A method for simultaneously determining fumonisin, isorhizin and toxin in food

Abstract

The invention discloses a method for simultaneously determining fumonisin, isorhizomenic acid and toxin in food, wherein an extract is used to extract a sample to obtain an extract; the extract is first passed through an Oasis PRiME HLB 6cc solid phase extraction column, and after the filtrate is collected, the filtrate is passed through a Pribalab MFC336 solid phase purification column, and the filtrate is collected to obtain a purified solution containing toxin; the Oasis PRiME HLB 6cc solid phase extraction column is washed and impurities are removed, and then eluted to collect the eluate containing fumonisin and isorhizomenic acid, and the collected eluate is combined with the purified solution containing toxin; the obtained product is detected by liquid chromatography-tandem mass spectrometry to determine the content of the three toxins. The method has high detection sensitivity, and the detection limits of fumonisin, isorhizomenic acid and toxin are 0.014, 0.016 and 0.006 μg / L respectively; the accuracy is high, which is better than the existing detection technology.

Description

Technical Field

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

[0002] Pseudomonas cocovenenans subsp. farinofermentans (Pseudomonas cocovenenans for short) is a common foodborne pathogen in the environment. Cereals and potato products, fermented rice and flour products, spoiled white fungus, black fungus and other foods are easily infected with this pathogen. People who accidentally eat food contaminated by Pseudomonas cocovenenans will suffer from food poisoning. The symptoms are mild, such as nausea and vomiting, abdominal pain and bloating, dizziness, and general weakness. In severe cases, jaundice, ascites, subcutaneous hemorrhage, convulsions, hematuria and even liver failure and kidney failure may occur. There is no specific antidote, and severe cases can lead to death. Pseudomonas cocovenenans produces a variety of toxins, among which fumonisin is the one that attracts the most attention. Its median lethal dose (LD50) for intravenous injection in mice is 2.5. 50 ) is 1.14 mg / kg. Pseudomonas cocovenenans produces both fumonisin and isomers of fumonisin, which are isomers of fumonisin and have a toxicity of about 25-50% of that of fumonisin. Fumonisin will be converted into isomers of fumonisin under light conditions. Toxin is also a toxin produced by Pseudomonas cocovenenans, and its LD 50 It is 1.7mg / kg. These toxins are extremely toxic. Fumaric acid inhibits the activity of adenine nucleotide transferase on mitochondria, leading to cell apoptosis and damage; toxin enters the cell, receives hydrogen from the reducing coenzyme and then transfers oxygen molecules, producing a large amount of hydrogen peroxide and showing toxicity. If a poisoning incident of Pseudomonas cocovenenans occurs, the treatment plans will be very different due to the different pathogenic mechanisms of fumaric acid and toxin. If you only focus on fumaric acid and ignore the existence of toxin, it may lead to unpredictable errors. In order to thoroughly understand the culprit of Pseudomonas cocovenenans poisoning, it is not enough to just detect fumaric acid (including isoflavone acid). It is also extremely important to confirm the detection of toxin.

[0003] At present, the detection methods for fumonisin include liquid chromatography and liquid chromatography-tandem mass spectrometry. The first and second methods in the national standard method GB 5009.189-2023 use high performance liquid chromatography and liquid chromatography-tandem mass spectrometry, respectively, with method detection limits of 5μg / kg and 1μg / kg, respectively. However, there is no national standard detection method for toxin and isoflavone. Zhang Wei et al. from Henan Provincial Center for Disease Control and Prevention (Ultra-high performance liquid chromatography-triple quadrupole / composite linear ion trap tandem mass spectrometry for rapid detection of fumonisin in food poisoning samples. Chinese Journal of Health Inspection, 2021, 31(15): 1811-1817; Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry for rapid detection of fumonisin and fumonisin in fermented rice noodles. Chinese Journal of Health Inspection, 2022, 32(3): 284-287.) Bond Elut Plexa PCX solid phase extraction method and QuEChERS method with C18, PSA powder and anhydrous sodium sulfate as purifiers combined with high performance liquid chromatography-tandem mass spectrometry were used to detect fumonisin and fumonisin in food poisoning samples and fermented rice noodles. In addition, a method for determining fumonisin and toxin in straw fermented feed by liquid chromatography tandem quadrupole linear ion trap mass spectrometry (LC-MS / MS-QTRAP) has also been reported (Suo Decheng, Xiao Zhiming, Zhang Jing, et al., Determination of fumonisin and toxin in straw fermented feed by liquid chromatography tandem quadrupole linear ion trap mass spectrometry. China Feed, 2022, 19: 93-98.), the detection limits of fumonisin and toxin in this method are 0.7 μg / kg and 1.5 μg / kg, respectively. In terms of patents, Huizhou Food and Drug Inspection Institute has published two patents for the simultaneous detection of fumonisin and toxin (China authorized patent CN 112505223 B, application for patent CN 115616116 A), but these two patents only changed the instrumental determination method from high-performance liquid chromatography-ultraviolet detection to liquid chromatography-tandem mass spectrometry, and there was no significant improvement or innovation in the sample pretreatment. Both patents used commercialized dSPE EMR-Lipid and dSPE EMR-Lipid+NaCl / MgSO 4As a purifier to implement the QuEChERS pretreatment method. It should be noted that there are contradictions between the patent and the literature. The patent specification states that the QuEChERS method using C18 and PSA powder will adsorb the target compounds, thereby reducing the recovery rate of fumonisin and toxin, and the dSPE EMR-Lipid adsorbent is selected, while the literature uses C18 and PSA powder as purifiers. The dSPE EMR-Lipid adsorbent is mainly used to remove lipids, while the lipid content in Tremella fuciformis, black fungus, cornmeal and wet rice flour is very low, and carbohydrates are their main components. Tremella fuciformis contains rich colloid and cellulose, and cornmeal wet rice flour contains a lot of starch. The effect of using only adsorbents for removing lipids to purify samples is questionable.

[0004] The chemical structures of fumonisin and fumonisin are quite different, and their physical and chemical properties are quite different. Fumonisin is a long-chain fatty acid structure with weak acidity. It is easily soluble in organic solvents such as petroleum ether, n-hexane, ether, chloroform, methanol, and alkaline aqueous solutions, and is unstable to acidic conditions, oxidants, and sunlight. Fumonisin is an alkaline compound containing multiple nitrogen atoms, which is soluble in water, methanol, acetonitrile, etc., and insoluble in non-polar solvents such as petroleum ether. If exactly the same extraction and purification methods are used, it may be difficult to take into account the extraction efficiency and recovery rate of both, making the sample measurement inaccurate. Summary of the invention

[0005] Aiming at the defects of lack of detection method, low method sensitivity and low detection accuracy of toxins produced by Pseudomonas cocovenenans, the present invention proposes a method for simultaneously detecting bongkrekic acid, isobongkrekic acid and toxoflavin in food.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for simultaneously determining fumonisin, isorhizin and toxin in food, characterized in that it comprises the following steps:

[0008] (1) Sample extraction: weigh the sample and extract the sample using an extraction solution to obtain an extraction solution;

[0009] (2) Sample purification: The extract obtained in step (1) is first passed through an Oasis PRiME HLB 6cc solid phase extraction column, and the filtrate is collected. The filtrate is then passed through a PriboFast MFC 336 solid phase purification column, and the filtrate is collected to obtain a purified solution containing cypermethrin;

[0010] Washing and removing impurities from the Oasis PRiME HLB 6cc solid phase extraction column, then eluting, collecting the eluate containing fumonisin and isoflavone, and combining the eluate containing fumonisin and isoflavone with the purified solution containing fumonisin;

[0011] (3) Sample determination: The product obtained in step (2) is detected by liquid chromatography-tandem mass spectrometry to determine the contents of fumonisin, isorhizin and fumonisin.

[0012] In some embodiments of the present invention, in step (1), the extracting solution is a methanol-water solution, and the volume ratio of methanol to water is 25:8.

[0013] In some embodiments of the present invention, in step (2), the washing liquid used for washing and removing impurities from the Oasis PRiME HLB 6cc solid phase extraction column is a methanol-water solution, and the volume ratio of methanol to water is 1:1; the eluent used for elution is methanol.

[0014] In some embodiments of the present invention, in step (3), the product obtained in step (2) is first blown dry, and the residue is redissolved with a re-solution and then tested on a machine, wherein the re-solution is a methanol-water solution, and the volume ratio of methanol to water is 1:9.

[0015] In some embodiments of the present invention, in step (3), the chromatographic column used in the liquid chromatography is ACQUITY UPLC BEH C18,

[0016] Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile.

[0017] The injection volume was 5 μL, the column temperature was 40°C, and the gradient elution program was:

[0018]

[0019]

[0020] In some embodiments of the present invention, in step (3), the mass spectrometry scanning mode is the Scheduled MRM mode, wherein the MRM detection window is 180s, the retention times of fumonisin, isofumonisin and toxin are set to 5.69min, 5.86min and 1.19min, respectively, and the scanning modes are negative ion scanning and positive ion scanning, respectively.

[0021] In some embodiments of the present invention, in the mass spectrometry conditions, ion source: electrospray ion source; scanning mode: positive and negative ion switching scanning; nebulizer gas pressure: 50.0psi; auxiliary gas pressure: 50.0psi; curtain gas pressure: 40.0psi; collision gas pressure: 6.0psi; ion source temperature: 550°C; scanning time: 50ms; collision chamber outlet voltage: 11.0V; collision chamber inlet voltage: 10.0V.

[0022] In some embodiments of the present invention, the ion selection parameters, retention time, and electrospray voltage of the target are:

[0023]

[0024] Specifically, the present invention adopts an external standard method to perform the quantitative analysis.

[0025] The calculation formula for the content of fumonisin (isofumonisin) in the sample is: X 米 =C 米 ×15 / 6, where the unit of X meter is μg / kg and the unit of C meter is ng / mL;

[0026] The formula for calculating the content of cypermethrin in the sample is: X 黄 =C 黄 ×15 / 3, where X 黄 The unit of is μg / kg, and the unit of C yellow is ng / mL.

[0027] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0028] The present invention adopts liquid chromatography-tandem mass spectrometry and selects a positive and negative ion switching scanning mode to propose a method for simultaneously detecting fumonisin, isofumonisin and toxin in food. The method can simultaneously screen and confirm fumonisin and toxin in poisoned samples, find out the culprit of food poisoning, and enable poisoned patients to receive correct treatment at an early stage. In addition, the method innovatively selects and optimizes the extraction and purification methods based on the molecular structure and physicochemical properties of fumonisin and toxin, and truly realizes the simultaneous determination of fumonisin and toxin in food, and the determination results are accurate and reliable. The method has high detection sensitivity, and the detection limits of fumonisin, isofumonisin and toxin are 0.014, 0.016 and 0.006 μg / L respectively; the method has high accuracy, which is superior to the existing detection technology, and provides reliable technical guarantee for food poisoning detection and the analysis and determination of extremely trace fumonisin, isofumonisin and toxin in easily contaminated food. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a flow chart for the simultaneous detection of fumonisin (isofumonisin) and toxin in food.

[0030] Figure 2 This is the total ion current diagram of fumonisin, isofumonisin and toxin.

[0031] Figure 3 The effect of different extraction solutions on the recovery rates of fumonisin, isofumonisin and toxin.

[0032] Figure 4 This is a comparison chart of the column changes before and after using the Oasis PRiME HLB 6cc solid phase extraction cartridge and the PriboFast MFC 336 cleanup column.

[0033] Figure 5 For use with HLB, MAX, MCX, Al 2 O 3 -NH 2 、Carb-NH 2 The recovery of fumonisin and toxin after the small column combined with PriboFast MFC 336 cleanup column.

[0034] Figure 6 This is a chromatogram-mass spectrum of a black fungus sample. DETAILED DESCRIPTION

[0035] In the following examples or comparative examples, the instruments, materials and reagents used are described as follows:

[0036] Experimental instruments: High performance liquid chromatography tandem mass spectrometer 6500+ (AB Company, USA), Multi reax universal oscillator (Heidolph Company, Germany), 3-30K centrifuge (Sigma Company, Germany)

[0037] Experimental materials: Oasis PRiME HLB 6cc solid phase extraction cartridge (Waters, USA), PriboFastMFC 336 purification cartridge (Qingdao Pribo Biotechnology Co., Ltd.)

[0038] Experimental reagents: methanol (chromatographic grade, Merck, Germany). Experimental water: Millipore ultrapure water.

[0039] Standard substances: fumonisin (Tanmo Quality Inspection, purity 98.5%, uncertainty 0.5%, valid until May 2024), isofumonisin (Pribolab, 100μg / mL, uncertainty 6.2%, valid until May 22, 2025), and toxin (Pribolab, purity 99%, uncertainty 1%, valid until August 2024).

[0040] Example 1: Detection process and result calculation

[0041] The flow chart of simultaneous detection of fumonisin, isorhizin and toxin in food of the present invention is as follows Figure 1 As shown, the specific steps are as follows:

[0042] 1.1 Sample processing

[0043] Weigh 1 g (accurate to 0.01 g) of the crushed and mixed sample (black fungus, white fungus, rice flour) into a 50 mL plastic centrifuge tube, add 10 mL of a mixed solution of methanol: water = 25:8, vortex extract for 40 min, centrifuge at 9500 rpm for 5 min, and draw the supernatant into a 15 mL plastic centrifuge tube. Add 5 mL of a mixed solution of methanol: water = 25:8, ultrasonic extract for 10 min, centrifuge at 9500 rpm for 3 min, combine the two extracts, and wait for purification. Take 6 mL of the extract and directly pass it through the PRiME HLB (6 cc) small column (no activation required), collect the filtrate in the plastic tube supporting the PriboFast MFC 336 purification column, with the tube mouth facing upwards, and slowly push the PriboFast MFC 336 purification column into the plastic tube, and the purified filtrate is collected above the purification column for standby use. The PRiME HLB (6cc) column was washed with 1:1 methanol-water solution (3 mL), the washing liquid was discarded, and then eluted with 5 mL of methanol, and the eluate was collected. 3 mL of the purification solution was combined with the eluate, and the mixture was placed in a 40°C water bath and blown under nitrogen until nearly dry. The residue was re-dissolved with 1:9 methanol-water solution (1 mL), centrifuged or filtered, and then subjected to liquid chromatography-tandem mass spectrometry.

[0044] 1.2 Chromatographic conditions

[0045] Chromatographic column: ACQUITY UPLC BEH C18 (1.7 μm × 100 mm × 2.1 mm);

[0046] Mobile phase: A water (containing 0.1% formic acid); B acetonitrile; injection volume: 5.0 μL; column temperature: 40°C;

[0047] The gradient elution program is shown in Table 1.

[0048] Table 1

[0049]

[0050] 1.3 Mass spectrometry conditions

[0051] Ion source: electrospray ion source; scanning mode: positive and negative ion switching scanning; quantitative detection mode: Scheduled MRM mode, MRM detection window in Scheduled MRM mode: 180s; nebulizer gas pressure: 50.0psi; auxiliary gas pressure: 50.0psi; air curtain gas pressure: 40.0psi; collision gas pressure: 6.0psi; ion source temperature: 550℃; scanning time: 50ms; collision chamber exit voltage: 11.0V; collision chamber entrance voltage: 10.0V; ion selection parameters, retention time, and electrospray voltage of the target are shown in Table 2. The total ion current of fumonisin, isofumonisin, and cypermethrin is shown in Table 2. Figure 2 shown. Figure 2 The peaks in the sample are fumonisin, fumonisin and isofumonisin in the order of elution, with retention times of 1.19 min, 5.69 min and 5.86 min, respectively. It can be seen that the separation degree of fumonisin and isofumonisin is good, achieving the purpose of simultaneous identification and analysis of the three substances with one injection.

[0052] Table 2 Target ion selection parameters, retention time settings and electrospray voltage

[0053]

[0054] 1.4 Matrix-matched standard curve

[0055] Take a blank sample that does not contain fumonisin, isofumonisin and toxin, prepare a blank matrix solution according to the sample treatment method in Example 1, accurately measure an appropriate amount of fumonisin, isofumonisin and toxin standard working solution, dilute with the blank matrix solution, prepare matrix-matched standard solutions with concentrations of 0.5, 1, 2, 5, 10, 20, and 50 μg / L of fumonisin, and prepare them before use.

[0056] 1.5 Result calculation

[0057] This method uses matrix-matched standard curve external standard method for quantification.

[0058] The calculation formula for the content of fumonisin (isofumonisin) in the sample is: X 米 =C 米 ×15 / 6, where X 米 Indicates the content of fumonisin (isofumonisin) in the sample, in μg / kg, C 米 It represents the concentration of fumonisin in the injection solution obtained by linear regression of the matrix-matched standard curve, in ng / mL;

[0059] The formula for calculating the content of cypermethrin in the sample is: X 黄 =C 黄 ×15 / 3, where X黄 It indicates the content of cypermethrin in the sample, in μg / kg. C yellow indicates the concentration of cypermethrin in the injection solution obtained by linear regression of the matrix matching standard curve, in ng / mL.

[0060] Example 2: Method spike recovery and precision

[0061] Take 1 portion of each sample (tremella, black fungus, wet rice flour), grind it evenly and divide it into 4 portions (each portion has a mass of 1g), keep one portion as a control, and add standard mixed solutions of low, medium and high contents (0.5, 2, 10ng / mL) to the other 3 portions. The spiked and control samples are implemented and the results are calculated according to the sample detection method in Example 1. The sample spike recovery rate is equal to the ratio of the amount obtained after the spiked sample is detected to the amount added. The precision of the method is to implement 6 parallel experiments for each spiked level, and calculate the relative standard deviation (RSD) of the six measurement results.

[0062] Table 3 Method spike recovery and precision

[0063]

[0064] As can be seen from Table 3, the recoveries of the three substances in the spiked samples with different levels of addition ranged from 83.1 to 95.9%, indicating that the recoveries were good and the precisions at different addition levels for different types of samples were also satisfactory.

[0065] Example 3: Linear Relationship, Detection Limit and Quantification Limit

[0066] According to the matrix matching standard curve: take a blank sample without the analyte, prepare a blank matrix solution according to the sample treatment method in Example 1, accurately measure an appropriate amount of the mixed standard solution, dilute it with the blank matrix solution, and prepare a series of 0.5, 1, 2, 5, 10, 20, 50 μg / L concentrations of fumonisin, isorhizomenic acid and toxin. Draw a standard curve with the concentration of the analyte as the horizontal coordinate and the peak area as the vertical coordinate. The detection limit is the concentration corresponding to the signal-to-noise ratio (S / N) of fumonisin (isofumonisin) and toxin in the matrix = 3, and the quantitative limit is the concentration corresponding to the S / N of fumonisin (isofumonisin) and toxin in the matrix = 10. Linear relationship, detection limit and quantitative limit are shown in Table 4.

[0067] Table 4 Linear relationship, detection limit and quantification limit

[0068]

[0069] Example 4

[0070] The detection process of this embodiment is basically the same as that of embodiment 1, except that Tremella fuciformis is used as the sample, and mixed solutions of methanol: water (volume ratio) of 25:2, 25:4, 25:8, 25:16, and 1:1 are used as the extract in the sample pretreatment, and the recovery rates of fumonisin and cypermethrin are investigated. Figure 3 .Depend on Figure 3 It can be seen that when the methanol: water (volume ratio) of the extract is 25:8, the recovery rates of both are optimal and satisfactory. When the methanol: water ratio is higher than 25:8, the retention of fumonisin on PRiME HLB becomes poor, and it is not completely adsorbed on the PRiME HLB column, resulting in the loss of fumonisin and a decrease in the recovery rate. Increasing the specific gravity of water in the extract will make the dry sample swell more, and the volume of the obtained sample extract will decrease, resulting in lower recovery of fumonisin and cypermethrin. Increasing the proportion of water in the extract will cause more protein and starch impurities to be extracted, making the resistance of the sample through the PRiME HLB column and PriboFast MFC 336 purification column larger, increasing the difficulty of operation.

[0071] Example 5

[0072] The detection process of this embodiment is basically the same as that of Example 1, except that black fungus is used as the sample, and the extract is not further passed through the PriboFast MFC 336 purification column after purification by the PRiME HLB column. The effect of column adsorption of impurities and the changes in the recovery rates of fumonisin and fumonisin after purification by only using the PRiME HLB column and the combined use of the PRiME HLB column and the PriboFast MFC 336 purification column are compared. Figure 4 It shows that after the sample is purified by the PriboFast MFC 336 purification column, the filler on the column changes from white to yellow-brown, indicating that the filler has adsorbed a large amount of impurities. In the actual operation process, it is also found that the solution purified by PriboFast MFC 336 is clear and transparent, and it is easy to blow dry in the subsequent nitrogen blowing process. There is no precipitate in the solution after re-dissolution, and it will not cause adverse effects on the instrument. The recovery rate results show that the recovery rate of fumonisin after the combined use of PRiME HLB column and PriboFast MFC 336 purification column is greater than 80%, and it remains basically stable. However, the spiked recovery rate of toxin without PriboFast MFC 336 purification is only about 50%, which is lower than the recovery rate of the detection method described in Example 2.

[0073] Example 6

[0074] The detection process of this embodiment is basically the same as that of embodiment 1, except that the first solid phase extraction column used is different. In this embodiment, the spiked extract (a 25:8 methanol-water mixed solution, with the concentration of fumonisin and toxin being 5 μg / L) is firstly passed through a solid phase extraction column (HLB, MAX, MCX, Al 2 O 3 -NH 2 、Carb-NH 2 ) and then purified by PriboFast MFC 336 column to investigate the recovery of fumonisin and cypermethrin. Figure 5 .Depend on Figure 5 It can be seen that the recovery rate of fumonisin and cypermethrin by HLB column is acceptable, but the effect of HLB column is slightly worse than that of PRiME HLB column, and HLB column needs to be pre-activated with methanol and water before use. For MAX and MCX columns, the recovery of fumonisin is not ideal, because fumonisin is retained on MAX column and not eluted, while MCX column has poor retention and causes sample loss; cypermethrin is not retained on MAX and MCX columns and flows directly into PriboFast MFC 336 purification column, so the recovery rate is relatively good. For Al 2 O 3 -NH 2 and Carb-NH 2 The two were well retained in the small column, but were not eluted under the experimental conditions of this embodiment and remained on the small column, resulting in a very low recovery rate and inaccurate test results.

[0075] Example 7: Practical application of the method

[0076] According to the detection process of Example 1, 30 portions of black fungus, 30 portions of white fungus and 30 portions of rice noodles were tested and analyzed. It was found that both fumonisin and isorbamic acid were detected in one portion of black fungus, and the test results were 0.051 mg / kg and 0.15 mg / kg respectively. Figure 6 ), and cypermethrin was not detected.

Claims

1. A method for simultaneously determining fumonisin, isorhizin and fumonisin in food, characterized in that: The following steps are involved: (1) Sample extraction: weigh the sample and extract the sample using an extraction solution to obtain an extraction solution; (2) Sample purification: The extract obtained in step (1) is first passed through an Oasis PRiME HLB 6cc solid phase extraction column, and the filtrate is collected. The filtrate is then passed through a PriboFast MFC 336 solid phase purification column, and the filtrate is collected to obtain a purified solution containing cypermethrin; The extract is a methanol-water solution, and the volume ratio of methanol to water is 25:8; Washing and removing impurities from the Oasis PRiME HLB 6cc solid phase extraction column, then eluting, collecting the eluate containing fumonisin and isoflavone, and combining the eluate containing fumonisin and isoflavone with the purified solution containing fumonisin; The washing solution used for washing and removing impurities from the Oasis PRiME HLB 6cc solid phase extraction column was a methanol-water solution, with a volume ratio of methanol to water of 1:1; the eluent used for elution was methanol; (3) Sample determination: The product obtained in step (2) is detected by liquid chromatography-tandem mass spectrometry to determine the contents of fumonisin, isorhizin and fumonisin.

2. The method for simultaneously determining fumonisin, isorhizin and fumonisin in food according to claim 1, characterized in that: In step (3), the product obtained in step (2) is first blown dry, and the residue obtained is redissolved with a re-solution and then tested on a machine. The re-solution is a methanol-water solution, and the volume ratio of methanol to water is 1:

9.

3. The method for simultaneously determining fumonisin, isorhizin and fumonisin in food according to claim 1, characterized in that: In step (3), the chromatographic column used in the liquid chromatography is ACQUITY UPLC BEH C18, Mobile phase A was water containing 0.1% formic acid, and mobile phase B was acetonitrile. The injection volume was 5 μL, the column temperature was 40°C, and the gradient elution program was:

4. The method for simultaneously determining fumonisin, isorhizin and toxin in food according to claim 1, characterized in that: In step (3), the mass spectrometry scanning mode is the Scheduled MRM mode, wherein the MRM detection window is 180s, the retention times of fumonisin, isofumonisin and toxin are set to 5.69min, 5.86min and 1.19min, respectively, and the scanning modes are negative ion scanning and positive ion scanning, respectively.

5. The method for simultaneously determining fumonisin, isorhizin and fumonisin in food according to claim 4, characterized in that: In the mass spectrometry conditions, ion source: electrospray ion source; scanning mode: positive and negative ion switching scanning; nebulizer gas pressure: 50.0psi; auxiliary gas pressure: 50.0psi; curtain gas pressure: 40.0psi; Collision gas pressure: 6.0 psi; ion source temperature: 550°C; scan time: 50 ms; collision chamber exit voltage: 11.0 V; collision chamber entrance voltage: 10.0 V.

6. The method for simultaneously determining fumonisin, isorhizin and toxin in food according to claim 5, characterized in that: The ion selection parameters, retention time, and electrospray voltage of the target are:

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