Aflatoxin separation ionization element and preparation method and kit thereof

Abstract

The invention discloses an aflatoxin separation ionization element and a preparation method and a kit thereof, wherein a method for preparing the aflatoxin separation ionization element, according to an embodiment of the invention, comprises: polymerizing 1,3,5-tris(4-aminophenyl)benzene and 1,3,5-triformylphloroglucinol to obtain an organic polymer; coating the organic polymer on a substrate surface to obtain the separation ionization element; using the separation ionization element for aflatoxin extraction in food, and using the separation ionization element as an ion source element for open mass spectrometry detection. The element can simultaneously realize aflatoxin extraction and direct mass spectrometry detection, the extraction method is simple, the mass spectrometry detection speed is fast, and the sensitivity is high.

Description

Technical Field

[0001] The present invention relates to the field of analysis, specifically, to an aflatoxin separation and ionization element and a preparation method and a kit thereof, and more specifically, to a method for preparing an aflatoxin separation and ionization element, and the aflatoxin separation and ionization element prepared therefrom, a method for detecting aflatoxin content, a kit for detecting aflatoxin and uses thereof. Background Art

[0002] There are many types of mycotoxins, among which aflatoxins are the most toxic type of mycotoxins discovered so far. Aflatoxins (AFs) are secondary metabolites with similar structures and physicochemical properties mainly produced by Aspergillus flavus, Aspergillus parasiticus (A. niger) and Aspergillus fumigatus. More than 20 types of AFs have been discovered so far. Among them, the most common ones in food are aflatoxin B1 (Aflatoxin B1, AFB1), aflatoxin B2 (Aflatoxin B2, AFB2), aflatoxin G1 (Aflatoxin G1, AFG1), aflatoxin G2 (Aflatoxin G2, AFG2), aflatoxin M1 (Aflatoxin M1, AFM1) and aflatoxin M2 (Aflatoxin M2, AFM2). During the growth, production, processing, storage and transportation of food, due to abnormalities in climate, storage and transportation environment, and transportation conditions, it is easy for fungi to breed and produce toxic and harmful aflatoxins.

[0003] The existing detection methods include thin layer analysis, enzyme-linked immunosorbent assay, capillary electrophoresis, high performance liquid chromatography, liquid chromatography-mass spectrometry, etc. Among them, liquid chromatography-mass spectrometry has become the most widely used detection method due to its low detection limit, high sensitivity and high precision. However, since the food matrix is ​​relatively complex and the aflatoxin residues are usually trace residues, the pretreatment technology requirements are relatively high. Common pretreatment methods such as supercritical fluid extraction, accelerated solvent extraction, solid phase extraction, and immunoaffinity chromatography have relatively cumbersome steps and may cause the loss of the target, resulting in inaccurate quantification.

[0004] Therefore, the method for detecting aflatoxin content needs to be improved. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, one object of the present invention is to provide an aflatoxin separation and ionization element and a preparation method and a kit thereof, which can be used for aflatoxin extraction in food and can be used as an ion source element for open mass spectrometry detection. The element can simultaneously realize aflatoxin extraction and direct mass spectrometry detection, with a simple extraction method, fast mass spectrometry detection speed and high sensitivity.

[0006] According to one aspect of the present invention, the present invention provides a method for preparing an aflatoxin separation ionization element. According to an embodiment of the present invention, the method comprises:

[0007] Dissolve 1,3,5-tri(4-aminophenyl)benzene in tetrahydrofuran to obtain a first solution; dissolve 1,3,5-triformylphloroglucinol in tetrahydrofuran to obtain a second solution; add the second solution to the first solution, add acetic acid, and perform a polymerization reaction to obtain an organic polymer; polish and clean the substrate to obtain a pretreated substrate; and immerse the pretreated substrate in a silicone gel solution, take it out, insert it into the organic polymer, and perform a drying and aging treatment after taking it out to obtain the aflatoxin separation and ionization element.

[0008] According to the method for preparing aflatoxin separation and ionization element of an embodiment of the present invention, an organic polymer is prepared on the surface of a substrate. The polymer has the advantages of large specific surface area and good selectivity, can achieve specific extraction of aflatoxin, has a large adsorption capacity, and a simple extraction method. In addition, the separation and ionization element can be used as a substrate directly for open solid substrate electrospray ionization mass spectrometry detection, without the need for a chromatographic separation process, thereby greatly improving the detection speed.

[0009] In addition, the method for preparing an aflatoxin separation and ionization element according to the above embodiment of the present invention may also have the following additional technical features:

[0010] According to an embodiment of the present invention, in the first dissolving solution, the ratio of 1,3,5-tris(4-aminophenyl)benzene (TAPB) to tetrahydrofuran (THF) is 100 mg: 15-25 mL.

[0011] According to an embodiment of the present invention, in the second dissolving solution, the ratio of 1,3,5-triformylphloroglucinol (Tp) to tetrahydrofuran is 60 mg: 6-10 mL.

[0012] According to an embodiment of the present invention, the polymerization reaction is carried out at 55-65° C. and 140-160 rpm / min shaking conditions for 5-7 hours.

[0013] According to an embodiment of the present invention, the substrate is a conical wooden stick, and the tip diameter of the substrate is less than 300 μm.

[0014] According to an embodiment of the present invention, the temperature of the drying and aging treatment is 55-65° C. and the time is 3-5 hours.

[0015] According to an embodiment of the present invention, the silicone adhesive solution is a mixed solution of neutral silicone sealant: benzene in a volume ratio of 1:0.8-1.2.

[0016] According to an embodiment of the present invention, the substrate is a conical wooden stick, and the tip diameter of the substrate is less than 300 μm.

[0017] According to another aspect of the present invention, the present invention provides an aflatoxin separation and ionization element. According to an embodiment of the present invention, the aflatoxin separation and ionization element is prepared by the aforementioned method. Therefore, the substrate surface of the aflatoxin separation and ionization element of the embodiment of the present invention has an organic polymer layer, and the polymer has the advantages of large specific surface area and good selectivity, can achieve specific extraction of aflatoxin, has a large adsorption capacity, and a simple extraction method, and the separation and ionization element can be used as a substrate directly for open solid substrate electrospray mass spectrometry detection, without the need for a chromatographic separation process, which greatly improves the detection speed.

[0018] According to another aspect of the present invention, the present invention provides a method for detecting aflatoxin content. According to an embodiment of the present invention, the method comprises: extracting a sample to be tested to obtain an extract; extracting aflatoxin in the extract using the aforementioned aflatoxin separation and ionization element to obtain a separation and ionization element enriched with aflatoxin; and analyzing the separation and ionization element enriched with aflatoxin to obtain a qualitative / quantitative detection result of aflatoxin in the sample to be tested.

[0019] According to the method for detecting aflatoxin content in an embodiment of the present invention, the aflatoxin in the sample to be tested is extracted using the aforementioned aflatoxin separation and ionization element. The extraction has high specificity and a large adsorption capacity, which reduces the interference of the complex matrix of the sample on subsequent detection. In addition, the method is easy to operate, simple and rapid, and has high recovery rate, reproducibility, sensitivity and accuracy of detection.

[0020] According to an embodiment of the present invention, the extraction process includes: mixing the sample to be tested with acetonitrile and sodium chloride to obtain a sample mixture; vortexing the sample mixture for 3-8 minutes, centrifuging it at a speed of 8000-12000r / min for 5-15 minutes at 4°C to obtain a supernatant; and evaporating and concentrating the supernatant, and then re-dissolving it in water to obtain the extract.

[0021] According to an embodiment of the present invention, the extraction time of the extraction process is 15-30 minutes, preferably, 20 minutes.

[0022] According to an embodiment of the present invention, the eluent for the elution treatment is acetonitrile, and the elution time is 4-10 min, preferably 6 min.

[0023] According to an embodiment of the present invention, the analysis and processing method is performed using high performance liquid chromatography-tandem mass spectrometry detection.

[0024] According to an embodiment of the present invention, the chromatographic conditions in the analysis and detection of the high performance liquid chromatography-tandem mass spectrometry are: chromatographic column: Xbridge C18, specification: 2.1mm×100mm, particle size: 3.5μm; injection volume: 5μL; column temperature: 30°C; flow rate: 0.35μL / min; mobile phase: A: acetonitrile / methanol (V:V, 1:1) solution, B: 0.1% formic acid water; gradient elution conditions are: 0-1min, 97% B; 1-2min, (97%-90%) B; 2-5min, (90%-88%) B; 5-7min, (88%-1%) B; 7-9min, 1% B; 9-11.1min, (1%-97%) B; 11.1-12min, 97% B.

[0025] According to an embodiment of the present invention, the mass spectrometry conditions in the analysis and detection of the high performance liquid chromatography-tandem mass spectrometry are: scanning mode: ESI + ; Detection method: multiple reaction monitoring; Electrospray voltage: 4500V; Nebulizer gas pressure: 35bar; Auxiliary gas pressure: 10bar; Ion source temperature: 550℃; Dwell time: 100ms.

[0026] According to an embodiment of the present invention, the analysis and processing method is performed using open solid substrate electrospray mass spectrometry detection.

[0027] According to an embodiment of the present invention, the open solid substrate electrospray ionization mass spectrometry detection includes: fixing the separation ionization element that has enriched the test compound at the front end of the mass spectrometer injection port, and ionizing the test substance by applying high voltage electricity and spraying solvent and entering the mass spectrometer for analysis and detection.

[0028] According to an embodiment of the present invention, the high voltage applied to the aflatoxin separation and ionization element is: 2.5-5Kv; the spray solvent is: a methanol solution containing (0-1)% acetic acid, which is continuously provided at a speed of 5μL / min using a controllable microflow pump.

[0029] According to an embodiment of the present invention, the aflatoxin is at least one selected from aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), aflatoxin M1 (AFM1) and aflatoxin M2 (AFM2).

[0030] According to an embodiment of the present invention, the sample to be tested is milk or dairy products.

[0031] According to another aspect of the present invention, the present invention provides a kit for detecting aflatoxin. According to an embodiment of the present invention, the kit includes the aforementioned aflatoxin separation and ionization element. Thus, the kit of the embodiment of the present invention utilizes the aforementioned aflatoxin separation and ionization element to extract and enrich aflatoxin in the sample to be tested, and has high adsorption specificity and large adsorption capacity, thereby improving the sensitivity and accuracy of the kit for aflatoxin detection, and is particularly suitable for the detection of aflatoxin in complex matrices.

[0032] According to an embodiment of the present invention, the kit further comprises: an extractant: acetonitrile and sodium chloride; a microporous filter membrane; and a standard solution: aflatoxin mixed standard solution.

[0033] According to an embodiment of the present invention, the aflatoxin is at least one selected from aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1 and aflatoxin M2.

[0034] According to another aspect of the present invention, the present invention provides the use of the aforementioned kit for detecting the content of aflatoxin in milk or dairy products. Since milk and dairy products, as animal-derived foods, may contain aflatoxin residues, the kit of the embodiment of the present invention is particularly suitable for the detection of milk and dairy products, and has high detection accuracy and sensitivity.

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

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

[0037] Figure 1 A schematic diagram showing the scanning electron microscope image results of an aflatoxin separation and ionization element according to an embodiment of the present invention is shown;

[0038] Figure 2A schematic diagram showing the effects of different elution conditions on the detection results according to one embodiment of the present invention, wherein A is the extraction time, B is the type of elution solvent, C is the acidity and alkalinity of the elution solvent, and D is the elution time;

[0039] Figure 3 A schematic diagram showing the results of the reusability of the aflatoxin separation ionization element according to one embodiment of the present invention;

[0040] Figure 4 A schematic diagram of an aflatoxin separation and ionization element for open mass spectrometry detection according to an embodiment of the present invention is shown, wherein A is the aflatoxin separation and ionization element, B is a high voltage power supply, C is a controllable microfluidic pump, and D is a mass spectrometer. DETAILED DESCRIPTION

[0041] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.

[0042] In the description of the present invention, the terms "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and do not require that the present invention must be constructed and operated in a specific orientation. Therefore, they should not be understood as limitations on the present invention.

[0043] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, the meaning of "plurality" is two or more.

[0044] 1. Aflatoxin separation ionization element and preparation method thereof

[0045] According to one aspect of the present invention, the present invention provides a method for preparing an aflatoxin separation and ionization element. According to the method for preparing an aflatoxin separation and ionization element according to an embodiment of the present invention, an organic polymer is prepared on the surface of a substrate to achieve highly selective extraction of aflatoxin, high specificity, large adsorption capacity, and a simple extraction method.

[0046] In order to facilitate understanding of the method for preparing aflatoxin separation ionization element, refer to Figure 4According to an embodiment of the present invention, the method is explained and described, and the method includes:

[0047] S100 reaction solution preparation

[0048] According to an embodiment of the present invention, 1,3,5-tris(4-aminophenyl)benzene (TAPB) is dissolved in tetrahydrofuran (THF) to obtain a first solution; 1,3,5-triformylphloroglucinol (Tp) is dissolved in tetrahydrofuran (THF) to obtain a second solution.

[0049] According to an embodiment of the present invention, the ratio of 1,3,5-tris(4-aminophenyl)benzene (TAPB) to tetrahydrofuran (THF) is 100 mg: 15-25 mL.

[0050] According to an embodiment of the present invention, the dissolution condition is ultrasonic dissolution for 20 minutes, so that TAPB and THF are fully dissolved and contacted, which is beneficial to improve the yield of subsequent organic polymers.

[0051] According to an embodiment of the present invention, the ratio of 1,3,5-triformylphloroglucinol (Tp) to tetrahydrofuran (THF) is 60 mg: 6-10 mL, preferably 60 mg: 8 mL. Thus, the organic polymer formed under this concentration condition has a stable structure and a relatively high yield.

[0052] According to an embodiment of the present invention, the dissolution treatment is carried out under ultrasonic dissolution for 2 minutes, thereby promoting rapid dissolution of Tp.

[0053] S200 Polymerization

[0054] According to an embodiment of the present invention, the second dissolving solution is added to the first dissolving solution, and acetic acid is added to carry out a polymerization reaction to obtain an organic polymer.

[0055] According to an embodiment of the present invention, the polymerization reaction is carried out at 55-65° C. and 140-160 rpm / min shaking conditions for 5-7 hours, thereby achieving a fast reaction rate, a short reaction time, and a high yield of organic polymers.

[0056] According to an embodiment of the present invention, a tetrahydrofuran solution of 1,3,5-triformyl phloroglucinol is added to the first dissolving solution while shaking, and then acetic acid is added, and the reaction is continued on a magnetic stirrer. Thus, adding the tetrahydrofuran solution of 1,3,5-triformyl phloroglucinol to the first dissolving solution is conducive to sufficient contact between Tp and TAPB.

[0057] According to an embodiment of the present invention, based on 100 mg of 1,3,5-tris(4-aminophenyl)benzene (TAPB), the amount of acetic acid added is 0.5-1.5 mL, preferably 1 mL. Thus, the catalytic effect is good and the reaction rate is fast.

[0058] S300 base coating

[0059] According to an embodiment of the present invention, after the substrate is immersed in a silicone gel solution, it is inserted into the organic polymer, taken out and subjected to a drying and aging treatment, so as to obtain the aflatoxin separation and ionization element.

[0060] According to an embodiment of the present invention, the substrate is a conical wooden stick. Therefore, the wooden stick is used as a raw material, which has low cost, excellent performance, and is easy to cut to a suitable size. The microstructure of the surface of the wooden stick is a porous structure, which is easy to cross-link polymers, and the functional material layer formed has a large specific surface area.

[0061] According to an embodiment of the present invention, the temperature of the drying and aging treatment is 55-65° C. and the time is 3-5 hours, thereby ensuring that the organic polymer coated on the extraction substrate is fully aged and dried.

[0062] According to an embodiment of the present invention, the tip diameter of the substrate is less than 300 μm. Therefore, it is convenient to perform open solid substrate electrospray mass spectrometry analysis, fix the prepared separation ionization element at the horizontal front end position of the mass spectrometer injection port, and elute the aflatoxin target adsorbed on the surface of the substrate by applying a spray solvent, and ionize at the tip through the action of a high-voltage power supply to form a Taylor cone spray, so as to directly enter the mass spectrometry detection.

[0063] According to an embodiment of the present invention, the silicone adhesive solution is a mixed solution of neutral silicone sealant: benzene in a volume ratio of 1:0.8-1.2.

[0064] According to an embodiment of the present invention, the substrate is immersed in a silicone gel solution for 30 seconds, slowly pulled out and immediately inserted into a test tube containing an organic polymer powder for 60 seconds, then slowly taken out, placed in an oven for aging, and then ultrasonically cleaned with methanol and dried with a nitrogen flow to obtain an organic polymer modified aflatoxin separation ionization element.

[0065] According to another aspect of the present invention, the present invention provides an aflatoxin separation and ionization element. According to an embodiment of the present invention, the aflatoxin separation and ionization element is prepared by the aforementioned method. Thus, the aflatoxin separation and ionization element has high specificity for aflatoxin adsorption and a large adsorption amount.

[0066] 2. Methods for detecting aflatoxin content

[0067] According to another aspect of the present invention, the present invention provides a method for detecting aflatoxin content. According to the method for detecting aflatoxin content of an embodiment of the present invention, the aflatoxin in the sample to be tested is extracted using the aforementioned aflatoxin separation and ionization element, the extraction has high specificity, large adsorption capacity, reduces the interference of the complex matrix of the sample on subsequent detection, and the method is easy to operate, simple and fast, and has high recovery rate, reproducibility, sensitivity and accuracy of detection.

[0068] In order to facilitate understanding of the method for detecting aflatoxin content, according to an embodiment of the present invention, the method is explained and described, and the method comprises:

[0069] S100 Extraction Processing

[0070] According to an embodiment of the present invention, the sample to be tested is subjected to extraction treatment to obtain an extract solution, thereby extracting aflatoxin compounds from the sample through the extraction treatment.

[0071] According to an embodiment of the present invention, the extraction process is carried out using a mixed solution of acetonitrile and sodium chloride. The inventors compared different extraction solvents and found that the extraction process using acetonitrile and sodium chloride has a high recovery rate of aflatoxin and a high precision of the test results. Through the extraction process, impurities in the sample can be removed, such as the interference of protein with the detection can be removed. Taking into account the stability of the recovery rate, according to an embodiment of the present invention, based on 1g of sample, the amount of acetonitrile added is 1-10mL, and the amount of sodium chloride added is 0.1-2g. As a result, the recovery rate of aflatoxin is high and the accuracy of the test results is higher.

[0072] According to an embodiment of the present invention, the extraction process includes: mixing the sample to be tested with acetonitrile and sodium chloride to obtain a sample mixture; vortexing the sample mixture for 3-8 minutes, centrifuging at 8000-12000 r / min for 5-15 minutes at 4°C to obtain a supernatant; and evaporating and concentrating the supernatant, adding water to redissolve it, and obtaining the extract. This is conducive to fully extracting aflatoxin in the sample to be tested.

[0073] S200 Extraction Process

[0074] According to an embodiment of the present invention, the aforementioned aflatoxin separation and ionization element is used to extract aflatoxin in the extract to obtain a test solution. The inventors have found that the use of the aflatoxin separation and ionization element for extraction treatment, compared with the traditional solid phase extraction column, does not require the steps of filling the solid phase extraction column and multiple elutions, and the entire extraction process is easy to operate, simple and fast, and can achieve the purpose of detecting trace aflatoxin residues in the sample to be tested, while the detection accuracy, recovery rate, and reproducibility are all high.

[0075] According to an embodiment of the present invention, the extraction time of the extraction process is 15-30 minutes, preferably 20 minutes, which is conducive to fully adsorbing the target from the extraction liquid onto the separation ionization element, thereby improving the accuracy of the detection result.

[0076] S300 Analysis and Testing

[0077] According to an embodiment of the present invention, the separation and ionization element enriched with the test compound is subjected to analysis and processing to obtain qualitative / quantitative detection results of the test compound. The analysis and processing includes chromatography tandem mass spectrometry detection and open solid substrate electrospray mass spectrometry detection.

[0078] (1) Chromatography tandem mass spectrometry

[0079] According to an embodiment of the present invention, the separation ionization element enriched with the test compound is eluted, and the aflatoxin content in the eluted solution is analyzed and detected by high performance liquid chromatography-tandem mass spectrometry.

[0080] According to an embodiment of the present invention, the eluent of the elution treatment is acetonitrile. Therefore, acetonitrile as the eluent is conducive to fully eluting the target from the separation ionization element, thereby improving the accuracy of the detection result.

[0081] According to an embodiment of the present invention, the elution time of the elution treatment is 4-10 minutes, preferably 6 minutes, which is conducive to fully eluting the target object from the separation and ionization element and improving the accuracy of the detection result.

[0082] According to an embodiment of the present invention, the chromatographic conditions in the analysis and detection of the high performance liquid chromatography-tandem mass spectrometry are: chromatographic column: Xbridge C18, specification: 2.1mm×100mm, particle size: 3.5μm; injection volume: 5μL; column temperature: 30°C; flow rate: 0.35μL / min; mobile phase: A: acetonitrile / methanol (V:V, 1:1) solution, B: 0.1% formic acid water.

[0083] Further, according to an embodiment of the present invention, the gradient elution program of the HPLC-MS / MS analysis and detection is: 0-1min, 97% B; 1-2min, (97%-90%) B; 2-5min, (90%-88%) B; 5-7min, (88%-1%) B; 7-9min, 1% B; 9-11.1min, (1%-97%) B; 11.1-12min, 97% B. Thus, under the gradient elution conditions, the target compound can be peaked within 9min, the detection speed is improved, and the peak shape of the chromatographic peak is good, and the detection accuracy, recovery rate and reproducibility are good.

[0084] According to an embodiment of the present invention, the mass spectrometry conditions in the analysis and detection of the high performance liquid chromatography-tandem mass spectrometry are: scanning mode: ESI + ; Detection method: multiple reaction monitoring; Electrospray voltage: 4500V; Nebulizer gas pressure: 35bar; Auxiliary gas pressure: 10bar; Ion source temperature: 550℃; Dwell time: 100ms.

[0085] According to an embodiment of the present invention, other mass spectrometry parameters of the mass spectrometer are as follows:

[0086] Table 1

[0087]

[0088] * is the quantitative ion

[0089] Furthermore, the analysis and detection of the embodiments of the present invention also include conventional detection steps in analytical chemistry, such as drawing standard curves, etc. Those skilled in the art can operate according to the specific circumstances, and will not be repeated here. For example, the preparation method of the aflatoxin standard curve is as follows: accurately draw aflatoxin standard, prepare a 0.1 mg / mL standard stock solution with acetonitrile, then accurately weigh an appropriate amount of aflatoxin standard stock solution, and dilute it with acetonitrile to a 10 μg / mL standard intermediate solution; accurately weigh an appropriate amount of standard intermediate solution. Dilute with the initial mobile phase to a series of concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100, 200 ng / mL. Use the aforementioned HPLC-MS / MS system for analysis and detection to obtain a quantitative standard curve for aflatoxin.

[0090] (2) Open solid substrate electrospray ionization mass spectrometry detection

[0091] According to an embodiment of the present invention, the open solid substrate electrospray ionization mass spectrometry detection includes: fixing the separation ionization element that has enriched the test compound at the front end of the mass spectrometer injection port, and ionizing the test substance by applying high voltage electricity and spraying solvent and entering the mass spectrometer for analysis and detection.

[0092] According to some specific embodiments of the present invention, the aflatoxin separation and ionization element after adsorption is mounted on a three-dimensional mobile platform, the wooden tip points to the mass spectrometer (MS) inlet, and is adjusted to a position 5 mm away from the MS inlet. Specifically, a controllable microflow pump can be used to continuously provide a methanol solution containing (0-1)% acetic acid at a rate of 5 μL / min as an elution solvent, and a mobile high-voltage power supply applies a high voltage of 2.5-5 kV to the wooden tip through a copper clamp, and the target generates spray ionization at the wooden tip for mass spectrometry analysis. According to some specific embodiments of the present invention, the mass spectrometer performs target analysis in positive ion mode and MRM mode.

[0093] According to an embodiment of the present invention, the aflatoxin is at least one selected from AFB1, AFB2, AFG1, AFG2, AFM1 and AFM2.

[0094] According to an embodiment of the present invention, the sample to be tested is milk or dairy products. As animal-derived foods, milk or dairy products may contain aflatoxin residues. The method for detecting aflatoxin in the embodiment of the present invention is particularly suitable for detecting milk or dairy products.

[0095] 3. Test kit

[0096] According to another aspect of the present invention, the present invention provides a kit for detecting aflatoxin. According to an embodiment of the present invention, the kit includes the aforementioned aflatoxin separation and ionization element. Thus, the kit of the embodiment of the present invention utilizes the aforementioned aflatoxin separation and ionization element to specifically adsorb aflatoxin in the sample to be tested, and the adsorption specificity is high and the adsorption amount is large, thereby improving the sensitivity and accuracy of the kit for aflatoxin detection, and is particularly suitable for the detection of aflatoxin in complex matrices.

[0097] According to an embodiment of the present invention, the kit further comprises: an extractant: acetonitrile and sodium chloride; a microporous filter membrane; and a standard solution: aflatoxin mixed standard solution.

[0098] According to an embodiment of the present invention, the aflatoxin is at least one selected from aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1 and aflatoxin M2.

[0099] According to another aspect of the present invention, the present invention provides the use of the aforementioned kit for detecting the content of aflatoxin in milk or dairy products. Since milk and dairy products, as animal-derived foods, may contain aflatoxin residues, the kit of the embodiment of the present invention is particularly suitable for the detection of milk and dairy products, and has high detection accuracy and sensitivity.

[0100] The present invention is described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and should not be construed as limiting the present invention.

[0101] Below in conjunction with embodiment, the scheme of the present invention will be explained.It will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.Unrepresented specific techniques or conditions in the embodiment, according to the technology or conditions described in the document in this area or according to the product specification sheet.Reagents used or instruments that do not indicate manufacturers are conventional products that can be obtained commercially, for example, can be purchased from Sigma company.

[0102] Example 1

[0103] This embodiment takes trace amounts of aflatoxin in milk samples as an example, and uses the aflatoxin separation and ionization element of the embodiment of the present invention to perform detection, as follows:

[0104] 1. Experimental methods

[0105] (1) Preparation of organic polymer materials:

[0106] Weigh 100 mg of 1,3,5-tri(4-aminophenyl)benzene (TAPB) in a round-bottom flask, then add 20 mL of tetrahydrofuran (THF), sonicate for 20 min, then put it into a magnetic stirrer and insert a magnetic rotor. Next, weigh 60 mg of 1,3,5-triformylphloroglucinol (Tp) and dissolve it in 8 mL of tetrahydrofuran, sonicate for 2 min to dissolve. Then, add the tetrahydrofuran solution of Tp to the above reaction system, add it while shaking, add 1 mL of acetic acid, and continue to react on a magnetic stirrer for 6 h (60 ° C, 150 rpm / min). After the reaction is completed, the obtained material is washed alternately with ethanol and acetonitrile 3 times, and placed in an oven to dry for use.

[0107] (2) Preparation of aflatoxin separation ionization element:

[0108] A 5 cm long wooden stick was selected as the substrate, the surface was polished smooth and uniform, and then ultrasonically cleaned with ultrapure water and dried at 60 ° C for use. The bonding method was used for preparation, and the specific steps are as follows: (a) Weigh a small amount of dried organic polymer powder for grinding and transfer to a 2.5 mL test tube; (b) The substrate was vertically immersed in a silicone glue solution (neutral silicone sealant: benzene, 1:1) for 30 seconds, then slowly pulled out from the slurry, immediately inserted into a test tube containing COP powder for 60 seconds, then slowly taken out, and then placed in a 60 ° C oven for aging for 4 hours; (c) After aging, the coating of the wooden stick was ultrasonically cleaned with methanol for 10 minutes and dried with nitrogen flow; (d) The excess coating was carefully scraped off with a blade, and the length of the coating was retained to be 2.5 cm. Finally, an organic polymer-modified aflatoxin separation and ionization element was obtained for subsequent experiments.

[0109] (3) Sample milk sample pretreatment:

[0110] Take 10.00 mL of commercially available milk and put it into a 50 mL centrifuge tube. After adding 10 mL of acetonitrile and 2.00 g of sodium chloride, vortex and shake for 5 min. Centrifuge at 10000 r / min for 10 min at 4 °C. Transfer 10 mL of the supernatant to another centrifuge tube, evaporate and concentrate, add ultrapure water to dissolve to 5 mL, and obtain the sample solution for subsequent experiments.

[0111] (4) Extraction process

[0112] Place 2.00 mL of sample solution in a 4 mL sample bottle with a sealing pad, insert the prepared aflatoxin separation ionization element, and place the coating on a shaker for extraction at 150 rpm for 20 minutes. Then immediately place the aflatoxin separation ionization element in a vial with 2 mL of acetonitrile eluent, ultrasonically elute for 6 minutes, collect the eluent, and filter the eluent through a 0.22 μm microporous filter membrane for HPLC-MS / MS analysis.

[0113] (5) On-machine measurement

[0114] Chromatographic column: Xbridge C18, specification: 2.1mm×100mm, particle size: 3.5μm; injection volume: 5μL; column temperature: 30℃; flow rate: 0.35μL / min; mobile phase: A: acetonitrile / methanol (V:V, 1:1) solution, B: 0.1% formic acid water. Gradient elution program: 0-1min, 97% B; 1-2min, (97%-90%) B; 2-5min, (90%-88%) B; 5-7min, (88%-1%) B; 7-9min, 1% B; 9-11.1min, (1%-97%) B; 11.1-12min, 97% B.

[0115] Mass spectrometry conditions:

[0116] Scanning method: ESI + ; Detection method: multiple reaction monitoring; Electrospray voltage: 4500 V; Nebulizer gas pressure: 35 bar; Auxiliary gas pressure: 10 bar; Ion source temperature: 550 ° C; Dwell time: 100 ms; Other mass spectrometry parameters are shown in Table 1.

[0117] (6) Preparation of standard solution and determination of standard curve, detection limit and quantification limit

[0118] Accurately draw aflatoxin standard, prepare a 0.1 mg / mL standard stock solution with acetonitrile, then accurately weigh an appropriate amount of aflatoxin standard stock solution, dilute it with acetonitrile to a 10 μg / mL standard intermediate solution; accurately weigh an appropriate amount of standard intermediate solution. Dilute it with the initial mobile phase to a series of concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100 and 200 ng / mL, use high performance liquid chromatography-tandem triple quadrupole mass spectrometry to detect each standard curve solution, analyze it with the workstation software, and draw a standard curve.

[0119] 2. Experimental results:

[0120] (1) SEM characterization

[0121] The separation ionization element was characterized using a Hitachi S-4800 scanning electron microscope to observe the surface morphology, with an acceleration voltage of 10 kV and magnifications of 30 and 2000 times. Figure 1 It can be clearly observed that the organic polymer material is tightly bound and evenly distributed on the surface of the separation ionization element. Under a larger magnification, it can be observed that the organic polymer material on the surface of the separation ionization element presents a highly cross-linked skeleton structure, providing action sites for the strong adsorption of the separation ionization element.

[0122] (2) Extraction time

[0123] The effects of extraction time of 5, 10, 15, 20, 25, and 30 min on the adsorption capacity were investigated, and three parallel tests were conducted at each time. Figure 2 As shown in Figure A, when the extraction time is less than 20 min, the peak area of ​​6AFs increases with the increase of extraction time, but when the extraction time is greater than 20 min, the peak area of ​​6AFs remains almost unchanged with the increase of extraction time. This may be because when the extraction time is less than 20 min, the adsorption of the separation ionization element has not reached saturation, while when the extraction time exceeds 20 min, the adsorption sites of the separation ionization element tend to be saturated. Therefore, 20 min is selected as the preferred extraction time.

[0124] (3) Elution solvent type

[0125] The type of elution solvent is directly related to the degree of desorption of aflatoxin in the separation ionization element. A good elution solvent can maximize the elution of the target from the separation ionization element. Five different elution solvents, methanol, ethanol, ethyl acetate, acetonitrile, and acetone, were investigated. The results are shown in Figure 2. Figure 2 As shown in Figure B, when acetonitrile is used as the elution solvent, the peak area of ​​the target is the largest and the elution amount is the highest. Therefore, using acetonitrile as the elution solvent can more effectively elute the target.

[0126] (4) Acidity and alkalinity of elution solvent

[0127] The acidity and alkalinity of the elution solvent were adjusted by adding different amounts of formic acid and ammonia water to investigate the effect of the acidity and alkalinity of the elution solvent on the elution performance. Figure 2 As shown in C, the addition of formic acid and ammonia water reduced the elution effect to a certain extent, and it was chosen not to adjust the acidity and alkalinity of the elution solvent.

[0128] (5) Elution time

[0129] The length of the elution time determines whether the six AFs adsorbed on the separation ionization element can be completely eluted, which directly affects the elution volume. The effects of different elution times (2, 4, 6, 8, 10 min) on the elution volume were investigated. The results are as follows: Figure 2As shown in D, when the elution time is short (0-6min), the elution effect is poor. When the elution time is greater than or equal to 6min, the elution amount is high and tends to be stable, indicating that aflatoxin has been basically eluted from the separation ionization element. Therefore, 6min is selected as the preferred elution time.

[0130] (6) Number of times of repeated use

[0131] To evaluate the reusability of the separation and ionization element, five cycles of adsorption experiments were conducted on spiked milk samples with an addition amount of 10 ng / mL, with the recovery rate as the indicator. Figure 3 As shown in the figure, with the increase of the number of uses, the recovery rate has decreased to a certain extent, but the recovery rate of the 5-cycle adsorption experiment is greater than 75%. After 5 cycles, the signal ratio is about 83.67% of the first use. In this case, the separation ionization element shows good regeneration performance and can be recycled many times.

[0132] (7) Preparation of standard curve and determination of detection limit and quantification limit

[0133] Aflatoxin standard solution was prepared in the manner described above, and the detection limit (S / N=3) and quantification limit (S / N=10) of aflatoxin were detected by liquid chromatography-tandem mass spectrometry according to the above detection chromatography-mass spectrometry conditions. Under the optimal conditions, the six aflatoxin analysis methods established were evaluated. As shown in Table 2, the six aflatoxins showed a good linear relationship in the range of 0.05-100 ng / g, and the correlation coefficients were all greater than 0.9995. The LODs and LOQs of this method were 0.01-0.04 ng / g and 0.03-0.13 ng / g, respectively.

[0134] Table 2 Linearity, LOD, LOQ, repeatability and reproducibility analysis of six AFs residues in milk

[0135]

[0136] (8) Precision of the detection method

[0137] Precision refers to the degree of agreement between the test values ​​of repeated tests under the same experimental conditions. The relative standard deviation (RSD%) is used as an indicator. The smaller the RSD value, the better the precision of the test. After the aflatoxin separation ionization element treats the 50ng / mL target solution, the elution is carried out for HPLC-MS / MS detection. The measurement is continued for 3 days, and the intra-day precision and inter-day precision are calculated. The results are shown in Table 2. The intra-day and inter-day precision (RSD) are 4.39-8.21% and 5.79-9.12%, respectively. The detection method of the embodiment of the present invention shows good precision.

[0138] (9) Measurement of actual samples

[0139] Four different brands of milk samples were collected from local supermarkets and analyzed using the detection kit and detection method of this embodiment. No detectable AFs residues were found in these milk samples. Afterwards, three different concentrations of AFs at 5, 10 and 50 ng / g were added to the actual milk samples for recovery determination. As shown in Table 3, the recovery of AFs was between 79.99% and 92.71%, and the RSD% was 3.12% to 9.54%.

[0140] Table 3 Recovery of AFs in real milk samples

[0141]

[0142]

[0143] Example 2

[0144] This embodiment takes trace amounts of aflatoxin in milk samples as an example, and uses the aflatoxin separation and ionization element of the embodiment of the present invention to perform detection based on open solid substrate electrospray ionization mass spectrometry.

[0145] (1) Sample milk sample pretreatment:

[0146] Take 10.00 mL of commercially available milk and put it into a 50 mL centrifuge tube. After adding 10 mL of acetonitrile and 2.00 g of sodium chloride, vortex and shake for 5 min. Centrifuge at 10000 r / min for 10 min at 4 °C. Transfer 10 mL of the supernatant to another centrifuge tube, evaporate and concentrate, add ultrapure water to dissolve to 5 mL, and obtain the sample solution for subsequent experiments.

[0147] (2) Extraction process

[0148] Place 2.00 mL of sample solution in a 4 mL sample bottle with a sealing mat, insert the prepared aflatoxin separation ionization element, and make sure that the coating is completely immersed in the sample solution. Place the element on a shaker and extract at 150 rpm for 20 min.

[0149] (3) On-machine measurement

[0150] The aflatoxin separation and ionization element that has completed the extraction and adsorption process is mounted on a three-dimensional mobile platform, with the wooden tip pointing to the mass spectrometer (MS) inlet and adjusted to a position 5 mm away from the MS inlet. A controllable microflow pump is used to continuously provide a methanol solution containing 0.01% acetic acid at a rate of 5 μL / min as an elution solvent. A mobile high-voltage power supply applies a high voltage of 4 kV through a copper clamp to the wooden tip, and the target generates spray ionization at the wooden tip for mass spectrometry analysis. See the schematic diagram. Figure 4The mass spectrometer was an AB SCIEXQTRAP 5500 ion trap mass spectrometer, and the target analysis was performed in positive ion mode and MRM mode, and the results were obtained by collecting mass spectrometry signals.

[0151] (4) Standard curve and content determination

[0152] Aflatoxin standard solutions with concentrations of 0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50 and 100 ng / mL were used to detect each standard curve solution using an open solid substrate electrospray ionization mass spectrometer. The workstation software was used to analyze the results, and a standard curve was drawn with the area of ​​the spray curve as the ordinate. The content was calculated based on the standard curve.

[0153] In summary, the method for detecting aflatoxin content in the embodiment of the present invention utilizes the aforementioned aflatoxin separation and ionization element to extract aflatoxin in the sample to be tested, and the extraction has high specificity and large adsorption capacity, which reduces the interference of the complex matrix of the sample on subsequent detection. In addition, the method is easy to operate, simple and rapid, and has high recovery rate, reproducibility, sensitivity and accuracy of detection.

[0154] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0155] Although the embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the claims and their equivalents.

Claims

1. A method for preparing an aflatoxin separation ionization element, characterized in that: include: Dissolving 1,3,5-tri(4-aminophenyl)benzene in tetrahydrofuran to obtain a first solution, wherein the ratio of 1,3,5-tri(4-aminophenyl)benzene to tetrahydrofuran is 100 mg:20 mL; Dissolving 1,3,5-triformylphloroglucinol in tetrahydrofuran to obtain a second solution, wherein the ratio of 1,3,5-triformylphloroglucinol to tetrahydrofuran is 60 mg:8 mL; adding the second dissolving solution to the first dissolving solution, and then adding acetic acid to carry out a polymerization reaction to obtain an organic polymer, wherein the polymerization reaction is carried out at 55-65° C. and 140-160 rpm / min shaking conditions for 5-7 hours; grinding and cleaning the substrate to obtain a pre-treated substrate; and The pretreated substrate is immersed in a silicone gel solution, taken out, inserted into the organic polymer, and then taken out for drying and aging treatment to obtain the aflatoxin separation and ionization element. The drying and aging treatment is carried out at a temperature of 55-65°C and for a period of 3-5 hours. The silicone adhesive solution is a mixed solution of neutral silicone sealant and benzene in a volume ratio of 1:0.8-1.

2. The substrate is a conical wooden stick, and the tip diameter of the substrate is less than 300 μm.

2. An aflatoxin separation ionization element, characterized in that: The aflatoxin separation and ionization element is prepared by the method according to claim 1.

3. A method for detecting aflatoxin content, characterized in that: include: Performing extraction treatment on the sample to be tested to obtain an extract; Using the aflatoxin separation and ionization element described in claim 2 to extract aflatoxin in the extract, so as to obtain a separation and ionization element enriched with aflatoxin; as well as The separation and ionization element enriched with aflatoxin is analyzed and processed to obtain the qualitative / quantitative detection result of the aflatoxin in the sample to be tested.

4. The method according to claim 3, characterized in that: The extraction process comprises: Mixing the sample to be tested with acetonitrile and sodium chloride to obtain a sample mixture; The sample mixture is vortexed for 3-8 minutes, and centrifuged at 8000-12000 r / min for 5-15 minutes at 4°C to obtain a supernatant; and The supernatant is evaporated and concentrated, and then redissolved in water to obtain the extract.

5. The method according to claim 3, characterized in that: The extraction time of the extraction process is 15-30 minutes.

6. The method according to claim 5, characterized in that The extraction time of the extraction process is 20 minutes.

7. The method according to claim 3, characterized in that The analysis and processing method is carried out by high performance liquid chromatography-tandem mass spectrometry detection.

8. The method according to claim 7, characterized in that The high performance liquid chromatography-tandem mass spectrometry detection comprises: eluting the separation ionization element enriched with aflatoxin, and analyzing and detecting the aflatoxin content in the eluate by using high performance liquid chromatography-tandem mass spectrometry.

9. The method according to claim 8, characterized in that The eluent of the elution treatment is acetonitrile, and the elution time is 4-10 min.

10. The method according to claim 7, characterized in that The chromatographic conditions in the HPLC-MS / MS detection are: Chromatographic column: Xbridge C18, specification: 2.1mm×100mm, particle size: 3.5μm; Injection volume: 5 μL; Column temperature: 30°C; Flow rate: 0.35 μL / min; Mobile phase: A: acetonitrile / methanol, V:V, 1:1 solution, B: 0.1% formic acid in water; The gradient elution conditions were: 0-1 min, 97% B; 1-2 min, 97%-90% B; 2-5 min, 90%-88% B; 5-7 min, 88%-1% B; 7-9 min, 1% B; 9-11.1 min, 1%-97% B; 11.1-12 min, 97% B.

11. The method according to claim 7, characterized in that The mass spectrometry conditions in the HPLC-MS / MS detection are: Scanning method: ESI + ; Detection method: Multiple reaction monitoring; Electrospray voltage: 4500 V; Atomizing gas pressure: 35 bar; Auxiliary gas pressure: 10bar; Ion source temperature: 550°C; Dwell time: 100ms.

12. The method according to claim 3, characterized in that The analytical processing method is carried out using open solid substrate electrospray mass spectrometry detection.

13. The method according to claim 12, characterized in that The open solid substrate electrospray mass spectrometry detection includes: fixing the separation ionization element enriched with aflatoxin at the front end of the mass spectrometer injection port, and directly applying high voltage electricity and spraying solvent on the element to ionize the substance to be tested and enter the mass spectrometer for analysis and detection.

14. The method according to claim 13, characterized in that The high voltage voltage applied to the aflatoxin separation and ionization element is: 2.5-5Kv; The spray solvent is a methanol solution containing 0-1% acetic acid, which is continuously provided at a speed of 5 μL / min using a controllable microflow pump.

15. The method according to any one of claims 3 to 14, characterized in that: The aflatoxin is at least one selected from aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1 and aflatoxin M2.

16. The method according to claim 3, characterized in that The sample to be tested is milk or dairy products.

17. A kit for detecting aflatoxin, characterized in that: comprising the aflatoxin separation ionization element according to claim 2, Extraction solvent: acetonitrile and sodium chloride; Microporous membranes; and Standard solution: Aflatoxin mixed standard solution, Wherein, the aflatoxin is at least one selected from aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1 and aflatoxin M2.

18. Use of the kit according to claim 17 for detecting aflatoxin content in milk or dairy products.

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