QuEChERS-LC-MS / MS-based method for simultaneously determining various mycotoxins in Chinese prickly ash

Abstract

The invention provides a QuEChERS-LC-MS / MS. The method for simultaneously determining various mycotoxins in Sichuan pepper based on QuEChERS-LC-MS / MS. The method specifically comprises the following steps: S1, crushing dry Sichuan pepper, sequentially adding ultrapure water, an extracting agent and a salting-out agent, and centrifuging to take supernate, so as to obtain pretreated Sichuan pepper extract; s2, adding the pretreated zanthoxylum bungeanum extracting solution into a purifying agent, performing vortex and centrifugation, taking supernate, and filtering to obtain a purified to-be-detected sample; s3, preparing fungaltoxin mixed standard solutions with different concentrations, and drawing a standard curve; and S4, determining the content of various fungaltoxins in the pepper by adopting a liquid chromatography-tandem triple quadrupole mass spectrometer. The determination method provided by the invention is convenient, simple and accurate, can effectively extract, purify and detect various mycotoxins in Sichuan pepper, and realizes high-throughput detection of 12 mycotoxins in dry red Sichuan pepper and dry green Sichuan pepper.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and particularly relates to a method for simultaneously determining multiple mycotoxins in Zanthoxylum bungeanum based on QuEChERS-LC-MS / MS. Background Art

[0002] Sichuan peppercorns, derived from the dried mature fruit of the Zanthoxylum plant of the Zanthoxylum family, are known as one of the traditional Chinese "eight major condiments" for their unique aroma and numbing taste. At the same time, it is also a long-standing Chinese medicinal material and edible spice, with a cultivation and consumption history of more than two thousand years in China. Based on the difference in the color of the outer skin of Sichuan peppercorns, they can be divided into two categories: red Sichuan peppercorns and green Sichuan peppercorns. Sichuan peppercorns are rich in chemical components, including alkaloids, essential oils, flavonoids, and coumarins, which show a variety of biological activities, including pain relief, protection of gastrointestinal health, and certain anti-tumor, anti-cancer and anti-inflammatory effects. The harvest season of Sichuan pepper is in summer, when frequent rainfall and high temperature create a high-humidity environment, which provides favorable conditions for the growth of fungi and may promote the production of mycotoxins. Mycotoxins are a group of secondary metabolites released by toxic fungi during their growth and reproduction stages. Ingestion of food contaminated with mycotoxins may cause serious health problems such as cancer, hepatitis and gastroenteritis. Sichuan pepper has a complex composition, including volatile oils, alkaloids, coumarins, flavonoids and terpenoids. These components may interfere with the precision, accuracy and repeatability of mycotoxin detection, thereby increasing the difficulty of detection. In addition, given the diverse chemical structures of mycotoxins and their extremely low content (usually measured in micrograms per kilogram), the effective extraction of these toxins from food also faces many challenges. In order to ensure the accuracy and reliability of the test results, proper pretreatment of pepper samples is an indispensable key step in the detection of mycotoxins. The QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method is a technology for extracting pesticide residues from fruits and vegetables proposed by the United States Department of Agriculture in 2003. The QuEChERS process mainly includes extraction, salting out and purification steps. Usually, acetonitrile, methanol, acetone or other organic solvents are used to extract the sample. Then, a salt agent is added to promote salting out and stratification. Finally, the extract is added to the adsorbent for cleaning, and the supernatant is taken for detection. Compared with other pretreatment methods, the QuEChERS method has the advantages of fewer processing steps, fast speed, simple operation, less organic solvent consumption, and compliance with green chemistry requirements. In the QuEChERS method, the selection of a suitable cleanup agent is crucial for removing interfering substances from the sample and improving the detection sensitivity and accuracy. Traditional adsorbents, such as primary secondary amines (PSA), octadecyl silica (C18), and graphitized carbon black (GCB), are widely used in various fields such as food analysis, environmental monitoring, and drug residue analysis. In recent years, the new carbon-based adsorbent ENVI-Carb has attracted much attention due to its excellent adsorption performance. The adsorption mechanism of ENVI-Carb is mainly attributed to its unique carbon surface structure. ENVI-Carb is mainly composed of graphite and nonporous carbon and presents a hexagonal atomic arrangement in which carbon atoms are interconnected in a layered manner. This structural arrangement gives it a high selectivity for planar aromatic compounds, hexagonal molecules, and hydrocarbon chains that can form a large number of surface contacts. ENVI-Carb can effectively remove pigments such as chlorophyll and carotenoids from food and natural products. Although the purification properties of ENVI-Carb have been documented in the literature, no study has yet applied it to the purification of samples containing mycotoxins. In the current technological context, due to the complexity and challenges of the mycotoxin detection process, methods for the simultaneous detection of multiple mycotoxins in Zanthoxylum bungeanum are relatively scarce and inefficient. Usually, these methods can only detect a few mycotoxins at the same time, and the detection range is relatively limited. Therefore, developing an accurate and simple pretreatment method for the detection of multiple mycotoxins in Zanthoxylum bungeanum is of great significance for effectively monitoring mycotoxin contamination in Zanthoxylum bungeanum. Summary of the invention

[0003] Technical problem to be solved: In view of the above technical problems, the purpose of the present invention is to provide a method for simultaneously determining multiple fungal toxins in red pepper and green pepper based on QuEChERS technology-liquid chromatography tandem triple quadrupole mass spectrometry. The determination method provided by the present invention is convenient, simple and accurate, so that various fungal toxins in pepper can be effectively extracted, purified and detected. The recovery rate and detection limit of the experiment meet the experimental requirements, and high-throughput detection of 12 fungal toxins in dried red pepper and dried green pepper is achieved.

[0004] Technical solution: A method for simultaneously determining multiple mycotoxins in Zanthoxylum bungeanum based on QuEChERS-LC-MS / MS, comprising the following steps: S1. Crush the dried pepper into pepper powder, add ultrapure water and shake for 1-10 min, add the extractant and shake for 1-10 min, then add the salting agent and vortex for 1-2 min, centrifuge at 6000 r / min for 1-3 min, take the supernatant, and obtain the pretreated pepper extract; S2. Add the pretreated Zanthoxylum bungeanum extract to the purifier, vortex for 1-3 min, centrifuge at 6000 r / min for 1-2 min at room temperature, take the supernatant, filter with a membrane, and obtain the purified sample to be tested; S3. Prepare mixed standard solutions of mycotoxins of different concentrations and draw standard curves; S4. Liquid chromatography-tandem triple quadrupole mass spectrometry was used to detect the samples to be tested, and the contents of various fungal toxins in Zanthoxylum bungeanum were obtained according to the standard curve. Furthermore, the pepper is red pepper and green pepper; the fungal toxins are aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, stericocyanin, citrinin, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, HT-2 toxin and T-2 toxin. Furthermore, in step S1, the mass volume ratio of pepper powder to ultrapure water is 1 g:(1-10) mL; and the volume ratio of ultrapure water to the extractant is 1:1. Furthermore, the extractant is a mixed solution of acetonitrile and formic acid in a volume ratio of 99:1. Furthermore, in step S1, the salting-out agent is a mixture of anhydrous sodium sulfate and sodium chloride, and the mass ratio of the pepper powder, anhydrous sodium sulfate and sodium chloride is 1:6:1.5. Furthermore, in step S2, the purifier is a mixture of ENVI-Carb and PSA, and the mass volume ratio of ENVI-Carb, PSA and pretreated Zanthoxylum bungeanum extract is 50 mg:200 mg:2 mL. Furthermore, the liquid chromatography conditions in step S4 are: Chromatographic column: Hypersil GOLD aQ column, 50 mm × 2.1 mm, 1.9 μm; Mobile phase: A-aqueous solution: 5mmol / L ammonium acetate, 0.1% formic acid; B-methanol; Column temperature: 40°C; Flow rate: 0.4 mL / min; Injection volume: 2 μL; Elution time: 5min; Gradient elution: 0-2 min, 10%→80% B; 2-2.5 min, 80%→100% B; 2.5-3.5 min, 100% B; 3.5-3.6 min, 100%→10% B; 3.6-5 min, 10% B. Furthermore, the triple quadrupole mass spectrometry conditions in step S4 are: Monitoring mode: Multiple reaction monitoring; Ionization method: electrospray ionization ESI; Capillary voltage: 5500V, -4500V; Ion source temperature: 550°C; Ion source gas 1: 276 kPa; Ion source gas 2: 276 kPa; Curtain gas: 276kPa. Beneficial effects: 1. The QuEChERS pretreatment method used in the present invention contains only two steps, which significantly reduces the sample preparation process. It not only supports the effective extraction of polar analytes, but also shows excellent selectivity and detectability, and is directly compatible with liquid chromatography combined with mass spectrometry. Compared with traditional methods, this method greatly reduces the amount of extractant required and sample preparation time, while achieving a higher recovery rate. In addition, this detection method is also suitable for trace analysis of fungal toxins in complex samples due to its wide applicability, showing strong versatility. 2. The main extractant of the present invention is acetonitrile, which ensures the efficient extraction of all target fungal toxins in the pepper sample by virtue of its advantages of reducing the co-extraction of lipophilic impurities (such as fat) and its wide solubility in molecules of different polarities; adding 1% formic acid to acetonitrile not only optimizes the extraction efficiency of fungal toxins, but also significantly promotes the molecular separation effect in subsequent chromatographic analysis and enhances the ionization efficiency in the mass spectrometry detection process, thereby achieving the enhancement of the analysis signal peak response and the improvement of the sensitivity of fungal toxin detection. 3. In the extraction and purification process of the present invention, anhydrous sodium sulfate is selected as the salting-out agent. Its excellent water removal performance not only ensures the efficient dehydration of the sample extract, but also promotes the full contact and reaction between the purifier and the extract, and significantly improves the purification effect; compared with the traditional salting-out agent anhydrous magnesium sulfate, anhydrous sodium sulfate avoids the solvent evaporation loss and agglomeration caused by easy heat release, thereby more effectively promoting the removal of matrix components in the pepper sample, and laying a solid foundation for the accurate detection of target analytes such as fungal toxins. 4. The present invention designs a purifier system composed of ENVI-Carb and PSA, wherein ENVI-Carb effectively reduces interfering substances in a complex Zanthoxylum bungeanum matrix with its strong adsorption capacity for pigments and planar compounds; and PSA exhibits an excellent removal effect on components such as organic acids and fatty acids, further purifying the sample; by regulating the dosage ratio of the two purifiers, the present invention achieves efficient removal of interfering substances in Zanthoxylum bungeanum samples, creating an ideal experimental environment for the detection of fungal toxins. 5. The present invention proposes an innovative sample pretreatment method, which effectively inhibits the interference of the Zanthoxylum bungeanum sample matrix on the detection results, and significantly reduces the negative impact of the complex matrix of Zanthoxylum bungeanum on the analysis of fungi toxins; combined with the optimization of liquid chromatography-tandem triple quadrupole mass spectrometry detection conditions, the present invention successfully achieves high-sensitivity and high-selectivity simultaneous detection of up to 12 fungal toxins in dried red and green Zanthoxylum bungeanum, has good specificity, selectivity and precision, can achieve low quantitative limit and high linear range detection, and the detection time for one sample is only 5 minutes; this method not only shows the advantages of simple operation, significant purification efficiency and high-throughput analysis, but also embodies the dual characteristics of economy and environmental protection, and provides strong technical support and theoretical basis for the detection of fungi toxins in Zanthoxylum bungeanum and its related products. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 A flow chart of the detection of mycotoxins in Zanthoxylum bungeanum of the present invention; Figure 2 The chromatograms are of 12 mycotoxins; Figure 3 The figure is a comparison chart of the recovery test results of Example 1 and Comparative Example 1, wherein A is dried red pepper; B is dried green pepper; Figure 4 The sample extraction color comparison diagram of Example 1 and Comparative Example 1; Figure 5 The figure is a comparison chart of the recovery test results of Example 1 and Comparative Example 2, wherein A is dried red pepper; B is dried green pepper; Figure 6 It is a comparison chart of the recovery test results of Example 1 and Comparative Example 3, wherein A is dried red pepper; B is dried green pepper. DETAILED DESCRIPTION The present invention is further described below in conjunction with the accompanying drawings and embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments: 1. Optimization of extraction agent The extraction solvents were acetonitrile, acetonitrile:formic acid = 99:1, acetonitrile:formic acid = 95:5, acetonitrile:acetic acid = 99:1, and acetonitrile:acetic acid = 95:5, and the recovery rates of various fungal toxins in Zanthoxylum bungeanum were determined according to the following steps. A method for simultaneously determining multiple mycotoxins in Zanthoxylum bungeanum based on QuEChERS-LC-MS / MS, comprising the following steps: S1. Crush the dried pepper into pepper powder, weigh 1g of dried red pepper powder or dried green pepper powder in a 50mL centrifuge tube, add 10mL of ultrapure water, shake the sample with a shaker for 10min, add 10mL of extractant, shake the sample with a shaker for 10min, then add 6g of anhydrous sodium sulfate and 1.5g of sodium chloride, vortex for 2min, centrifuge at 6000r / min for 3min, take the supernatant, and obtain the pretreated pepper extract; S2. Add 2 mL of pretreated Zanthoxylum bungeanum extract to the purifier (PSA 200 mg, ENVI-Carb 50 mg), vortex for 3 min, centrifuge at 6000 r / min for 2 min at room temperature, take the supernatant, filter with a membrane, and obtain the purified sample to be tested (blank matrix solution); S3. Use the purified sample to be tested to prepare the fungal toxin standard solution into a mixed standard working solution at seven concentration levels (0.1-200 μg / kg for dried red and green peppercorns), and establish a standard curve. S4. Liquid chromatography-tandem triple quadrupole mass spectrometry was used to detect the samples to be tested, and the contents of various fungal toxins in Zanthoxylum bungeanum were obtained according to the standard curve. The liquid chromatography conditions are: Chromatographic column: Hypersil GOLD aQ column, 50 mm × 2.1 mm, 1.9 μm; Mobile phase: A-aqueous solution: 5mmol / L ammonium acetate, 0.1% formic acid; B-methanol; Column temperature: 40°C; Flow rate: 0.4 mL / min; Injection volume: 2 μL; Elution time: 5min; Gradient elution is shown in Table 1: Table 1 Mobile phase gradient elution program The triple quadrupole mass spectrometry conditions are: Monitoring mode: Multiple reaction monitoring; Ionization method: electrospray ionization (ESI); Capillary voltage: 5500 V (ESI+), -4500 V (ESI-); Ion source temperature: 550°C; Ion source gas 1: 276 kPa; Ion source gas 2: 276 kPa; Curtain gas: 276kPa. Table 2 Test results of different extractants The test results are shown in Table 2. When the extractant is acetonitrile:formic acid = 99:1, a higher recovery rate is obtained, and the recovery rates of 12 fungal toxins are between 80.98% and 104.42%. Therefore, the extractant is determined to be acetonitrile:formic acid = 99:1. 2. Optimization of extraction time The extraction solvent was determined to be acetonitrile:formic acid = 99:1, and the extraction times were selected to be 2, 4, 6, 8, 10, and 15 min. The other experimental steps and detection conditions remained unchanged, and the recovery rate was tested. Table 3 Test results of different extraction times The test results are shown in Table 3. When the extraction time was 10 min, a higher recovery rate was obtained, and the recovery rates of 12 mycotoxins were between 73.81% and 85.11%. Therefore, 10 min was selected as the extraction time. 3. Optimization of Purifier The extraction agent was determined to be acetonitrile:formic acid = 99:1, the extraction time was 10 min, and the purifiers were selected to be 100 mg PSA + 50 mg ENVI-Carb, 200 mg PSA + 50 mg ENVI-Carb, 100 mg C18 + 50 mg ENVI-Carb, 200 mg C18 + 50 mg ENVI-Carb, 300 mg PSA + 50 mg ENVI-Carb, 300 mg C18 + 50 mg ENVI-Carb. The other experimental steps and detection conditions remained unchanged, and the recovery rate was tested. Table 4 Test results of different purifiers The test results are shown in Table 4. When the purifier is 200 mg PSA + 50 mg ENVI-Carb, the highest extraction efficiency can be obtained, and the recovery rate of 12 fungal toxins is 78.55-90.60%. Therefore, the purifier is determined to be 200 mg PSA + 50 mg ENVI-Carb. 4. Optimization of purification time The extraction agent was determined to be acetonitrile:formic acid = 99:1, the extraction time was 10 min, the purification agent was 200 mg PSA + 50 mg ENVI-Carb, the purification time was selected to be 1, 2, 3, 4, 5 min, and the other experimental steps and detection conditions remained unchanged for recovery rate detection. Table 5 Test results of different purification time The test results are shown in Table 5. When the purification time is 3 minutes, a higher recovery rate is obtained, and the recovery rates of 12 fungal toxins are between 75.06% and 87.27%. Therefore, the purification time is determined to be 3 minutes. Example 1 A method for simultaneously determining multiple mycotoxins in Zanthoxylum bungeanum based on QuEChERS-LC-MS / MS, comprising the following steps: S1. Crush the dried pepper into pepper powder, weigh 1g of dried red pepper powder or dried green pepper powder in a 50mL centrifuge tube, add 10mL of ultrapure water, shake the sample with a shaker for 10min, add 10mL of extractant (acetonitrile: formic acid = 99:1), shake the sample with a shaker for 10min, then add 6g of anhydrous sodium sulfate and 1.5g of sodium chloride as salting-out agents, vortex for 2min, centrifuge at 6000r / min for 3min, take the supernatant, and obtain the pretreated pepper extract; S2. Add 2 mL of pretreated Zanthoxylum bungeanum extract to the purifier (PSA 200 mg, ENVI-Carb 50 mg), vortex for 3 min, centrifuge at 6000 r / min for 2 min at room temperature, take the supernatant, filter with a membrane, and obtain the purified sample to be tested (blank matrix solution); S3. Use the purified sample to be tested to prepare the fungal toxin standard solution into a mixed standard working solution at seven concentration levels (0.1-200 μg / kg for dried red and green peppercorns), and establish a standard curve. S4. Liquid chromatography-tandem triple quadrupole mass spectrometry was used to detect the samples to be tested, and the contents of various fungal toxins in Zanthoxylum bungeanum were obtained according to the standard curve. The liquid chromatography conditions are: Chromatographic column: Hypersil GOLD aQ column, 50 mm × 2.1 mm, 1.9 μm; Mobile phase: A-aqueous solution: 5mmol / L ammonium acetate, 0.1% formic acid; B-methanol; Column temperature: 40°C; Flow rate: 0.4 mL / min; Injection volume: 2 μL; Elution time: 5min; Gradient elution program: 0-2 min, 10%→80% B; 2-2.5 min, 80%→100% B; 2.5-3.5 min, 100% B; 3.5-3.6 min, 100%→10% B; 3.6-5 min, 10% B. The triple quadrupole mass spectrometry conditions are: Monitoring mode: Multiple reaction monitoring; Ionization method: electrospray ionization (ESI); Capillary voltage: 5500 V (ESI+), -4500 V (ESI-); Ion source temperature: 550°C; Ion source gas 1: 276 kPa; Ion source gas 2: 276 kPa; Curtain gas: 276kPa. Using the above detection method, the compound ion pairs and mass spectrometry parameters of the mycotoxin to be detected are shown in Table 6 below: Table 6 Compound ion pairs and mass spectrometry parameters of 12 fungal toxins * Q, quantitative ion; C, qualitative ion. The standard curves of 12 fungal toxins were drawn based on the absorption peaks of liquid chromatography-tandem triple quadrupole mass spectrometry and the concentrations of the spiked solutions (Table 7). As can be seen from Table 7, the R 2 The linear relationship of the method was good. The detection limits of the method were 0.2-0.6 μg / kg and 0.1-0.7 μg / kg for dry red pepper and dry green pepper, respectively. The quantification limits of the method were 0.6-1.9 μg / kg and 0.4-2.2 μg / kg for dry red pepper and dry green pepper, respectively. Table 7 Linear range and correlation coefficient (R 2 ), limit of detection (LOD) and limit of quantification (LOQ) Tests of spike recovery and matrix effect Three different spike levels (1, 5, and 10 μg / kg for dried red and green peppercorns) were selected, and the peppercorn samples were pretreated using the method developed above in this protocol to investigate the recoveries of 12 fungal toxins. The pretreatment method developed in this scheme was used to extract the blank matrix solution and prepare the standard curve. The matrix effect was evaluated by the ratio K of the slope of the matrix matching standard curve and the slope of the organic solvent (acetonitrile) standard curve. If the K value was between 0.8 and 1.2, it indicated that the matrix effect was not obvious. When K was greater than 1.2, it was a matrix enhancement effect, and when K was less than 0.8, it was a matrix inhibition effect. The matrix effect of 12 fungal toxins was investigated. The recoveries, relative standard deviations and matrix effects of the 12 mycotoxins in Example 1 were calculated. The results are shown in Table 8. Table 8 Recovery, relative standard deviation and matrix effect of 12 kinds of mycotoxins It can be seen from Table 8 that the matrix effect of red pepper is 0.88-1.11, and the matrix effect is not obvious; the matrix effect of green pepper is 0.85-1.07, and the matrix effect is not obvious. Comparative Example 1 The difference between this comparative example and Example 1 is that the purifier is replaced with 100 mg PSA, 100 mg C18, 100 mg GCB, 100 mg ENVI-Carb, and the detection conditions remain unchanged, as follows: A method for simultaneously determining multiple mycotoxins in Zanthoxylum bungeanum based on QuEChERS-LC-MS / MS, comprising the following steps: S1. Crush the dried pepper into pepper powder, weigh 1g of dried red pepper powder or dried green pepper powder in a 50mL centrifuge tube, add 10mL of ultrapure water, shake the sample with a shaker for 10min, add 10mL of extractant (acetonitrile:formic acid=99:1), shake the sample with a shaker for 10min, then add 6g of anhydrous sodium sulfate and 1.5g of sodium chloride, vortex for 2min, centrifuge at 6000r / min for 3min, take the supernatant, and obtain the pretreated pepper extract; S2. Add 2 mL of the pretreated Zanthoxylum bungeanum extract to the purifier (100 mg PSA, 100 mg C18, 100 mg GCB, 100 mg ENVI-Carb), vortex for 3 min, centrifuge at 6000 r / min for 2 min at room temperature, take the supernatant, filter through a filter membrane, and obtain the purified sample to be tested (blank matrix solution); S3. Using the purified test samples, seven concentration levels (0.1-200 μg / kg for dried red pepper and dried green pepper) were used to prepare the mycotoxin standard solution into a mixed standard working solution, and a standard curve was established. S4. Liquid chromatography-tandem triple quadrupole mass spectrometry was used to detect the samples to be tested, and the contents of various fungal toxins in Zanthoxylum bungeanum were obtained according to the standard curve. Comparative Example 2 The difference between this comparative example and Example 1 is that the salting-out agent is replaced by 6g MgSO4+1.5g NaAc, 6g MgSO4+1.5g NaCl, 6g Na2SO4+1.5g NaAc, and the detection conditions remain unchanged, as follows: A method for simultaneously determining multiple mycotoxins in Zanthoxylum bungeanum based on QuEChERS-LC-MS / MS, comprising the following steps: S1. Crush the dried pepper into pepper powder, weigh 1g of dried red pepper powder or dried green pepper powder in a 50mL centrifuge tube, add 10mL of ultrapure water, shake the sample with a shaker for 10min, add 10mL of extractant (acetonitrile:formic acid=99:1), shake the sample with a shaker for 10min, then add salting-out agent (6g MgSO4+1.5gNaAc, 6g MgSO4+1.5gNaCl, 6g Na2SO4+1.5g NaAc), vortex for 2min, centrifuge at 6000r / min for 3min, take the supernatant, and obtain the pretreated pepper extract; S2. Add 2 mL of pretreated Zanthoxylum bungeanum extract to the purifier (PSA 200 mg, ENVI-Carb 50 mg), vortex for 3 min, centrifuge at 6000 r / min for 2 min at room temperature, take the supernatant, filter with a membrane, and obtain the purified sample to be tested (blank matrix solution); S3. Use the purified sample to be tested to prepare the fungal toxin standard solution into a mixed standard working solution at seven concentration levels (0.1-200 μg / kg for dried red and green peppercorns), and establish a standard curve. S4. Liquid chromatography-tandem triple quadrupole mass spectrometry was used to detect the samples to be tested, and the contents of various fungal toxins in Zanthoxylum bungeanum were obtained according to the standard curve. Comparative Example 3 The difference between this comparative example and Example 1 is that the extractant is replaced with acetonitrile, methanol, and acetone, and the detection conditions remain unchanged, as follows: A method for simultaneously determining multiple mycotoxins in Zanthoxylum bungeanum based on QuEChERS-LC-MS / MS, comprising the following steps: S1. Crush the dried pepper into pepper powder, weigh 1g of dried red pepper powder or dried green pepper powder in a 50mL centrifuge tube, add 10mL of ultrapure water, shake the sample with a shaker for 10min, add 10mL of extractant (acetonitrile, methanol, acetone), shake and mix the sample with a shaker for 10min, then add 6g of anhydrous sodium sulfate and 1.5g of sodium chloride as salting-out agent, vortex for 2min, centrifuge at 6000r / min for 3min, take the supernatant, and obtain the pretreated pepper extract; S2. Add 2 mL of pretreated Zanthoxylum bungeanum extract to the purifier (PSA 200 mg, ENVI-Carb 50 mg), vortex for 3 min, centrifuge at 6000 r / min for 2 min at room temperature, take the supernatant, filter with a membrane, and obtain the purified sample to be tested (blank matrix solution); S3. Use the purified sample to be tested to prepare the fungal toxin standard solution into a mixed standard working solution at seven concentration levels (0.1-200 μg / kg for dried red and green peppercorns), and establish a standard curve. S4. Liquid chromatography-tandem triple quadrupole mass spectrometry was used to detect the samples to be tested, and the contents of various fungal toxins in Zanthoxylum bungeanum were obtained according to the standard curve. For different purifiers, the test results are as follows Figure 3 A. For dried red peppercorns, after PSA purification, the recoveries of six mycotoxins (DON, 3-AcDON, 15-AcDON, OTA, ST, T-2) were limited to 44.93% to 58.41%, while the recoveries of the other six mycotoxins were in the range of 60% to 70%; except for OTA (64.19%) and ST (67.53%), the recoveries of other mycotoxins treated with C18 adsorbent were generally between 20% and 50%. Figure 3 As shown in Figure B, for dried green peppercorns, the recovery rates of mycotoxins were low regardless of whether they were treated with C18 or PSA. Specifically, the recovery rates after C18 treatment ranged from 31.8% to 54.2%, while the recovery rates after PSA treatment ranged from 43.8% to 68.2%. When GCB was used as a purifier, the recoveries of mycotoxins in both Zanthoxylum samples were low, especially AFB1, AFB2, T-2, and ST. Although GCB has excellent adsorption capacity for pigments, it also adsorbs planar mycotoxins, such as aflatoxins and sterigmatocystin, which interferes with the detection of mycotoxins and leads to low recoveries. Further observation Figure 4 It can be found that after ENVI-Carb treatment, the color of the extract solution became significantly lighter and almost transparent. In the two pepper samples, the recovery rate of mycotoxins was stable between 64.8% and 75.9% when ENVI-Carb was used as the adsorbent ( Figure 3 ENVI-Carb is a highly efficient adsorbent that is unique in that it forms hexagonal ring structures on the carbon surface that are interconnected and arranged in layers within the graphite flakes. The non-porous nature of the adsorbent allows for faster handling and absorption without the need for analytes to diffuse into the pores. In addition, ENVI-Carb is effective in removing interfering compounds such as pigments and polyphenols. For different salting-out agents, Figure 5It can be seen that when NaCl and NaAc were used as salting-out agents, respectively (i.e., 6gNa2SO4+1.5g NaCl and 6gNa2SO4+1.5gNaAc), there was no significant difference in the recovery of mycotoxins in the two pepper samples. This shows that under the action of these two salting-out agents, the extraction efficiency of mycotoxins in pepper samples is comparable. When MgSO4 was used as a salting-out agent, the recovery of mycotoxins decreased, which may be because MgSO4 releases heat during the hydration reaction, resulting in the degradation of mycotoxins with poor thermal stability, thereby reducing their recovery. At the same time, MgSO4 is easy to aggregate in the solution, which may encapsulate the target mycotoxins and affect the recovery rate. On the other hand, when Na2SO4 and NaCl were used in combination, the recovery rates of the 12 mycotoxins all reached more than 70%. This result shows that the combined use of the two can significantly improve the extraction efficiency of mycotoxins. Therefore, 6g Na2SO4 and 1.5g NaCl were finally selected as salting-out agents. For different extractants, the test results are as follows Figure 6 When methanol is used as an extractant, more impurities are carried over during the extraction process, which significantly interfere with the detection of mycotoxins, resulting in a low recovery rate. In contrast, due to its low polarity, acetone is not ideal for the extraction of mycotoxins with higher polarity, which also leads to a lower recovery rate. After comparing a variety of solvents, we found that acetonitrile produces relatively little matrix interference during the extraction process and can dissolve mycotoxins of different polarities well. Furthermore, when 1% formic acid is added to acetonitrile, this mixed solvent is not only conducive to better chromatographic separation in LC-MS / MS analysis, but also enhances the ionization effect, thereby obtaining better analytical performance. Therefore, the extraction agent was determined to be acetonitrile:formic acid = 99:1. The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the spirit and technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A method for simultaneously determining multiple mycotoxins in Zanthoxylum bungeanum based on QuEChERS-LC-MS / MS, characterized in that: The following steps are involved: S1. Crush the dried pepper into pepper powder, add ultrapure water and shake for 1-10 min, add the extractant and shake for 1-10 min, then add the salting agent and vortex for 1-2 min, centrifuge at 6000 r / min for 1-3 min, take the supernatant, and obtain the pretreated pepper extract; S2. Add the pretreated Zanthoxylum bungeanum extract to the purifier, vortex for 1-3 min, centrifuge at 6000 r / min for 1-2 min at room temperature, take the supernatant, filter with a membrane, and obtain the purified sample to be tested; S3. Prepare mixed standard solutions of mycotoxins of different concentrations and draw standard curves; S4. Liquid chromatography coupled with a triple quadrupole mass spectrometer was used to detect the samples, and the contents of various fungal toxins in Zanthoxylum bungeanum were obtained according to the standard curve.

2. A method for simultaneously determining multiple mycotoxins in Zanthoxylum bungeanum based on QuEChERS-LC-MS / MS according to claim 1, characterized in that: The peppers are red peppers and green peppers; the fungal toxins are aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, stericocyanin, citrinin, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, HT-2 toxin and T-2 toxin.

3. A method for simultaneously determining multiple mycotoxins in Zanthoxylum bungeanum based on QuEChERS-LC-MS / MS according to claim 1, characterized in that: In the step S1, the mass volume ratio of pepper powder to ultrapure water is 1 g:(1-10) mL; the volume ratio of ultrapure water to the extractant is 1:

1.

4. A method for simultaneously determining multiple mycotoxins in Zanthoxylum bungeanum based on QuEChERS-LC-MS / MS according to claim 3, characterized in that: The extractant is a mixed solution of acetonitrile and formic acid in a volume ratio of 99:

1.

5. A method for simultaneously determining multiple mycotoxins in Zanthoxylum bungeanum based on QuEChERS-LC-MS / MS according to claim 1, characterized in that: In step S1, the salting-out agent is a mixture of anhydrous sodium sulfate and sodium chloride, and the mass ratio of the pepper powder, anhydrous sodium sulfate and sodium chloride is 1:6:1.

5.

6. A method for simultaneously determining multiple mycotoxins in Zanthoxylum bungeanum based on QuEChERS-LC-MS / MS according to claim 1, characterized in that: In the step S2, the purifier is a mixture of ENVI-Carb and PSA, and the mass volume ratio of ENVI-Carb, PSA and pretreated Zanthoxylum bungeanum extract is 50 mg:200 mg:2 mL.

7. The method for simultaneously determining multiple mycotoxins in Zanthoxylum bungeanum based on QuEChERS-LC-MS / MS according to claim 1, characterized in that: The liquid chromatography conditions in step S4 are: Chromatographic column: Hypersil GOLD aQ column, 50 mm × 2.1 mm, 1.9 μm; Mobile phase: A-aqueous solution: 5mmol / L ammonium acetate, 0.1% formic acid; B-methanol; Column temperature: 40°C; Flow rate: 0.4 mL / min; Injection volume: 2 μL; Elution time: 5min; Gradient elution: 0-2 min, 10%→80% B; 2-2.5 min, 80%→100% B; 2.5-3.5 min, 100% B; 3.5-3.6 min, 100%→10% B; 3.6-5 min, 10% B.

8. The method for simultaneously determining multiple mycotoxins in Zanthoxylum bungeanum based on QuEChERS-LC-MS / MS according to claim 1, characterized in that: The triple quadrupole mass spectrometry conditions in step S4 are: Monitoring mode: Multiple reaction monitoring; Ionization method: electrospray ionization ESI; Capillary voltage: 5500V, -4500V; Ion source temperature: 550°C; Ion source gas 1: 276 kPa; Ion source gas 2: 276 kPa; Curtain gas: 276kPa.

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