,3,5-tris(4-aminophenyl)benzene at a concentration of 5 mg / mL; and The secondary reaction solution and acetic acid are mixed with the pre-polymerization solution after pre-reaction to carry out a polymerization reaction, so as to form an adsorption layer on the surface of the acidified steel sheet and obtain an integrated extraction device. The secondary reaction solution is a 2 mL tetrahydrofuran solution containing 30.0 mg 1,3,5-tricarboxymethyl phloroglucinol. The conductive layer has a thickness of 0.1-0.5 mm, and the extraction layer has a thickness of 5-30 μm.

2. The apparatus of claim 1, wherein, The extraction element is in the shape of an isosceles triangle, with the legs of the isosceles triangle having a length of 1.5-2.5 cm and the base having a length of 0.5-1.5 cm.

3. The apparatus of claim 1, wherein, Further includes: A conductive solid sample holder is fitted onto a support frame and is connected to the extraction element and the high-voltage power supply.

4. The apparatus of claim 1, wherein, Further includes: A movable insulating ruler is provided, which is connected to the support frame and located at the upper end of the extraction element. The length of the insulating ruler is adjustable, and the projection of the total length of the insulating ruler in the horizontal direction is longer than the projection of the extraction element in the horizontal direction.

5. A mass spectrometer, characterized by, include: Mass spectrometer detector, the mass spectrometer detector including an inlet; as well as The separation and ionization integrated mass spectrometry ionization device for the detection of zearalenone compounds according to any one of claims 1-4, wherein the extraction element of the mass spectrometry ionization device is arranged opposite to the sample inlet.

6. Use of the mass spectrometry ionization device according to any one of claims 1-4 and the mass spectrometer according to claim 5 in detecting the content of zearalenone compounds in zeaxanthin.

7. Use according to claim 6, characterized in that, The zearalenone toxin is at least one of zearalenone (ZEA), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), α-zearalenol (α-ZAL), and β-zearalenol (β-ZAL).

8. A method for detecting the content of zearalenone compound in a sample to be tested, characterized in that, include: The sample to be tested is brought into contact with the extraction element of the mass spectrometer of claim 5 in order to extract zearalenone compounds from the sample to be tested; The extraction element is mounted on a support frame, and a spray desorption solvent is dropped onto the surface of the extraction element to desorb the extracted zearalenone compounds. as well as A high voltage is applied to the extraction element by a high voltage power supply, the target analyte is ionized and enters a mass spectrometer for detection, so as to obtain the content of zearalenone compounds in the sample to be tested.

9. The method of claim 8, wherein, The extraction was carried out with shaking at 1200 rpm for 20-50 minutes; The volume of the sample to be tested is 5-25 mL; The distance between the tip of the extraction element and the inlet of the mass spectrometer detector is 3-8 mm; The mass ionization conditions of the integrated mass spectrometry ionization device are as follows: the spray voltage of the high-voltage power supply is -2.0~-4.0kV; The spray desorption solvent is a methanol solution containing 0-0.4% formic acid, and the volume of the desorption solvent is 10-30 μL; The detection conditions of the mass spectrometer detector are as follows: Detection method: Multiple reaction monitoring (MRM); Nebulizer gas pressure: 55 psi; Auxiliary gas pressure: 50 psi; Air curtain pressure: 20 psi; Ion source temperature: 550 ℃; Dwell time: 100 ms.

10. The method of claim 9, wherein, The extraction was carried out with shaking at 1200 rpm for 30 minutes; The volume of the sample to be tested is 10 mL; The distance between the tip of the extraction element and the inlet of the mass spectrometer detector is 5 mm; The mass ionization conditions of the integrated mass spectrometry ionization device are as follows: the spray voltage of the high-voltage power supply is -3.5 kV; The spray desorption solvent is a methanol solution containing 0-0.4% formic acid, and the volume of the desorption solvent is 20 μL; The detection conditions of the mass spectrometer detector are as follows: Detection method: Multiple reaction monitoring (MRM); Nebulizer gas pressure: 55 psi; Auxiliary gas pressure: 50 psi; Air curtain pressure: 20 psi; Ion source temperature: 550 ℃; Dwell time: 100 ms.

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