Method for simultaneously detecting aflatoxin b1 and three precursors thereof and early warning method for aflatoxin b1

Abstract

The present application belongs to the technical field of 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 for aflatoxin B1. The detection method comprises the following steps: taking a crushed sample to be detected, extracting AFB1 and its precursors in the sample to obtain a sample extract, purifying and concentrating the sample extract by IL@CTP-DSPE, and then performing ultra-high performance liquid chromatography-mass spectrometry or high performance liquid chromatography-ultraviolet detection analysis to obtain qualitative and quantitative results; by using the detection method, the dynamic changes of the content of the precursor early warning indicator and AFB1 can be accurately tracked, correlation analysis is performed in combination with a heat map, and early warning before AFB1 pollution occurs is realized. The method can simultaneously and effectively extract AFB1 and its three precursors in a dynamic sample, and the established detection method has high accuracy and high sensitivity; the present application first proposes an early warning strategy of taking multiple precursors as early warning indicators together, thereby guaranteeing the quality and safety of grains.

Description

[0001] This invention belongs to the field of hazardous pollutant risk warning and detection technology, specifically involving a method for simultaneously and effectively extracting and sensitively and accurately detecting aflatoxin B1 and its three typical precursors in mycelium and grain sample matrices, as well as a method for early warning of aflatoxin B1. Background Technology

[0002] Aflatoxins (AFs) are a class of secondary metabolites produced by Aspergillus flavus and Aspergillus parasiticus under natural conditions, severely contaminating food crops such as corn, rice, and wheat. The dihydrofuran ring and coumarin structure give AFs high toxicity at low levels to humans and animals, with aflatoxin B1 (AFB1) being the most toxic.

[0003] Early warning systems, which monitor the dynamic changes in factors related to aflatoxin (AF) production, can identify and predict potential AF pollution risks, making them an effective strategy for controlling and preventing AF pollution. In recent years, precursors in the aflatoxin biosynthesis pathway have attracted considerable interest as early risk warning markers, including versicolorin A (Ver A), versicolorin B (Ver B), and sterigmatocystin (ST). In the process of acetic acid generating AFB1 through a multi-step enzymatic reaction, some typical precursors, such as averantin (AVN), are the second stable precursor in the AF biosynthesis pathway; ST is a key precursor in the AFB1 synthesis branch; and O-methylsterigmatocystin (OMST) is the final precursor in the AFB1 synthesis pathway. The production and concentration of these precursors and AFB1 are dynamic processes. In the early stages of mold contamination, the levels of precursors and AFB1 are extremely low. As the mold growth process progresses, various precursors with different structures often coexist with significant concentration differences. Furthermore, the production of AFB1 and its precursors is a result of quantitative to qualitative changes during mold proliferation. During this period, the composition and quality of the grain are in dynamic flux. Grains such as wheat, rice, and corn, rich in starch, protein, fat, and pigments, have complex matrices, making sample purification more challenging to detect AFB1 or its precursors during this dynamic process. Therefore, sensitively and accurately monitoring the dynamic changes of AFB1 and its various precursors in grains to provide early warning of AFB1 contamination is a highly challenging task, and improving the extraction and enrichment capabilities in sample pretreatment is key to solving this problem.

[0004] Wu et al. prepared a poly(methacrylic acid-co-diethenyl-benzene) monolithic column for in-tube solid-phase microextraction to determine 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 one 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 the 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 determine the dynamic changes in the biosynthesis of AFB1 and its various precursors in mycelium. This method has no purification or enrichment steps and is only applicable to mycelial samples with simple matrices. However, it cannot represent real grain samples and cannot accurately reflect the AFB1 warning situation in actual grain samples (Xie H, Wang X, Zhang L, et al. Monitoring metadata production of aflatoxin biosynthesis by Orbitrap Fusion mass spectrometry and D-optimal mixture design method[J]. Analytical Chemistry, 2018, 90(24):14331-14338). To date, there are few publicly published methods for tracking the dynamic changes of AFB1 and its precursors in moldy grains.Furthermore, highly sensitive AFB1 and precursor detection methods can identify potential contamination earlier and extend the warning time window, which is crucial for accurately establishing the dynamic relationship between AFB1 and its precursors. The improvement in sensitivity obviously depends on the performance of the extraction medium.

[0005] In summary, using AFB1 precursors as early risk warning markers is an effective strategy for preventing AFB1 contamination. However, grain samples have complex matrices, and AFB1 and its precursors have significant differences in structure and polarity. Furthermore, the content of precursors is often extremely low in the early stages of mold growth, and the types and contents of precursors and AFB1 are in a dynamic process throughout the mold growth process. Therefore, it is necessary to develop sensitive and accurate detection methods suitable for monitoring the dynamic changes of precursors and AFB1 during Aspergillus flavus contamination, so as to provide technical support for analyzing the correlation between precursors and AFB1 production and establishing an early warning system for AFB1. Summary of the Invention

[0006] To address the shortcomings of existing AFB1 early warning methods based on precursors as markers, which only involve simple rice sample matrices and cannot accurately reflect the early warning situation of AFB1 in complex actual grain samples, and which have limited sensitivity and cannot simultaneously and efficiently extract AFB1 and its precursors with different structures and polarities, this invention provides a highly sensitive detection method for AFB1 and its three precursors in mycelia and grain samples, and an early warning method for aflatoxin B1 based on this detection method. The detection method is based on optimized ionic liquid@covalent organic polymer (IL@CTP) dispersion solid-phase extraction (DSPE) technology combined with ultra-high performance liquid chromatography-liquid chromatography-mass spectrometry (UHPLC-MS / MS). The early warning method is performed on artificially infected Aspergillus flavus mycelia and corn samples using the IL@CTP-DSPE-UHPLC-MS / MS method. Furthermore, this sample pretreatment can also be coupled with high performance liquid chromatography-ultraviolet (HPLC-UV) for the simultaneous detection of four target compounds in rice, wheat, and corn samples.

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

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

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

[0010] Step 2: The covalent organic polymer CTP obtained in Step 1 and 1-methylimidazole were reacted in ethanol at 25°C, followed by the addition of 1-bromobutane to obtain ionic liquid@covalent organic polymer (IL@CTP). The molar ratio of 1-methylimidazole to 1-bromobutane was 1:1. The reaction time of the covalent organic polymer and 1-methylimidazole was 15-18 h, and the reaction time after the addition of 1-bromobutane was 24-26 h.

[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 and reacted for 14-18 hours; after cooling, it is washed and then dried under vacuum 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 15-18 hours, then an equimolar amount of 1-bromobutane is added, and the mixture is stirred for 24-26 hours. After washing the product, it is dried under vacuum to obtain IL@CTP.

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

[0013] This invention targets AFB1 and its three precursors (OMST, AVN, and ST), and establishes a sensitive and accurate detection method for four target compounds in mycelium and moldy corn by combining IL@CTP-based DSPE technology with ultra-high performance liquid chromatography-mass spectrometry or high performance liquid chromatography-ultraviolet spectrometry. The method includes the following steps:

[0014] AFB1 and its three precursors OMST, AVN and ST were extracted from the sample using an extractant to obtain a sample extract. The sample extract was then subjected to IL@CTP-based dispersive solid-phase extraction and UHPLC-MS / MS analysis to obtain quantitative results for the four target compounds (AFB1 and its three precursors OMST, AVN and ST).

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

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

[0017] The desorption time in the dispersion solid-phase extraction process is 10–360 s.

[0018] In the optimized IL@CTP-based dispersive solid-phase extraction method, the ratio of IL@CTP to desorbent is 2 mg:1 mL. The adsorption time of IL@CTP is 10 min. Acetone is used as the desorbent. The desorption time is 120 s. Furthermore, this invention found that the highest adsorption efficiency for the four target compounds AFB1, OMST, AVN, and ST was achieved when the IL impregnation amount was 6%.

[0019] This invention combines the optimized DSPE technology with UHPLC-MS / MS for the determination of AFB1, OMST, AVN, and ST in samples (such as mycelium and moldy grains). First, the four target compounds are extracted from the grains. Then, the optimized IL@CTP-based DSPE technology is used to extract the four target compounds. Next, UHPLC-MS / MS is used to detect the content of the four target compounds. Furthermore, this DSPE technology is combined with HPLC-UV for the simultaneous determination of the four target compounds in samples (such as rice, wheat, and corn).

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

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

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

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

[0024] The beneficial effects of this invention are as follows:

[0025] (1) Existing technologies combining Fe3O4 / ZIFs-based magnetic solid-phase extraction with high-performance liquid chromatography can simultaneously determine two precursors, AVN and ST, which have different structures and polarities. However, it cannot effectively extract AFB1 simultaneously, even when using an acidic desorbent (0.1 mol·L⁻¹). -1Formic acid / acetonitrile ratio of 1:9 (v / v) also failed to effectively desorb AFB1. However, this invention utilizes IL@CTP, a medium rich in functional groups, as the extraction medium. This medium exhibits various interactions and pore-filling effects with the target analytes, enabling the simultaneous and efficient extraction of AFB1, OMST, AVN, and ST, which possess different structures and polarities. Furthermore, the raw materials are inexpensive and readily available, and the synthesis conditions are mild.

[0026] (2) This invention establishes for the first time a detection method for simultaneously determining 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) This 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 to 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 growth, realizing early warning of AFB1 contamination and providing an effective early warning method for early AFB1 contamination in grains. Attached Figure Description

[0029] Figure 1 The 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. Infrared spectrum of IL@CTP; B. Nitrogen adsorption-desorption curves of CTP and IL@CTP; C. Pore size distribution of CTP and IL@CTP; D. Scanning electron microscope images of CTP and IL@CTP and surface scans of C, N, and Br elements in IL@CTP; E. Potential diagrams 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 target analytes. B. Desorbent type, C. Desorbent volume, D. Desorption time, E. Adsorbent mass, F. Effect of adsorption time on extraction efficiency.

[0033] Figure 5 Heatmap showing the dynamic changes and correlation analysis of AFB1 and its precursors in mycelium.

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

[0035] Figure 7 Heatmap of dynamic changes and correlation analysis of AFB1 and its precursors in stored maize.

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

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of the embodiments of this invention will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0038] This invention first prepares and screens adsorbents, then optimizes the extraction parameters of AFB1, OMST, AVN, and ST using the screened adsorbents. Finally, the optimized parameters are combined with UHPLC-MS / MS for the determination of AFB1, OMST, AVN, and ST in mycelium and moldy corn samples. The optimized extraction technique combined with UHPLC-MS / MS is used to establish an early warning method for AFB1 contamination. This invention also relates to combining optimized parameters with HPLC-UV for the simultaneous determination of four target compounds in samples (such as rice, wheat, and corn). The structural formulas of AFB1, OMST, AVN, and ST are as follows: Figure 1 As shown. The IL@CTP-DSPE-UHPLC-MS / MS and IL@CTP-DSPE-HPLC-UV methods of this invention can both be used for the detection of AFB1, OMST, AVN and ST in cereals (such as rice, wheat and corn). Among them, 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, Henan University of Technology. Other commercially available products can also be used as substitutes for this Aspergillus flavus strain.

[0040] Preparation method of Aspergillus flavus spore suspension: Take an appropriate amount of Aspergillus flavus spore solution and spread it on PDA medium. Incubate it upside down at 30℃ for 5 days. After the incubation, add an appropriate amount of sterile water and collect Aspergillus flavus spores. Count the spores using a hemocytometer to obtain Aspergillus flavus spore suspension.

[0041] The rice, corn, and wheat were purchased from local markets in Zhengzhou.

[0042] Example 1: Preparation of IL@CTP

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

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

[0045] The prepared CTP and IL@CTP were characterized as follows: Figure 3 As shown, the infrared spectrum at 1700 cm⁻¹ -1 and 1390cm -1 The characteristic peaks at 1600-1450 cm⁻¹ are attributed to C=N and CN vibrations, respectively, indicating the presence of a triazine structure in IL@CTP. -1 The peak in the region corresponds to the C=C vibration and the asymmetric CH stretching vibration of the imidazole ring, at 1182 cm⁻¹. -1 The characteristic peaks at the point of induction are attributed to the stretching vibrations of the imidazole ring, confirming the successful impregnation of ILs. The N2 adsorption / desorption isotherms of CTP and IL@CTP are distinct Type I curves. The curves rise rapidly in the low relative pressure region (P / P0 = 0.001), indicating the presence of a microporous structure. A hysteresis loop appears when P / P0 > 0.4, indicating the presence of a mesoporous structure. Scanning electron microscopy shows that both CTP and IL@CTP are spherical, ranging in size from 1.0 to 1.5 μm, with particle aggregation causing irregular shapes in some particles. The presence of Br in IL@CTP indicates successful impregnation of ILs into CTP. The Zeta potentials of CTP and IL@CTP are -4.6 ± 0.5 mV and 6.8 ± 0.6 mV, respectively, with the introduction of positively charged ILs causing the change in Zeta potential. The specific surface areas of CTP and IL@CTP are 941.3 m² and 941.3 m², respectively. 2 / g and 854.2m 2The median micropore sizes were 0.67 nm and 0.5 nm, respectively, and the reduction in specific surface area and micropore size also proves the success of IL impregnation. The high surface area of ​​IL@CTP means abundant functional groups and adsorption sites, which is beneficial for the adsorption of target analytes by IL@CTP. In addition, the mesopores (approximately 2.2 nm) of IL@CTP allow analytes to diffuse into the mesopore interior and adsorb on the inner surface of the mesopore.

[0046] Example 2: Experimental Study of Adsorbents with Different Il Impregnation Amounts

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

[0048] Prepare a mixed aqueous solution of AFB1, OMST, AVN, and ST, with a concentration of 20 ng / mL for each of the four target compounds. Take 20 mL of the mixed aqueous solution of AFB1, OMST, AVN, and ST, add 2 mg of CTP and the five ILs with different IL impregnation amounts to CTP, shake at 220 rpm for 5 min, centrifuge at 10000 rpm for 5 min, filter the supernatant through a 0.22 μm PTFE filter membrane, and analyze by HPLC-UV.

[0049] The adsorption efficiencies were compared, and the results are as follows: Figure 4 As shown in Figure A.

[0050] from Figure 4 As shown in Figure A, among the impregnation amounts examined, IL impregnation at 6% resulted in the highest adsorption efficiency for the four target analytes. Compared to CTP, IL@CTP with an IL impregnation amount of 6% exhibited adsorption efficiencies for the four target analytes that were 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 embodiment primarily studies the effect of the type of desorbent on the extraction recovery rate. Methanol, ethanol, acetonitrile, and acetone were used as desorbents. The specific operating steps of this embodiment are as follows:

[0053] (1) Crush the blank rice sample (rice without aflatoxin and its precursors), weigh 2.0 g of the sample, add 120 ng / g of four mixed standards of AFB1, OMST, AVN and ST, add 8 mL of extraction solvent (80% acetonitrile aqueous solution), vortex for 2 min, ultrasonically extract for 10 min, centrifuge at 10000 rpm for 5 min, transfer 4 mL of supernatant, concentrate under nitrogen blowing at 60 °C to about 0.1 mL, add 12 mL of primary water to redissolve the residue to obtain the sample solution.

[0054] (2) Add 2 mg IL@CTP to 12 mL of sample solution, shake at 220 rpm for 10 min, centrifuge at 10000 rpm for 5 min, and discard the supernatant. Add 1 mL each of the desorbents methanol, ethanol, acetonitrile, and acetone, sonicate for 120 s, centrifuge at 10000 rpm for 5 min, and blow the supernatant to near dryness under nitrogen at 60 °C. Redissolve in 200 μL of acetonitrile. Filter through a 0.22 μm PTFE membrane and analyze using HPLC-UV.

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

[0056] from Figure 4 As shown in Figure B, among the desorbents examined, acetone exhibited the highest extraction recovery rate. This is likely because acetone contains unsaturated carbonyl groups, giving it stronger hydrophobicity than other desorbents, effectively disrupting the π-π and hydrophobic interactions between the target analyte and the adsorbent.

[0057] Example 4: Experimental Study of Desorbents with Different Volumes

[0058] This embodiment mainly studies the effect of different volumes of desorbent on the extraction recovery rate. Acetone was used as the desorbent, with volumes of 0.25 mL, 0.5 mL, 1 mL, 1.5 mL, 2 mL, and 2.5 mL. The specific operating steps of this embodiment are as follows:

[0059] (1) Crush the blank rice sample, weigh 2.0 g of the sample, add 120 ng / g of four mixed standards of AFB1, OMST, AVN and ST, add 8 mL of extraction solvent (80% acetonitrile aqueous solution), vortex for 2 min, ultrasonically extract for 10 min, centrifuge at 10000 rpm for 5 min, transfer 4 mL of supernatant, concentrate under nitrogen blowing at 60 °C to about 0.1 mL, add 12 mL of primary water to redissolve the residue to obtain the sample solution.

[0060] (2) Add 2 mg IL@CTP to 12 mL of sample solution, shake at 220 rpm for 10 min, centrifuge at 10000 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 10000 rpm for 5 min, and blow the supernatant to near dryness under nitrogen at 60 °C. Redissolve in 200 μL of acetonitrile. Filter through a 0.22 μm PTFE membrane and analyze using HPLC-UV.

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

[0062] from Figure 4 As shown in Figure C, the extraction recovery rate gradually increases when the volume of acetone increases from 0.25 mL to 1 mL. Further increasing the volume of acetone does not change the extraction recovery rate, indicating that 1 mL of acetone can achieve complete desorption.

[0063] Example 5: Experimental Study on Different Desorption Times

[0064] This embodiment mainly studies the effect of different desorption times on the extraction recovery rate. 1 mL of acetone was used as the desorption agent, and the desorption times were 10 s, 30 s, 60 s, 120 s, 240 s, and 360 s. The specific operating steps of this embodiment are as follows:

[0065] (1) Crush the blank rice sample, weigh 2.0 g of the sample, add 120 ng / g of four mixed standards of AFB1, OMST, AVN and ST, add 8 mL of extraction solvent (80% acetonitrile aqueous solution), vortex for 2 min, ultrasonically extract for 10 min, centrifuge at 10000 rpm for 5 min, transfer 4 mL of supernatant, concentrate under nitrogen blowing at 60 °C to about 0.1 mL, add 12 mL of primary water to redissolve the residue to obtain the sample solution.

[0066] (2) Add 2 mg IL@CTP to 12 mL of sample solution, shake at 220 rpm for 10 min, centrifuge at 10000 rpm for 5 min, and discard the supernatant. Add 1 mL of acetone, sonicate for 10 s, 30 s, 60 s, 120 s, 240 s, and 360 s respectively, centrifuge at 10000 rpm for 5 min, and blow the supernatant to near dryness under nitrogen at 60 °C. Redissolve in 200 μL of acetonitrile. Filter through a 0.22 μm PTFE membrane and analyze using HPLC-UV.

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

[0068] from Figure 4As shown in Figure D, when the desorption time increases from 10s to 120s, the extraction recovery rate gradually increases. Further extending the desorption time does not change the extraction recovery rate, indicating that 120s can achieve complete desorption.

[0069] Example 6: Experimental Study on Different Adsorbent Masses

[0070] This embodiment mainly studies the effect of different adsorbent dosages on extraction recovery. 1 mL of acetone was used as the desorbent, and the desorption time was 120 s. The adsorbent masses were 1 mg, 2 mg, 3 mg, 4 mg, and 5 mg, respectively. The specific operating steps of this embodiment are as follows:

[0071] (1) Crush the blank rice sample, weigh 2.0 g of the sample, add 120 ng / g of four mixed standards of AFB1, OMST, AVN and ST, add 8 mL of extraction solvent (80% acetonitrile aqueous solution), vortex for 2 min, ultrasonically extract for 10 min, centrifuge at 10000 rpm for 5 min, transfer 4 mL of supernatant, concentrate under nitrogen blowing at 60 °C to about 0.1 mL, add 12 mL of primary water to redissolve the residue to obtain the 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, respectively. Shake at 220 rpm for 10 min, centrifuge at 10000 rpm for 5 min, and discard the supernatant. Add 1 mL of acetone, sonicate for 120 s, centrifuge at 10000 rpm for 5 min, and blow the supernatant to near dryness under nitrogen at 60 °C. Redissolve in 200 μL of acetonitrile. Filter through a 0.22 μm PTFE membrane and analyze using HPLC-UV.

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

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

[0075] Example 7: Experimental Study on Different Adsorption Times

[0076] This embodiment mainly studies the effect of different adsorption times on the extraction recovery rate. The desorbent used was 1 mL of acetone, the desorption time was 120 s, and the adsorbent mass was 2 mg. Adsorption times were set at 2 min, 5 min, 10 min, 15 min, 20 min, and 25 min. The specific operating steps of this embodiment are as follows:

[0077] (1) Crush the blank rice sample, weigh 2.0 g of the sample, add 120 ng / g of four mixed standards of AFB1, OMST, AVN and ST, add 8 mL of extraction solvent (80% acetonitrile aqueous solution), vortex for 2 min, ultrasonically extract for 10 min, centrifuge at 10000 rpm for 5 min, transfer 4 mL of supernatant, concentrate under nitrogen blowing at 60 °C to about 0.1 mL, add 12 mL of primary water to redissolve the residue to obtain the sample solution.

[0078] (2) Add 2 mg IL@CTP to 12 mL of sample solution, and shake at 220 rpm for 2 min, 5 min, 10 min, 15 min, 20 min, and 25 min respectively for adsorption. Centrifuge at 10000 rpm for 5 min and discard the supernatant. Add 1 mL of acetone, sonicate for 120 s, centrifuge at 10000 rpm for 5 min, and blow the supernatant with nitrogen at 60 °C until nearly dry. Redissolve in 200 μL of acetonitrile. Filter through a 0.22 μm PTFE membrane and analyze using HPLC-UV.

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

[0080] from Figure 4 As shown in Figure F, the extraction recovery rate gradually increased as the adsorption time increased from 2 min to 10 min. Further increasing the adsorbent mass did not improve the extraction recovery rate, but excessive adsorption time increased the operation time. This indicates that the optimal adsorption time is 10 min. Therefore, the optimized DSPE conditions are as follows: Add 2 mg IL@CTP to 12 mL of sample solution, shake at 220 rpm for 10 min, centrifuge at 10000 rpm for 5 min, and discard the supernatant. Add 1 mL of acetone, sonicate for 120 s, centrifuge at 10000 rpm for 5 min, and blow the supernatant to near dryness under nitrogen at 60 °C. Redissolve in 200 μL of acetonitrile. Filter through a 0.22 μm PTFE membrane.

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

[0082] Aspergillus flavus spore suspension was inoculated into Sabouraud broth to achieve an inoculum size of 5 × 10⁻⁶. 5The mycelium was cultured at 28℃ with shaking (200 rpm) for 200 mg / mL. The mycelium was collected by filtration and ground with liquid nitrogen. 200 mg of mycelium was aliquoted and added to 8 mL of extraction solvent (80% acetonitrile aqueous solution). The mixture was vortexed for 2 min, ultrasonically extracted for 10 min, centrifuged at 10000 rpm for 5 min, and 4 mL of the supernatant was collected. The supernatant was concentrated to near dryness under nitrogen blowing at 60℃, and the residue was reconstituted with 12 mL of primary water to obtain the sample solution. Extraction was then performed under optimized DSPE conditions, and the contents of AFB1, OMST, AVN, and ST were detected by UHPLC-MS / MS.

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

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

[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 the IL@CTP-DSPE-UHPLC-MS / MS method in mycelial samples

[0088]

[0089] The method of this invention exhibits good linearity (r) in the range of 0.03–200 ng / g. 2(≥0.9954). The limits of detection and quantitation for AFB1, OMST, AVN, and ST in mycelial samples were 0.01–0.02 ng / g and 0.03–0.06 ng / g, respectively. Intra-day and inter-day precision 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 is negligible.

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

[0091] Select intact corn kernels, pulverize the samples, and autoclave at 121℃ for 20 min. Weigh 2.0 g of the sample, add 8 mL of extraction solvent (80% acetonitrile aqueous solution), vortex for 2 min, sonicate for 10 min, centrifuge at 10000 rpm for 5 min, transfer 4 mL of supernatant, concentrate to near dryness under nitrogen blowing at 60℃, and redissolve the residue with 12 mL of primary water to obtain the sample solution. Then, extract under optimized DSPE conditions, and detect the contents of AFB1, OMST, AVN, and ST using UHPLC-MS / MS (UHPLC-MS / MS analytical 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 the IL@CTP-DSPE-UHPLC-MS / MS method in maize samples.

[0093]

[0094]

[0095] The method of this invention exhibits good linearity (r) in the range of 0.06–200 ng / g. 2 (≥0.9965). The limits of detection and quantitation for AFB1, OMST, AVN, and ST in stored maize samples were 0.02–0.03 ng / g and 0.06–0.1 ng / g, respectively. Intra-day and inter-day precision were both less than 7.9%. The matrix effect ranged from 83.2% to 117.7%. This indicates that the method of the present invention has high sensitivity and good repeatability, with a very small matrix effect.

[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 broth to achieve an inoculum size of 5 × 10⁻⁶. 5The control group was not inoculated. The cells were cultured at 28°C with shaking for 9 days (200 rpm). Samples were collected every 12 hours from day 1 to day 6, and every 24 hours from day 7 to day 9. Mycelia were obtained by filtration and ground with liquid nitrogen. 200 mg of mycelium was aliquoted and added to 8 mL of extraction solvent (80% acetonitrile aqueous solution). The mixture was vortexed for 2 min, ultrasonically extracted for 10 min, centrifuged at 10000 rpm for 5 min, and 4 mL of the supernatant was collected. The supernatant was concentrated to near dryness under nitrogen blowing at 60°C, and 12 mL of primary water was added to reconstitute the residue to obtain the sample solution as the intracellular sample. Additionally, 12 mL of Sabouraud dextrose liquid culture medium after the corresponding culture time was aliquoted as the extracellular sample. Extraction was then performed 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 conducted in triplicate. The dynamic changes and correlation analysis of AFB1 and its precursor levels (total levels inside and outside the cell) over 9 days are shown in the heatmap below. Figure 5 As shown.

[0098] The results showed that AFB1 content peaked at 5.5 days, followed by a decline. AVN, ST, and OMST contents peaked at 3.5, 4.5, and 5 days, respectively, before declining, indicating that the decrease in precursor content prepares for AFB1 production. Significant variations in AFB1 and precursor contents were observed between days 1 and 5.5, with a decline occurring after 5.5 days. This suggests that under suitable liquid culture conditions, AFB1 accumulation is very rapid, with precursors and AFB1 produced almost simultaneously, and the precursor content reaching its peak before AFB1. Correlation analysis heatmaps of the four analytes showed that OMST (Pearson's = 0.72) and ST (Pearson's = 0.12) were positively correlated with AFB1 content, while AVN (Pearson's = -0.082) showed no significant correlation with AFB1.

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

[0100] Select intact corn kernels and autoclave them at 121℃ for 20 minutes. Dilute the Aspergillus flavus spore suspension with sterile water at a ratio of 4 mL / g, mix thoroughly, and inoculate the corn kernels to achieve an inoculum size of 5 × 10⁻⁶. 5The corn was inoculated at 4°C for 48 hours, with stirring every 12 hours to ensure uniform rehydration. 25g of inoculated corn was weighed and placed in sterile petri dishes (9cm in diameter). These dishes were then placed in a desiccator containing saturated ammonium sulfate solution and incubated in the dark at 28°C for 19 days. Samples were collected daily. Collected samples were stored at -20°C before extraction. The inoculated corn samples were sterilized at 121°C for 30 minutes and dried in a 115°C oven for 1 hour. They were then pulverized and ground with liquid nitrogen. Uninoculated corn samples served as blank samples. Extraction was performed according to the above steps under optimized DSPE conditions. 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 conducted in triplicate. A photograph of artificially inoculated A. flavus corn stored in a constant temperature and humidity environment for 19 days is shown below. Figure 6 As shown in the figure. The heatmap of dynamic changes and correlation analysis of AFB1 and its precursors is shown in the figure. Figure 7 As 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 days 2 and 3, respectively, while AFB1 was undetectable at these times. OMST was first detected on day 5, and AFB1 was first detected on the same day at a concentration of 2.1 ng / g, while the appearance of the corn samples remained unchanged from days 1 to 5. By day 6, the AFB1 content was 14.1 ng / g, and by day 7 it had risen to 106.7 ng / g, exceeding the limits in China and the United States (<20 ng / g). At this point, a small amount of white mycelium could be observed on the surface of the corn samples. Figure 6Since AVN was detected as early as day 2, while AFB1 exceeded the limit on day 7, the results indicate that AVN can predict the occurrence of AFB1 contamination in stored maize 5 days in advance. Correlation analysis heatmaps showed that the structurally closely related OMST and ST levels were significantly correlated (Pearson's = 0.85), and both were positively correlated with AFB1 levels, with OMST (Pearson's = 0.62) showing a higher correlation with AFB1 than ST (Pearson's = 0.23). AVN levels were negatively correlated with AFB1 levels (Pearson's = -0.28). The dynamic changes and correlations of maize 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, peaking on days 9 (AVN), 13 (ST), and 15 (OMST), while AFB1 peaked on day 17. The successive appearance of these precursors also helps to predict the occurrence of AFB1 contamination. The early warning method proposed in this invention uses the precursors AVN, ST, and OMST together as early warning indicators of AFB1 pollution, so as to provide a wider early warning time window and more accurate early warning results.

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

[0103] (1) Rice sample

[0104] The rice sample was pulverized, and 2.0 g of the sample was weighed. 8 mL of extraction solvent (80% acetonitrile aqueous solution) was added, and the mixture was vortexed for 2 min, ultrasonically extracted for 10 min, and then centrifuged at 10000 rpm for 5 min. 4 mL of the supernatant was collected and concentrated to approximately 0.1 mL under nitrogen blowing at 60 °C. 12 mL of primary water was added to redissolve the residue to obtain the sample solution. Extraction was then performed under 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 / Vis detector. Column: Welch Ultimate XB-C30, 250 mm × 4.6 mm, 5 μm particle size; column temperature 35℃. Mobile phase: acetonitrile / water (55 / 45, v / v), flow rate 1.0 mL / min. -1 The UV detection wavelength was set to 362nm (0-6min) and 30nm (6-17min), and the injection volume was 20μL.

[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 the IL@CTP-DSPE-HPLC-UV method in rice samples

[0108]

[0109] The method of this invention exhibits good linearity in the range of 0.6–240 ng / g (r 2 (≥0.9984). The limits of detection and limits of quantitation for AFB1, OMST, AVN, and ST in rice samples were 0.2–0.8 ng / g and 0.6–2.4 ng / g, respectively. Intra-day and inter-day precision were both less than 7.2%. This indicates that the method of the present invention has satisfactory sensitivity and good repeatability.

[0110] After pulverizing the rice sample, 2.0 g of the sample was weighed and spiked at three levels to detect AFB1, OMST, AVN, and ST. The sample was processed using the method described above. Three parallel measurements were performed for each spiked concentration, and the average value was taken. The recovery rate of the sample was calculated based on the actual amount added and the measured results. The results are shown in Table 5. Representative chromatograms of the rice sample before and after extraction are shown below. Figure 8 As shown in Figure A.

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

[0112]

[0113] Level 1: The spiking amounts of AFB1, OMST, AVN, and ST were 1.2, 2.4, 1.2, and 1.2 ng / g, respectively; Level 2: The spiking amount for all levels was 6 ng / g; Level 3: The spiking amount for all levels was 24 ng / g; ND: Not detected; SD: Standard deviation.

[0114] The results showed that AFB1, OMST, AVN, and ST were not detected in the rice samples. Furthermore, the recoveries of the spiked samples ranged from 86.7% to 104.4%, and the precision ranged from 1.5% to 6.9%. The experimental results indicate 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 pulverized, and 2.0 g of the sample was weighed. 8 mL of extraction solvent (80% acetonitrile aqueous solution) was added, vortexed for 2 min, and ultrasonically extracted for 10 min. The mixture was then centrifuged at 10000 rpm for 5 min, and 4 mL of the supernatant was collected. The supernatant was concentrated to approximately 0.1 mL under nitrogen blowing at 60 °C, and 12 mL of primary water was added to redissolve the residue to obtain the sample solution. Extraction was then performed under optimized DSPE conditions, and the contents of AFB1, OMST, AVN, and ST were detected by HPLC-UV (HPLC-UV conditions for 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 the IL@CTP-DSPE-HPLC-UV method in wheat samples

[0118]

[0119]

[0120] The method of this invention exhibits good linearity in the range of 0.6–240 ng / g (r 2 (≥0.9982). The limits of detection and limits of quantitation for AFB1, OMST, AVN, and ST in wheat samples were 0.2–0.8 ng / g and 0.6–2.4 ng / g, respectively. Intra-day and inter-day precision were both less than 7.8%, indicating that the method of this invention has good sensitivity and repeatability.

[0121] After pulverizing the wheat sample, 2.0 g of the sample was weighed and spiked at three levels to detect AFB1, OMST, AVN, and ST. The sample was processed using the method described above. Three parallel measurements were performed for each spiked concentration, and the average value was taken. The recovery rate of the sample was calculated based on the actual amount added and the measured results. The results are shown in Table 7. Representative chromatograms of the wheat sample before and after extraction are shown below. Figure 8 As shown in B.

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

[0123]

[0124] Level 1: The spiking amounts of AFB1, OMST, AVN, and ST were 1.2, 2.4, 1.2, and 1.2 ng / g, respectively; Level 2: The spiking amount for all levels was 6 ng / g; Level 3: The spiking amount for all levels was 24 ng / g; ND: Not detected; SD: Standard deviation.

[0125] The results showed that AFB1, OMST, AVN, and ST were not detected in the wheat samples. Furthermore, the recoveries of the spiked samples ranged from 83.3% to 98.3%, with RSDs ranging from 1.5% to 6.7%. The experimental results indicate 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 pulverized, and 2.0 g of the sample was weighed. 8 mL of extraction solvent (80% acetonitrile aqueous solution) was added, vortexed for 2 min, and ultrasonically extracted for 10 min. The mixture was then centrifuged at 10000 rpm for 5 min, and 4 mL of the supernatant was collected. The supernatant was concentrated to near dryness under nitrogen blowing at 60℃, and 12 mL of primary water was added to redissolve the residue to obtain the sample solution. Extraction was then performed under optimized DSPE conditions, and the contents of AFB1, OMST, AVN, and ST were detected by HPLC-UV (HPLC-UV conditions for 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 the IL@CTP-DSPE-HPLC-UV method in maize samples.

[0129]

[0130]

[0131] The method of this invention exhibits good linearity in the range of 1.2–240 ng / g (r 2 (≥0.9958). The limits of detection and limits of quantitation for AFB1, OMST, AVN, and ST in corn samples were 0.4–2.0 ng / g and 1.2–6.0 ng / g, respectively. Intra-day and inter-day precision were both less than 7.6%, indicating that the method of this invention has good sensitivity and repeatability.

[0132] After crushing the corn sample, 2.0 g of the sample was weighed and spiked at three levels to detect AFB1, OMST, AVN, and ST. The sample was processed using the method described above. Three parallel measurements were performed for each spiked concentration, and the average value was taken. The recovery rate of the sample was calculated based on the actual amount added and the measured results. The results are shown in Table 9. Representative chromatograms of the corn sample before and after extraction are shown below. Figure 8 As shown in C.

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

[0134]

[0135] Level 1: The spiking amounts of AFB1, OMST, AVN, and ST were 1.2, 6, 1.2, and 1.2 ng / g, respectively; Level 2: The spiking amount for all was 6 ng / g; Level 3: The spiking amount for all was 24 ng / g; ND: Not detected; SD: Standard deviation.

[0136] The results showed that 4.5 ng / g of AFB1 was detected in the maize sample, while OMST, AVN, and ST were not detected. Furthermore, the recoveries of the spiked samples ranged from 79.2% to 94.9%, with RSDs ranging from 3.6% to 8.7% (n=3). The experimental results indicate that the established analytical method has satisfactory accuracy and repeatability and can be used to determine AFB1, OMST, AVN, and ST in maize samples.

[0137] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for simultaneous detection of aflatoxin B1 and its three precursors OMST, AVN, and ST, characterized in that, AFB1 and its three precursors OMST, AVN and ST were extracted from the sample using an 80% acetonitrile aqueous solution as the extractant. The sample extract was then dispersed and extracted using an ionic liquid@covalent organic polymer, followed by ultra-high performance liquid chromatography-mass spectrometry or high performance liquid chromatography-ultraviolet detection analysis to obtain quantitative results for AFB1 and its three precursors OMST, AVN and ST. The sample is mycelium or grain sample; the extraction is ultrasonic extraction; The ionic liquid@covalent organic polymer is prepared according to the following steps: Step 1: 1,3,5-Triphenylbenzene, cyanochloroethylene, and anhydrous aluminum chloride are reacted in an organic solvent at 70°C to obtain a covalent organic polymer; the molar ratio of 1,3,5-triphenylbenzene, cyanochloroethylene, and anhydrous aluminum chloride is 1:1:2; dichloromethane is selected as the organic solvent; the reaction time is 14h to 18h. 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 to continue the reaction to obtain an ionic liquid@covalent organic polymer; 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; the ionic liquid impregnation amount in the ionic liquid@covalent organic polymer is 4% or 6%; The desorbent in the dispersion solid-phase extraction process is acetone or acetonitrile, and the ratio of ionic liquid@covalent organic polymer to desorbent is 1-5 mg. The adsorption time for ionic liquids @ covalent organic polymers is 2–40 min, and the desorption time is 10–360 s. The amount of ionic liquid is 0.25–2.5 mL.

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

3. An ionic liquid@covalent organic polymer, characterized in that, It is prepared using steps 1 and 2 of the method described in claim 1.

4. A method for early warning of aflatoxin B1 contamination, characterized in that, The precursors AVN, ST, and OMST are used together as early warning indicators of AFB1 contamination. Based on the method described in claim 1 or 2, the dynamic changes of AFB1, OMST, AVN, and ST in the sample are detected, 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 sample.

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