Method for detecting emerging mycotoxins in agricultural products

Abstract

A method for detecting emerging mycotoxins in agricultural products is provided, comprising: Step 1: Extracting the agricultural product using an extraction solvent to prepare a sample solution to be tested, wherein the extraction solvent contains, by volume, at least 85% or more of at least one organic solvent selected from the group consisting of alcohols and nitriles having 1 to 4 carbon atoms, relative to 100% of the extraction solvent; Step 2: Separating the emerging mycotoxins in the sample solution to be tested using supercritical fluid chromatography, wherein the supercritical fluid chromatography uses a gradient elution of mobile phase A and mobile phase B, mobile phase A being a supercritical fluid, and mobile phase B containing at least one organic solvent selected from the group consisting of alcohols and nitriles having 1 to 4 carbon atoms; Step 3: Detecting the emerging mycotoxins separated in step 2 using a mass spectrometer detector, wherein the mass spectrometer detector is in multiple reaction monitoring mode.

Description

Technical Field

[0001] This invention relates to a method for detecting emerging mycotoxins in agricultural products, belonging to the field of agricultural product contaminant analysis and detection. Background Technology

[0002] In the field of food safety and the analysis and detection of contaminants in agricultural products, the monitoring of mycotoxins has always been a core challenge. Traditionally, regulatory and research focus has been on traditional toxins such as aflatoxin, for which clear limits have been established. However, with the advancement of analytical techniques and toxicological understanding, the potential health risks of a class of contaminants known as "emerging mycotoxins" are becoming increasingly prominent.

[0003] Emerging mycotoxins refer to mycotoxins that are prevalent in agricultural products but are not yet included in routine risk screening and regulation, and for which there are no relevant limit standards. Among them, Fusarium toxins and Alternaria toxins are the main emerging contaminants in grains and other agricultural products, widely distributed and often co-contaminating with traditional toxins; some toxins are highly toxic. Although Alternaria and Fusarium belong to different fungal groups, their toxins are closely related in ecological distribution, toxic effects, and food safety risks. Both are soil-dwelling fungi and often coexist in farmland and grain storage, forming a competitive symbiosis. Their coexistence can lead to an "acute-chronic toxicity superposition." However, for these two major classes of mycotoxins, only a few toxins such as deoxynivalenol (DON) from Fusarium have limit standards globally; there are currently no regulations restricting Alternaria toxins. Therefore, there is an urgent need to develop detection methods and standards for the rapid and accurate detection of emerging mycotoxins.

[0004] Currently, detection technologies for mycotoxins mainly include thin-layer chromatography, enzyme-linked immunosorbent assay (ELISA), gas chromatography-mass spectrometry (GC-MS), high-performance liquid chromatography (HPLC), and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS). Among these, rapid screening methods based on immunological principles, such as ELISA and its improved forms, while widely used in on-site initial screening, have inherent limitations in the stability of their recognition elements (antibodies), their tolerance to complex matrices, and their specific recognition ability for antigen structural analogs. For example, CN116794303A proposes a chemiluminescent immunoassay method that optimizes the reaction interface by using novel materials such as gold nanoparticles loaded on mesoporous silica, improving sensitivity and stability. CN121022988A proposes a novel biosensor method based on mycotoxin-specific nucleic acid aptamers, which has outstanding advantages such as ultra-high sensitivity and rapid detection. However, these methods essentially still rely on antibodies or nucleic acid aptamers targeting single or a few known toxins, and cannot be flexibly extended to structurally diverse emerging toxins. They can only serve as preliminary screening tools and are difficult to achieve high-throughput, accurate quantification of multiple types and a wide range of polarities of toxins. As a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) method, recent studies have explored the analysis of 43 mycotoxins in complex food matrices through the design of multiple independent pretreatment processes (refer to Rapid and sensitive UHPLC-MS / MS methods for dietary sample analysis of 43 mycotoxins in China total diet study, Journal of Advanced Research 39 (2022) 15–47). Furthermore, CN118209655A proposes a technique to increase the detection range of specific emerging toxins, such as ergot alkaloids, to 11 species by employing a Captiva EMR-Lipid purification column. Nevertheless, when dealing with Fusarium toxins and Alternaria toxins, especially their mixtures, traditional HPLC modes using aqueous / organic phases often necessitate complex pretreatment or purification methods to ensure the separation of compounds with different properties. This not only results in long analysis cycles and low throughput but also makes efficient separation of isomers such as Fusarium toxin B / B1 difficult, potentially leading to mutual interference during mass spectrometry detection and limiting the accuracy of quantification. Summary of the Invention

[0005] The problem the invention aims to solve

[0006] To date, rapid methods such as immunology and biosensors have fallen short of providing highly specific and accurate analysis for emerging mycotoxins. On the other hand, conventional methods based on liquid chromatography-mass spectrometry (LC-MS) have room for improvement in handling the separation of emerging toxins and isomers with a wide range of polarities. Given this situation, there is an urgent need to develop methods capable of separating and detecting multiple emerging mycotoxins and their isomers in a short time.

[0007] Therefore, the problem to be solved by the present invention is to provide a method for accurate qualitative and quantitative analysis of emerging mycotoxins in agricultural products without complicated pretreatment and in a short time.

[0008] Solution for solving the problem

[0009] The inventors conducted in-depth research to achieve the above objectives and found that by extracting agricultural products with a specific extraction solvent and separating emerging mycotoxins in supercritical fluid chromatography using a specific mobile phase, and then detecting them using a mass spectrometer in multiple reaction monitoring mode, the above problems can be solved, thus completing the present invention.

[0010] That is, the present invention includes the following methods.

[0011] [1] A method for detecting emerging mycotoxins in agricultural products, characterized in that the method comprises:

[0012] Step 1: Extract the agricultural product using an extraction solvent to prepare a sample solution to be tested, wherein, relative to 100% of the extraction solvent, the extraction solvent contains at least one organic solvent selected from the group consisting of alcohols and nitrile compounds having 1 to 4 carbon atoms in a volume ratio of more than 85%.

[0013] Step 2: The emerging mycotoxin in the sample solution is separated using supercritical fluid chromatography, wherein the supercritical fluid chromatography uses mobile phase A and mobile phase B for gradient elution, mobile phase A is a supercritical fluid, and mobile phase B contains at least one organic solvent selected from the group consisting of alcohols and nitriles having 1 to 4 carbon atoms.

[0014] Step 3: Detect the emerging mycotoxin separated in Step 2 using a mass spectrometer, wherein the mass spectrometer is in multiple reaction monitoring (MRM) mode.

[0015] The emerging mycotoxin is selected from at least one of the following groups: beauveria bassiana, fucoidan A, fucoidan A1, fucoidan B, fucoidan B1, T-2 toxin, HT-2 toxin, T-2 triol, T-2 tetraol, novofusarene alcohol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, zearalenone, zearalenone, α-zearalenol, β-zearalenol, α-zearalenol, β-zearalenol, albiziol, albiziol monomethyl ether, tebuconazole, albiziolene, and mycophenolic acid.

[0016] [2] The method according to [1] is characterized in that the extraction solvent contains at least one organic solvent selected from the group consisting of methanol, ethanol, isopropanol and acetonitrile.

[0017] [3] The method according to [1] or [2] is characterized in that the extraction solvent further contains a carboxylic acid having 1 to 4 carbon atoms, wherein the carboxylic acid has a volume concentration of 0.05% to 0.25% in the extraction solvent.

[0018] [4] The method according to [3] is characterized in that the carboxylic acid is formic acid and / or acetic acid.

[0019] [5] According to the method of [1], the mobile phase A contains supercritical carbon dioxide and the mobile phase B contains at least one organic solvent selected from the group consisting of methanol, ethanol, isopropanol and acetonitrile.

[0020] [6] According to the method of [1] or [5], the mobile phase B further comprises:

[0021] Carboxylic acids with 1 to 4 carbon atoms at a volume concentration of 0.02% to 0.2%; and / or,

[0022] Ammonium salts of carboxylic acids with 1 to 4 carbon atoms, having a molar concentration of 1 to 25 mM.

[0023] [7] The method according to [6] is characterized in that the carboxylic acid having 1 to 4 carbon atoms is formic acid and / or acetic acid, and the ammonium salt of the carboxylic acid having 1 to 4 carbon atoms is ammonium formate and / or ammonium acetate.

[0024] [8] According to the method described in [1], the stationary phase in the supercritical fluid chromatography is selected from silica gel modified with polar groups, and the polar groups are selected from diol, amino, and cyano groups.

[0025] [9] According to the method of [1], the gradient elution is characterized in that:

[0026] For 30% to 50% of the total run time from the start of gradient elution, the proportion of mobile phase B is 3% to 10%.

[0027] Subsequently, for 10% to 30% of the total operating time, the proportion of mobile phase B is 20% to 40%.

[0028] The proportion of mobile phase B is then maintained at 3% to 10% until the end of the total running time.

[0029] Wherein, the sum of the proportions of mobile phase A and mobile phase B is 100%, and the proportion is a volume ratio.

[0030] The proportion of mobile phase A and the total flow rate of mobile phase B are greater than 0.5 mL / min and less than 2.5 mL / min.

[0031] The total running time is between 6 minutes and 20 minutes.

[0032]

[10] According to the method described in [1], the ion source of the mass spectrometer detector is an atmospheric pressure chemical ionization source or an electrospray ionization source.

[0033]

[11] The method according to [1] is characterized in that the agricultural product is grain.

[0034] The effects of the invention

[0035] The method of this invention, by using supercritical fluid chromatography-tandem mass spectrometry (SFC-MS / MS), enables the separation and detection of emerging fungal toxins and related metabolites of the genera *Alternaria* and *Fusarium* in a short time.

[0036] Furthermore, the method of this invention, by combining supercritical fluid chromatography with highly sensitive tandem mass spectrometry, can effectively improve the separation efficiency of complex matrix samples, achieving more accurate quantitative analysis. According to the method of this invention, even for complex matrix samples, analysis can be performed after simple solvent extraction without complex pretreatment or purification, thus further improving detection efficiency.

[0037] Furthermore, the method of the present invention uses a specific extraction solvent to extract agricultural products, enabling the thorough extraction of various emerging mycotoxins and related metabolites contained in agricultural products with a simple extraction operation. This allows for the simultaneous separation and detection of multiple emerging mycotoxins and related metabolites within a short time. According to the detection method of the present invention, the time from sample preparation to completion of detection can be controlled within 50 minutes or less, for example, within 30 minutes.

[0038] In addition, the method of the present invention reduces the amount of mobile phase used compared with traditional liquid chromatography-mass spectrometry (LC-MS) techniques by using supercritical fluid chromatography, which is beneficial to environmental protection. Attached Figure Description

[0039] Figure 1 Chromatograms of 24 emerging fungal toxins and related metabolites were analyzed by SFC-MS / MS. Detailed Implementation

[0040] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0041] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0042] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0043] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0044] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0045] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0046] One embodiment of the present invention is a method for detecting emerging mycotoxins in agricultural products, wherein the emerging mycotoxins are selected from Fusarium toxins and Alternaria toxins. Specifically, the emerging mycotoxins are selected from at least one of the following groups: Beauveria bassiana, Fusarium toxin A, Fusarium toxin A1, Fusarium toxin B, Fusarium toxin B1, T-2 toxin, HT-2 toxin, T-2 triol, T-2 tetraol, Nystatin, Deoxynystatin, 3-acetyldeoxynystatin, 15-acetyldeoxynystatin, Zearalenone, Zearalenone, α-Zaralenone, β-Zaralenone, α-Zaralenol, β-Zaralenol, Alternaria, Alternaria monomethyl ether, Tengtoxin, Alternariae, and Mycophenolic acid.

[0047] The method includes the following steps:

[0048] Step 1: Extract the agricultural product using an extraction solvent to prepare a sample solution to be tested, wherein, relative to 100% of the extraction solvent, the extraction solvent contains at least one organic solvent selected from the group consisting of alcohols and nitrile compounds having 1 to 4 carbon atoms in a volume ratio of more than 85%.

[0049] Step 2: The emerging mycotoxin in the sample solution is separated using supercritical fluid chromatography, wherein the supercritical fluid chromatography uses mobile phase A and mobile phase B for gradient elution, mobile phase A is a supercritical fluid, and mobile phase B contains at least one organic solvent selected from the group consisting of alcohols and nitriles having 1 to 4 carbon atoms.

[0050] Step 3: Detect the emerging mycotoxins separated in Step 2 using a mass spectrometer detector, wherein the mass spectrometer detector is in multiple reaction monitoring mode.

[0051] In this invention, the agricultural products used as the detection targets are not particularly limited and can be plants, animals, microorganisms, and their primary products obtained in agricultural activities. Representatively, agricultural products can be grains. Grains are generally divided into cereals (also known as cereal grains), legumes, and tubers. Cereals include rice (indica rice, japonica rice, glutinous rice), wheat (wheat, barley, oats, rye), corn, sorghum, millet, sorghum, buckwheat, etc.; legumes include soybeans, broad beans, peas, mung beans, red beans, kidney beans, etc.; tubers include potatoes, yams, taro, cassava, etc. In particular, the detection method of this invention can be suitable for detecting emerging mycotoxins in cereals.

[0052] The detection method of this invention can simultaneously detect multiple emerging mycotoxins, therefore the target analyte can be multiple emerging mycotoxins. Of course, the target analyte can also be a single emerging mycotoxin, which goes without saying. In this invention, the emerging mycotoxin used as the detection target is selected from Fusarium toxins and Alternaria toxins, and can be any one of the toxins belonging to the Fusarium or Alternaria toxin genera listed below, or more than two. It should be noted that Fusarium toxins and Alternaria toxins are named after the genera *Fusarium* and *Alternaria*, respectively, but are not necessarily unique to fungi of the *Fusarium* or *Alternaria* genera. Any compound corresponding to these toxins can be used in the detection method of this application. Therefore, these toxins used as the detection targets of the detection method of this application are not limited to those produced by fungi of the *Fusarium* and *Alternaria* genera, but can also be produced by fungi other than the *Fusarium* and *Alternaria* genera or other organisms, or manufactured by chemical or biochemical methods.

[0053] Examples of Fusarium toxins include: cyclic ester peptide toxins, such as...

[0054] ;

[0055] Trichothecene toxins, such as

[0056] ;

[0057] Estrogen toxins, such as

[0058] .

[0059] Examples of Alternaria toxins include:

[0060] .

[0061] Details of these compounds are described in Table 1.

[0062] [Table 1]

[0063]

[0064] From the perspective of effectively utilizing the separation effect of supercritical fluid chromatography, the emerging mycotoxin is preferably selected from at least one of the following groups: beautifier, enstatin A, enstatin A1, enstatin B, enstatin B1, T-2 toxin, HT-2 toxin, T-2 triol, T-2 tetraol, novofusarene alcohol, deoxynofusarene alcohol, 3-acetyldeoxynofusarene alcohol, 15-acetyldeoxynofusarene alcohol, zearalenone, zearalenone, α-zearalenone alcohol, β-zearalenone alcohol, α-zearalenol, β-zearalenol, cross-linked phenol, cross-linked phenol monomethyl ether, tebuconazole, cross-linked mycotoxin, and mycophenolic acid.

[0065] The following is a detailed description of each step in the method of this application.

[0066] Step 1

[0067] Step 1 is the extraction step, in which the agricultural product being tested is extracted using an extraction solvent to prepare a sample solution for testing. The extraction solvent contains, by volume, at least 85% or more of at least one organic solvent selected from the group consisting of alcohols and nitrile compounds having 1 to 4 carbon atoms. Using such an extraction solvent, emerging mycotoxins can be extracted efficiently. From the viewpoint of improving extraction efficiency, the volume ratio of at least one organic solvent selected from the group consisting of alcohols and nitrile compounds having 1 to 4 carbon atoms is preferably 90% or more, more preferably 95% or more, and particularly preferably 99% or more, relative to 100% of the extraction solvent.

[0068] From the viewpoint of improving extraction efficiency, methanol, ethanol, isopropanol, and acetonitrile are preferred among alcohols and nitrile compounds with 1 to 4 carbon atoms, and methanol and acetonitrile are more preferred, with acetonitrile being particularly preferred.

[0069] The extraction solvent may also contain solvents other than alcohols and nitriles with 1 to 4 carbon atoms. Examples of other solvents include one or more organic solvents that are not alcohols or nitriles with 1 to 4 carbon atoms, such as water, tetrahydrofuran, acetone, chloroform, dichloromethane, dimethyl sulfoxide, and formamide. From the viewpoint of improving extraction efficiency and reducing impurities, it is preferable that the volume ratio of other solvents in the extraction solvent is less than 10%, more preferably less than 5%, and particularly preferably no other solvents are used.

[0070] From the viewpoint of improving extraction efficiency and the sensitivity and accuracy of subsequent mass spectrometry analysis, it is preferable that the extraction solvent also contains a carboxylic acid with 1 to 4 carbon atoms, wherein the volume concentration (i.e., volume percentage concentration) of the carboxylic acid in the extraction solvent is 0.05% to 0.25%. From the same viewpoint, the carboxylic acid is more preferably a monocarboxylic acid with 1 to 4 carbon atoms, further preferably formic acid and / or acetic acid, and most preferably formic acid. From the same viewpoint, the volume concentration of the carboxylic acid in the extraction solvent is more preferably 0.06% to 0.2%, further preferably 0.08% to 0.15%. In a particularly preferred embodiment, the extraction solvent is an acetonitrile solution containing 0.08% to 0.15% formic acid by volume.

[0071] Before extraction from agricultural products, they can be pulverized as needed. Commonly known pulverizing equipment such as mortars and pestles, grinders, and crushers can be used. The extraction method is not particularly limited, as long as the analyte and extraction solvent are sufficiently in contact. Typically, extraction can be performed by immersing the analyte in the extraction solvent. The extraction time depends on the type and relative amount of the analyte and the extraction solvent, and is typically from 5 to 40 minutes. Considering both accuracy and speed of analysis, an extraction time of 10 to 35 minutes, preferably 20 to 30 minutes, is preferred. During extraction, heating, stirring, shaking, or ultrasonic treatment can be performed as needed. After extraction, solid-liquid separation can be performed as needed through settling, filtration, centrifugation, etc., and the resulting liquid fraction can be used as the sample solution to be tested.

[0072] It should be noted that, depending on the properties, state, and composition of the sample solution, it can be directly used in subsequent step 2, or it can be diluted with a conventional solvent before being used in subsequent step 2. From the perspective of saving procedures, it is preferable to directly use the sample solution obtained in step 1 in subsequent step 2.

[0073] The detection method of the present invention, by using the aforementioned extraction solvent in step 1, enables sufficient extraction in a short time with simple operation. Furthermore, combined with step 2, which uses supercritical fluid chromatography with advantages such as high efficiency and speed, the time from sample preparation to detection completion is controlled within 50 minutes or less. In some preferred embodiments, this time can be controlled within 30 minutes.

[0074] Step 2

[0075] Step 2 is a chromatographic separation step, wherein the emerging mycotoxin in the sample solution is separated using supercritical fluid chromatography. Supercritical fluid chromatography uses a gradient elution with mobile phase A and mobile phase B, wherein mobile phase A is a supercritical fluid, and mobile phase B contains at least one organic solvent selected from the group consisting of alcohols and nitrile compounds having 1 to 4 carbon atoms.

[0076] Supercritical fluid chromatography (SFC) is a chromatographic method that uses supercritical fluid as the mobile phase. Supercritical fluids possess properties between those of gases and liquids. Compared to traditional liquid chromatography (LC) and gas chromatography (GC), SFC combines the advantages of both and exhibits unique separation selectivity. In this invention, the use of a supercritical fluid chromatography column for the separation of emerging mycotoxins not only effectively separates various emerging mycotoxins and their metabolites with complex structures and significant polarity differences, but also effectively separates isomers.

[0077] In supercritical fluid chromatography, the supercritical fluid can be supercritical carbon dioxide or supercritical ethane, etc. In some preferred embodiments of the present invention, supercritical carbon dioxide is preferred as the aforementioned mobile phase A because it is mild, non-toxic, chemically inert, readily available, and environmentally friendly. Mobile phase B is a modifier (sometimes also called an entrainer) used to adjust the polarity of the supercritical fluid. From the viewpoint of improving detection efficiency and accuracy, alcohols and nitrile compounds with 1 to 4 carbon atoms used as mobile phase B are preferably methanol, ethanol, isopropanol, or acetonitrile, more preferably methanol or isopropanol, and particularly preferably methanol.

[0078] From the viewpoint of improving separation selectivity and enhancing the sensitivity and accuracy of mass spectrometry analysis, mobile phase B is preferably substantially free of organic solvents or water, except for alcohols and nitrile compounds with 1 to 4 carbon atoms. "Substantially free" means that it is not actively added, but is allowed to be present at the level of impurities, for example, within a volume or weight ratio of 0.1%.

[0079] From the viewpoint of improving separation selectivity and enhancing the sensitivity and accuracy of mass spectrometry analysis, it is preferable that the mobile phase B also contains a carboxylic acid with 1 to 4 carbon atoms and / or an ammonium salt of that carboxylic acid. It should be noted that "containing a carboxylic acid with 1 to 4 carbon atoms and / or an ammonium salt of that carboxylic acid" includes: containing a carboxylic acid with 1 to 4 carbon atoms, or containing an ammonium salt of a carboxylic acid with 1 to 4 carbon atoms, or containing both.

[0080] In the case of a carboxylic acid having 1 to 4 carbon atoms, from the viewpoint of improving separation selectivity and enhancing the sensitivity and accuracy of mass spectrometry analysis, the volume concentration (i.e., volume percentage concentration) of the carboxylic acid in the mobile phase B is 0.02% to 0.2%, more preferably 0.04% to 0.16%, and even more preferably 0.06% to 0.12%. The carboxylic acid may be used alone or in combination with two or more. It should be noted that when multiple carboxylic acids are present simultaneously, this volume concentration refers to the total volume concentration of all carboxylic acids. From the viewpoint of improving separation selectivity and enhancing the sensitivity and accuracy of mass spectrometry analysis, the carboxylic acid is more preferably a monocarboxylic acid having 1 to 4 carbon atoms, more preferably formic acid and / or acetic acid, and most preferably formic acid.

[0081] In the case of ammonium salts containing carboxylic acids having 1 to 4 carbon atoms, from the viewpoint of improving separation selectivity and enhancing the sensitivity and accuracy of mass spectrometry analysis, the molar concentration (i.e., molar concentration, also known as molar-volume concentration) of the ammonium salt in the mobile phase B is 1 mM to 25 mM, more preferably 2 mM to 20 mM, and even more preferably 5 mM to 15 mM. The ammonium salt can be used alone or in combination with two or more. It should be noted that when multiple ammonium salts containing carboxylic acids having 1 to 4 carbon atoms are simultaneously present, this molar concentration refers to the total molar concentration of all such ammonium salts. "Ammonium salt of carboxylic acids having 1 to 4 carbon atoms" refers to a salt formed by a carboxylate anion having 1 to 4 carbon atoms and an ammonium cation; in other words, the 1 to 4 carbon atoms refers only to the number of carbon atoms in the carboxylate anion. The ammonium cation can be NH4+. + The ammonium salt of the carboxylic acid having 1 to 4 carbon atoms is preferred to be an ammonium salt of a monocarboxylic acid having 1 to 4 carbon atoms, further preferred to be ammonium formate and / or ammonium acetate, and most preferably ammonium formate.

[0082] In a particularly preferred embodiment, mobile phase A can be supercritical carbon dioxide, and mobile phase B can be a methanol solution containing 0.08% to 0.12% formic acid by volume and 8 to 12 mM ammonium formate by molar concentration, more preferably a methanol solution containing 0.08% to 0.12% formic acid by volume.

[0083] The chromatographic column (i.e., supercritical fluid chromatography column) used in the supercritical fluid chromatography of this invention is not particularly limited; for example, a packed column can generally be used. In some embodiments, the inner diameter of the SFC column is preferably 1-8 mm, more preferably 2-5 mm, and the length is preferably 50-300 mm, more preferably 100-250 mm. The stationary phase in the supercritical fluid chromatography column can be selected from polar group-modified silica gel; for polar groups, it can be selected from glycol, amino, and cyano groups. In this invention, amino-modified silica gel is preferably used as the stationary phase in the supercritical fluid chromatography column. In addition, the stationary phase can be porous spherical silica gel with a particle size of, for example, 1-5 µm. From the viewpoint of improving the separation selectivity for emerging mycotoxins, amino-modified silica gel columns are preferred. As the supercritical fluid chromatography column, commercially available SFC analytical columns can be used.

[0084] In this invention, supercritical fluid chromatography employs gradient elution. There are no particular limitations as long as it can separate emerging mycotoxins; for example, it can be set as follows:

[0085] For 30% to 50% of the total run time from the start of gradient elution, the proportion of mobile phase B is 3% to 10%.

[0086] Subsequently, for 10% to 30% of the total operating time, the proportion of mobile phase B is 20% to 40%.

[0087] The proportion of mobile phase B is then maintained at 3% to 10% until the end of the total running time.

[0088] Wherein, the sum of the proportions of mobile phase A and mobile phase B is 100%, and the proportion is a volume ratio.

[0089] The total flow rate of mobile phase A and mobile phase B is greater than 0.5 mL / min and less than 2.5 mL / min.

[0090] The total running time is between 6 minutes and 20 minutes.

[0091] In a preferred embodiment, the gradient elution conditions can be:

[0092] From 0 to 4 minutes, the proportion of mobile phase B increases from 3% to 10% to 20% to 40%.

[0093] From 6 min to 8 min, the proportion of mobile phase B was maintained at 20-40%.

[0094] Immediately after 8 minutes, reduce the proportion of mobile phase B from 20-40% to 3-10%;

[0095] The proportion of mobile phase B was maintained at 3%~10% until the end of 10 minutes.

[0096] In this invention, there are no particular limitations on back pressure and column temperature; they can be selected from common conditions depending on the type of supercritical fluid used. For example, when supercritical carbon dioxide is used as the mobile phase, the back pressure can be 8 MPa or more, preferably 10 to 40 MPa, more preferably 10 to 25 MPa, and the column temperature can be 31°C or more, preferably 35°C to 60°C, more preferably 38°C to 55°C.

[0097] The supercritical fluid chromatography system used in this invention can be commercially available, such as the "Nexera UC" supercritical fluid chromatography system from Shimadzu Corporation.

[0098] Step 3

[0099] Step 3 is the detection step, in which the emerging mycotoxin separated in step 2 is detected using a mass spectrometer detector for qualitative or quantitative analysis. In this invention, the mass spectrometer detector uses multiple reaction monitoring (MRM) mode; therefore, the mass spectrometer detector can also be referred to as a tandem mass spectrometry analysis device. Representatively, a triple quadrupole mass spectrometer or a quadrupole-time-of-flight (Q-TOF) mass spectrometer can be used. The triple quadrupole mass spectrometer is preferred for quantitative analysis because it can accurately, sensitively, and reliably determine the content of target substances in complex systems.

[0100] The mass spectrometer detector can be an electrospray ionization (ESI) source or an atmospheric pressure chemical ionization (APCI) source. From the perspective of efficiently ionizing emerging mycotoxins and their metabolites and improving the accuracy of MRM detection, ESI is preferred.

[0101] There are no particular restrictions on the MRM ion pairs and quantitative ions of various emerging fungal toxins and their metabolites, as long as the purpose of qualitative / quantitative analysis can be achieved. For example, those described in the embodiments of this application can be used, but are not limited thereto.

[0102] According to the detection method of the present invention, no complex pretreatment is required; analysis can be performed simply by solvent extraction. This method enables rapid and accurate qualitative and quantitative analysis of emerging mycotoxins and their metabolites, and in particular, it enables rapid and accurate simultaneous qualitative and quantitative analysis of multiple emerging mycotoxins and their metabolites.

[0103] Example

[0104] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents, instruments, and chromatographic columns whose manufacturers are not specified are all commercially available conventional products.

[0105] In the embodiments, unless otherwise specified, the following experimental equipment and analytical conditions were used.

[0106] Experimental equipment

[0107] The supercritical fluid chromatography system (LC-30AD SF supercritical CO2 fluid pump, LC-30AD entrainer pump, SFC-30A back pressure control unit, SIL-30AC autosampler, CTO-20AC column oven (equipped with a high-pressure six-way switching valve) and triple quadrupole mass spectrometer detector LCMS-8050 (with ESI source) were used; CBM-20A system controller and LabSolutions LCMS Ver.5.120 workstation were also used.

[0108] Analysis conditions

[0109] Liquid phase conditions:

[0110] Chromatographic column: Shim-pack UC-NH2 2.1×150 mm, 3 µm

[0111] Column temperature: 40℃

[0112] Mobile phase: A SF-CO2, B 0.1% formic acid methanol solution

[0113] Flow rate: 1 mL / min

[0114] Injection volume: 5µL

[0115] BPR temperature: 50℃

[0116] BPR pressure: 10MPa

[0117] The gradient elution procedure is shown in Table 2.

[0118] [Table 2]

[0119]

[0120] Mass spectrometry conditions

[0121] Ion source: ESI

[0122] Interface voltage: 4kV

[0123] Nebulizing gas: Nitrogen 3L / min

[0124] Heating gas: Air 10L / min

[0125] Drying gas: Nitrogen 10 L / min

[0126] DL tube temperature: 250℃

[0127] Interface temperature: 300℃

[0128] Heating module temperature: 400℃

[0129] Scanning mode: Multiple Response Monitoring (MRM)

[0130] The multiple reaction monitoring parameters for 24 emerging mycotoxins and related metabolites are shown in Table 3 below.

[0131] [Table 3]

[0132]

[0133] Example 1

[0134] Using the experimental equipment and analytical conditions described above, a mixed solution of mycotoxins containing the 24 target compounds shown in Table 3 was analyzed. The results are as follows: Figure 1 As shown, by using the detection method of this application, all 24 compounds were well retained and separated on the SFC chromatographic column, thus enabling accurate qualitative / quantitative analysis.

[0135] Example 2

[0136] For the 24 target compounds shown in Table 3, a series of standard solutions were prepared (concentrations of 1 ng / mL, 2 ng / mL, 5 ng / mL, 8 ng / mL, 10 ng / mL, and 20 ng / mL, with an isotopic internal standard concentration of 5 ppb). 5 µL injections were performed using an autosampler, and quantification was conducted using the internal standard method to investigate the linearity of the method. As shown in Table 4, the results indicate that, on the SFC-MS / MS analysis system, good linearity was obtained for all 24 compounds in the range of 1–20 ng / mL, with a linear correlation coefficient r0. 2 The limits of detection were all greater than 0.9912 and ranged from 0.007 ng / mL to 1.695 ng / mL. Therefore, the detection method described in this application allows for accurate quantitative analysis of each of these 24 compounds.

[0137] [Table 4]

[0138]

[0139] Example 3

[0140] Using 5 mL of extraction solvent, mycotoxins and their metabolites in 1 g of blank corn matrix spiked sample (5 ppb) were extracted by ultrasonic extraction for 30 min. After centrifugation, the spiked recovery rates of different solvents were investigated. The results are shown in Table 5.

[0141] [Table 5]

[0142]

[0143] In Table 5, “—” indicates that the spiked recovery rate could not be measured. In Table 5, “80% methanol, 0.1% formic acid” means “80% (v / v) methanol aqueous solution containing 0.1% (v / v) formic acid”. Other similar expressions have the same meaning.

[0144] As shown in Table 5, the spiked recoveries of the 24 target analytes could not be determined when using 80% methanol extraction. When using 80% methanol containing 0.1% formic acid, the spiked recoveries of enstatin A, enstatin A1, enstatin B, and enstatin B1 were below 70%, and the spiked recoveries of other target analytes could not be determined. When using 80% acetonitrile, the spiked recoveries of T-2 triol, T-2 tetraol, and β-zearalenone were below 70%, and the spiked recovery of 15-acetyldeoxynivalenol could not be determined. When using 80% acetonitrile or 80% acetonitrile containing 0.1% formic acid, the spiked recovery of β-zearalenone was below 70%. For cases where spiked recoveries could not be determined or were below 70%, the accuracy of the qualitative analysis was considered low.

[0145] In contrast, extraction with acetonitrile solution containing 0.1% formic acid yielded excellent recoveries (92.6%–128.8%) for all targets, enabling accurate quantitative analysis of 24 targets simultaneously. Furthermore, extraction with 90% methanol, methanol, 90% acetonitrile, acetonitrile, 90% methanol containing 0.1% formic acid, methanol containing 0.1% formic acid, and 90% acetonitrile containing 0.1% formic acid all achieved relatively good recoveries for multiple targets, with no instances of unmeasurable or below-70% recoveries, allowing for accurate quantitative analysis of more than two of the 24 targets simultaneously.

[0146] Therefore, by using an extraction solvent consisting of at least one organic solvent selected from the group consisting of alcohols and nitriles with 1 to 4 carbon atoms in a volume ratio of more than 85%, sample solutions for SFC-MS / MS analysis can be prepared, enabling the simultaneous and accurate analysis of multiple emerging mycotoxins in complex matrices derived from grains and other sources.

[0147] Example 4

[0148] Grains spiked with 1 ng / mL, 2 ng / mL, and 5 ng / mL were extracted using a 0.1% formic acid acetonitrile solution. The recoveries at different spiking concentrations were investigated, and the results are shown in Tables 6 and 7. All 24 compounds showed good recoveries, especially compounds other than T-2 triol and T-2 tetraol, which achieved excellent recoveries ranging from 70% to 130%. Therefore, the method of this invention can achieve simultaneous and accurate analysis of multiple emerging mycotoxins in complex matrices derived from grains and other sources.

[0149] [Table 6]

[0150]

[0151] [Table 7]

[0152]

[0153] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0154] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method of detecting emerging mycotoxins in agricultural products, characterized in that, The method includes: Step 1: Extract the agricultural product using an extraction solvent to prepare a sample solution to be tested, wherein, relative to 100% of the extraction solvent, the extraction solvent contains at least one organic solvent selected from the group consisting of alcohols and nitrile compounds having 1 to 4 carbon atoms in a volume ratio of more than 85%. Step 2: The emerging mycotoxin in the sample solution is separated using supercritical fluid chromatography, wherein the supercritical fluid chromatography uses mobile phase A and mobile phase B for gradient elution, mobile phase A is a supercritical fluid, and mobile phase B contains at least one organic solvent selected from the group consisting of alcohols and nitriles having 1 to 4 carbon atoms. Step 3: Detect the emerging mycotoxin separated in Step 2 using a mass spectrometer, wherein the mass spectrometer is in multiple reaction monitoring (MRM) mode. The emerging mycotoxin is selected from at least one of the following groups: beauveria bassiana, fucoidan A, fucoidan A1, fucoidan B, fucoidan B1, T-2 toxin, HT-2 toxin, T-2 triol, T-2 tetraol, novofusarene alcohol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, zearalenone, zearalenone, α-zearalenol, β-zearalenol, α-zearalenol, β-zearalenol, albiziol, albiziol monomethyl ether, tebuconazole, albiziolene, and mycophenolic acid.

2. The method of claim 1, wherein, The extraction solvent contains at least one organic solvent selected from the group consisting of methanol, ethanol, isopropanol, and acetonitrile.

3. The method according to claim 1 or 2, characterized in that, The extraction solvent also contains a carboxylic acid having 1 to 4 carbon atoms, and the carboxylic acid in the extraction solvent has a volume concentration of 0.05% to 0.25%.

4. The method of claim 3, wherein, The carboxylic acid is formic acid and / or acetic acid.

5. The method of claim 1, wherein, The mobile phase A contains supercritical carbon dioxide, and the mobile phase B contains at least one organic solvent selected from the group consisting of methanol, ethanol, isopropanol, and acetonitrile.

6. The method according to claim 1 or 5, characterized in that, The mobile phase B also contains: Carboxylic acids with 1 to 4 carbon atoms at a volume concentration of 0.02% to 0.2%; and / or, Ammonium salts of carboxylic acids with 1 to 4 carbon atoms, having a molar concentration of 1 to 25 mM.

7. The method of claim 6, wherein, The carboxylic acid having 1 to 4 carbon atoms is formic acid and / or acetic acid, and the ammonium salt of the carboxylic acid having 1 to 4 carbon atoms is ammonium formate and / or ammonium acetate.

8. The method of claim 1, wherein, The stationary phase in the supercritical fluid chromatography is selected from silica gel modified with polar groups, wherein the polar groups are selected from diol groups, amino groups, and cyano groups.

9. The method of claim 1, wherein, The ion source of the mass spectrometer detector is an atmospheric pressure chemical ionization source or an electrospray ionization source.

10. The method of claim 1, wherein, The agricultural product in question is grain.

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