A method for determining 14 kinds of mycotoxins in peanuts by high performance liquid chromatography tandem mass spectrometry

Abstract

The application discloses a method for determining 14 kinds of mycotoxins in peanuts based on high performance liquid chromatography tandem mass spectrometry, and belongs to the technical field of food detection. The method comprises the steps of solution preparation, test sample solution preparation and high performance liquid chromatography tandem mass spectrometry detection. An acetonitrile-water-acetic acid mixed solution is used as an extraction solution, the peanut sample is ground and crushed, low-temperature ultrasonic extraction, centrifugation, concentration, redissolution and filter membrane filtration are performed, a GL Sciences InertSustain AQ C18 chromatographic column is used for separation, and a triple quadrupole mass spectrometer is used for detection in a multiple reaction monitoring mode. The application realizes synchronous determination of the 14 kinds of mycotoxins, has the advantages of simple pretreatment, high detection efficiency, good sensitivity and high accuracy, and can be used for accurate detection of mycotoxins in peanuts.

Description

Technical Field

[0001] This invention relates to the field of food testing technology, and in particular to a method for determining 14 mycotoxins in peanuts by high performance liquid chromatography-tandem mass spectrometry. Background Technology

[0002] Peanuts, as an important oilseed and economic crop in my country, are not only a core raw material for edible oil production but also a significant source of high-quality protein, dietary fiber, and various minerals in the daily diet, occupying a crucial position in the national dietary structure and agricultural economic system. However, the high oil and protein content of peanuts makes them highly susceptible to contamination by fungi such as Aspergillus, Penicillium, and Fusarium throughout the entire industrial chain, from planting and harvesting to storage and processing. These fungi multiply rapidly under suitable temperature and humidity conditions, producing various highly toxic mycotoxins that pose a serious threat to food safety.

[0003] Mycotoxins are a class of naturally occurring toxic secondary metabolites with various harmful effects, including carcinogenicity, teratogenicity, mutagenicity, and hepatotoxicity and nephrotoxicity. They are chemically stable and difficult to completely destroy through ordinary processing and cooking methods. Among them, aflatoxin B1, ochratoxin A, and fumonisins have been listed as key food contaminants by the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO), and their residue levels in food are strictly limited. If peanuts and peanut products are contaminated with mycotoxins, it will not only affect product quality and market circulation but may also accumulate through the food chain, posing a long-term potential threat to consumers' health. Therefore, establishing accurate and comprehensive mycotoxin detection methods is a crucial step in ensuring the safety of peanut food.

[0004] Currently, the main methods for detecting mycotoxins in food include thin-layer chromatography (TLC), enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS / MS). Among these, TLC is cumbersome to operate and has low sensitivity, and has been gradually phased out. Although ELISA has the advantages of being rapid and having high throughput, its specificity and accuracy are insufficient, and it is prone to false positive results, so it is only suitable for preliminary screening. HPLC is widely used in routine testing due to its good separation effect and accurate quantification, but it has poor selectivity when simultaneously detecting multiple mycotoxins and has limited ability to detect trace amounts of toxins.

[0005] Therefore, developing a comprehensive, simple, and high-performance method for the simultaneous detection of mycotoxins is of great practical significance and application value for improving the peanut food safety testing system, preventing food safety risks, and protecting consumer health. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a detection method based on high performance liquid chromatography-tandem mass spectrometry to achieve simultaneous, rapid and accurate determination of 14 fungal toxins, thereby improving detection efficiency and detection coverage.

[0007] To achieve the above objectives, the technical solution adopted by this invention is: a method for determining 14 mycotoxins in peanuts by high performance liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0008] Step S1, Solution Preparation: Prepare the mobile phase, extract, standard stock solution, standard working solution, and internal standard solution separately; the mobile phase consists of an organic phase and an inorganic phase, the organic phase being chromatographic acetonitrile, and the inorganic phase being a 0.1% (v / v) formic acid aqueous solution; the extract is a mixed solution of acetonitrile, water, and acetic acid in a volume ratio of 35:64.5:0.5; the internal standard solution is a 100 ng / mL stable isotope internal standard mixture used for matrix effect correction;

[0009] Step S2, Preparation of Test Solution: Select peanut samples, remove impurities, pulverize using a high-speed grinder, pass through a 20-mesh sieve, and store in a sealed bag at room temperature for later use, ensuring sample homogeneity; weigh 5g of the pulverized sample, place it in a 50mL centrifuge tube, add 20mL of extraction solution, and extract ultrasonically at room temperature for 30min, shaking once every 5min during extraction to ensure full contact between the sample and the extraction solution and promote toxin dissolution; after extraction, add 100mg N-propylethylenediamine, 50mg C18 adsorbent, and 30mg graphitized carbon black to the centrifuge tube, and vortex for 2min; then place the centrifuge tube in a centrifuge and centrifuge at 5000rpm for 5min, and transfer the supernatant to a nitrogen blow-off tube; after nitrogen blowing or freeze-drying the supernatant at 40℃, add 1mL of extraction solution and 10μL of mixed internal standard solution, vortex for 1min to reconstitute, mix thoroughly, and filter through a 0.22μm polytetrafluoroethylene filter membrane, collecting the filtrate as the test solution for detection;

[0010] Step S3, Instrument Detection: Ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (AB SCIEXTRIPLE QUAD 5500+) was used for detection. Through synergistic optimization of chromatographic and mass spectrometric parameters, effective separation and high-sensitivity detection of 14 toxins were achieved.

[0011] Step S4, Result Analysis: Based on the detection results of the standard working solution, a matrix matching standard curve is plotted with the ratio of the target toxin peak area to the internal standard peak area as the ordinate (y) and the target toxin concentration as the abscissa (x). The internal standard method is used to quantitatively analyze 14 fungal toxins in the test solution. Qualitative confirmation is performed by the ratio of retention time and characteristic ion pairs to ensure the accuracy of the detection results.

[0012] Preferably, the method for preparing the formic acid aqueous solution in step S1 includes the following steps: adding 900 mL of ultrapure water and 1 mL of formic acid to a 1 L volumetric flask in sequence, and then making up to the mark with ultrapure water.

[0013] Preferably, the method for preparing the extract in step S1 includes the following steps: adding 400 mL of ultrapure water, 350 mL of acetonitrile and 5 mL of acetic acid to a 1 L volumetric flask in sequence, and then making up to the mark with ultrapure water.

[0014] Preferably, the method for preparing the standard stock solution in step S1 includes the following steps: preparing 1 mg / mL single standard stock solutions of AFB1, AFB2, AFG1, AFG2, ZEN, CIT, and OTA respectively using chromatographic methanol as solvent; preparing 1 mg / mL single standard stock solutions of FB1, FB2, and FB3 respectively using 50% (v / v) acetonitrile aqueous solution as solvent; and preparing 1 mg / mL single standard stock solutions of T-2, ST, DON, and PAT respectively using chromatographic acetonitrile as solvent.

[0015] Preferably, the method for preparing the standard working solution in step S1 includes the following steps: take each single standard stock solution and mix them together, use the extract as the diluent, add the stable isotope internal standard mixture to make the final concentration of each internal standard 1 ng / mL, and dilute stepwise to prepare a mixed standard working solution with 14 toxin concentrations of 0.1, 1, 5, 10, 20, 50, 75 and 100 ng / mL, which is prepared and used immediately.

[0016] Preferably, the method for preparing the internal standard solution in step S1 includes the following steps: weighing... , , Dissolve 1 mg of each standard in chromatographic methanol and dilute to 100 mL to obtain a 10 μg / mL single-standard internal standard stock solution; then take 1 mL of each single-standard internal standard stock solution and dilute to 100 mL with chromatographic methanol to obtain... , , The concentration of each component was 100 ng / mL in the mixed internal standard solution; the ultrasonic extraction power in step S2 was 300 W and the frequency was 40 kHz.

[0017] Preferably, the chromatographic conditions for detection in step S3 are as follows: GL Sciences InertSustainAQ C18 column (3μm 3.0×100mm); column temperature 30℃; injection volume 2μL; flow rate 0.4mL / min; gradient elution program designed for the retention characteristics of 14 toxins: 0-2min: maintain 20% organic phase to elute highly polar toxins such as DON and PAT first; 2-8min: linearly increase organic phase from 20% to 60% to achieve separation of moderately polar toxins; 8-12min: increase organic phase to 90% to elute less polar toxins such as AFB1, AFB2, and T-2; 12-15min: maintain 90% organic phase to ensure complete elution of strongly hydrophobic toxins; 15-16min: rapidly decrease to 20%; 16-18min: equilibrate the column.

[0018] Preferably, the mass spectrometry conditions for the detection in step S3 are as follows: the ion source is an electrospray ion source; positive and negative ion mode switching detection is used, with AFB1, AFB2, AFG1, AFG2, FB1, FB2, FB3, T-2, ST, and ZEN monitored in positive ion mode, with a spray voltage of 5500V; CIT, OTA, DON, and PAT monitored in negative ion mode, with a spray voltage of -4500V; ion source temperature of 550℃; nebulizer gas pressure of 50psi; auxiliary gas pressure of 50psi; curtain gas pressure of 35psi; and the scanning method is multi-stage reaction monitoring (MRM), which screens specific characteristic ion pairs for each toxin and optimizes the collision energy to ensure the specificity and sensitivity of the detection.

[0019] Due to the application of the above technical solution, the present invention has the following beneficial effects:

[0020] (1) The method for determining 14 fungal toxins in peanuts by high performance liquid chromatography-tandem mass spectrometry disclosed in this invention is the first to achieve simultaneous determination of 14 highly hazardous fungal toxins in peanuts, covering major toxin categories such as aflatoxins, fumonisins, and trichothecenes, which solves the limitation of existing standards that only detect 2-4 toxins and meets the needs of simultaneous screening of multiple residues.

[0021] (2) The method for determining 14 fungal toxins in peanuts by high performance liquid chromatography-tandem mass spectrometry disclosed in this invention adopts a pretreatment mode of "exclusive extract + composite adsorbent d-SPE purification". The ratio of the extract is optimized for peanut matrix to achieve efficient extraction of 14 toxins with different polarities. The PSA / C18 / GCB composite adsorbent accurately removes interfering substances such as oil, protein, and pigment. Combined with stable isotope internal standard correction, the matrix effect inhibition rate is high, which greatly improves the detection accuracy.

[0022] (3) The method for determining 14 fungal toxins in peanuts by high performance liquid chromatography-tandem mass spectrometry disclosed in this invention does not require complex column chromatography steps in the pretreatment process, and the whole process is short; the chromatographic elution time is short, which enables rapid detection of large batches of samples, and the detection efficiency is significantly improved compared with existing methods.

[0023] (4) The high-performance liquid chromatography-tandem mass spectrometry method disclosed in this invention for the determination of 14 mycotoxins in peanuts exhibits high recovery rates for each toxin, small relative standard deviations, and good accuracy and reproducibility. The limits of detection are as low as 0.01 ng / mL-3 ng / mL, and the limits of quantitation are 0.03 ng / mL-10 ng / mL. The sensitivity is significantly better than existing methods, meeting the requirements for trace toxin detection. This method is applicable to the detection of mycotoxins in peanuts and peanut products (such as peanut oil and peanut butter). It is simple to operate, cost-effective, and can be widely applied in food safety supervision, food enterprise quality control, and third-party testing institutions, possessing significant promotional value. Attached Figure Description

[0024] Figure 1 This is the total ion chromatogram of the 14 fungal toxin standard solutions in this invention. Detailed Implementation

[0025] The following description is intended to disclose the invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art.

[0026] Example 1 Solution Preparation

[0027] Mobile phase preparation: The organic phase is 100% acetonitrile (chromatographic grade); the inorganic phase is 0.1% formic acid aqueous solution. To prepare the mobile phase, add 900 mL of ultrapure water (resistivity 18.2 MΩ·cm) to a 1 L volumetric flask, then add 1 mL of formic acid (chromatographic grade), stir well, and then dilute to the mark with ultrapure water. After ultrasonic degassing for 15 min, it is ready for use.

[0028] Preparation of extract: Add 400 mL of ultrapure water, 350 mL of acetonitrile (chromatographic grade) and 5 mL of acetic acid (chromatographic grade) to a 1 L volumetric flask in sequence. Dilute to the mark with ultrapure water, shake well and set aside. The volume ratio of acetonitrile, water and acetic acid in the extract is 35:64.5:0.5 and the pH is 3.2.

[0029] Preparation of standard stock solutions: Accurately weigh 10 mg each of AFB1, AFB2, AFG1, AFG2, ZEN, CIT, and OTA standards (purity ≥98%), dissolve them in chromatographic methanol, and dilute to 10 mL to obtain a 1 mg / mL single-standard stock solution; weigh 10 mg each of FB1, FB2, and FB3 standards, dissolve them in an acetonitrile:water mixture (50:50, v / v), and dilute to 10 mL; weigh 10 mg each of T-2, ST, DON, and PAT standards, dissolve them in chromatographic acetonitrile, and dilute to 10 mL. All stock solutions should be stored at -20°C protected from light and have a shelf life of 6 months.

[0030] Preparation of mixed internal standard solution: Weigh out , , Dissolve 1 mg of each standard (purity ≥98%) in chromatographic methanol and dilute to 100 mL to obtain a single standard internal standard stock solution of 10 μg / mL; then take 1 mL of each single standard internal standard stock solution and dilute to 100 mL in chromatographic methanol to obtain a mixed internal standard solution of 100 ng / mL, and store at -20℃ protected from light.

[0031] Preparation of standard working solutions: Take an appropriate amount of each single standard stock solution, use the extract as a diluent, add the mixed internal standard solution to make the final concentration of the internal standard 1 ng / mL, and dilute stepwise to prepare mixed standard working solutions with concentrations of 0.1, 1, 5, 10, 20, 50, 75, and 100 ng / mL. Prepare and use immediately.

[0032] Example 2: Preparation and Detection of Test Solution

[0033] Sample pretreatment: Select 20 moldy peanut samples, remove impurities and insect-infested parts, crush them with a high-speed grinder, pass them through a 20-mesh sieve, and store them in sealed bags at room temperature for later use.

[0034] Preparation of test solution: According to the steps in the technical solution of this invention, weigh 5g (accurate to 0.0001g) of the pulverized sample, add 20mL of extraction solution, and sonicate at room temperature for 30min (300W, 40kHz), shaking once every 5min during the extraction; add 100mg PSA, 50mg C18 and 30mg GCB, vortex for 2min, centrifuge at 5000rpm for 5min; take the supernatant and blow dry with nitrogen at 40℃, add 1mL of extraction solution and 10μL of mixed internal standard solution to reconstitute, and filter through a 0.22μm polytetrafluoroethylene filter membrane to obtain the test solution.

[0035] Instrument detection: The standard working solution and the test solution were detected according to the set chromatographic-mass spectrometry conditions. The instrument was an AB SCIEX TRIPLE QUAD 5500+ ultra-high performance liquid chromatography-mass spectrometry system, and the column was a GLSciences InertSustain AQ C18 (3µm 3.0×100 mm). The column temperature was 30℃, the injection volume was 2µL, the flow rate was 0.4 mL / min, and the elution gradient is shown in Table 1. The mass spectrometry parameters and multi-stage reaction detection parameters are shown in Tables 2 and 3.

[0036] Record the chromatograms and mass spectrometry data. A typical total ion chromatogram of 14 fungal toxin standard solutions is shown below. Figure 1 The peaks of each toxin were symmetrical, well separated, and without overlap. A comparison table of the retention times of the 14 fungal toxins is shown in Table 4.

[0037] Table 1 Gradient elution program

[0038]

[0039] Table 2 Mass Spectrometry Parameters

[0040]

[0041] Table 3 Multi-stage reaction monitoring parameters

[0042]

[0043] Table 4 Retention times of 14 fungal toxins

[0044]

[0045] Example 3 Methodological Validation

[0046] Matrix-matched standard curve: Blank peanut samples were pretreated according to the above method. Mixed standard working solutions of different concentrations were prepared using matrix extract and analyzed by HPLC-MS / MS. A standard curve was plotted with the peak area ratio of the target toxin to the internal standard as the ordinate (y) and the concentration as the abscissa (x). The regression equation and correlation coefficient (r) were calculated. The results showed that the correlation coefficients of all 14 mycotoxins were greater than 0.998, indicating excellent linearity (see Table 5).

[0047] Table 5 Peanut matrix matched standard curve and limits of detection and quantitation

[0048]

[0049] Limits of detection (LOD) and limits of quantitation (LOQ): The LOD was defined as 3 times (S / N=3) the baseline noise at the corresponding retention time of the target analyte in the blank sample, and the LOQ was defined as 10 times (S / N=10). Results showed that the LOD ranged from 0.01 ng / mL to 3 ng / mL, and the LOQ ranged from 0.03 ng / mL to 10 ng / mL. The sensitivity was significantly better than existing methods (the LOD of existing methods is typically 0.1 ng / mL to 5 ng / mL), meeting the requirements for trace detection (see Table 5).

[0050] Recovery and precision: Mixed standard solutions at three concentration levels (10, 20, and 50 ng / mL) were added to blank peanut samples. Six parallel tests were performed for each concentration, with continuous measurements over 3 days. The recovery rate and relative standard deviation (RSD) were calculated. The results showed that the recovery rate at each concentration level was 85.95%–110.30%, and the RSD was ≤4.8%, indicating that the method had good accuracy and reproducibility, meeting the requirements of food testing standards (see Table 6).

[0051] Matrix effect evaluation: The matrix effect was evaluated by the slope ratio of the matrix-matched standard curve and the solvent standard curve. A slope ratio between 0.92 and 1.08 indicates that the matrix effect was effectively suppressed, further verifying the synergistic effect of the composite adsorbent purification and internal standard correction.

[0052] Table 6. Recovery rates and precision of 14 mycotoxins in peanut matrix

[0053]

[0054] Example 4: Actual Sample Testing

[0055] The method of this invention was used to test 20 moldy peanut samples. The results showed that AFB1, AFB2, AFG1, and AFG2 were detected in all samples, with detection ranges of 6.9-119.0 ng / mL, 1.7-56.8 ng / mL, 0.32-15.4 ng / mL, and 0.43-5.15 ng / mL, respectively. In some samples, the AFB1 content exceeded the national standard limit (20 ng / mL), requiring close monitoring. The contents of 10 other mycotoxins, including FB1, FB2, and T-2, were all below the detection limit. These results provide reliable data support for peanut food safety risk assessment and verify the practical application value of this method.

[0056] The above embodiments are only for illustrating the technical concept and features of the present invention. Their purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be used to limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for determining 14 mycotoxins in peanuts by high performance liquid chromatography-tandem mass spectrometry, characterized in that, Includes the following steps: Step S1, Solution Preparation: Prepare the mobile phase, extract, standard stock solution, standard working solution, and internal standard solution separately; the mobile phase consists of an organic phase and an inorganic phase, the organic phase being chromatographic acetonitrile, and the inorganic phase being a 0.1% (v / v) formic acid aqueous solution; the extract is a mixed solution of acetonitrile, water, and acetic acid in a volume ratio of 35:64.5:0.5; the internal standard solution is a 100 ng / mL stable isotope internal standard mixture used for matrix effect correction; Step S2, Preparation of Test Solution: Select peanut samples, remove impurities, pulverize using a high-speed grinder, pass through a 20-mesh sieve, and store in a sealed bag at room temperature for later use, ensuring sample homogeneity; weigh 5g of the pulverized sample, place it in a 50mL centrifuge tube, add 20mL of extraction solution, and extract ultrasonically at room temperature for 30min, shaking once every 5min during extraction to ensure full contact between the sample and the extraction solution and promote toxin dissolution; after extraction, add 100mg N-propylethylenediamine, 50mg C18 adsorbent, and 30mg graphitized carbon black to the centrifuge tube, and vortex for 2min; then place the centrifuge tube in a centrifuge and centrifuge at 5000rpm for 5min, and transfer the supernatant to a nitrogen blow-off tube; after nitrogen blowing or freeze-drying the supernatant at 40℃, add 1mL of extraction solution and 10μL of mixed internal standard solution, vortex for 1min to reconstitute, mix thoroughly, and filter through a 0.22μm polytetrafluoroethylene filter membrane, collecting the filtrate as the test solution for detection; Step S3, Instrument Detection: Ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC-MS / MS) was used for detection. Through synergistic optimization of chromatographic and mass spectrometric parameters, effective separation and high-sensitivity detection of 14 toxins were achieved. The chromatographic conditions were as follows: GL Sciences InertSustain AQ C18 column; column temperature 30℃; injection volume 2 μL; flow rate 0.4 mL / min; gradient elution program designed based on the retention characteristics of the 14 toxins: 0-2 min. Maintain 20% organic phase to elute highly polar toxins such as DON and PAT first; linearly increase the organic phase from 20% to 60% over 2-8 minutes to achieve separation of moderately polar toxins; increase the organic phase to 90% over 8-12 minutes to elute less polar toxins such as AFB1, AFB2, and T-2; maintain 90% organic phase for 12-15 minutes to ensure complete elution of strongly hydrophobic toxins; rapidly decrease the organic phase to 20% over 15-16 minutes, and equilibrate the column over 16-18 minutes; the mass spectrometry conditions for the detection are: electrospray ionization source; positive and negative ion mode switching detection. The system monitors AFB1, AFB2, AFG1, AFG2, FB1, FB2, FB3, T-2, ST, and ZEN in positive ion mode with a spray voltage of 5500V; and CIT, OTA, DON, and PAT in negative ion mode with a spray voltage of -4500V. The ion source temperature is 550℃; the atomizing gas pressure is 50psi; the auxiliary gas pressure is 50psi; and the curtain gas pressure is 35psi. The scanning method is multi-stage reaction monitoring, which screens specific characteristic ion pairs for each toxin and optimizes the collision energy to ensure the specificity and sensitivity of the detection. Step S4, Result Analysis: Based on the detection results of the standard working solution, a matrix matching standard curve is plotted with the ratio of the target toxin peak area to the internal standard peak area as the ordinate and the target toxin concentration as the abscissa. The internal standard method is used to quantitatively analyze 14 fungal toxins in the test solution. Qualitative confirmation is performed by the ratio of retention time and characteristic ion pairs to ensure the accuracy of the detection results.

2. The method for determining 14 mycotoxins in peanuts by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The method for preparing the formic acid aqueous solution in step S1 includes the following steps: adding 900 mL of ultrapure water and 1 mL of formic acid to a 1 L volumetric flask in sequence, and then making up to the mark with ultrapure water.

3. The method for determining 14 mycotoxins in peanuts by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The preparation method of the extract in step S1 includes the following steps: add 400 mL of ultrapure water, 350 mL of acetonitrile and 5 mL of acetic acid to a 1 L volumetric flask in sequence, and then dilute to the mark with ultrapure water.

4. The method for determining 14 mycotoxins in peanuts by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The method for preparing the standard stock solutions in step S1 includes the following steps: preparing 1 mg / mL single standard stock solutions of AFB1, AFB2, AFG1, AFG2, ZEN, CIT, and OTA respectively using chromatographic methanol as solvent; preparing 1 mg / mL single standard stock solutions of FB1, FB2, and FB3 respectively using 50% (v / v) acetonitrile aqueous solution as solvent; and preparing 1 mg / mL single standard stock solutions of T-2, ST, DON, and PAT respectively using chromatographic acetonitrile as solvent.

5. The method for determining 14 mycotoxins in peanuts by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The method for preparing the standard working solution in step S1 includes the following steps: take each single standard stock solution and mix them together. Use the extract as the diluent and add a stable isotope internal standard mixture to make the final concentration of each internal standard 1 ng / mL. Dilute stepwise to prepare a mixed standard working solution with 14 toxin concentrations of 0.1, 1, 5, 10, 20, 50, 75 and 100 ng / mL. Prepare and use immediately.

6. The method for determining 14 mycotoxins in peanuts by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The method for preparing the internal standard solution in step S1 includes the following steps: weighing AFB1- 13 C 17 OTA 13 C 20 ZEN- 13 C 18 Dissolve 1 mg of each standard in chromatographic methanol and dilute to 100 mL to obtain a 10 μg / mL single-standard internal standard stock solution; then take 1 mL of each single-standard internal standard stock solution and dilute to 100 mL with chromatographic methanol to obtain AFB1- 13 C 17 OTA 13 C 20 ZEN- 13 C 18 The concentration of each component is 100 ng / mL in the mixed internal standard solution.

7. The method for determining 14 mycotoxins in peanuts by high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that, The ultrasonic extraction in step S2 has a power of 300W and a frequency of 40kHz.

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