Method for simultaneously detecting rice yeast acid, isorice yeast acid and toxoflavin

Abstract

The invention discloses a method for simultaneously detecting rice yeast acid, isorice yeast acid and toxoflavin, and belongs to the technical field of food detection. The method for simultaneously detecting the rice yeast acid, the isorice yeast acid and the toxoflavin comprises the following steps: mixing a to-be-detected sample and an extracting solution, extracting, and purifying to obtain a to-be-detected sample solution; detecting the to-be-detected sample solution by adopting a liquid chromatography-tandem mass spectrometry method; the extracting solution is a mixed solution of acetonitrile and acetic acid. According to the method provided by the invention, the sample pretreatment process is simplified, the problem of simultaneous extraction of the rice yeast acid, the isorice yeast acid and the toxoflavin with relatively large polarity difference is solved, simultaneous determination through one-time extraction is realized, the test time is shortened, the contents of the rice yeast acid, the isorice yeast acid and the toxoflavin in the fresh wet powder can be simply and efficiently detected and confirmed, and the method is suitable for large-scale popularization and application. The measurement result is accurate and reliable.

Description

Technical Field

[0001] The invention relates to the technical field of food detection, in particular to a method for simultaneously detecting fumonisin, isorhizin and toxin. Background Art

[0002] In recent years, food poisoning incidents caused by eating foods contaminated by Burkholderia gladiolipv. cocovenenans (BGC), such as homemade fermented cornmeal, fresh wet powder and black fungus, have occurred frequently, with a high mortality rate, which has attracted widespread attention. BGC will produce a variety of toxins such as Bongkrekic acid (BA), Iso-bongkrekic acid (IBA) and Toxoflavin (TF) after contaminating food under certain conditions. It is the foodborne pathogen with the highest mortality rate discovered so far. It is reported that BA is a mitochondrial toxin. Its lethality is based on causing mitochondrial dysfunction, cell damage and even apoptosis, and ultimately causing damage to solid organs such as the heart, brain, and liver. The LD of BA in mice and humans is 2.34. 50 The values ​​range from 1 to 3.16 mg / kg and 0.68 to 6.84 mg / kg, respectively. There is no specific antidote. IBA is an isomer of BA, and its toxicity is about 1 / 2 to 1 / 4 of BA. In addition to being converted into IBA under alkaline conditions, BA also produces IBA in the initial stage of sunlight degradation. TF produces toxicity by promoting the active electron transfer between reduced coenzyme I (NADH) and oxygen, and bypassing the hydrogen peroxide produced by the cytochrome system. Its LD 50 BGC has strong drug resistance. Food contaminated by BGC cannot be eaten safely even after being cooked using common cooking methods. Ingestion by mistake can cause acute poisoning, damage to multiple organs, and even death.

[0003] At present, most reports on the detection methods of BA, IBA and TF focus on the determination of one or two of them, such as the determination of fumonisin, fumonisin, fumonisin and isofumonisin by liquid chromatography-tandem mass spectrometry, and the determination of BA by immunosensor based on the obtained specific monoclonal antibodies. There are very few research results on the simultaneous detection of BA, IBA and TF, but recently Fang Lanyun et al. based on ultra-high performance liquid chromatography-tandem mass spectrometry, used solid phase extraction columns and solid phase cleanup columns in series, and carried out sample loading-washing-elution treatment to achieve the simultaneous determination of the three toxins. However, the sample processing process of this method consumes a lot of organic reagents, and the operation process is cumbersome and time-consuming. Therefore, it is urgent to establish an analytical method that is simple to operate, accurate and can detect BA, IBA and TF simultaneously. Summary of the invention

[0004] The object of the present invention is to provide a method for simultaneously detecting fumonisin, isorbamic acid and toxin, so as to solve the problems existing in the above-mentioned prior art.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention: a method for simultaneously detecting fumonisin, isorhizin and toxin, comprising the following steps:

[0007] The sample to be tested and the extract are mixed and extracted and then purified to obtain a solution of the sample to be tested;

[0008] The sample solution to be tested is detected by liquid chromatography-tandem mass spectrometry;

[0009] The extract is a mixed solution of acetonitrile and acetic acid.

[0010] Furthermore, the volume fraction of acetic acid in the extract is 1 to 3%.

[0011] Furthermore, the fillers used for the purification include ethylenediamine-N-propylsilane, C18 and anhydrous sodium sulfate.

[0012] Furthermore, the mass ratio of ethylenediamine-N-propylsilane, C18 and anhydrous sodium sulfate is (30-120):(40-80):(30-80).

[0013] Furthermore, the mass ratio of ethylenediamine-N-propylsilane, C18 and anhydrous sodium sulfate is 30:60:60, 80:40:40 or 120:60:30.

[0014] Furthermore, the method for preparing the test sample solution specifically includes: adding the extract to the test sample, mixing evenly, standing for 30 minutes, shaking for 30 minutes, and then centrifuging, taking the supernatant for purification and filtering through a membrane to obtain the test sample solution.

[0015] Furthermore, the sample to be tested is fresh wet powder.

[0016] Furthermore, the detection conditions of the liquid chromatography-tandem mass spectrometry are as follows:

[0017] Liquid chromatography analysis conditions:

[0018] Column: Hypersil GOLD C18, 2.1×100 mm, 1.9 μm;

[0019] Mobile phase: Phase A is water containing 0.1 vol.% formic acid, and phase B is acetonitrile;

[0020] Flow rate: 0.3 mL / min;

[0021] Injection volume: 4 μL;

[0022] Column temperature: 35 °C;

[0023] The gradient elution program is as follows: 0-2 min, mobile phase A 70-30 vol.%, mobile phase B 30-70 vol.%; 2-6 min, mobile phase A 30-5 vol.%, mobile phase B 70-95 vol.%; 6-8 min, mobile phase A 5 vol.%, mobile phase B 95 vol.%; 8-8.1 min, mobile phase A 5-70 vol.%, mobile phase B 95-30 vol.%; 8.1-10 min, mobile phase A 70 vol.%, mobile phase B 30 vol.%;

[0024] Mass spectrometry analysis conditions:

[0025] Ion source: electrospray ion source;

[0026] Air curtain air pressure: 35.0psi;

[0027] Collision gas pressure: 9.0 psi;

[0028] Ion source temperature: 550°C;

[0029] Scanning mode: positive and negative ion switching scanning;

[0030] Quantitative detection method: MRM mode.

[0031] The present invention discloses the following technical effects:

[0032] (1) The method of the present invention can realize the simultaneous detection of the contents of fumonisin, isorhizin and toxin, and has the advantages of simple detection method and high efficiency.

[0033] (2) The method provided by the present invention simplifies the sample pretreatment process, solves the problem of simultaneous extraction of fumonisin, isorhizomelic acid and toxin with large polarity differences, realizes simultaneous determination in one extraction, shortens the test time, and can simply and efficiently detect and confirm the content of fumonisin, isorhizomelic acid and toxin in fresh wet powder, and the determination results are accurate and reliable.

[0034] (3) The method of the present invention has high detection sensitivity and accuracy. The detection limits of fumonisin, isorhizin and toxin are 0.2 μg / kg, 0.2 μg / kg and 0.5 μg / kg, respectively, which are superior to existing detection technologies and provide technical support and guarantee for food safety risk monitoring and rapid screening and detection of food poisoning samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 The total ion current diagram of fumonisin, isofumonisin and toxin;

[0037] Figure 2 The effects of different extraction solvents on the recovery of fumonisin, isofumonisin and toxin;

[0038] Figure 3 The effects of solid phase extraction columns (C18, HLB, Al-N) and anion exchange column PAX on the recovery of fumonisin, isoflavone and toxin;

[0039] Figure 4 The effects of different purification columns on the recovery of fumonisin, isofumonisin and toxin;

[0040] Figure 5 The effects of different filler ratios on the recovery of fumonisin, isofumonisin and toxin in the homemade QuEChERS purification tubes;

[0041] Figure 6 This study was to investigate the changes in the contents of fumonisin, isofumonisin and toxin in fresh wet powder after inoculation with Burkholderia gladiolipv. Cocovenenan 83756 at different times. DETAILED DESCRIPTION

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0044] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0045] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0046] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0047] The instrument used in the present invention is as follows:

[0048] ACQUITY UPLC instrument, Triple-Quad 5500System-QTRAP Ready mass spectrometer, KS 4000i shaker, G-16 high-speed centrifuge, Milli-Q water purifier, QUINTIX313-1CN electronic balance.

[0049] The experimental materials used in the present invention are as follows:

[0050] 0.22 μm nylon filter, anhydrous sodium sulfate, C18 powder, PSA powder, commercial QuEChERS1 (Shenzhen Comma Biotechnology Co., Ltd.), commercial QuEChERS2 (Anpuyun Laboratory Supplies (Shanghai) Co., Ltd., 100 mg ethylenediamine-N-propylsilane + 40 mg C18 + 20 mg GCB), MFC 160, MFC 220 and MFC 228 purification columns (Qingdao Puruibang Biotechnology Co., Ltd.), anion exchange column PAX (Anpuyun Laboratory Supplies (Shanghai) Co., Ltd.), C18, AL-N, HLB and GCB solid phase columns (Shenzhen Comma Biotechnology Co., Ltd.). Burkholderia gladioli pv. Cocovenenan 83756 was provided by the Microbiology Laboratory of Food Testing Department, Shenzhen Institute of Metrology and Quality Inspection.

[0051] The experimental reagents used in the present invention are as follows:

[0052] Methanol, acetonitrile (all chromatographic grade), formic acid, acetic acid (all chromatographic grade, ≥98%), and other reagents were of analytical grade, and the experimental water was ultrapure water.

[0053] Standard substances: fumonisin, isofumonisin, and toxin (all 100 μg / mL).

[0054] Example 1

[0055] A method for simultaneously detecting fumonisin, isofumonisin and toxin:

[0056] (1) Preparation of standard solution:

[0057] Standard stock solution of fumonisin (20.0 μg / mL): Accurately transfer 1.0 mL of fumonisin standard to a 5.00 mL volumetric flask and dilute to the mark with methanol.

[0058] Isomycin standard stock solution (20.0 μg / mL): Accurately transfer 1.0 mL of isomycin standard to a 5.00 mL volumetric flask and dilute to the mark with acetonitrile / water (volume ratio 1:1, containing 0.1 vol.% ammonia water).

[0059] Toxoplasmin standard stock solution (20.0 μg / mL): Accurately transfer 1.0 mL of toxoplasmin standard into a 5.00 mL volumetric flask and dilute to the mark with methanol.

[0060] Mixed standard working solution 1 (1.00 μg / mL): Accurately pipette 0.050 mL of 20.0 μg / mL standard stock solutions of fumonisin, isofumonisin and toxin, and dilute to 1 mL with methanol.

[0061] Mixed standard working solution 2: Accurately pipette 0.050mL, 0.050mL and 0.125mL of the standard stock solutions of fumonisin, isofumonisin and toxin, respectively, and dilute to 1mL with methanol.

[0062] (2) Preparation of blank matrix solution:

[0063] Weigh 5 g (accurate to 0.1 g) of the fresh wet powder sample that does not contain fumonisin, isorbamic acid and toxin in a 50 mL plastic centrifuge tube, add 20 mL of acetonitrile solution containing 3 vol.% acetic acid (as extraction solvent), let stand for 30 min, shake for extraction for 30 min, centrifuge at 9000 r / min for 4 min, draw 5 mL of supernatant into a QuEChERS purification centrifuge tube, shake for 30 min, centrifuge at 9000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm organic filter membrane to obtain a blank matrix solution.

[0064] The fillers in the QuEChERS purification tube are ethylenediamine-N-propylsilane (PSA), C18 and anhydrous sodium sulfate, with contents of 30 mg, 60 mg and 60 mg respectively.

[0065] (3) Linear relationship, detection limit and quantification limit

[0066] Accurately measure an appropriate amount of mixed standard working solution 2, dilute it with blank matrix solution, and prepare a series of mixed standard curves with concentrations of 0.1, 0.5, 1.0, 5.0, 10.0, and 20.0 ng / mL for fumonisin and isorhizin, and concentrations of 0.25, 1.25, 2.50, 12.5, 25.0, and 50.0 ng / mL for toxin. Draw a standard curve with the concentration of the analyte as the horizontal axis and the peak area as the vertical axis.

[0067] The detection limit was the concentration corresponding to the signal-to-noise ratio S / N=3 for fumonisin, isomonisin and toxin in the matrix, and the quantification limit was the concentration corresponding to the S / N=10 for fumonisin, isomonisin and toxin in the matrix.

[0068] Chromatographic conditions:

[0069] Chromatographic column: Hypersil GOLD C18 column (100 mm × 2.1 mm, 1.9 μm, Thermo Scientific);

[0070] Mobile phase: A: water containing 0.1 vol.% formic acid, B: acetonitrile;

[0071] Flow rate: 0.3 mL / min;

[0072] Column temperature: 35 °C;

[0073] Injection volume: 4 μL;

[0074] The gradient elution program is shown in Table 1.

[0075] Table 1 Gradient elution program

[0076]

[0077] Mass spectrometry conditions:

[0078] Ion source: electrospray ion source;

[0079] Scanning mode: positive and negative ion switching scanning;

[0080] Quantitative detection method: MRM mode;

[0081] Air curtain air pressure: 35.0psi;

[0082] Collision gas pressure: 9.0 psi;

[0083] Ion source temperature: 550°C.

[0084] The ion selection parameters, retention time, and electrospray voltage of the target compounds are shown in Table 2; the total ion currents of fumonisin, isofumonisin, and toxin are shown in Figure 1 .

[0085] Table 2 Target ion selection parameters, retention time settings and electrospray voltage

[0086]

[0087] Wherein, * represents the quantitative ion.

[0088] Figure 1 The peaks in the sample are Toxoflavin, Bongkrekic and Isobongkrekic in the order of elution, with retention times of 0.93min, 3.17min and 3.40min, respectively. It can be seen that Bongkrekic and Isobongkrekic have good separation, and the purpose of simultaneous identification and analysis of the three substances can be achieved with one injection.

[0089] Table 3 Linear relationship, detection limit and quantification limit

[0090]

[0091] The results showed that good linear relationships were observed for fumonisin and isorbamic acid in the range of 0.1-20.0 ng / mL, and for toxin in the range of 0.25-50.0 ng / mL.

[0092] (4) Recovery rate and precision

[0093] Take several 5g (accurate to 0.1g) blank fresh wet powder samples, add low, medium and high levels of standard mixed solutions, of which fumonisin and isofumonisin are added at 0.40μg / kg, 2.00μg / kg and 4.00μg / kg, and toxin is added at 1.00μg / kg, 5.00μg / kg and 10.0μg / kg. Perform 3 levels and 6 parallel repeated determination experiments according to the same analytical steps to determine the recovery rate and intra-day relative standard deviation (RSD). The intra-day precision experiment is calculated by analyzing 3 parallel samples of 3 added levels for three consecutive days.

[0094] Spiked recovery rate = (spike sample measurement value - control sample measurement value) / theoretical spike amount × 100%.

[0095] Table 4 Spiked recovery and precision

[0096]

[0097] It can be seen from Table 4 that at the three spike levels, the recovery and precision of the method of the present invention are satisfactory.

[0098] (5) Sample processing:

[0099] Weigh 5 g (accurate to 0.1 g) of the crushed and mixed fresh wet powder sample into a 50 mL plastic centrifuge tube, add 20 mL of acetonitrile solution containing 3 vol.% acetic acid (as extraction solvent), let stand for 30 min, shake for extraction for 30 min, centrifuge at 9000 r / min for 4 min, draw 5 mL of supernatant into a QuEChERS purification centrifuge tube, shake for 30 min, centrifuge at 9000 r / min for 5 min, take the supernatant and filter it through a 0.22 μm organic filter membrane to obtain the sample solution to be tested.

[0100] The fillers in the QuEChERS purification tube are ethylenediamine-N-propylsilane (PSA), C18 and anhydrous sodium sulfate, with contents of 30 mg, 60 mg and 60 mg respectively.

[0101] Liquid chromatography-tandem mass spectrometry was used to detect the sample solution.

[0102] (6) Result calculation

[0103] The contents of fumonisin, isofumonisin and toxin in the samples were calculated based on the drawn standard curve and chromatographic detection results.

[0104] Example 2

[0105] The same as Example 1, except that the extraction solvents in step (2) are respectively methanol, acetonitrile, 50% methanol (methanol solution with a volume fraction of 50% methanol), 50% acetonitrile (acetonitrile solution with a volume fraction of 50%), ammonia methanol (1.0 mL ammonia, 80 mL methanol, add water to 100 mL), 0.1% formic acid water (formic acid solution with a volume fraction of 0.1%) + acetonitrile (15 + 85, v: v), 0.1% ammonia solution (ammonia solution with a volume fraction of 0.1%) + acetonitrile (15 + 85, v: v), 3% acetic acid acetonitrile (i.e. acetonitrile solution containing 3 vol.% acetic acid), 3% acetic acid methanol (acetic acid methanol solution with a volume fraction of 3%). The blank matrix solution obtained by extraction with the same extraction solvent is diluted with the mixed standard working solution to prepare a standard curve.

[0106] The recovery rates of fumonisin, isofumonisin and toxin (added level was 20.0 μg / kg) were determined and calculated. The results are shown in Figure 2 .

[0107] from Figure 2It can be seen that when 3% acetic acid and acetonitrile are used as the extraction solvent, the recovery rates of the three toxins (BA, IBA and TF) are the best, which are 99.7%, 105% and 92.7% respectively. Compared with pure acetonitrile and 50% acetonitrile, the extraction effects of the three toxins are improved when 0.1% formic acid water + acetonitrile is used as the extraction solution. The extraction ability of 0.1% ammonia solution + acetonitrile for weakly alkaline toxin is significantly higher than that of 0.1% formic acid water + acetonitrile, but the extraction effect for weakly acidic fumonisin and isofumonisin is poor. Methanol is a protonic solvent that can form hydrogen bonds and improve the separation of acid-base electronegative compounds. Among the extraction solvents containing methanol, acidified methanol has the best comprehensive recovery effect.

[0108] Example 3

[0109] The same as Example 1, except that the QuEChERS purification centrifuge tube for preparing the blank matrix solution is replaced with a solid phase extraction column and an anion exchange column (C18, HLB, Al-N, PAX) pre-activated with methanol and water.

[0110] The recovery rates of fumonisin, isofumonisin and toxin (added level was 20.0 μg / kg) were determined. The results are shown in Figure 3 .

[0111] from Figure 3 It can be seen that the recovery of cypermethrin by the four solid phase extraction columns is not ideal. The reason may be that the molecular weight of cypermethrin is small, and it is not retained on the solid phase extraction column and cannot be effectively enriched and purified. Among them, the recovery rates of C18, HLB and PAX for fumonisin and isomonisin are acceptable. Al-N only has a small recovery of cypermethrin, because the aluminum oxide in Al-N can remove lipophilic compounds, and cypermethrin and isomonisin are just lipophilic substances. In summary, under the experimental conditions of the present invention, the above four solid phase extraction columns cannot achieve simultaneous purification and enrichment of the three toxins.

[0112] Example 4

[0113] The same as Example 1, except that the purification columns used in preparing the blank matrix solution were GCB, MFC 160, MFC 220, MFC 228, commercial QuEChERS 1, commercial QuEChERS 2 and homemade QuEChERS purification tube.

[0114] The filling materials in the homemade QuEChERS purification tube are ethylenediamine-N-propylsilane (PSA), C18 and anhydrous sodium sulfate, with contents of 60 mg, 80 mg and 80 mg respectively. 。

[0115] The recovery rates of fumonisin, isofumonisin and toxin (added level was 20.0 μg / kg) were determined. The results are shown in Figure 4 .

[0116] from Figure 4 It can be seen that the homemade QuEChERS purification tube containing 60mg PSA, 80mg C18 and 80mg anhydrous sodium sulfate has the best recovery effect on the three toxins. The reason is that PSA removes fatty acids, carbohydrates, lipids and some pigments through hydrogen bonding, and C18 has an adsorption effect on non-polar components and can remove some lipid components, thus effectively removing the interference of coexisting impurities and improving the extraction efficiency of target compounds. Secondly, the purification columns MFC 160, MFC 220 and MFC 228 have poor enrichment and purification performance for the three toxins, while GCB has a strong adsorption effect on the three toxins but almost no recovery.

[0117] Example 5

[0118] The same as Example 1, the only difference is that the purification column used in preparing the blank matrix solution is a self-made QuEChERS purification tube with different filler ratios.

[0119] The fillers of QuEChERS (60+80+0) are ethylenediamine-N-propylsilane (PSA) and C18, with contents of 60 mg and 80 mg, respectively;

[0120] The fillers of QuEChERS) (30+60+60) are ethylenediamine-N-propylsilane (PSA), C18 and anhydrous sodium sulfate, with contents of 30 mg, 60 mg and 60 mg respectively.

[0121] The fillers of QuEChERS (40+40+60) are ethylenediamine-N-propylsilane (PSA), C18 and anhydrous sodium sulfate, with contents of 40 mg, 40 mg and 60 mg respectively.

[0122] The fillers of QuEChERS (60+80+80) are ethylenediamine-N-propylsilane (PSA), C18 and anhydrous sodium sulfate, with contents of 60 mg, 80 mg and 80 mg respectively.

[0123] The fillers of QuEChERS (80+40+40) are ethylenediamine-N-propylsilane (PSA), C18 and anhydrous sodium sulfate, with contents of 80 mg, 40 mg and 40 mg respectively.

[0124] The fillers of QuEChERS (120+60+30) are ethylenediamine-N-propylsilane (PSA), C18 and anhydrous sodium sulfate, with contents of 120 mg, 60 mg and 30 mg respectively.

[0125] The recovery rates of fumonisin, isofumonisin and toxin (added level was 20.0 μg / kg) were determined. The results are shown in Figure 5 .

[0126] from Figure 5 It can be seen that among the 6 homemade QuEChERS purification tubes with different proportions, the homemade QuEChERS purification tube containing 30mg PSA, 60mg C18 and 60mg anhydrous sodium sulfate has the best recovery effect on the three toxins (TF, BA and IBA), which are 85.0%, 106% and 88.8% respectively. The homemade QuEChERS purification tube containing 40mg PSA, 40mg C18 and 60mg anhydrous sodium sulfate has a good enrichment and purification effect on fumonisin and isofumonisin, but the recovery rate of toxin is only 68.9%. In summary, the homemade QuEChERS purification tube (filler is ethylenediamine-N-propylsilane (PSA), C18 and anhydrous sodium sulfate, the content is 30mg, 60mg and 60mg respectively) has the best simultaneous enrichment and purification effect on the three toxins.

[0127] Example 6

[0128] According to the detection process of Example 1, the changes in the content of the three toxins in the fresh wet powder culture medium sample inoculated with Burkholderia gladiolipv. Cocovenenan83756 at different culture times (0h, 4h, 8h, 12h, 16h, 20h, 24h, 36h, 48h, 60h and 72h) were measured. The results are shown in Figure 6 .

[0129] from Figure 6 It can be seen that after 16 hours of culture, fumonisin, isofumonisin and cypermethrin began to be detected. With the increase of culture time, the content of fumonisin and isofumonisin also increased and reached the maximum value at 60 hours of culture, which was 28.9 mg / kg and 2.75 mg / kg respectively, and then the content of the two showed a downward trend. The content of cypermethrin showed a continuous increase with the increase of culture time, and its content was as high as 133 mg / kg at 72 hours of culture.

[0130] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for simultaneously detecting fumonisin, isorhizin and fumonisin, characterized in that: The following steps are involved: The sample to be tested and the extract are mixed and extracted and then purified to obtain a solution of the sample to be tested; The sample solution to be tested is detected by liquid chromatography-tandem mass spectrometry; The extract is a mixed solution of acetonitrile and acetic acid.

2. The method according to claim 1, characterized in that The volume fraction of acetic acid in the extract is 1-3%.

3. The method according to claim 1, characterized in that The fillers used for the purification include ethylenediamine-N-propylsilane, C18 and anhydrous sodium sulfate.

4. The method according to claim 3, characterized in that The mass ratio of the ethylenediamine-N-propylsilane, C18 and anhydrous sodium sulfate is (30-120):(40-80):(30-80).

5. The method according to claim 1, characterized in that The method for preparing the test sample solution specifically comprises: adding the extract to the test sample, mixing evenly, standing and shaking, and then centrifuging, taking the supernatant for purification and membrane filtering to obtain the test sample solution.

6. The method according to claim 1, characterized in that The sample to be tested is fresh wet powder.

7. The method according to claim 1, characterized in that The detection conditions of the liquid chromatography-tandem mass spectrometry are as follows: Liquid chromatography analysis conditions: Column: Hypersil GOLD C18, 2.1×100 mm, 1.9 μm; Mobile phase: Phase A is water containing 0.1 vol.% formic acid, and phase B is acetonitrile; Flow rate: 0.3 mL / min; Injection volume: 4 μL; Column temperature: 35°C; The gradient elution program is as follows: 0-2 min, mobile phase A 70-30 vol.%, mobile phase B 30-70 vol.%; 2-6 min, mobile phase A 30-5 vol.%, mobile phase B 70-95 vol.%; 6-8 min, mobile phase A 5 vol.%, mobile phase B 95 vol.%; 8-8.1 min, mobile phase A 5-70 vol.%, mobile phase B 95-30 vol.%; 8.1~10min, mobile phase A 70vol.%, mobile phase B 30vol.%; Mass spectrometry analysis conditions: Ion source: electrospray ion source; Air curtain air pressure: 35.0psi; Collision gas pressure: 9.0 psi; Ion source temperature: 550°C; Scanning mode: positive and negative ion switching scanning; Quantitative detection method: MRM mode.

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