Method for simultaneously detecting aflatoxin B1 and three precursors thereof and early warning method of aflatoxin B1

Abstract

The invention belongs to the technical field of risk early warning detection of harmful pollutants, and particularly relates to a method for simultaneously detecting aflatoxin B1 and three precursors thereof and an early warning method of the aflatoxin B1. The detection method comprises the following steps: taking a crushed sample to be detected, extracting AFB1 and a precursor thereof in the sample to obtain a sample extracting solution, purifying and concentrating the sample extracting solution through IL (at) CTP-DSPE, and then carrying out ultra-high performance liquid chromatography-mass spectrometry or high performance liquid chromatography-ultraviolet detection analysis to obtain qualitative and quantitative results; by utilizing the detection method, the dynamic change of the content of the precursor early warning indicator and the content of the AFB1 can be accurately tracked, correlation analysis is carried out in combination with a heat map, and early warning before AFB1 pollution occurs is realized. According to the method, AFB1 and three precursors thereof in a dynamic sample can be effectively extracted at the same time, and the established detection method is high in accuracy and sensitivity; according to the invention, an early-warning strategy that various precursors are used as early-warning indicators together is proposed for the first time, and the quality safety of grains is guaranteed.

Description

[0001] The present invention belongs to the technical field of risk early warning detection of harmful pollutants, and specifically relates to a method for simultaneously and effectively extracting and sensitively and accurately detecting aflatoxin B1 and three typical precursors thereof in mycelium and grain sample matrices, and an early warning method for aflatoxin B1. Background Art

[0002] Aflatoxins (AFs) are a class of secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus under natural conditions, which seriously contaminate food crops such as corn, rice and wheat. The dihydrofuran ring and coumarin structure make AFs highly toxic to humans and animals at low levels, among which aflatoxin B1 (AFB1) is the most toxic.

[0003] Early warning can identify and predict potential AFs contamination risks by monitoring the dynamic changes of related factors before AFs are produced, which is an effective strategy for controlling and preventing AFs contamination. In recent years, precursors in the aflatoxin biosynthesis pathway have attracted great interest as markers for early risk warning, including versicolorin A (Ver A), versicolorin B (Ver B) and sterigmatocystin (ST). In the process of acetic acid generating AFB1 through multi-step enzymatic reactions, some typical precursors such as averantin (AVN) are the second stable precursor in the AFs biosynthesis pathway, ST is the key precursor of the AFB1 synthesis branch pathway, and O-methylsterigmatocystin (OMST) is the last precursor of the AFB1 synthesis pathway. The production and content of these precursors and AFB1 is a dynamic process. In the early stage of mold contamination, the content of precursors and AFB1 is extremely low; as the mold process develops, multiple precursors with different structures usually coexist and the concentrations vary greatly; in addition, the production of AFB1 and its precursors is the result of quantitative change to qualitative change during the proliferation of mold, during which the composition and quality of grains are in dynamic change. Grains such as wheat, rice and corn are rich in starch, protein, fat and pigments, which make the matrix complex. If AFB1 or its precursors in grains are to be detected during this dynamic change, sample purification is more difficult. Therefore, it is a very challenging task to sensitively and accurately monitor the dynamic changes of AFB1 and multiple precursors in grains to warn of the occurrence of AFB1 contamination early, and improving the extraction and enrichment capabilities in sample pretreatment is the core of solving this problem.

[0004] Wu et al. prepared a poly(methacrylic acid-co-divinylbenzene) monolithic column for in-tube solid phase microextraction determination of AFB1 and ST in rice (Wu F, Xu C, Jiang N, et al. Poly(methacrylic acid-co-diethenyl-benzene) monolithic microextraction column and its application to simultaneous enrichment and analysis of mycotoxins[J]. Talanta, 2017, 178: 1-8). This method only involves AFB1 and a precursor ST, and does not focus on the dynamic changes of AFB1 and ST. The inventor's research group combined Fe3O4 / ZIFs-based magnetic solid-phase extraction with high performance liquid chromatography to effectively extract and detect AVN and ST in rice. However, AFB1 could not be effectively extracted at the same time due to its strong polarity, and the dynamic relationship between precursors AVN and ST and AFB1 could not be given (Zhang Y, Man Y, Li J, et al. Fe3O4 / ZIFs-based magnetic solid-phase extraction for the effective extraction of two precursors with diverse structures in aflatoxinB1 biosynthetic pathway [J]. Talanta, 2023, 259, 124534). Li's team developed a liquid-liquid extraction technique combined with high-resolution mass spectrometry to measure the dynamic changes of AFB1 and its multiple precursor biosynthesis in mycelium. This method has no purification and enrichment steps and is only applicable to mycelium samples with simple matrices. However, it cannot represent real grain samples and cannot truly reflect the AFB1 warning situation in actual grain samples (Xie H, Wang X, Zhang L, et al. Monitoring metabolite production of aflatoxin biosynthesis by Orbitrap Fusion massspectrometry and D-optimal mixture design method [J]. Analytical Chemistry, 2018, 90 (24): 14331-14338). So far, there are few publicly published methods for tracking the dynamic changes of AFB1 and its precursors in moldy grains.In addition, highly sensitive AFB1 and precursor detection methods can identify potential contamination earlier and expand the early warning time window, which is very important for accurately establishing the dynamic relationship between AFB1 and precursors, and the improvement in sensitivity obviously depends on the performance of the extraction medium.

[0005] In summary, using the precursor of AFB1 as an early risk warning marker is an effective strategy to prevent AFB1 contamination. However, the matrix of grain samples is complex, the structure and polarity of AFB1 and its precursors are very different, and the content of precursors is often extremely low in the early stage of moldy growth. The types and contents of precursors and AFB1 are in a dynamic change process throughout the moldy growth process. Therefore, it is necessary to develop sensitive and accurate detection methods suitable for monitoring the dynamic changes of precursors and AFB1 during aflatoxin contamination, so as to provide technical support for analyzing the correlation between the production of precursors and AFB1 and establishing early warning of AFB1. Summary of the invention

[0006] In view of the existing AFB1 early warning method based on precursors as markers, only a simple rice sample matrix is ​​involved, which cannot truly reflect the early warning of AFB1 in actual grain samples with complex matrices, and the problem that the sensitivity is limited and AFB1 and its precursor substances with different structures and polarities cannot be extracted simultaneously and efficiently, the present invention provides a detection method with high sensitivity and the ability to simultaneously determine AFB1 and its three precursors in mycelium and grain samples, and an early warning method for aflatoxin B1 based on the detection method, wherein the detection method is based on the optimized ionic liquid @ covalent organic polymer (IL @ CTP) dispersed solid phase extraction (DSPE) technology combined with ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) instrument. The early warning method is based on the IL @ CTP-DSPE-UHPLC-MS / MS method and is carried out on mycelium and corn samples artificially infected with Aspergillus flavus. In addition, the sample pretreatment can also be combined with high performance liquid chromatography-ultraviolet (HPLC-UV) to simultaneously detect four target substances in rice, wheat and corn samples.

[0007] The present invention specifically adopts the following technical solutions:

[0008] The present invention provides an ionic liquid@covalent organic polymer (IL@CTP), wherein the IL@CTP is prepared by the following steps:

[0009] Step 1: 1,3,5-triphenylbenzene, cyanuric chloride and anhydrous aluminum chloride are reacted in an organic solvent at 70° C. to obtain a covalent organic polymer CTP, wherein the organic solvent can be selected from dichloromethane, the molar ratio of 1,3,5-triphenylbenzene, cyanuric chloride and anhydrous aluminum chloride is 1:1:2, and the reaction time is 14h to 18h.

[0010] Step 2: The covalent organic polymer CTP and 1-methylimidazole obtained in step 1 are reacted in ethanol at 25°C, and then 1-bromobutane is added to react to obtain ionic liquid @ covalent organic polymer (IL@CTP). The molar ratio of 1-methylimidazole to 1-bromobutane is 1:1. The reaction time of the covalent organic polymer and 1-methylimidazole is 15h to 18h, and the reaction time after adding 1-bromobutane is 24h to 26h.

[0011] The specific operation is as follows: 1,3,5-triphenylbenzene and cyanuric chloride are dissolved in dichloromethane, anhydrous aluminum chloride is added, and the mixture is heated to 70°C for reaction for 14h to 18h; after cooling, the mixture is washed and then vacuum dried to obtain CTP; the CTP is weighed and dispersed in ethanol, 1-methylimidazole is added dropwise, and the mixture is stirred at 25°C for 15h to 18h, an equal molar amount of 1-bromobutane is added, and the mixture is stirred for 24h to 26h. The product is washed and vacuum dried to obtain IL@CTP.

[0012] The present invention also provides a method for simultaneously detecting aflatoxin B1 and its three precursors OMST, AVN and ST, comprising:

[0013] The present invention uses AFB1 and its three precursors (OMST, AVN and ST) as targets, combines IL@CTP-based DSPE technology with ultra-high performance liquid chromatography-mass spectrometry or high performance liquid chromatography-ultraviolet to establish a detection method for sensitively and accurately determining the four targets in mycelium and moldy corn, comprising the following steps:

[0014] AFB1 and its three precursors OMST, AVN and ST in the sample were extracted with an extractant to obtain a sample extract. The sample extract was subjected to IL@CTP-based dispersed solid phase extraction and then analyzed by UHPLC-MS / MS to obtain the quantitative results of the four target substances (AFB1 and its three precursors OMST, AVN and ST).

[0015] In a further embodiment, the desorbent in the dispersed solid phase extraction process is methanol, ethanol, acetone or acetonitrile. The amount of IL@CTP used in the dispersed solid phase extraction process and the desorbent used are in a ratio of 1-5 mg:0.25-2.5 mL.

[0016] The adsorption time of IL@CTP in the dispersed solid phase extraction process is 2 to 40 minutes.

[0017] The desorption time in the dispersed solid phase extraction process is 10 to 360 seconds.

[0018] In the optimized IL@CTP-based dispersed solid phase extraction method, the dosage ratio of IL@CTP to desorbent is 2 mg:1 mL. The adsorption time of IL@CTP is 10 min. The desorbent is acetone. The desorption time is 120 s. In addition, the present invention found that when the IL impregnation amount is 6%, the adsorption efficiency of the four target substances AFB1, OMST, AVN and ST is the highest.

[0019] In the embodiment of the present invention, the above optimized DSPE technology is combined with UHPLC-MS / MS for the determination of AFB1, OMST, AVN and ST in samples (such as mycelium and moldy grains); the four target substances in the grains are first extracted, and then the above optimized IL@CTP-based DSPE technology is used to extract the four target substances; then the content of the four target substances is detected by UHPLC-MS / MS. In addition, the DSPE technology is also combined with HPLC-UV for the simultaneous determination of the four target substances in samples (such as rice, wheat and corn).

[0020] The specific steps for extracting AFB1, OMST, AVN and ST from samples are as follows:

[0021] The sample was ground, and the crushed sample was weighed, and an 80% acetonitrile aqueous solution was added, mixed, and then ultrasonically extracted. After centrifugation, the supernatant was removed, concentrated to near dryness by nitrogen blowing, and the residue was redissolved with water. The ratio of the crushed sample to the 80% acetonitrile aqueous solution was 1 g: 4 mL.

[0022] The present invention also provides an AFB1 pollution early warning method, comprising:

[0023] The precursors AVN, ST and OMST are used together as early warning indicators of AFB1 contamination. The dynamic changes of AFB1, OMST, AVN and ST in the samples are detected based on the above method, and then the correlation between OMST, AVN and ST and the occurrence of AFB1 contamination is analyzed to provide early warning of AFB1 contamination in the samples.

[0024] The beneficial effects of the present invention are:

[0025] (1) The existing Fe3O4 / ZIFs-based magnetic solid phase extraction combined with high performance liquid chromatography can simultaneously determine two precursors AVN and ST with different structures and polarities, but it cannot effectively extract AFB1 at the same time, even when an acidic desorbent (0.1 mol·L -1Formic acid / acetonitrile = 1:9 (v / v)) cannot desorb AFB1 well. However, the present invention uses IL@CTP with rich functional groups as the extraction medium, which can have multiple interactions and pore filling effects with the target, and can simultaneously and efficiently extract AFB1, OMST, AVN and ST with different structures and polarities, and the raw materials are cheap and easy to obtain, and the synthesis conditions are mild.

[0026] (2) The present invention is the first to establish a detection method for the simultaneous determination of AFB1 and its three precursors, which can sensitively and accurately detect AFB1, OMST, AVN and ST in mycelium, rice, wheat, corn and moldy corn samples.

[0027] (3) The present invention is the first to use the established IL@CTP-DSPE-UHPLC-MS / MS method to monitor the dynamic changes of AFB1 and its precursors OMST, AVN and ST, and analyze the correlation between the precursors and the occurrence of AFB1 contamination.

[0028] (4) Compared with the IL@CTP-DSPE-HPLC-UV method, the IL@CTP-DSPE-UHPLC-MS / MS method has higher sensitivity and can effectively detect target substances with very low content in the early stage of mold and mildew, thus achieving early warning of AFB1 contamination and providing an effective early warning method for AFB1 contamination in grains. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Structural formulas of AFB1, OMST, AVN and ST.

[0030] Figure 2 :Schematic diagram of the structure of IL@CTP.

[0031] Figure 3 :Characterization of CTP and IL@CTP. A. IR spectrum of IL@CTP; B. Nitrogen adsorption-desorption curves of CTP and IL@CTP; C. Pore size distribution of CTP and IL@CTP; D. SEM images of CTP and IL@CTP and surface scan of C, N, and Br elements of IL@CTP; E. Potential diagram of CTP and IL@CTP.

[0032] Figure 4 : Optimization of IL impregnation amount and extraction conditions. A. Adsorption efficiency of CTP and IL@CTP for four targets. B. Desorbent type, C. Desorbent volume, D. Desorption time, E. Adsorbent mass, F. Effect of adsorption time on extraction efficiency.

[0033] Figure 5 : Heat map of dynamic changes and correlation analysis of AFB1 and precursors in mycelium.

[0034] Figure 6 : Photographs of corn infested with A. flavus stored for 19 days.

[0035] Figure 7 : Heat map of dynamic changes and correlation analysis of AFB1 and precursors in stored corn.

[0036] Figure 8 : Chromatograms of rice (A), wheat (B), and corn (C) samples before and after DSPE extraction. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution in the embodiment of the present invention will be clearly described below in conjunction with the drawings in the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, not all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] The embodiment of the present invention first prepares and screens adsorbents, then optimizes the parameters for extracting AFB1, OMST, AVN and ST with the screened adsorbents, and finally combines the optimized parameters with UHPLC-MS / MS for the determination of AFB1, OMST, AVN and ST in mycelium and moldy corn samples, and combines the optimized extraction technology with UHPLC-MS / MS to establish an early warning method for AFB1 contamination. The present invention also relates to combining the optimized parameters with HPLC-UV for the simultaneous determination of four target substances in samples (such as rice, wheat and corn). The structural formulas of AFB1, OMST, AVN and ST are as follows: Figure 1 The IL@CTP-DSPE-UHPLC-MS / MS and IL@CTP-DSPE-HPLC-UV methods of the present invention can be used for the detection of AFB1, OMST, AVN and ST in cereals (such as rice, wheat and corn), among which the IL@CTP-DSPE-UHPLC-MS / MS method has higher sensitivity and accuracy and is more suitable for the dynamic monitoring of AFB1 and its precursors in samples.

[0039] The Aspergillus flavus strain (NRRL3357) used in the examples was provided by the School of Bioengineering of Henan University of Technology, and the Aspergillus flavus strain may also be replaced by other commercially available products.

[0040] Preparation method of Aspergillus flavus spore suspension: absorb an appropriate amount of Aspergillus flavus spore liquid and apply it to PDA culture medium, invert and culture at 30°C for 5 days, add an appropriate amount of sterile water and collect Aspergillus flavus spores, use a hemocytometer to count the spores, and obtain Aspergillus flavus spore suspension.

[0041] Rice, corn, and wheat were purchased from the local market in Zhengzhou.

[0042] Example 1: Preparation of IL@CTP

[0043] IL@CTP is prepared according to the following steps:

[0044] 3.68g of 1,3,5-triphenylbenzene and 2.22g of cyanuric chloride were dissolved in 150mL of dichloromethane, and then 3.00g of anhydrous aluminum chloride was gradually added, and the mixture was heated to 70°C for reaction for 16h. After natural cooling, it was washed three times with dichloromethane, methanol and water in sequence, and then vacuum dried at 60°C for 12h. The obtained product is CTP. The CTP was weighed and dispersed in 20mL of ethanol, and 0.33g of 1-methylimidazole was added dropwise. It was magnetically stirred at 25°C for 15h, and 1-bromobutane (0.55g) in an amount equimolar to 1-methylimidazole was added. Stirring was continued for 24h, and the product was washed three times with ethanol and vacuum dried at 60°C for 12h to obtain the product IL@CTP. The structural schematic diagram of IL@CTP is shown in the figure Figure 2 shown.

[0045] The prepared CTP and IL@CTP were characterized as follows Figure 3 As shown in the infrared spectrum, 1700cm -1 and 1390cm -1 The characteristic peaks at 1600-1450cm are attributed to C=N and CN vibrations, indicating that IL@CTP has a triazine structure. -1 The peak in the region corresponds to the C=C vibration and the asymmetric CH stretching vibration of the imidazole ring, 1182 cm -1 The characteristic peak at is attributed to the stretching vibration of the imidazole ring, which proves the successful impregnation of ILs. The N2 adsorption / desorption isotherms of CTP and IL@CTP are obvious type I curves. The curve rises rapidly in the low relative pressure region (P / P0=0.001), indicating the existence of a microporous structure. A hysteresis loop appears when P / P0>0.4, indicating the existence of a mesoporous structure. Scanning electron microscopy shows that both CTP and IL@CTP are spherical, with a size of 1.0-1.5μm, and the aggregation of particles causes some particles to have irregular shapes. The presence of Br element in IL@CTP indicates that ILs have been successfully impregnated into CTP. The Zeta potentials of CTP and IL@CTP are -4.6±0.5mV and 6.8±0.6mV, respectively. The introduction of positively charged ILs causes changes in the Zeta potential. The specific surface areas of CTP and IL@CTP are 941.3m 2 / g and 854.2m 2 / g, and the median values ​​of micropore diameters are 0.67nm and 0.5nm, respectively. The reduction in specific surface area and micropore diameter also proves that IL impregnation is successful. The high surface area of ​​IL@CTP means abundant functional groups and adsorption sites, which is conducive to the adsorption of IL@CTP on the target. In addition, the mesopores of IL@CTP (about 2.2nm) allow the analyte to diffuse into the mesopores and adsorb on the inner surface of the mesopores.

[0046] Example 2: Experimental study of adsorbents with different IL impregnation amounts

[0047] This example mainly studies the effect of different IL impregnation amounts on adsorption efficiency. The amount of IL raw material was calculated according to the theoretical nitrogen content provided by IL, and five IL@CTPs with IL impregnation amounts (theoretical nitrogen content provided by IL / (CTP+IL) mass) of 2wt%, 4wt%, 6wt%, 8wt% and 10wt% were prepared. They were used as adsorbents for adsorption of AFB1, OMST, AVN and ST respectively. The specific operation steps are as follows:

[0048] A mixed aqueous solution of AFB1, OMST, AVN and ST was prepared for use, wherein the concentration of each of the four target substances was 20 ng / mL. 20 mL of the mixed aqueous solution of AFB1, OMST, AVN and ST was measured, and 2 mg of CTP and the above five IL@CTPs with different IL impregnation amounts were added respectively. After oscillation at 220 rpm for 5 min, the mixture was centrifuged at 10000 rpm for 5 min. The supernatant was filtered through a 0.22 μm PTFE filter membrane and analyzed by HPLC-UV.

[0049] The adsorption efficiencies obtained were compared. Figure 4 As shown in A.

[0050] from Figure 4 It can be seen in Figure A that among the investigated impregnation amounts, the adsorption efficiency of the four targets was the highest when the IL impregnation amount was 6%. Compared with CTP, the adsorption efficiency of the four targets by IL@CTP with an IL impregnation amount of 6% was increased by 35.7%-53.4%, indicating that IL impregnation significantly improved the adsorption performance of CTP.

[0051] Example 3: Effect of desorbent type on extraction efficiency

[0052] This example mainly studies the effect of the type of desorbent on the extraction recovery rate. The desorbents used are methanol, ethanol, acetonitrile and acetone. The specific operating steps of this example are as follows:

[0053] (1) A blank rice sample (rice without aflatoxin and its precursors) was crushed, 2.0 g of the sample was weighed, 120 ng / g of a mixed standard of AFB1, OMST, AVN and ST was added, 8 mL of an extractant (80% acetonitrile aqueous solution) was added, vortexed for 2 min, ultrasonically extracted for 10 min, centrifuged at 10000 rpm for 5 min, 4 mL of the supernatant was removed, concentrated to about 0.1 mL at 60° C. with nitrogen blowing, and 12 mL of primary water was added to re-dissolve the residue to obtain a sample solution.

[0054] (2) Add 2 mg IL@CTP to 12 mL sample solution, shake at 220 rpm for 10 min, centrifuge at 10,000 rpm for 5 min, and discard the supernatant. Add 1 mL of desorbent methanol, ethanol, acetonitrile, and acetone, respectively, sonicate for 120 s, centrifuge at 10,000 rpm for 5 min, and blow the supernatant to near dryness with nitrogen at 60 °C. Add 200 μL acetonitrile to re-dissolve. Filter through a 0.22 μm PTFE filter membrane and analyze by HPLC-UV.

[0055] The obtained extraction recoveries were compared and the results were as follows: Figure 4 As shown in B.

[0056] from Figure 4 As can be seen in Figure B, among the desorbents investigated, acetone has the highest extraction recovery rate. This may be because acetone contains unsaturated carbonyl groups and has stronger hydrophobicity than other desorbents, which effectively destroys the π-π and hydrophobic interactions between the target and the adsorbent.

[0057] Example 4: Experimental study of different volumes of desorbent

[0058] This example mainly studies the effect of different volumes of desorbent on the extraction recovery rate. The desorbent is acetone, and the volumes of acetone are 0.25mL, 0.5mL, 1mL, 1.5mL, 2mL, and 2.5mL respectively. The specific operation steps of this example are as follows:

[0059] (1) The blank rice sample was crushed, 2.0 g of the sample was weighed, 120 ng / g of 4 mixed standards of AFB1, OMST, AVN and ST were added, 8 mL of the extractant (80% acetonitrile aqueous solution) was added, vortexed for 2 min, ultrasonically extracted for 10 min, centrifuged at 10000 rpm for 5 min, 4 mL of the supernatant was removed, concentrated to about 0.1 mL at 60°C with nitrogen blowing, and 12 mL of primary water was added to re-dissolve the residue to obtain a sample solution.

[0060] (2) Add 2 mg IL@CTP to 12 mL sample solution, shake at 220 rpm for 10 min, centrifuge at 10,000 rpm for 5 min, and discard the supernatant. Add 0.25 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL, and 2.5 mL of acetone, respectively, sonicate for 120 s, centrifuge at 10,000 rpm for 5 min, and blow the supernatant to near dryness with nitrogen at 60 °C. Add 200 μL acetonitrile to reconstitute. Filter through a 0.22 μm PTFE filter membrane and analyze by HPLC-UV.

[0061] The obtained extraction recoveries were compared and the results were as follows: Figure 4 As shown in C.

[0062] from Figure 4 As can be seen in C, when the acetone volume increases from 0.25 mL to 1 mL, the extraction recovery rate gradually increases. When the acetone volume is further increased, the extraction recovery rate remains unchanged, indicating that 1 mL of acetone can achieve complete desorption.

[0063] Example 5: Experimental study of different desorption times

[0064] This example mainly studies the effect of different desorption times on the extraction recovery rate. The desorbent is 1 mL of acetone, and the desorption times are 10 s, 30 s, 60 s, 120 s, 240 s, and 360 s, respectively. The specific operation steps of this example are as follows:

[0065] (1) The blank rice sample was crushed, 2.0 g of the sample was weighed, 120 ng / g of 4 mixed standards of AFB1, OMST, AVN and ST were added, 8 mL of the extractant (80% acetonitrile aqueous solution) was added, vortexed for 2 min, ultrasonically extracted for 10 min, centrifuged at 10000 rpm for 5 min, 4 mL of the supernatant was removed, concentrated to about 0.1 mL at 60°C with nitrogen blowing, and 12 mL of primary water was added to re-dissolve the residue to obtain a sample solution.

[0066] (2) Add 2 mg IL@CTP to 12 mL sample solution, shake at 220 rpm for 10 min, centrifuge at 10,000 rpm for 5 min, and discard the supernatant. Add 1 mL acetone, sonicate for 10 s, 30 s, 60 s, 120 s, 240 s, and 360 s, centrifuge at 10,000 rpm for 5 min, and blow the supernatant to near dryness with nitrogen at 60 °C. Add 200 μL acetonitrile to reconstitute. Filter through a 0.22 μm PTFE filter membrane and analyze by HPLC-UV.

[0067] The obtained extraction recoveries were compared and the results were as follows: Figure 4 As shown in D.

[0068] from Figure 4As can be seen in D, when the desorption time increases from 10s to 120s, the extraction recovery rate gradually increases, and the extraction recovery rate remains unchanged when the desorption time is further extended, indicating that complete desorption can be achieved in 120s.

[0069] Example 6: Experimental study of different adsorbent qualities

[0070] This example mainly studies the effect of different amounts of adsorbent on the extraction recovery rate. The desorbent is 1 mL of acetone, and the desorption time is 120 s. The adsorbent mass is 1 mg, 2 mg, 3 mg, 4 mg, and 5 mg respectively. The specific operation steps of this example are as follows:

[0071] (1) The blank rice sample was crushed, 2.0 g of the sample was weighed, 120 ng / g of 4 mixed standards of AFB1, OMST, AVN and ST were added, 8 mL of the extractant (80% acetonitrile aqueous solution) was added, vortexed for 2 min, ultrasonically extracted for 10 min, centrifuged at 10000 rpm for 5 min, 4 mL of the supernatant was removed, concentrated to about 0.1 mL at 60°C with nitrogen blowing, and 12 mL of primary water was added to re-dissolve the residue to obtain a sample solution.

[0072] (2) Add 1 mg, 2 mg, 3 mg, 4 mg, and 5 mg of IL@CTP to 12 mL of sample solution, shake at 220 rpm for 10 min, centrifuge at 10,000 rpm for 5 min, and discard the supernatant. Add 1 mL of acetone, sonicate for 120 s, centrifuge at 10,000 rpm for 5 min, and blow the supernatant to near dryness with nitrogen at 60 °C. Add 200 μL of acetonitrile to reconstitute. Filter through a 0.22 μm PTFE filter membrane and analyze by HPLC-UV.

[0073] The obtained extraction recoveries were compared and the results were as follows: Figure 4 As shown in E.

[0074] from Figure 4 It can be seen from E that when the adsorbent mass increases from 1 mg to 2 mg, the extraction recovery rate gradually increases. Further increasing the adsorbent mass does not improve the extraction recovery rate, and excessive adsorbent dosage is not conducive to desorption, indicating that the optimal adsorbent mass is 2 mg.

[0075] Example 7: Experimental study of different adsorption times

[0076] This example mainly studies the effect of different adsorption times on the extraction recovery rate. The desorbent is 1 mL of acetone, the desorption time is 120 s, and the adsorbent mass is 2 mg. The adsorption time is 2 min, 5 min, 10 min, 15 min, 20 min, and 25 min respectively. The specific operation steps of this example are as follows:

[0077] (1) The blank rice sample was crushed, 2.0 g of the sample was weighed, 120 ng / g of 4 mixed standards of AFB1, OMST, AVN and ST were added, 8 mL of the extractant (80% acetonitrile aqueous solution) was added, vortexed for 2 min, ultrasonically extracted for 10 min, centrifuged at 10000 rpm for 5 min, 4 mL of the supernatant was removed, concentrated to about 0.1 mL at 60°C with nitrogen blowing, and 12 mL of primary water was added to re-dissolve the residue to obtain a sample solution.

[0078] (2) Add 2 mg IL@CTP to 12 mL sample solution, oscillate and adsorb at 220 rpm for 2 min, 5 min, 10 min, 15 min, 20 min, and 25 min, centrifuge at 10,000 rpm for 5 min, and discard the supernatant. Add 1 mL acetone, sonicate for 120 s, centrifuge at 10,000 rpm for 5 min, and blow the supernatant to near dryness with nitrogen at 60 °C. Add 200 μL acetonitrile to reconstitute. Filter through a 0.22 μm PTFE filter membrane and analyze by HPLC-UV.

[0079] The obtained extraction recoveries were compared and the results were as follows: Figure 4 As shown in F.

[0080] from Figure 4 As can be seen in F, when the adsorption time increases from 2 min to 10 min, the extraction recovery rate gradually increases. Further increasing the adsorbent mass does not improve the extraction recovery rate, but too much adsorption time increases the operation time. It shows that the optimal adsorption time is 10 min. Therefore, the optimized DSPE conditions are: add 2 mg IL@CTP to 12 mL sample solution, oscillate and adsorb at 220 rpm for 10 min, centrifuge at 10000 rpm for 5 min, and discard the supernatant. Add 1 mL acetone, ultrasonicate for 120 s, centrifuge at 10000 rpm for 5 min, and blow the supernatant to near dryness at 60 ° C with nitrogen. Add 200 μL acetonitrile to re-dissolve. Filter through a 0.22 μm PTFE filter membrane.

[0081] Example 8: Performance evaluation of the IL@CTP-DSPE technology combined with UHPLC-MS / MS for the determination of AFB1, OMST, AVN and ST in mycelial samples

[0082] The Aspergillus flavus spore suspension was inoculated into Sabouraud liquid medium to make the inoculum reach 5×10 5 / mL, shake culture at 28℃ (200r / min), collect mycelium by suction and grind with liquid nitrogen. Take 200mg mycelium, add 8mL of extractant (80% acetonitrile aqueous solution), vortex for 2min, ultrasonic extraction for 10min, centrifuge at 10000rpm for 5min, remove 4mL of supernatant, concentrate to near dryness with nitrogen blowing at 60℃, add 12mL of first-grade water to re-dissolve the residue to obtain the sample solution. Then extract under the optimized DSPE conditions, and detect the contents of AFB1, OMST, AVN and ST by UHPLC-MS / MS.

[0083] UHPLC-MS / MS analysis conditions: Vanquish UHPLC system was coupled to TSQ Altis triple quadrupole mass spectrometer. Chromatographic separation was performed using a C18 column (Agilent, 100 mm × 2.1 mm, 2.5 μm), and the mobile phase consisted of A (0.1% formic acid in water) and B (methanol). Flow rate: 0.3 mL min -1 , injection volume 5μL, column temperature 35℃. The gradient elution program was set as follows: 0-2min, 40% B; 3-7min, 40-60% B; 3-7min, 60% B; 7-8min, 60-40% B; 8-10min, 40% B. The analytes were detected in positive ion mode using multiple reaction monitoring MS / MS acquisition mode. The main ion source parameters are as follows: spray voltage 3500V; sheath gas 35Arb; auxiliary gas 5Arb; sweep gas 0Arb. Other related parameters are: ion transfer tube temperature: 350℃; vaporization chamber temperature 350℃; collision gas (CID): 1.5mTorr. The mass spectrometry parameters of the four targets are shown in Table 1.

[0084] Table 1. Physicochemical properties and MS / MS parameters of the four targets

[0085]

[0086] The analytical performance of the DSPE-UHPLC-MS / MS method in mycelial samples is shown in Table 2.

[0087] Table 2. Analytical performance of IL@CTP-DSPE-UHPLC-MS / MS method in mycelium samples

[0088]

[0089] The method of the present invention has good linearity (r 2≥0.9954). The detection limits and quantification limits of AFB1, OMST, AVN and ST in mycelial samples were 0.01-0.02 ng / g and 0.03-0.06 ng / g, respectively. The intra-day and inter-day precisions were both less than 6.1%. The matrix effect was 91.7%-104.3%. This indicates that the method of the present invention has high sensitivity and good repeatability, and the matrix effect can be ignored.

[0090] Example 9: Performance evaluation of the method based on IL@CTP-DSPE technology combined with UHPLC-MS / MS for the determination of AFB1, OMST, AVN and ST in stored corn samples

[0091] Select corn with intact appearance, crush the sample, and sterilize it at 121℃ for 20min. Weigh 2.0g of sample, add 8mL of extractant (80% acetonitrile aqueous solution), vortex for 2min, ultrasonically extract for 10min, centrifuge at 10000rpm for 5min, remove 4mL of supernatant, concentrate to near dryness with nitrogen blowing at 60℃, add 12mL of primary water to dissolve the residue to obtain sample solution. Then extract under optimized DSPE conditions, and detect the contents of AFB1, OMST, AVN and ST by UHPLC-MS / MS (UHPLC-MS / MS analysis conditions in Example 8). The analytical performance of the established IL@CTP-DSPE-UHPLC-MS / MS method in corn samples is shown in Table 3.

[0092] Table 3. Analytical performance of IL@CTP-DSPE-UHPLC-MS / MS method in corn samples

[0093]

[0094]

[0095] The method of the present invention has good linearity (r 2 ≥0.9965). The detection limit and quantification limit of AFB1, OMST, AVN and ST in stored corn samples were 0.02-0.03 ng / g and 0.06-0.1 ng / g, respectively. The intra-day and inter-day precisions were less than 7.9%. The matrix effect was 83.2%-117.7%. This shows that the method of the present invention has high sensitivity and good repeatability, and the matrix effect is very small.

[0096] Example 10: Study on the dynamic changes of AFB1 and its precursor content in liquid culture medium

[0097] The Aspergillus flavus spore suspension was inoculated into 50 mL of Sabouraud liquid medium to make the inoculum volume reach 5 × 10 5 / mL, the control group was not inoculated, and the culture was shaken at 28°C for 9 days (200r / min). From the first day to the sixth day, samples were collected every 12h, and from the seventh day to the ninth day, samples were collected every 24h. The mycelium was filtered and ground with liquid nitrogen. 200mg was taken, 8mL of extractant (80% acetonitrile aqueous solution) was added, vortexed for 2min, ultrasonically extracted for 10min, centrifuged at 10000rpm for 5min, 4mL of supernatant was removed, concentrated to near dryness with nitrogen blowing at 60°C, and 12mL of first-class water was added to re-dissolve the residue to obtain the sample solution as the intracellular sample. In addition, Sabouraud liquid medium (12mL) after the corresponding culture time was taken as the extracellular sample. Then, extraction was carried out under the optimized DSPE conditions, and the contents of AFB1, OMST, AVN and ST were detected by UHPLC-MS / MS (UHPLC-MS / MS analysis conditions in Example 8). The experiment was set up in 3 parallels. The dynamic change pattern and correlation analysis heat map of the content of AFB1 and its precursor (the sum of the content inside and outside the cell) within 9 days are shown in the figure. Figure 5 shown.

[0098] The results showed that the AFB1 content reached the highest on day 5.5 and then decreased. The AVN, ST and OMST contents reached the highest on days 3.5, 4.5 and 5, respectively, and then decreased. The decrease in the precursor content prepared for the production of AFB1. The AFB1 and precursor contents changed greatly from day 1 to day 5.5, and entered a decreasing stage after day 5.5, indicating that the accumulation of AFB1 was very rapid under the conditions of suitable liquid culture medium, and the precursor was produced almost simultaneously with AFB1, and the precursor content reached a peak before AFB1. The correlation analysis heat map of the four analytes showed that OMST (Pearson's = 0.72) and ST (Pearson's = 0.12) were positively correlated with the AFB1 content, and AVN (Pearson's = -0.082) had no significant correlation with AFB1.

[0099] Example 11: Dynamic changes in the content of AFB1 and its precursors in stored corn and early warning strategies based on AFB1 precursors

[0100] Select corn with intact appearance and sterilize it at 121℃ for 20min. Dilute the Aspergillus flavus spore suspension with sterile water at a ratio of 4mL / g, mix well and inoculate it into the corn to make the inoculation amount reach 5×10 5 / g, stored at 4°C for 48h, stirred with a glass rod every 12h to rehydrate the corn evenly. Weigh 25g of inoculated corn and put them into sterile culture dishes (9cm in diameter). Then, put the culture dish into a dryer filled with saturated ammonium sulfate solution and culture it in a dark environment at 28°C for 19 days. Take a sample once a day. The collected samples were stored at -20°C before extraction. The contaminated corn samples were sterilized at 121°C for 30min and dried in a 115°C drying oven for 1h. Then crush them with a grinder and grind them with liquid nitrogen. Corn samples that were not inoculated with Aspergillus flavus were used as blank samples. Extraction was carried out according to the above steps, extraction was carried out under optimized DSPE conditions, and the contents of AFB1, OMST, AVN and ST were detected by UHPLC-MS / MS (UHPLC-MS / MS analysis conditions in Example 8). The experiment was set up in 3 parallels. Photos of corn artificially infected with A. flavus stored in a constant temperature and humidity environment for 19 days are shown in Figure 2. Figure 6 The dynamic changes and correlation analysis heat map of AFB1 and its precursors are shown in Figure 7 shown.

[0101] The results showed that under appropriate conditions, the production and accumulation of AFB1 and its three precursors changed very rapidly. AVN and ST were first detected on the 2nd and 3rd days, respectively, while AFB1 was at undetectable levels. OMST was first detected on the 5th day, and AFB1 was first detected on the same day with a concentration of 2.1ng / g, while the appearance of the corn samples remained unchanged from days 1 to 5. By the 6th day, the AFB1 content was 14.1ng / g, and by the 7th day it had risen to 106.7ng / g, which exceeded the limit standards in China and the United States (<20ng / g). At the same time, a small amount of white hyphae could be observed on the surface of the corn samples ( Figure 6). Since AVN was detected as early as the second day, while AFB1 exceeded the limit on the seventh day, the results showed that AVN could predict the occurrence of AFB1 contamination in stored corn 5 days in advance. The heat map of correlation analysis showed that the levels of OMST and ST, which are closely related in structure, were significantly correlated (Pearson's = 0.85), and both were positively correlated with the level of AFB1, among which OMST (Pearson's = 0.62) had a higher correlation with AFB1 than ST (Pearson's = 0.23). The level of AVN was negatively correlated with the level of AFB1 (Pearson's = -0.28). The dynamic changes and correlations of corn samples inoculated with aflatoxin showed that AVN was detected first, OMST had the highest correlation with AFB1, followed by ST. These precursors coexisted for 6 days, reaching peaks on the 9th day (AVN), 13th day (ST), and 15th day (OMST), respectively, while AFB1 reached its peak on the 17th day. The successive appearance of the precursors also helps to warn the occurrence of AFB1 contamination. The early warning method proposed in the present invention uses the precursors AVN, ST and OMST as early warning indicators of AFB1 contamination to provide a wider early warning time window and more accurate early warning results.

[0102] Example 12: Performance evaluation of the method based on IL@CTP-DSPE technology combined with HPLC-UV for the determination of AFB1, OMST, AVN and ST in cereal samples and its application to actual cereal samples

[0103] (1) Rice samples

[0104] The rice sample was crushed, 2.0 g of the sample was weighed, 8 mL of the extractant (80% acetonitrile aqueous solution) was added, vortexed for 2 min, ultrasonically extracted for 10 min, centrifuged at 10000 rpm for 5 min, 4 mL of the supernatant was removed, concentrated to about 0.1 mL at 60 ° C by nitrogen blowing, and 12 mL of primary water was added to re-dissolve the residue to obtain a sample solution. Then, the extraction was carried out under the optimized DSPE conditions, and the contents of AFB1, OMST, AVN and ST were detected by HPLC-UV.

[0105] HPLC-UV analysis conditions: Shimadzu LC-20A liquid chromatograph, equipped with LC-20AD pump, CTO-10A column oven, SIL-20A autosampler and SPD-20A UV / visible detector. Chromatographic column: Welch Ultimate XB-C30, 250 mm × 4.6 mm, 5 μm particle size; column temperature 35 °C. Mobile phase was acetonitrile / water (55 / 45, v / v), flow rate 1.0 mL min -1 The UV detection wavelength was set to 362 nm (0-6 min) 30 nm (6-17 min), and the injection volume was 20 μL.

[0106] The analytical performance of the established IL@CTP-DSPE-HPLC-UV method in rice samples is shown in Table 4.

[0107] Table 4. Analytical performance of IL@CTP-DSPE-HPLC-UV method in rice samples

[0108]

[0109] The method of the present invention has good linearity (r 2 ≥0.9984). The detection limit and quantification limit of AFB1, OMST, AVN and ST in rice samples were 0.2-0.8 ng / g and 0.6-2.4 ng / g, respectively. The intra-day and inter-day precisions were both less than 7.2%, indicating that the method of the present invention has satisfactory sensitivity and good repeatability.

[0110] After the rice sample was crushed, 2.0 g of the sample was weighed, and the amount of AFB1, OMST, AVN and ST was spiked at three levels and processed according to the above method. Each spiked sample was measured three times in parallel and the average value was taken. The spike recovery of the sample was calculated based on the actual addition amount and the measured results. The results are shown in Table 5. The representative chromatograms of the rice sample before and after extraction are shown in Table 5. Figure 8 As shown in A.

[0111] Table 5. Detection of AFB1, OMST, AVN and ST in rice by IL@CTP-DSPE-HPLC-UV (n=3)

[0112]

[0113] Level 1: The spiked amounts of AFB1, OMST, AVN and ST were 1.2, 2.4, 1.2 and 1.2 ng / g respectively; Level 2: The spiked amounts were all 6 ng / g; Level 3: The spiked amounts were all 24 ng / g; ND: not detected; SD: standard deviation.

[0114] The results showed that AFB1, OMST, AVN and ST were not detected in rice samples. In addition, the sample spike recovery was 86.7% to 104.4%, and the precision was 1.5% to 6.9%. The experimental results showed that the established analytical method has satisfactory accuracy and repeatability and can be used to determine AFB1, OMST, AVN and ST in rice samples.

[0115] (2) Wheat samples

[0116] The wheat sample was crushed, 2.0 g of the sample was weighed, 8 mL of the extractant (80% acetonitrile aqueous solution) was added, vortexed for 2 min, ultrasonically extracted for 10 min, centrifuged at 10000 rpm for 5 min, 4 mL of the supernatant was removed, concentrated to about 0.1 mL by nitrogen blowing at 60 ° C, and 12 mL of primary water was added to re-dissolve the residue to obtain the sample solution. Then, the extraction was carried out under the optimized DSPE conditions, and the contents of AFB1, OMST, AVN and ST were detected by HPLC-UV (HPLC-UV conditions in rice samples). The analytical performance of the established IL@CTP-DSPE-HPLC-UV method in wheat samples is shown in Table 6.

[0117] Table 6. Analytical performance of IL@CTP-DSPE-HPLC-UV method in wheat samples

[0118]

[0119]

[0120] The method of the present invention has good linearity (r 2 ≥0.9982). The detection limit and quantification limit of AFB1, OMST, AVN and ST in wheat samples were 0.2-0.8 ng / g and 0.6-2.4 ng / g, respectively. The intra-day and inter-day precisions were both less than 7.8%, indicating that the method of the present invention has good sensitivity and repeatability.

[0121] After the wheat sample was crushed, 2.0 g of the sample was weighed, and the amount of AFB1, OMST, AVN and ST was spiked at three levels and processed according to the above method. Each spiked sample was measured three times in parallel and the average value was taken. The spike recovery of the sample was calculated based on the actual addition amount and the measured results. The results are shown in Table 7. The representative chromatograms of wheat samples before and after extraction are shown in Table 7. Figure 8 As shown in B.

[0122] Table 7. Detection of AFB1, OMST, AVN and ST in wheat by IL@CTP-DSPE-HPLC-UV (n=3)

[0123]

[0124] Level 1: The spiked amounts of AFB1, OMST, AVN and ST were 1.2, 2.4, 1.2 and 1.2 ng / g respectively; Level 2: The spiked amounts were all 6 ng / g; Level 3: The spiked amounts were all 24 ng / g; ND: not detected; SD: standard deviation.

[0125] The results showed that AFB1, OMST, AVN and ST were not detected in wheat samples. In addition, the sample spike recovery was 83.3% to 98.3%, and the RSD was 1.5% to 6.7%. The experimental results showed that the established analytical method has satisfactory accuracy and repeatability and can be used to determine AFB1, OMST, AVN and ST in wheat samples.

[0126] (3) Corn samples

[0127] The corn sample was crushed, 2.0 g of the sample was weighed, 8 mL of the extractant (80% acetonitrile aqueous solution) was added, vortexed for 2 min, ultrasonically extracted for 10 min, centrifuged at 10000 rpm for 5 min, 4 mL of the supernatant was removed, concentrated to near dryness with nitrogen blowing at 60 ° C, and 12 mL of primary water was added to re-dissolve the residue to obtain a sample solution. Then, the extraction was carried out under the optimized DSPE conditions, and the contents of AFB1, OMST, AVN and ST were detected by HPLC-UV (HPLC-UV conditions in rice samples). The analytical performance of the established IL@CTP-DSPE-HPLC-UV method in corn samples is shown in Table 8.

[0128] Table 8. Analytical performance of IL@CTP-DSPE-HPLC-UV method in corn samples

[0129]

[0130]

[0131] The method of the present invention has good linearity (r 2 ≥0.9958). The detection limit and quantification limit of AFB1, OMST, AVN and ST in corn samples were 0.4-2.0 ng / g and 1.2-6.0 ng / g, respectively. The intra-day and inter-day precisions were both less than 7.6%, indicating that the method of the present invention has good sensitivity and repeatability.

[0132] After the corn sample was crushed, 2.0 g of the sample was weighed, and the amount of AFB1, OMST, AVN and ST was spiked at three levels and processed according to the above method. Each spiked sample was measured three times in parallel and the average value was taken. The spike recovery of the sample was calculated based on the actual addition amount and the measured results. The results are shown in Table 9. The representative chromatograms of corn samples before and after extraction are shown in Table 9. Figure 8 As shown in C.

[0133] Table 9. Detection of AFB1, OMST, AVN and ST in corn by IL@CTP-DSPE-HPLC-UV (n=3)

[0134]

[0135] Level 1: The spiked amounts of AFB1, OMST, AVN and ST were 1.2, 6, 1.2 and 1.2 ng / g respectively; Level 2: The spiked amounts were all 6 ng / g; Level 3: The spiked amounts were all 24 ng / g; ND: not detected; SD: standard deviation.

[0136] The results showed that 4.5 ng / g of AFB1 was detected in the corn samples, while OMST, AVN and ST were not detected. In addition, the sample spike recovery rate was 79.2% to 94.9%, and the RSD was 3.6% to 8.7% (n=3). The experimental results showed that the established analytical method has satisfactory accuracy and repeatability and can be used to determine AFB1, OMST, AVN and ST in corn samples.

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An ionic liquid@covalent organic polymer, characterized in that: Prepared according to the following steps: Step 1: reacting 1, 3, 5-triphenylbenzene, cyanuric chloride and anhydrous aluminum chloride in an organic solvent at 70° C. to obtain a covalent organic polymer; Step 2: The covalent organic polymer obtained in step 1 and 1-methylimidazole are reacted in ethanol at 25° C., and then 1-bromobutane is added for reaction to obtain an ionic liquid@covalent organic polymer.

2. An ionic liquid@covalent organic polymer according to claim 1, characterized in that: In step 1, the organic solvent is dichloromethane; the molar ratio of 1, 3, 5-triphenylbenzene, cyanuric chloride and anhydrous aluminum chloride is 1:1:2, and the reaction time is 14h to 18h.

3. An ionic liquid@covalent organic polymer according to claim 1, characterized in that: In step 2, the molar ratio of 1-methylimidazole to 1-bromobutane is 1:1; the reaction time of the covalent organic polymer and 1-methylimidazole is 15 h to 18 h, and the reaction is carried out for 24 h to 26 h after adding 1-bromobutane.

4. A method for simultaneously detecting aflatoxin B1 and its three precursors OMST, AVN and ST, characterized in that: AFB1 and its three precursors OMST, AVN and ST are extracted from the sample to obtain a sample extract, and the sample extract is subjected to dispersed solid phase extraction using the ionic liquid@covalent organic polymer described in any one of claims 1 to 4, and then analyzed using an ultra-high performance liquid chromatography-mass spectrometer or a high performance liquid chromatography-ultraviolet detection to obtain quantitative results of AFB1 and its three precursors OMST, AVN and ST.

5. The method according to claim 4, characterized in that The desorbent in the dispersed solid phase extraction process is methanol, ethanol, acetone or acetonitrile.

6. The method according to claim 5, characterized in that The ratio of the amount of ionic liquid@covalent organic polymer to the amount of desorbent in the dispersed solid phase extraction process is 1-5 mg:0.25-2.5 mL.

7. The method according to claim 4, characterized in that The adsorption time of the ionic liquid@covalent organic polymer in the dispersed solid phase extraction process is 2 to 40 minutes.

8. The method according to claim 4, characterized in that The desorption time in the dispersed solid phase extraction process is 10 to 360 s.

9. The method according to claim 4, characterized in that The impregnation amount of the ionic liquid in the ionic liquid@covalent organic polymer is 6%; the dosage ratio of the ionic liquid@covalent organic polymer to the desorbent is 2 mg:1 mL; the adsorption time of IL@CTP is 10 min; the desorbent is acetone; and the desorption time is 120 s.

10. A method for early warning of aflatoxin B1 pollution, characterized in that: The precursors AVN, ST and OMST are used together as early warning indicators of AFB1 contamination. The dynamic changes of AFB1, OMST, AVN and ST in the sample are detected based on the method described in any one of claims 5 to 9, and then the correlation between OMST, AVN and ST and the occurrence of AFB1 contamination is analyzed to provide an early warning of AFB1 contamination in the sample.

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