Method for detecting various types of mycotoxins in tea samples and its application

Abstract

The present invention provides a method for detecting multiple types of mycotoxins in tea samples and its application. Based on the technical difficulty of lacking high-throughput mycotoxin detection in the field, the present invention provides a solution. The present invention discloses an ultra-high performance liquid chromatography-tandem mass spectrometry method for detecting mycotoxins in tea samples, which is established based on optimized extraction steps, improved QuEChERS purification technology combined with ultra-high performance liquid chromatography-tandem mass spectrometry. The technical solution of the present invention can realize the one-time detection of multiple mycotoxins, and the types of mycotoxins are at least 16.

Description

Technical Field

[0001] The present invention relates to the technical field of chromatographic analysis of trace chemical substances; more specifically, the present invention relates to a method for detecting various types of mycotoxins in tea samples and application thereof. Background Art

[0002] Mycotoxins contaminate various foods, beverages and grain crops. About 1 / 4 of grain production is contaminated by mycotoxins to varying degrees. All crops have the probability of being contaminated by toxin-producing fungi and then containing biological toxins during growth, post-harvest storage and processing. Mycotoxins not only contaminate food and cause food poisoning, but most of them are also carcinogenic, teratogenic and mutagenic, posing a huge threat to human survival and health. Although the conditions for food growth and storage can be controlled to reduce the occurrence of contamination, and some mycotoxins can be removed during food processing, they cannot be completely removed. Therefore, it is very necessary to detect the content of mycotoxins in food production projects.

[0003] There are many kinds of tea, and there are thousands of famous teas. According to the color and taste differences formed by different processing techniques and fermentation degrees, they are mainly divided into six basic tea categories: green tea, white tea, yellow tea, oolong tea, black tea and dark tea. Due to its unique flavor and health functions, it is the most consumed beverage in the world besides water. However, mycotoxins are one of the potential chemical pollutants in the process of tea tree planting, tea production, storage and consumption. In particular, the unique pile fermentation process in post-fermented tea involves a variety of microorganisms, including: Aspergillus niger, Aspergillus clavatus, Aspergillus glaucus and other Aspergillus species, Rhizopus, Penicillium, Trichoderma, yeast and thermophilic bacteria; on the one hand, microorganisms are conducive to the formation of unique aroma and quality of tea, on the other hand, due to the humid processing environment and the complexity of the industrial environment, it may lead to contamination by harmful toxin-producing fungi, increasing the risk of mycotoxin production and accumulation.

[0004] At present, more and more attention is paid to the quality and safety of tea at home and abroad. Most of the research focuses on the detection of heavy metals and pesticide residues, and there are also many studies on functional active ingredients in tea. With the in-depth investigation and research on the contamination of mycotoxins in tea at home and abroad, it is found that the contamination of mycotoxins that may exist in such products is diverse, and a variety of mycotoxins may cause contamination to tea during planting, processing and storage. LC-MS / MS is widely used in the determination of mycotoxins in food. Due to the complexity of the tea matrix, there is still a lack of effective means in this field to reduce the tea matrix effect, especially for the LC-MS / MS method for the simultaneous detection of multiple mycotoxins. At present, there is a lack of research on the simultaneous detection of multiple mycotoxins in fermented tea, and there is also a lack of research data on the risk assessment of mycotoxins in different types of fermented tea. In particular, for such mycotoxins as α-zearalenol, β-zearalenol, α-zearalenol, and β-zearalenol, there is still a lack of relevant contamination data, and no suitable means have been found in the tea matrix to achieve simultaneous detection with other multiple toxins through a one-time operation.

[0005] In summary, the current detection methods for various mycotoxins in tea at home and abroad are cumbersome and inefficient, and rarely achieve high-throughput detection of multiple mycotoxins. Summary of the invention

[0006] The object of the present invention is to provide an ultra-high performance liquid chromatography-tandem mass spectrometry method for detecting fungal toxins in tea samples. The method is established based on optimized extraction steps, improved QuEChERS purification technology combined with ultra-high performance liquid chromatography-tandem mass spectrometry.

[0007] In a first aspect of the present invention, a method for detecting multiple types (including two or more types) of mycotoxins in a tea sample is provided, the method comprising:

[0008] (1) extracting an extract containing (or potentially containing) mycotoxins from a tea sample, wherein the extraction is performed using a polar organic solvent, and formic acid is added to the polar organic solvent;

[0009] (2) purifying the extract of (1) using a QuEChERS purification method to obtain a purified extract;

[0010] (3) detecting the purified extract of the above (2) by high performance liquid chromatography-tandem mass spectrometry to determine the fungal toxin content;

[0011] Wherein, the mycotoxins include two or more selected from the following group: α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), α-zearalenol (α-ZAL), β-zearalenol (β-ZAL), zearalenone (ZEN), aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), deoxynivalenol (DON), 15-acetylated deoxynivalenol (15-Ac DON), 3-acetyl deoxynivalenol (3-Ac DON), ochratoxin A (OTA), neo-orange alcohol (NEO), T-2 toxin (T-2), citrinin (CIT).

[0012] In a preferred embodiment, the fungal toxins include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 types.

[0013] In another preferred embodiment, in step (1), the polar organic solvent is selected from: acetonitrile, methanol, and ethyl acetate.

[0014] In another preferred embodiment, in step (1), the concentration of formic acid in the polar organic solution is 0.1% to 1.4%; preferably 0.2% to 1.2%; more preferably 0.5% to 1.1% (such as 0.6%, 0.8%, 0.9%, 1%).

[0015] In another preferred embodiment, in step (1), the concentration of the polar organic solvent is 95-100%; more preferably 98-100% (such as 99%).

[0016] In another preferred embodiment, in step (1), the formic acid used for addition (before adding the sample) is 95-100%; more preferably, the formic acid is 98-100% (such as 99%).

[0017] In another preferred embodiment, in step (1), when extracting the tea sample, 5 to 16 ml of a polar organic solvent containing formic acid is used per gram of tea sample; preferably, 8 to 12 ml of a polar organic solvent containing formic acid is used per gram of tea sample; more preferably, 9 to 11 ml of a polar organic solvent containing formic acid is used per gram of tea sample; for example, 10 ml of formic acid acetonitrile is used per gram of tea sample.

[0018] In another preferred embodiment, in step (3), high performance liquid chromatography-tandem mass spectrometry is used for detection; preferably, an aqueous solution containing ammonium acetate is used as mobile phase A, and methanol is used as mobile phase B.

[0019] In another preferred embodiment, in step (3), the high performance liquid chromatography-tandem mass spectrometry is ultra high performance liquid chromatography-tandem mass spectrometry.

[0020] In another preferred embodiment, Agilent Extend-C 18 A chromatographic column (150 mm×3.0 mm, 3.5 μm) was used as a chromatographic column for ultra-high performance liquid chromatography.

[0021] In another preferred embodiment, the time for the gradient elution program is 20±5 minutes (preferably 20±3 minutes; more preferably 20±2 minutes or 20±1 minute).

[0022] In another preferred embodiment, in step (3), detection and analysis are performed under the conditions of mass spectrometry optimization parameters (Table 1).

[0023] In another preferred embodiment, in step (3), the determination of the fungal toxin content includes quantitative determination or qualitative determination.

[0024] In another preferred embodiment, in step (3), high performance liquid chromatography-tandem mass spectrometry is used for detection, and ultra high performance liquid chromatography-triple quadrupole tandem mass spectrometry is used for detection.

[0025] In another preferred embodiment, in step (3) high performance liquid chromatography-tandem mass spectrometry detection, the mass spectrometry conditions are set to:

[0026] When measuring AFB1, the product ion with an m / z value of 241.100 was used as the quantification ion;

[0027] When measuring AFB2, the product ion with an m / z value of 287.200 was used as the quantification ion;

[0028] When measuring AFG1, the product ion with an m / z value of 200.100 was used as the quantification ion;

[0029] When measuring AFG2, the product ion with an m / z value of 189.100 was used as the quantification ion;

[0030] When measuring ZEN, the product ion with an m / z value of 175.030 was used as the quantitative ion;

[0031] When measuring α-ZEL, the product ion with an m / z value of 275.200 was used as the quantitative ion;

[0032] When measuring β-ZEL, the product ion with an m / z value of 275.200 was used as the quantitative ion;

[0033] When measuring α-ZAL, the product ion with an m / z value of 277.200 was used as the quantitative ion;

[0034] When measuring β-ZAL, the product ion with an m / z value of 277.200 was used as the quantification ion;

[0035] When measuring DON, the product ion with an m / z value of 265.000 was used as the quantitative ion;

[0036] When measuring 15-Ac DON, the product ion with an m / z value of 137.000 was used as the quantitative ion;

[0037] When measuring 3-Ac DON, the product ion with an m / z value of 307.160 was used as the quantitative ion;

[0038] When measuring OTA, the product ion with an m / z value of 239.100 was used as the quantitative ion;

[0039] When measuring NEO, the product ion with an m / z value of 185.100 was used as the quantification ion;

[0040] When measuring T-2, the product ion with an m / z value of 215.000 was used as the quantitative ion;

[0041] When measuring CIT, the product ion with an m / z value of 249.000 was used as the quantitative ion;

[0042] The quantifier utilizes collision energy to generate appropriate product ions.

[0043] In another preferred embodiment, in step (2), in the QuEChERS purification method, the purification tube used comprises the following fillers: carboxyl multi-walled carbon nanotubes (MWCNTs-COOH), styrene divinyl benzene copolymer (Hydrophilic-Lipophilic Balance, HLB), silica gel (Silica Gel, SG); preferably, in terms of weight ratio, carboxyl multi-walled carbon nanotubes: styrene divinyl benzene copolymer: silica gel is 1: (1-10): (1-10); preferably, it is 1: (6-9): (5-10).

[0044] In another preferred embodiment, in step (2), the mass ratio of carboxylated multi-walled carbon nanotubes: styrene divinylbenzene copolymer: silica gel is 1:(7-8):(7-8).

[0045] In another preferred embodiment, in step (2), the mass ratio of carboxylated multi-walled carbon nanotubes: styrene divinylbenzene copolymer: silica gel is 1:7.5:7.5.

[0046] In another preferred embodiment, the method further includes the step of crushing the tea sample before step (1).

[0047] In another preferred embodiment, after step (1), the method further comprises the step of obtaining a supernatant by centrifugation.

[0048] In another preferred embodiment, the method further comprises: using a standard curve method to perform qualitative and / or quantitative analysis; preferably, before step (3) detection, preparing a matrix spike solution: taking a blank sample solution that does not contain toxins, and then adding a toxin standard solution to prepare matrix spiked sample (standard) solutions of different concentrations; preferably, after step (3) detection, using the detection conditions of step (3) to measure the matrix spiked sample solutions of different concentrations to obtain a matrix spiked curve, and using a matrix external standard method to achieve qualitative and / or quantitative analysis.

[0049] In another preferred embodiment, the standard curve is a matrix-spiked curve. Preferably, the determination method of step (3) is used to determine matrix-spiked sample solutions of different concentrations to obtain a matrix-spiked curve, and the matrix external standard method is used to simultaneously achieve qualitative and quantitative analysis.

[0050] In another preferred embodiment, the preparation of the matrix spiked solution includes: taking a blank sample solution without toxins, and then adding a toxin standard solution to prepare matrix spiked solutions of different concentrations.

[0051] In another aspect of the present invention, there is provided an application of any of the above methods for detecting multiple types (including two or more) of mycotoxins in tea samples; preferably, the detection includes quantitative detection or qualitative detection; preferably, the mycotoxins include two or more selected from the group consisting of: α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), α-zearalenol (α-ZAL), β-zearalenol (β-ZAL), zearalenone (ZEN), aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), deoxynivalenol (DON), 15-acetylated deoxynivalenol (15-Ac DON), 3-acetyl deoxynivalenol (3-Ac DON), 15-acetylated deoxynivalenol (15 ... DON), ochratoxin A (OTA), neosolaniol (NEO), T-2 toxin (T-2), citrinin (CIT).

[0052] In another aspect of the present invention, a kit for detecting the content of multiple types (including two or more types) of mycotoxins in a tea sample is provided, comprising:

[0053] (a) a polar organic solvent for extracting an extract containing (or potentially containing) mycotoxins from a tea sample, wherein formic acid is added to the polar organic solvent;

[0054] (b) a purification tube suitable for the QuEChERS purification method, wherein the purification tube comprises the following fillers: carboxylated multi-walled carbon nanotubes, styrene divinyl benzene copolymer, and silica gel;

[0055] (c) Reagents for high performance liquid chromatography-tandem mass spectrometry detection, comprising: an aqueous solution containing ammonium acetate (as mobile phase A) and methanol (as mobile phase B).

[0056] In a preferred embodiment, the kit further comprises: standard products of the plurality of types (including two or more types) of mycotoxins.

[0057] In another preferred embodiment, the kit further comprises: reference substances for the multiple types (including two or more) of mycotoxins.

[0058] In another preferred embodiment, the kit further includes: an elution reagent.

[0059] In another preferred embodiment, the kit further includes: instructions for use describing the method for detecting the content of mycotoxins in tea samples.

[0060] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 , recovery rate of mycotoxin extraction in tea matrix with acetonitrile at different formic acid ratios (percent, %).

[0062] Figure 2 , mass spectrometric response diagram of fumonisins in tea matrix with acetonitrile at different formic acid ratios;

[0063] a, mass spectrometric response of FB1 extracted with 0% formic acid and acetonitrile;

[0064] b, mass spectrometric response of FB1 extracted with 0.1% formic acid and acetonitrile;

[0065] c, mass spectrometry response of FB1 extracted with 1% formic acid and acetonitrile;

[0066] d, mass spectrometry response diagram of FB2 and FB3 extracted with 0% formic acid and acetonitrile;

[0067] e, mass spectrometric response diagram of FB2 and FB3 extracted with 0.1% formic acid and acetonitrile;

[0068] f. Mass spectrometric response diagram of FB2 and FB3 extracted with 1% formic acid and acetonitrile.

[0069] Figure 3 , purification recovery rate of mycotoxins in tea matrix by different multifunctional purification columns (percentage, %);

[0070] The abscissa a represents the Cleanert MC (Code# LC-MYT10-B) purification column;

[0071] The abscissa b represents the purification tube containing the mixed filler MWCNTs-COOH:HLB:SG of 1:7.5:7.5 (Chinese patent application CN 202011052151.3);

[0072] The abscissa c represents the CNW 301MFC (Code#SBEQ-CD301S) purification column;

[0073] The abscissa d represents the MFC 260 (Code#M2600-25T) purification column;

[0074] The abscissa e represents the Romerlab MycoSpin 400 (Code#COCMY2400) purification column.

[0075] Figure 4 , total ion chromatograms of 16 fungal toxins under optimized LC-MS / MS conditions. DETAILED DESCRIPTION

[0076] Based on the technical difficulty of lack of high-throughput mycotoxin detection in the field, the present invention provides a solution. The present invention discloses an ultra-high performance liquid chromatography-tandem mass spectrometry method for detecting mycotoxins in tea samples, which is established based on optimized extraction steps, improved QuEChERS purification technology combined with ultra-high performance liquid chromatography-tandem mass spectrometry. The technical solution of the present invention can realize the detection of multiple mycotoxins at one time, and the types of mycotoxins are at least 16.

[0077] Based on the high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis method, there are higher requirements for the sample to be tested, and eliminating or reducing the matrix effect is one of the key factors determining the determination method. The sample pretreatment step mainly includes extraction and purification, and common sample pretreatment methods include: solid phase extraction (SPE), magnetic solid phase extraction (MSPE), dispersed liquid-liquid microextraction (DLLME), QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe), immunoaffinity column (IAC) concentration, multifunctional purification column (MFC) purification, etc. At the same time, sample pretreatment and analysis techniques based on nanomaterials, biomaterials and synthetic materials are also considered to be important methods for detecting fungal toxins. In the present invention, the detection method is optimized, and a technical solution that is different from the prior art and can realize high-throughput detection of multiple fungal toxins is provided.

[0078] the term

[0079] As used in the present invention, the "tea sample" includes natural tea samples, artificially processed tea samples, or tea products.

[0080] As used in the present invention, the "high throughput" refers to the simultaneous detection of multiple mycotoxins in different types of tea (including green tea, oolong tea, black tea, dark tea, etc.) in one batch of tests, and the types of mycotoxins detected can reach at least 16.

[0081] As used in the present invention, the "mycotoxin" is a secondary metabolite produced by some fungi under suitable temperature and humidity conditions. Preferably, the "mycotoxins" described in the present invention include but may not be limited to two or more selected from the following group: α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), α-zearalenol (α-ZAL), β-zearalenol (β-ZAL), zearalenone (ZEN), aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), deoxynivalenol (DON), 15-acetylated deoxynivalenol (15-Ac DON), 3-acetyldeoxynivalenol (3-Ac DON), ochratoxin A (OTA), neosolani (NEO), T-2 toxin (T-2), citrinin (CIT).

[0082] As used in the present invention, "matrix" refers to components in a sample other than the target analyte. Since the matrix often significantly interferes with the analysis process of the analyte and affects the accuracy of the analysis results, these effects and interferences are called matrix effects.

[0083] As used in the present invention, "target analyte" is also referred to as "target substance"; unless otherwise specified, the "target analyte" refers to "mycotoxin".

[0084] Optimization of extraction methods for multiple mycotoxins in tea samples

[0085] For a variety of fungal toxins, to achieve good detection, a prerequisite is to extract as many types of toxins and as many quantities as possible from the tea sample, so as to provide support for subsequent accurate detection.

[0086] Those skilled in the art understand that the chemical structure of each mycotoxin is different, so it is a difficult problem in the art to extract them together and extract them once to obtain multiple mycotoxins that are conducive to subsequent detection and meet the detection standards. In particular, for such mycotoxins as α-zearalenol, β-zearalenol, α-zearalenol, β-zearalenol, etc., no suitable means have been found in the tea matrix to achieve simultaneous detection with other multiple toxins through a one-time operation. The present invention realizes the one-time effective extraction of multiple mycotoxins in tea samples through effective optimization of the extraction method and simple and easy means.

[0087] In the present invention, polar organic solvents are used to extract fungal toxins. Some polar organic solvents can be used in the present invention, including but not limited to: methanol, ethanol, and acetonitrile.

[0088] As a preferred embodiment of the present invention, acetonitrile is used as a polar organic solvent to extract tea samples to obtain an extract containing multiple mycotoxins. The inventors have observed that the matrix interference of the acetonitrile extract is small. Preferably, 95-100% acetonitrile is used as the extractant in this process; more preferably, the acetonitrile is 98-100% acetonitrile, such as 99-100% acetonitrile.

[0089] However, the inventors have also found that when multiple mycotoxins need to be extracted simultaneously, the extraction effect of mycotoxins sensitive to the polarity range is not ideal when acetonitrile is used as a single extractant. After in-depth analysis, the inventors have found a solution to effectively solve / reduce this problem. Therefore, as a more preferred embodiment of the present invention, when extracting with a polar organic solution, formic acid is added to the polar organic solvent.

[0090] In view of the fact that there are many kinds of mycotoxins to be determined, in order to achieve effective extraction as much as possible, the inventors have carefully analyzed the appropriate amount of formic acid to be added. As a preferred embodiment of the present invention, the concentration of formic acid in the polar organic solution is 0.1% to 1.4%; preferably 0.2% to 1.2%; more preferably 0.5% to 1.1% (such as 0.6%, 0.8%, 0.9%, 1%). Under such an amount of formic acid addition, the inventors found that the extraction effect of mycotoxins was greatly improved, and 16 kinds of mycotoxins could be extracted at the same time, and the interference caused by the matrix effect could be effectively avoided / reduced.

[0091] The pre-treatment step also includes a step of crushing the tea sample, which can crush the tea sample to a suitable powder size. A person skilled in the art can use a variety of methods to perform this step. Preferably, this step can be performed by mechanical treatment.

[0092] As a preferred embodiment of the present invention, after extracting the tea sample with acetonitrile and obtaining the extract, the method further comprises the step of centrifuging and obtaining the supernatant. Other methods of obtaining the desired substance from the extract may also be included in the present invention.

[0093] As a preferred embodiment of the present invention, when acetonitrile is used to extract tea samples, 5 to 15 mL of acetonitrile is used per gram of tea samples; more preferably, 8 to 12 mL of acetonitrile is used per gram of tea samples. In a specific embodiment, the inventors optimized the amount of extractant and sample; when the sample is extracted with about 10 times (mL:g) volume of extractant, the recovery rate is relatively high.

[0094] In a specific embodiment of the present invention, the pretreatment step specifically includes: taking a tea sample ground into powder into a polytetrafluoroethylene centrifuge tube, adding a polar organic solvent containing formic acid, ultrasonic-assisted extraction for 30±10 min, centrifuging at 4000±1000 r / min for 10±5 min (preferably, ultrasonic-assisted extraction for 30±5 min, centrifuging at 4000±500 r / min for 10±3 min; more preferably, ultrasonic-assisted extraction for 30±3 min, centrifuging at 4000±300 r / min for 10±2 min), and taking the supernatant for later use.

[0095] Optimization of purification methods for extraction products

[0096] In the pretreatment step before testing, after obtaining the extract containing multiple mycotoxins, a purification step is also included to obtain a purified extract that can be used for subsequent testing, thereby providing support for subsequent accurate testing.

[0097] In order to obtain a good purification effect, the inventors prepared the types of fillers in the purification tube for QuEChERS purification, including using carboxylated carbon nanotubes, HLB and silica gel as the main fillers. Furthermore, the inventors also optimized the dosage ratio of the fillers, and according to the weight ratio, MWCNTs-COOH:HLB:SG is 1:(1-10):(1-10); preferably 1:(6-9):(5-10); more preferably 1:(7-8):(7-8).

[0098] The purification recovery rate of the mixed filler of the present invention is significantly higher than that of commercial purification columns, and the effect is excellent.

[0099] HLB (Code#SBEQ-CA3100) is a hydrophilic-lipophilic balanced adsorbent. The inventors found that it has a good recovery rate for the target of the present invention. Silica gel (Code#236799) is chemically stable and has a very ideal effect when used as an adsorption filler in the present invention. MWCNTs-COOH (Code#190710134732) can effectively remove tea polyphenols, chlorophyll, and lutein.

[0100] In a specific embodiment of the present invention, the preparation of the purification tube includes: weighing the mixed filler according to the weight ratio of MWCNTs-COOH:HLB:SG (1:7.5:7.5) in a 2 ml centrifuge tube, vortexing for 30 seconds to mix the filler, and setting aside.

[0101] In a specific embodiment of the present invention, the purification process using the purification tube includes: taking 1 ml of the reserved tea matrix supernatant, adding it to the purification tube, shaking for 1 minute, centrifuging at 12000r / min for 5 minutes, taking the supernatant again through a 0.22μm organic filter membrane, and placing it in a brown injection bottle for LC-MS / MS determination.

[0102] Based on the new improvement of the present invention, the present invention also provides the use of the purification tube, which is used as a purification tube based on the QuEChERS purification method to purify different types of tea extracts. In the present invention, compared with some commercial purification tubes, the homemade purification tube is more suitable for high-throughput simultaneous purification of a system containing multiple (at least 16) mycotoxins.

[0103] High-throughput detection of multiple mycotoxins in tea samples

[0104] The purified extract obtained through the purification step can be used to qualitatively and / or quantitatively detect the target substance using high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS). Preferably, the high performance liquid chromatography-tandem mass spectrometry is ultra high performance liquid chromatography-tandem mass spectrometry.

[0105] As a preferred embodiment of the present invention, when performing the detection, an Agilent Extend-C 18 A chromatographic column (150 mm×3.0 mm, 3.5 μm) was used as a chromatographic column for ultra-high performance liquid chromatography.

[0106] The inventors' research work shows that [M+Na] + This can lead to incomplete fragmentation of the parent ion, and the cation after fragmentation is likely to appear in the Na + ions, thereby reducing the abundance of the target analyte signal. After in-depth research, the inventors have found a way to avoid this phenomenon as much as possible. The inventors have found that adding ammonium acetate to the aqueous phase can inhibit the formation of certain fungal toxins (aflatoxins and trichothecenes) [M+Na] + ions. Ammonium acetate can enhance the response of 15-Ac DON, NEO and T-2 toxins. Therefore, as a preferred embodiment of the present invention, when using high performance liquid chromatography-tandem mass spectrometry for detection, an aqueous solution containing ammonium acetate is used as mobile phase A and methanol is used as mobile phase B.

[0107] In other aspects, after determining the measurement conditions, known qualitative / quantitative calculation methods can be used to perform qualitative / quantitative analysis of the target. As a preferred embodiment of the present invention, a standard curve method is used to perform qualitative and / or quantitative analysis. Preferably, a matrix-spiked sample solution with different concentrations can be measured to obtain a matrix-spiked curve, and qualitative and quantitative analysis can be simultaneously achieved using a matrix external standard method.

[0108] As a preferred embodiment of the present invention, a standard curve method is used for qualitative and / or quantitative analysis; preferably, a matrix spiked solution can be prepared by taking a blank sample solution to be tested that does not contain a plurality of mycotoxins, and then adding a plurality of mycotoxins standard solutions to prepare matrix spiked sample (standard) solutions of different concentrations; preferably, the matrix spiked sample solutions of different concentrations are measured using the detection conditions of the high performance liquid chromatography-tandem mass spectrometry method to obtain a matrix spiked curve, and a matrix external standard method is used to achieve qualitative and / or quantitative analysis.

[0109] Applications and kits

[0110] At present, there is a lack of methods for the simultaneous detection of multiple mycotoxins in fermented tea in the art, especially for such mycotoxins as α-zearalenol, β-zearalenol, α-zearalenol, β-zearalenol, etc., and no suitable means for detection through a one-time operation has been found in the art. An effective mycotoxin extraction method and a tea matrix purification method are of great significance for the LC-MS / MS detection of multiple mycotoxins in tea. In the technical scheme of the present invention, a simple and efficient LC-MS / MS method for determining multiple mycotoxins in tea is established in combination with the QuEChERs pretreatment technology previously optimized by the inventor. It can be applied to the determination of various teas or tea products.

[0111] In a specific embodiment of the present invention, a specific operation step is provided, including the following:

[0112] A. Grinding samples: Select different fermented teas and grind the fermented tea samples;

[0113] B. Weighing samples: Weigh appropriate amounts of crushed fermented tea samples of different types;

[0114] C. Extraction: add an appropriate volume of polar solvent with formic acid added, shake and mix, perform ultrasound-assisted extraction, and obtain extract a;

[0115] D. Centrifugation: centrifuge the extract a obtained in step C to obtain a supernatant b;

[0116] E. Purification: Take an appropriate volume of supernatant b into the prepared purification tube, shake and mix, and centrifuge to obtain supernatant c;

[0117] F. Filtration: Filter the supernatant c in an organic filter membrane to obtain the target detection liquid d which is placed in a brown injection bottle;

[0118] G. Analysis: Place the target detection solution d in the Ultimate 3000 / TSQ Vantage TM The data were analyzed and processed using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry.

[0119] The amount of tea leaves weighed in step B is 1 g;

[0120] The polar solvent with certain acidity in step C is 1% formic acid acetonitrile;

[0121] The appropriate volume in step C is 10 mL;

[0122] The ultrasonic-assisted extraction conditions in step C are 55° C., 30 min;

[0123] The centrifugal conditions in step D are 4000 r / min, 10 min

[0124] The appropriate volume in step E is 1 mL

[0125] Prepare the purification tube as described in step E: According to the weight ratio, 120 mg of MWCNTs-COOH:HLB:SG (1:7.5:7.5) is weighed and mixed, placed in a 2 mL centrifuge tube and vortexed for 30 seconds to mix the filler, and set aside.

[0126] The centrifugal condition in step E is 12000r / min, 5min

[0127] The pore size of the organic filter membrane in step F is 0.22 μm;

[0128] The G step uses a mobile phase of (5mmol / L ammonium acetate-water)-methanol, and the mobile phase A is 5mmol / L ammonium acetate-water solution, and the mobile phase B is methanol. The separation is performed by a gradient elution method in which the concentration of the mobile phase B is gradually increased, and the gradient elution program is: 0-1min, 15% B, 1-3min, 15-25% B, 3.0-6.5min, 25-50% B, 6.5-12min, 50-100% B, 12-15min, 100% B, 15-17min, 100-15% B, 17-20min, 15% B;

[0129] The chromatographic column used in step G is Agilent Extend-C 18 Column (150 mm × 3.0 mm, 3.5 μm); column temperature: 30 °C; injection volume: 10 μL; flow rate: 0.35 mL / min;

[0130] In step G, the ion source is an electrospray ion source (ESI), and the scanning mode is a positive and negative ion scanning mode; the electrospray voltage is: 3000V (ESI + ) / 2500V(ESI -), detection mode was selected reaction monitoring (SRM); ion source temperature: 250°C; nebulizing gas was nitrogen with a flow rate of 650L / h; curtain gas was nitrogen with a flow rate of 40L / h; auxiliary heating gas was nitrogen with a temperature of 300°C; sheath gas pressure was 40psi; ion scanning gas pressure was 5psi; auxiliary gas pressure was 5psi.

[0131] The method of the present invention has good purification effect on tea matrix, fast analysis speed, high sensitivity and good reproducibility, and provides an effective way for simultaneous detection of multiple mycotoxins in tea. The method of the present invention has the characteristics of being fast, simple, cheap, effective, stable and safe.

[0132] Based on the new discovery of the inventors, the present invention also provides a kit for detecting the content of multiple fungal toxins in tea samples, comprising: a polar organic solvent for extracting an extract containing (or potentially containing) fungal toxins from the tea samples, wherein formic acid is added to the polar organic solvent; a purification tube suitable for the QuEChERS purification method, wherein the purification tube comprises the following fillers: carboxylated multi-walled carbon nanotubes, styrene divinylbenzene copolymer, and silica gel; and a reagent for high performance liquid chromatography-tandem mass spectrometry detection, comprising: an aqueous solution containing ammonium acetate (as mobile phase A) and methanol (as mobile phase B).

[0133] As a preferred embodiment of the present invention, the kit further comprises: standard substances of various mycotoxins; reference substances; elution reagents; and / or instructions for use of a method for detecting the contents of various mycotoxins in tea samples.

[0134] The method and kit of the present invention have the advantages of low cost, simple operation, high degree of matrix purification, high method sensitivity, and accurate quantitative and qualitative analysis.

[0135] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or conditions recommended by the manufacturer. Based on the examples in the present invention, other examples obtained based on the present invention by other technicians in the field without making creative achievements all belong to the protection scope of the present invention. All technical and scientific terms used herein have the same meaning as those commonly understood by technicians in the technical field of the present invention. DETAILED DESCRIPTION

[0137] 1. Experimental Materials and Methods

[0138] (1) Instruments, reagents and materials

[0139] Ultimate 3000 / TSQ Vantage TMUltra-performance liquid chromatography-triple quadrupole tandem mass spectrometry (UPLC-MS / MS); MILLI-Q water purification machine (Millipore, Billerica, MA, USA).

[0140] Multifunctional stainless steel grinding machine (FSJ-A05N6).

[0141] The methanol, ammonium acetate, formic acid and acetonitrile used in the experiment were all of chromatographic grade.

[0142] The materials mainly include analytical grade carboxyl multi-walled carbon nanotubes (MWCNTs-COOH), styrene divinylbenzene copolymer (Hydrophilic-Lipophilic Balance, HLB) and silica gel (SG) filler; as well as Cleanert MC, MFC260, Romerlab MycoSpin 400, CNW 301 MFC purification columns.

[0143] Toxin Standards:

[0144] Aflatoxin mixed standard (AFB1:AFB2:AFG1:AFG2=4:1:4:1, 10000344);

[0145] Zearalenone (ZEN, Z 2125);

[0146] α-Zeralenol (α-ZEL, Z 0166);

[0147] β-Zeralenol (β-ZEL, Z 2000);

[0148] α-Zealanol (α-ZAL, Z 0292);

[0149] β-Zealanol (β-ZAL, Z 0417);

[0150] Deoxynivalenol (DON, D 0156);

[0151] 15-acetylated deoxynivalenol (15-Ac DON, A 1556);

[0152] 3-Acetyldeoxynivalenol (3-Ac DON, A 6166);

[0153] Ochratoxin A (OTA, O 1877);

[0154] Neofusarium solani (NEO, BRM S 92001);

[0155] T-2 toxin (T-2, T 4887);

[0156] Citrinin (CIT, C 1017).

[0157] The tea samples mainly include green tea (Biluochun), oolong tea (Dahongpao), black tea (Wuyi black tea), and dark tea (Pu'er tea) purchased in supermarkets or tea shops.

[0158] (2) Chromatographic conditions

[0159] Column: Agilent Extend-C 18 Column (150mm×3.0mm, 3.5μm, Art.No.763954-302); column temperature: 30℃; injection volume: 10μL; flow rate: 0.35mL / min; mobile phase: phase A is 5mmol / L ammonium acetate-water, phase B is methanol; gradient elution program: 0-1min, 15% B, 1-3min, 15-25% B, 3.0-6.5min, 25-50% B, 6.5-12min, 50-100% B, 12-15min, 100% B, 15-17min, 100-15% B, 17-20min, 15% B.

[0160] (3) Mass spectrometry conditions

[0161] Electrospray ion source (ESI), scanning mode positive and negative ion scanning mode, electrospray voltage: 3000V (ESI + ) / 2500V(ESI - ), selected reaction monitoring (SRM); ion source temperature: 250°C; nebulizer gas is nitrogen, flow rate 650L / h; curtain gas is nitrogen, flow rate 40L / h; auxiliary heating gas is nitrogen, temperature 300°C; sheath gas pressure 40psi; ion scanning gas pressure 5psi; auxiliary gas pressure 5psi; other mass spectrometry parameters / acquisition conditions are shown in Table 1, where the marked a is the quantitative ion.

[0162] (4) Preparation of standard stock solution and working solution

[0163] The standard is diluted with pure acetonitrile to prepare a standard mother solution and stored in a -40°C refrigerator. After determining the measurement conditions, the known qualitative / quantitative calculation methods can be used to perform qualitative / quantitative analysis of the target. The standard curve method is used for qualitative and / or quantitative analysis. Preferably, the matrix-spiked sample solution with different concentrations can be measured to obtain a matrix-spiked curve, and the matrix external standard method can be used to simultaneously perform qualitative and quantitative analysis. In subsequent examples, tea samples that are confirmed to be free of mycotoxins will be used for matrix effect evaluation and method pretreatment optimization.

[0164] Preparation of matrix spike curve: Take a blank tea sample without mycotoxins, extract and purify it according to the established pretreatment method to obtain a tea matrix solution, use it to dilute the standard stock solution of the target mycotoxins to prepare a matrix-matched standard solution.

[0165] Prepare matrix spiked samples of different concentrations: weigh 1 g of blank tea sample without mycotoxins into a 50 mL centrifuge tube, add known concentration or low, medium, and high concentration mycotoxin standard stock solutions according to the relevant limit standards for mycotoxins, mix for 30 seconds and place in the dark at room temperature for 30 minutes. After the organic solvent evaporates, the state of the real tea sample can be better simulated. The obtained matrix spiked samples can be prepared and used immediately.

[0166] (5) Sample pretreatment method

[0167] The sample pretreatment steps mainly include extraction and purification, and common sample pretreatment methods include: solid phase extraction (SPE), magnetic solid phase extraction (MSPE), dispersive liquid-liquid microextraction (DLLME), QuEChERS (Quick, Easy, Cheap, Effect). The present invention provides an ultra-high performance liquid chromatography-tandem mass spectrometry method for detecting multiple mycotoxins in tea samples, and the method is established based on optimized extraction steps, improved QuEChERS purification technology and ultra-high performance liquid chromatography-tandem mass spectrometry.

[0168] The early stage of the pretreatment step also includes the step of crushing the tea sample: extracting the tea sample with formic acid (such as 1%) and acetonitrile, and after obtaining the extract, centrifuging to obtain the supernatant.

[0169] In the subsequent specific embodiments, the pretreatment step specifically includes: taking 1 g of the tea sample ground into powder and placing it in a 50 mL polytetrafluoroethylene centrifuge tube, adding 10 mL, 1% formic acid acetonitrile, ultrasonic-assisted extraction at 55°C for 30 min, and centrifuging at 4000 r / min for 10 min; taking 1 mL of the reserved tea matrix supernatant, adding it to a purification tube, shaking for 1 min, centrifuging at 12000 r / min for 5 min, taking the supernatant again and passing it through a 0.22 μm organic filter membrane, and placing it in a brown injection bottle for UPLC-MS / MS determination.

[0170] In a specific embodiment of the present invention, the preparation of a homemade purification tube (Chinese patent application CN 202011052151.3) includes: according to the weight ratio, 120 mg MWCNTs-COOH:HLB:SG (1:7.5:7.5) is weighed and mixed into a 2 mL centrifuge tube, and the mixture is vortexed for 30 seconds to mix the filler and set aside.

[0171] Example 1. Establishment of mass spectrometry conditions

[0172] In this embodiment, the mass spectrometry parameters are determined and optimized. The inventors found in the early stage of the study that [M+Na] + This can lead to incomplete fragmentation of the parent ion, and the cation after fragmentation is likely to appear in the Na + ions, thereby reducing the signal abundance of the target analyte.

[0173] After discovering this phenomenon, the inventors further studied and tried to avoid its occurrence as much as possible. The inventors analyzed various additives in order to change this situation. As a result, they found that adding ammonium acetate to the aqueous phase can inhibit the formation of certain fungal toxins (aflatoxins and trichothecenes) [M+Na] + Ions. Ammonium acetate can enhance the responses of 15-Ac DON, NEO, and T-2 toxins.

[0174] When the mobile phase was (5mmol / L ammonium acetate-water)-methanol, each analyte was monitored in the full scan mode with an m / z range of 50-1000 in the positive and negative ion modes to obtain the appropriate precursor ions. AFB1, AFB2, AFG1, AFG2, and OTA contain C=O or methoxy (CH3O-), which are easy to obtain hydrogen ions. They can show a good response in the positive ionization mode and are mainly [M+H] + The precursor ion is formed in the form of a hydroxyl group (-OH) in the ZEN molecules, which is easy to lose hydrogen ions. A better response can be obtained in the negative ionization mode. The precursor ion [MH] - Has the highest ion abundance; DON and 3-Ac DON precursor ion [M+CH3COO] - Has the highest ion abundance; the precursor ions of 15-Ac DON, NEO, and T-2 are mainly in the form of [M+NH4] + ; The most abundant CIT ion is [M+OH] - Different optimal parent ions were selected, and collision-induced dissociation (CID) was performed with different argon gases to obtain product ion spectra. The product ions and collision energies were automatically optimized on the mass spectrometer. The signal acquisition information of various fungal toxins optimized in SRM mode: characteristic ion pairs, collision energy and other parameter conditions are shown in Table 1. The corresponding product ions were selected as quantitative ions and marked as a .

[0175] Table 1. Retention time and MS parameters for mycotoxin analysis

[0176]

[0177]

[0178] tR : Retention Time;

[0179] CE: Collision Energy.

[0180] Example 2: Optimization of extractants for mycotoxins in tea samples

[0181] In this example, firstly, a known concentration (C Ai ) tea matrix, the extraction of mycotoxins was carried out using polar organic solvents.

[0182] According to the experimental results, the inventors found that as the concentration of acetonitrile increases, the extraction recovery rate of the target analyte also gradually increases, and the pure acetonitrile extract has little matrix interference in the subsequent analysis process. However, the inventors found that the extraction effect of single acetonitrile as an extractant for fungal toxins sensitive to the polarity range is not ideal. Therefore, it is necessary to find a solution to this problem.

[0183] After repeated screening, debugging of test conditions and changes in schemes, the inventors preliminarily determined that the effect of extraction could be enhanced and the types of detectable toxins could be expanded by adding auxiliary reagents to acetonitrile.

[0184] The inventors have conducted a lot of analysis and testing on various experimental reagents. Many reagents are not beneficial to improving the extraction effect, and some even reduce the extraction effect or cause deviation in the measurement results or fail to measure effectively. After many aspects of research and screening, the inventors found that formic acid is beneficial to enhancing the extraction effect overall.

[0185] The inventors added different proportions of formic acid to the extractant and compared the effects of different dosages. The extraction recovery rates of different mycotoxins by the extractant were calculated when the formic acid content was 0%, 0.1%, 1%, 2%, 5%, 10%, and 15%. The recovery rate was calculated according to the following equation (1), where C ai is the actual detected concentration of spiked mycotoxin i, C bi is the concentration of mycotoxin i detected in blank tea sample, C Ai is the theoretical concentration of spiked mycotoxin i.

[0186] Recovery rate (%) = (C ai -C bi ) / C Ai ×100——(1)

[0187] The recovery rate of toxins is shown in Table 2 and Figure 1 shown.

[0188] Table 2

[0189]

[0190] Figure 1 In the figure above, the recovery rates of different toxins are different. In the general method validation, the recovery rate of spiked samples needs to be within the range of 60-120%, and it is believed that the closer the recovery rate is to 100%, the more ideal the recovery effect of the method is. As summarized in Table 2, in order to achieve a good recovery rate for each toxin including fumonisin, it is relatively preferred to select about 1% formic acid.

[0191] The responses of fumonisin toxins to the test are shown in Table 3 and Figure 2 shown.

[0192] Table 3

[0193]

[0194] According to Table 3 and Figure 2 It can be seen that for the presence of fumonisins, the response is enhanced with the addition of formic acid in the extractant.

[0195] In summary, the inventors, through research, screening and optimization analysis, believe that adding formic acid to acetonitrile is beneficial to improving the extraction recovery rate of toxins. More importantly, the inventors have found a special optimization mode that can detect multiple mycotoxins at the same time. In the acetonitrile to which formic acid is added, the concentration of formic acid is preferably 0.1-5%. However, the inventors also found that when the formic acid concentration is 2% or 5%, the recovery rate of CIT exceeds 120%, indicating that other impurities in the recovery system also respond, resulting in the occurrence of false positives, that is, a matrix effect occurs. Therefore, a formic acid concentration of less than 2% (such as 0.1% to 1.4%) is suitable.

[0196] In subsequent experiments, the inventors selected acetonitrile with 1% formic acid added as the extractant of the present invention to meet the recovery rate and response of the above-mentioned fungal toxins.

[0197] Example 3: Comparison of pre-treatment and purification methods for mycotoxins in tea samples

[0198] In the existing work of the present invention, a self-made purification tube was prepared (Chinese patent application CN 202011052151.3), and it was verified that the purification tube can effectively detect aflatoxin in tea matrix. In this embodiment, the inventor analyzed the self-made purification tube and other various commercial purification devices, trying to explore a method for efficient purification of fungal toxins.

[0199] Weigh 5 groups of tea leaves according to the above steps, with 3 replicates in each group, where the spiked and extracted steps remain unchanged according to the above conditions, and the obtained extracts are added to different purification devices. For the homemade purification tube (Chinese patent application CN202011052151.3), take 1ml of supernatant in a 2ml purification tube (MWCNTs-COOH:HLB:SG is 1:7.5:7.5), shake and mix for 1min, centrifuge at 12000r / min for 5min, take the supernatant through a 0.22μm organic filter membrane, and put it in a brown injection bottle for LC-MS / MS detection. The purification process of other multifunctional purification columns (Cleanert MC, MFC 260, Romerlab MycoSpin 400, CNW 301MFC) refers to the existing literature and product-related instructions for purification, and also passes through a 0.22μm organic filter membrane and is placed in a brown injection bottle for LC-MS / MS detection. Finally, compare the test results of the five groups.

[0200] The results are as follows Figure 3 As shown in the figure, different multifunctional purification columns have different advantages in mycotoxin purification:

[0201] Cleanert MC purification column has good recovery rate for 3-Ac DON, 15-Ac DON, AFs, T-2, NEO, and ZEN (61.24-119.58%);

[0202] The CNW 301MFC purification column has a good recovery rate for 3-Ac DON, AFs, and ZEN (63.38-110.53%);

[0203] The MFC 260 purification column has a good recovery rate for T-2 purification (85.13%);

[0204] Romerlab MycoSpin 400 purification column has good recovery rate (64.95-116.93%) for DON, 3-Ac DON, AFBs, CIT, T-2, NEO, OTA, and ZEN;

[0205] In general, the homemade purification tube has a good recovery rate for the above-mentioned mycotoxins (74.26-119.98%); the mixed filler has an ideal purification recovery rate and has the potential to replace commercial multifunctional purification columns.

[0206] The analysis of the inventors also shows that the purification effect of each purification tube on fumonisin is not ideal. Therefore, the purification of fumonisin should be carried out through a single toxin purification method of immunoaffinity column, strong anion exchange column and specific adsorption fumonisin filler.

[0207] The present invention uses the medium concentration of 16 fungal toxins (the medium concentration of each toxin set in Table 4) under suitable chromatographic mass spectrometry conditions and pre-treatment methods to detect the effect as follows Figure 4 shown.

[0208] Example 4: Verification of the LC-MS / MS Detection Method of the Present Invention for Multiple Mycotoxins in Tea

[0209] In this embodiment, relevant parameters of the method of the present invention are verified, and the precision and accuracy of the method are verified.

[0210] (1) According to the above method for preparing the standard curve and matrix spiked curve, standard curves with the same linear range were prepared using tea matrix pretreatment solutions extracted with formic acid and acetonitrile at different ratios. The matrix effect was evaluated by comparing the slopes of the calibration curves (ka and kb represent the slopes of the tea matrix matching curve and the acetonitrile matching curve extracted with formic acid and acetonitrile at different ratios, respectively). The calculation method is shown in the following formula (2).

[0211] ME(%)=(K a / K b -1)×100——(2);

[0212] (2) The LODs and LOQs of the method established in the present invention are determined by adding a spike at the lowest concentration, and the response is 3 times and 10 times the average value of the chromatographic baseline noise, respectively. The accuracy and precision of the established method are tested by using the intra-day precision and inter-day precision analysis methods. In the validation of the LC-MS / MS method for 16 fungal toxins, the low, medium and high concentrations added, as well as the preparation range of the matrix standard curve, the detection limit, the quantification limit and other related information are shown in Table 4 below.

[0213] Table 4. Information on spiked concentrations, linear ranges, detection limits, and quantification limits of various mycotoxins

[0214]

[0215]

[0216] Four types of spiked samples (green tea, oolong tea, black tea, and dark tea) were prepared according to the above steps for preparing tea samples spiked with mycotoxins at low, medium, and high concentrations. They were pretreated according to the established method, and finally the recovery and repeatability of the method were analyzed and determined, with six replicates at each concentration.

[0217] The verification results are shown in Table 5: the detection limit and quantification limit of mycotoxins ranged from 0.015 to 15.00 μg.kg -1 and 0.03~30.00μg.kg -1 ; This method has a spike concentration range of 0.13 to 600 μg.kg-1 The recovery rate was 60.99-118.46%, and the relative standard deviations (RSDs) were small.

[0218] Table 5. Accuracy and precision of LC-MS / MS method in different tea matrices

[0219]

[0220]

[0221] Therefore, the inventors have achieved simultaneous detection of up to 16 fungal toxins in one operation by optimizing the detection means, with a low limit of detection of fungal toxins and stable and accurate results.

[0222] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for detecting multiple types of mycotoxins in tea samples, characterized in that: The method comprises: (1) extracting an extract containing mycotoxins from a tea sample, wherein the extraction is performed using a polar organic solution, and formic acid is added to the polar organic solution; the polar organic solution is an acetonitrile solution, and the concentration of formic acid in the polar organic solution is 0.1% to 1.4%; (2) Purifying the extract of (1) by using a QuEChERS purification method to obtain a purified extract; in the QuEChERS purification method, the purification tube used comprises the following fillers: carboxylated multi-walled carbon nanotubes, styrene divinyl benzene copolymer, and silica gel; in terms of weight ratio, the carboxylated multi-walled carbon nanotubes: styrene divinyl benzene copolymer: silica gel is 1: (6-9): (5-10); (3) detecting the purified extract of the above (2) by high performance liquid chromatography-tandem mass spectrometry to determine the fungal toxin content; Among them, the fungal toxins include: α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol, zearalenone, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, deoxynivalenol, 15-acetylated deoxynivalenol, 3-acetyldeoxynivalenol, ochratoxin A, neosolani, T-2 toxin and citrinin.

2. The method according to claim 1, characterized in that In step (1), the concentration of formic acid in the polar organic solution is 0.2% to 1.2%.

3. The method according to claim 2, characterized in that In step (1), the concentration of formic acid in the polar organic solution is 0.5% to 1.1%.

4. The method according to claim 1, characterized in that In step (3), high performance liquid chromatography-tandem mass spectrometry is used for detection; an aqueous solution containing ammonium acetate is used as mobile phase A, and methanol is used as mobile phase B.

5. The method according to claim 4, characterized in that In step (3), the high performance liquid chromatography-tandem mass spectrometry is ultra high performance liquid chromatography-tandem mass spectrometry; Agilent Extend-C 18 The chromatographic column is used as a chromatographic column for ultra-high performance liquid chromatography; the time of the gradient elution program is 20±5 minutes.

6. The method according to any one of claims 1, 4 or 5, characterized in that: In step (3) high performance liquid chromatography-tandem mass spectrometry detection, the mass spectrometry conditions are set as follows: When measuring AFB1, the product ion with an m / z value of 241.100 was used as the quantification ion; When measuring AFB2, the product ion with an m / z value of 287.200 was used as the quantification ion; When measuring AFG1, the product ion with an m / z value of 200.100 was used as the quantification ion; When measuring AFG2, the product ion with an m / z value of 189.100 was used as the quantification ion; When measuring ZEN, the product ion with an m / z value of 175.030 was used as the quantitative ion; When measuring α-ZEL, the product ion with an m / z value of 275.200 was used as the quantitative ion; When measuring β-ZEL, the product ion with an m / z value of 275.200 was used as the quantitative ion; When measuring α-ZAL, the product ion with an m / z value of 277.200 was used as the quantitative ion; When measuring β-ZAL, the product ion with an m / z value of 277.200 was used as the quantification ion; When measuring DON, the product ion with an m / z value of 265.000 was used as the quantitative ion; When measuring 15-Ac DON, the product ion with an m / z value of 137.000 was used as the quantitative ion; When measuring 3-Ac DON, the product ion with an m / z value of 307.160 was used as the quantitative ion; When measuring OTA, the product ion with an m / z value of 239.100 was used as the quantitative ion; When measuring NEO, the product ion with an m / z value of 185.100 was used as the quantification ion; When measuring T-2, the product ion with an m / z value of 215.000 was used as the quantitative ion; When measuring CIT, the product ion with an m / z value of 249.000 was used as the quantitative ion; The quantifier utilizes collision energy to generate appropriate product ions.

7. Use of the method according to any one of claims 1 to 6 for detecting multiple types of mycotoxins in tea samples; the detection comprises quantitative detection or qualitative detection; the mycotoxins comprise: α-Zeralenol, β-Zeralenol, α-Zeralanol, β-Zeralanol, Zearalenone, Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, Aflatoxin G2, Deoxynivalenol, 15-acetylated Deoxynivalenol, 3-acetyldeoxynivalenol, Ochratoxin A, Neosolaniol, T-2 toxin and Citrinin.

8. A kit for detecting the content of multiple types of mycotoxins in tea samples, comprising: (a) a polar organic solution for extracting an extract containing mycotoxins from a tea sample, wherein formic acid is added to the polar organic solution; The polar organic solution is an acetonitrile solution, and the concentration of formic acid in the polar organic solution is 0.1% to 1.4%; (b) A purification tube suitable for the QuEChERS purification method, wherein the purification tube comprises the following fillers: carboxylated multi-walled carbon nanotubes, styrene divinyl benzene copolymer, and silica gel; in terms of weight ratio, carboxylated multi-walled carbon nanotubes: styrene divinyl benzene copolymer: silica gel is 1: (6-9): (5-10); (c) Reagents for high performance liquid chromatography-tandem mass spectrometry detection, including: aqueous solution containing ammonium acetate, methanol; Among them, the fungal toxins include: α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol, zearalenone, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, deoxynivalenol, 15-acetylated deoxynivalenol, 3-acetyldeoxynivalenol, ochratoxin A, neosolani, T-2 toxin and citrinin.

9. The kit according to claim 8, characterized in that The kit also includes: Standards of the various types of mycotoxins; Reference substances of the aforementioned multiple mycotoxins; and / or, Elution reagent.

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