Detection method of zearalenone and derivatives thereof

Abstract

The invention relates to the technical field of analysis and detection, in particular to a method for detecting zearalenone (ZEN) and derivatives thereof. According to the detection method, zearalenone and nine derivatives thereof can be detected at the same time, the linear relation of the detection method is good, the detection limit meets the experimental requirements, the accuracy and precision are high, the stability is good, the purpose of detecting multiple modified ZEN mycotoxins at the same time is achieved, and the detection method has good application prospects. The mycotoxin type of the modified ZEN, which is already confirmed to easily exist in the corn at present, is covered, a technical basis is provided for subsequent scientific evaluation of the mycotoxin pollution degree in the corn and formulation of a ZEN limit standard in the corn by a management department, and the application prospect is wide.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, and in particular to a method for detecting zearalenone and its derivatives. Background Art

[0002] Corn is highly susceptible to fungal contamination during its growth process, resulting in mycotoxin contamination. Due to its highly toxic, naturally occurring, stable, and non-degradable nature, it is the primary hazard affecting corn quality and safety, seriously impacting consumer safety. Zearalenone (ZEN) is one of the most serious mycotoxins, exhibiting estrogenic, genotoxic, immunotoxic, genotoxic, and carcinogenic properties. ZEN contamination in corn and its products poses a serious threat to the safety of industries such as food processing and feed. Recent studies have revealed that, in addition to free mycotoxins, grains are also contaminated with "masked mycotoxins." Modified ZEN retain the original structure and toxicity of the mycotoxins. After hydrolysis of the macromolecules in the digestive tracts of animals and humans, they release large amounts of the original toxin, which poses a serious health hazard. Furthermore, modified ZEN can transform back into its free form during digestion, increasing uncertainty about toxin contamination levels and posing significant economic losses and health risks to both animals and humans. The potential hazards of masked mycotoxins have attracted considerable attention, and they are now considered as contributing factors when setting limits for some mycotoxins in products. Therefore, accurate monitoring of ZEN contamination levels is crucial to reducing and controlling the hazards of toxins in corn and related products and protecting the health of animals and humans.

[0003] Currently, the detection of modified ZEN in corn mainly uses high-performance liquid chromatography-tandem mass spectrometry / mass spectrometry. However, the modified forms of modified ZEN detected are relatively few, only the alcohol and enol metabolites of ZEN, and glycoside and sulfate metabolites are not involved. Therefore, omissions are easy to occur when analyzing the degree of ZEN contamination in corn, resulting in the inability to fully assess the contamination of zearalenone and its derivatives in corn. Summary of the Invention

[0004] In order to solve the above technical problems, the present invention provides a method for detecting zearalenone and its derivatives, comprising:

[0005] The sample to be tested containing zearalenone and / or zearalenone derivatives is mixed with an internal standard and an extract, and then extracted by shaking. Anhydrous magnesium sulfate and anhydrous sodium acetate are then added, and the mixture is shaken and mixed before centrifugation. The first supernatant is mixed with octadecylsilane and purified using a QuEChERS cleanup method (i.e., a fast, simple, inexpensive, efficient, durable, and safe sample pretreatment method). The purified second supernatant is taken, nitrogen-purged, and then redissolved with a redissolving solution, filtered, and then detected by liquid chromatography-tandem mass spectrometry. The extract and redissolving solution are acetonitrile aqueous solutions.

[0006] The present invention finds that the use of anhydrous magnesium sulfate and anhydrous sodium acetate can separate the acetonitrile phase from the aqueous phase while reducing the water content in the acetonitrile phase, thereby increasing the acetonitrile phase content and making it easier to extract the fungal toxins of the organophilic reagent.

[0007] Compared with the conventional method of direct purification of the extract, the present invention found that the purifier C 18 It can remove non-polar interfering substances while ensuring the recovery rate during the analysis process and reducing the loss of purification.

[0008] Preferably, the internal standard is [ 13 C 18 ]-ZEN internal standard.

[0009] Preferably, the amount of the internal standard added is 100 to 200 microliters.

[0010] Preferably, the extracting solution is a 70% to 90% (v / v) acetonitrile aqueous solution; and / or, the reconstitution solution is a 2% to 8% (v / v) (preferably 4% to 6%) acetonitrile aqueous solution.

[0011] In addition, the present invention also found that controlling the volume percentage of acetonitrile in the extract to 70% to 90% (most preferably 80%) can ensure effective extraction of ZEN and its derivatives in corn while extracting as little interfering impurities as possible.

[0012] Preferably, the extract does not contain formic acid.

[0013] Preferably, the mass volume ratio of the sample to be tested to the extract is 1 g: (3-5) mL;

[0014] and / or, the mass ratio of anhydrous magnesium sulfate to anhydrous sodium acetate is (3-5):1;

[0015] And / or, the volume mass ratio of the first supernatant to octadecylsilane is 1 mL: (40-60) mg.

[0016] Preferably, the chromatographic column of the liquid chromatography is C 18 The chromatographic column has the specifications of 100×2.1 mm and 2.6 μm.

[0017] Preferably, the mobile phase of the liquid chromatography comprises mobile phase A and mobile phase B, mobile phase A is 0.05% to 0.15% (v / v) formic acid aqueous solution, and mobile phase B is acetonitrile.

[0018] Preferably, the gradient elution conditions of liquid chromatography are as follows:

[0019]

[0020] Preferably, the column temperature of the liquid chromatography is 35° C. to 40° C.; and / or the flow rate is 0.2 to 0.4 mL / min.

[0021] Preferably, the mass spectrometry scanning mode is negative ion scanning, and the detection mode is multiple reaction monitoring.

[0022] Preferably, the ionization mode of mass spectrometry is electrospray ionization, with spray voltage: 4200-4800 V, atomization temperature: 520° C.-580° C.; curtain gas: 32-38 L / h, nebulizing gas: 45-55 mL / min, and auxiliary gas: 45-55 mL / min.

[0023] Preferably, the sample to be tested includes: zearalenone (ZEN), α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalanone (ZAN), α-zearalenol-4-glucoside (α-ZEL-Glu), β-zearalenol-4-glucoside (β-ZEL-Glu), zearalanone-4-glucoside (ZEN-4-O-β-D-glucopyranoside, ZEN-Glu), zearalenone-4-sulfate ammonium salt (Zearalenone4-Sulfate Ammonium Salt, ZEN-4-Sul) at least one.

[0024] Preferably, the sample to be tested is a crushed corn sample to be tested.

[0025] Preferably, the corn sample to be tested is prepared by pulverizing and passing through a 20-mesh sieve.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention provides a method for detecting zearalenone and its derivatives, which can simultaneously detect zearalenone and its nine derivatives. The detection method of the present invention has good linearity, a detection limit that meets experimental requirements, high accuracy and precision, and good stability, thereby achieving the purpose of simultaneously detecting multiple modified ZEN mycotoxins, covering the types of modified ZEN mycotoxins that have been confirmed to be easily present in corn. This method provides a technical basis for subsequent scientific assessments of the degree of mycotoxin contamination in corn and for management departments to formulate ZEN limit standards in corn, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the total ion current chromatogram of 10 fungal toxins.

[0029] Figure 2 This is the characteristic ion chromatogram of zearalenone.

[0030] Figure 3 This is the characteristic ion chromatogram of zearalanol.

[0031] Figure 4 This is the characteristic ion chromatogram of zearalenol and zearalanone.

[0032] Figure 5 This is the characteristic ion chromatogram of zearalenol-4-glucoside.

[0033] Figure 6 This is the characteristic ion chromatogram of zearalenone-4-glucoside.

[0034] Figure 7 This is the characteristic ion chromatogram of zearalenone-4-sulfate ammonium salt.

[0035] Figure 8 This is a diagram of the screening results of extraction solvents; different lowercase letters indicate statistical differences.

[0036] Figure 9 This is a figure showing the screening results of the amount of formic acid added to the extraction solvent; different lowercase letters indicate statistical differences.

[0037] Figure 10 This is a diagram of the screening results of purification materials.

[0038] Figure 11 This is a quantitative screening result chart.

[0039] Figure 12 This is the screening result diagram of the composite salting-out reagent. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. In the embodiments provided in this specification, if specific techniques or conditions are not specified, they are carried out according to the techniques or conditions described in the literature in this field, or according to the product specifications. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be purchased through regular channels. The water used in the following examples is all first-class water specified in GB / T6682.

[0041] Example 1

[0042] This embodiment provides a method for detecting zearalenone and its derivatives, and the steps are as follows:

[0043] 1. Reagents and Materials

[0044] 1.1 Reagents

[0045] Acetonitrile (CH3CN): chromatographic grade; methanol (CH3OH): chromatographic grade; acetic acid: chromatographic grade; formic acid: chromatographic grade.

[0046] 1.2 Standard solution

[0047] (1) Standard substances: 10 mycotoxins including zearalenone (ZEN), α-zearalanol (α-ZAL), β-zearalanol (β-ZAL), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalenone (ZAN), α-zearalenol-4-glucoside (α-ZEL-Glu), β-zearalenol-4-glucoside (β-ZEL-Glu), zearalenone-4-glucoside (ZEN-Glu), zearalenone-4-sulfate ammonium salt (ZEN-4-Sul), [ 13 C 18 The purity of the internal standard zearalenone was greater than 99%, and both were purchased from Qingdao Pribolab Company.

[0048] (2) Standard stock solution (100 μg / mL): Accurately weigh appropriate amounts of 10 mycotoxin standards into a 10 mL volumetric flask, dissolve them in methanol and make up to volume to prepare a standard stock solution with a mass concentration of 100 μg / mL. Store at -20°C in the dark.

[0049] (3) Mixed standard intermediate solution: Accurately pipette 0.1 mL of each of the 10 standard stock solutions into a 10 mL volumetric flask to prepare a mixed standard intermediate solution with a mass concentration of 1 μg / mL. Dilute to volume with methanol and store at -18°C in the dark.

[0050] (4) Mixed standard working solution: Accurately pipette an appropriate amount of the mixed standard intermediate solution into the same volumetric flask, and use the initial mobile phase solution to prepare a series of standard working solutions with different mass concentrations. The mass concentration gradient is 0.1, 0.5, 1, 5, 20, and 100 μg / L.

[0051] (5) Internal standard intermediate solution: Accurately draw 1.0 mL of 10 μg / L [ 13 C 18 ]-ZEN internal standard solution was diluted to volume with methanol in a 10 mL volumetric flask to prepare an internal standard intermediate solution with a mass concentration of 1 μg / mL and stored at -18°C in the dark.

[0052] (6) Internal standard working solution: Accurately draw 1.0 mL of 1.0 μg / L [ 13 C 18 ]-ZEN internal standard intermediate solution was diluted to volume with methanol in a 10 mL volumetric flask to prepare an internal standard working solution with a mass concentration of 0.1 μg / mL and stored at -18°C in the dark.

[0053] 2. Instruments and equipment

[0054] The liquid chromatography-mass spectrometry / mass spectrometer is equipped with an electrospray ionization source (ESI); an analytical balance with a mass of 0.01 g; an oscillator; a centrifuge with a speed of not less than 4000 r / min; a vortex mixer; a hammer mill; and a nitrogen blower.

[0055] 3. Operation method

[0056] 3.1 Sample preparation: Grind the sample with a hammer cyclone mill, pass it through a 20-mesh sieve, and mix it for later use.

[0057] 3.2 Sample Extraction: Weigh 5 g (accurate to 0.01 g) of ground sample into a 50 mL centrifuge tube. Add 100 μL of internal standard working solution, followed by 20 mL of 80% acetonitrile in water (v:v = 80:20). After vortexing for 40 minutes, add 6 g of anhydrous magnesium sulfate (MgSO4) and 1.5 g of anhydrous sodium acetate (CH3COONa). Vortex for 1 minute. Centrifuge at 4000 rpm / min for 5 minutes. Remove the supernatant and wait for purification.

[0058] 3.3 Sample purification: Pipette 10 mL of the sample to be purified into a 15 mL centrifuge tube and add 50.0 mg C 18The mixture was shaken for 1 min, centrifuged, and 4 mL of the supernatant was accurately transferred to a glass test tube. The mixture was dried with nitrogen at 40°C, and redissolved in 1.0 mL of 5% acetonitrile aqueous solution. The mixture was mixed, filtered through a 0.22 μm filter membrane, and subjected to liquid chromatography-tandem mass spectrometry.

[0059] 4. Instrument conditions

[0060] 4.1 HPLC conditions

[0061] Chromatographic column Kinetex C 18 Column (100 × 2.1 mm, 2.6 μm) or equivalent; column temperature: 35°C; injection volume: 2 μL; flow rate: 0.4 mL / min; mobile phase and gradient elution conditions are shown in Table 1. Mobile phase A: 0.1% (v / v) formic acid in water; mobile phase B: acetonitrile.

[0062] Table 1 Gradient elution conditions

[0063]

[0064]

[0065] 4.2 Mass spectrometry reference conditions Ionization mode: electrospray ionization (ESI); spray voltage: 4500 V; atomization temperature: 550°C; curtain gas: 35 L / h; nebulizing gas: 50.0 mL / min, auxiliary gas: 50.0 mL / min. Before use, adjust the flow rates of each gas to ensure that the mass spectrometry sensitivity meets the detection requirements; scanning mode: negative ion scanning; detection mode: multiple reaction monitoring. For detailed parameters, see Table 2.

[0066] Table 2 Multiple reaction monitoring parameters

[0067]

[0068]

[0069] Note: * indicates quantitative ion.

[0070] 5. Qualitative and quantitative determination

[0071] 5.1 Qualitative determination

[0072] The standard solution and sample were injected separately and measured under the above instrument conditions. Under the same experimental conditions, the chromatographic retention time of the analyte in the sample was the same as that of the mixed standard solution, and the selected ions appeared in the sample chromatogram after background subtraction. Comparison of the abundance ratio of the selected ions with the abundance ratio of the corresponding ions in the mixed matrix standard solution was performed. If the value was within the allowable range (see Table 3), the corresponding analyte was determined to be present in the sample.

[0073] Table 3 Maximum allowable deviation of relative ion abundance in qualitative analysis

[0074] Relative ion abundance ＞50 ＞20～50 ＞10～20 ≤10 Maximum allowed deviation ±20 ±25 ±30 ±50

[0075] 5.2 Quantitative determination

[0076] Inject the standard working solution separately, draw the standard working curve with the peak area as the ordinate and the mass concentration of the standard working solution as the abscissa, and quantify the samples using the standard working curve. The response values of the analytes in the sample solutions should be within the linear range of the instrument.

[0077] Calculation formula for mycotoxin content:

[0078]

[0079] X - the content of the tested ingredient in the test sample, in micrograms per kilogram (μg / kg);

[0080] c―The concentration of the measured component solution obtained from the standard working curve, in nanograms per milliliter (ng / mL);

[0081] v - the volume of the test solution, in milliliters (mL);

[0082] m-the weight of the test sample, in grams (g);

[0083] f― represents the dilution factor.

[0084] The blank value should be subtracted from the calculated result. The blank value is obtained through a blank test; a blank test is performed according to the test sample preparation steps, except that no sample is added. The calculated result should be rounded to two significant figures. In the test mass spectrum, no mass peaks should be detected that match the reference sample's chromatographic peaks in terms of retention time, mass-to-charge ratio, and relative abundance ratio of the selected qualifier ions.

[0085] Example 2

[0086] This example investigates the methodological aspects of the detection method of Example 1.

[0087] 1. Linear relationship and detection limit

[0088] Using 5% acetonitrile aqueous solution as the solvent, mixed standard working solutions with concentration gradients of 0.1, 0.5, 1.0, 5.0, 10, 20, 100, and 200 ng / mL were prepared according to the method for preparing the mixed standard working solution in Example 1. The extraction, cleanup, and detection conditions of Example 1 were followed. A linear regression fit was performed, with the peak area (Y) of the target mycotoxin chromatographic peak as the ordinate and the injection concentration (X, μg / L) as the abscissa. The limit of detection (LOD) was determined as the concentration corresponding to a characteristic ion signal-to-noise ratio (S / N) > 3. The results are shown in Table 4.

[0089] Table 4 Regression equation, linear range and detection limit

[0090]

[0091]

[0092] As can be seen from the table, the 10 mycotoxins in 5% acetonitrile solution showed a good linear relationship with their peak areas in the range of 0.1-100 ng / mL, with a correlation coefficient greater than 0.997. The detection limits of α-ZEL-Glu were 1.0 μg / kg, and the detection limits of ZEN, α-ZAL, β-ZAL, α-ZEL, β-ZEL, ZAN, β-ZEL-Glu, ZEN-Glu, and ZEN-4-Sul were 0.5 μg / kg, indicating high sensitivity.

[0093] 2. Method accuracy

[0094] Negative sample matrix solution: Take a blank sample with no mycotoxins detected (referred to as the negative sample) and prepare a negative sample matrix solution according to the preparation method of the test solution in Example 1. Preparation of the spiked test solution: Add the mixed standard intermediate solution to the negative sample matrix solution to a concentration level of 20 μg / L. Add three different concentration levels of mixed standard working solutions at low, medium, and high levels to the negative sample and process according to the preparation method of the test solution. The assay was performed according to the detection conditions of Example 1, and the spiked recovery and relative standard deviation (RSD) were calculated. The results are shown in Table 5.

[0095] Table 5 Spike recovery

[0096]

[0097]

[0098] As can be seen from the table, the recoveries of the 10 mycotoxins were all between 83.0% and 112.2%, and the relative standard deviations (RSDs) were between 2.0% and 11.4%, indicating a high accuracy.

[0099] 3. Method precision

[0100] Intra-day precision examines the reproducibility of assay results using the same reagents on the same day. Ten mycotoxin standard working solutions at a concentration of 20.0 μg / L were added to the negative sample matrix solution. Six injections were made consecutively. The assay was performed according to the detection conditions of Example 1. The relative standard deviation (RSD) was calculated. The results are shown in Table 6.

[0101] Table 6 Precision

[0102]

[0103]

[0104] As can be seen from the table, the relative standard deviations (RSDs) of the 10 mycotoxins were all less than 15.0%, indicating that the detection method of the present invention has good precision.

[0105] 4. Method stability

[0106] Ten mycotoxin standard working solutions at a concentration of 20.0 μg / L were added to the negative sample matrix solution. Samples were injected at different times over 48 hours and assayed according to the detection conditions of Example 1. The stability of the ten mycotoxins in the sample was investigated by calculating the relative standard deviations (RSDs) of the peak areas of the ten compounds. The results are shown in Table 7.

[0107] Table 7 Stability

[0108]

[0109] As can be seen from the table, the 10 mycotoxins were relatively stable within 48 hours, with relative standard deviations (RSD) less than 9.42%.

[0110] 5. Method Specificity

[0111] Ten kinds of ZEN and its derivatives were added to the negative sample matrix solution and the test was carried out according to the detection conditions of Example 1. The total ion current was shown in FIG. Figure 1 As can be seen from the figure, the detection of 10 ZEN and its derivatives is not interfered with, indicating that the method has good specificity. The characteristic ion chromatograms of 10 ZEN and its derivatives are shown in Figures 2 to 7 The retention time of zearalenone was 4.61min, the retention time of α-zearalanol was 4.02min, the retention time of β-zearalanol was 3.58min, the retention time of α-zearalenol was 4.11min, the retention time of β-zearalenol was 3.63min, the retention time of zearalenone was 4.55min, the retention time of α-zearalenol-4-glucoside was 2.51min, the retention time of β-zearalenol-4-glucoside was 2.24min, the retention time of zearalenone-4-glucoside was 2.80min, and the retention time of zearalenone-4-sulfate ammonium salt was 5.97min.

[0112] Example 3

[0113] Based on the detection conditions of Example 1, this example uses a single-factor variable experiment to investigate the effects of changes in various parameters on the detection results. Except for the optimization of mass spectrometry conditions using standard substance solutions, all other conditions that are optimized use spiked test sample solutions.

[0114] 1. Screening of mass spectrometry conditions

[0115] A mixed standard intermediate solution of 10 mycotoxins was diluted to a standard solution of a defined concentration and injected into the mass spectrometer at a flow rate of 10 μL / min. The standard substances were first subjected to primary mass spectrometry analysis (parent ion scanning) at the positive and negative sources to obtain molecular ion peaks. The corresponding parent ion peaks were then selected and their daughter ions were subjected to secondary mass spectrometry analysis (daughter ion scanning) to obtain fragment ion information. The resulting mass spectrometry parameters were then optimized to determine the appropriate ion mode, parent ion, daughter ion, and corresponding collision energy and cone voltage for each mycotoxin.

[0116] The ionization effects of the analyte in positive and negative source modes were compared. It was found that in negative source mode, the parent ions 317.0, 319.1, 321.1, 397.1, 479.1, and 481.1 of the analyte had a good response, but in positive source mode, the ionization effect was poor. Therefore, the detection method of the present invention further optimized the mass spectrometry method in negative source mode. Because α-ZAL and β-ZAL are isomers, they have the same parent ions and daughter ions. α-ZEL, β-ZEL, and ZAN are isomers, and their parent ions and daughter ions are the same. α-ZEL-Glu and β-ZEL-Glu are isomers, and their parent ions and daughter ions are the same, so further separation is required when optimizing the liquid phase conditions.

[0117] 2. Screening of extraction solvents

[0118] 100% acetonitrile (ACE), 80% acetonitrile water, 50% acetonitrile water, and 10% acetonitrile water were selected as extraction solvents, and the recovery rate of each extract was investigated. Figure 8 As shown in the figure, a comparison of different extraction solution concentrations revealed a certain impact on the mycotoxin extraction efficiency. With decreasing organic reagent concentration, the extraction recovery rate gradually decreased. However, extraction with only organic reagents was not conducive to sufficient infiltration of the corn sample. Considering both economic cost and extraction efficiency, 80% acetonitrile in water was selected as the extraction reagent.

[0119] 3. Screening of the amount of formic acid added to the extraction solvent

[0120] 0.1%, 0.5%, 1.0%, and 2.0% (v / v) formic acid (FA) were added to the above-determined extraction solvent to investigate the effect of formic acid on the extraction efficiency. Figure 9As shown in the figure, adding formic acid to the extract actually inhibited the ionization efficiency of the mycotoxin to be tested, and the recovery rate decreased. Taking into account the economic cost and extraction efficiency, 80% acetonitrile aqueous solution was selected without adding formic acid.

[0121] 4. Purification material screening

[0122] The extract was purified by QuEChERS method, and the purification of N-propylethylenediamine (PSA), octadecylsilane (C 18 ), graphitized carbon black (GCB), neutral alumina (AlO2) and their different combinations on the purification effect of the extract. 18 The addition amount of PSA and AlO2 is 50mg, and the addition amount of GCB is 25mg. Each purifier is used alone and 50mg C 18 +50mg PSA, 50mg C 18 +50mg PSA+50mg AlO2,50mg C 18 The purification effect of the purifier was analyzed by combining 50 mg PSA and 25 mg GCB.

[0123] The results are as follows Figure 10 As shown in the figure, through comparative analysis, it was found that different purification combinations can reduce the interference in the analysis process, but the purification combination with the addition of PSA will cause the recovery rate of the four mycotoxins α-ZEL-Glu, β-ZEL-Glu, ZEN-Glu and ZEN-4-Sul to be low. Therefore, the purification agent C was selected. 18 As a QuEChERS purification material.

[0124] 5. Screening of quantitative methods

[0125] The effect of corn matrix on the response values of 10 mycotoxins was further investigated. The matrix solution before purification was obtained by corn according to the extraction method. The matrix solution was prepared into a 20 μg / L matrix-matched standard solution after treatment at the following levels: no concentration, 2-fold concentration, 5-fold concentration, 10-fold concentration, and 20-fold concentration. The content was calculated using the standard solution prepared with 5% acetonitrile under the analytical conditions. The results are shown in Figure 2. Figure 11 As shown in the figure, it was found that corn matrix has an inhibitory effect on 10 fungal toxins. The inhibitory effect increases with the increase of matrix concentration. Therefore, in order to eliminate the influence of the matrix on the response value of the test component, [ 13 C 18 ]-ZEN internal standard, and the analytes were quantified by the internal standard method.

[0126] 6. Screening of composite salting-out reagents

[0127] 6g anhydrous MgSO4 and 1.5g anhydrous CH3COONa, 6g anhydrous MgSO4 and 1.5g sodium chloride (NaCl), 6g sodium sulfate (Na2SO4) and 1.5g anhydrous CH3COONa were selected as composite salting-out reagents for extraction, and after purification, liquid chromatography-tandem mass spectrometry was performed. The results are shown in Figure 2. Figure 12 As shown, the combination of 6g anhydrous MgSO4 and 1.5g anhydrous CH3COONa has the best extraction effect. The inventors also found through experiments that the combination of granular anhydrous magnesium sulfate and anhydrous sodium acetate is better than the powder type.

[0128] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A method for detecting zearalenone and its derivatives, characterized in that: include: The sample to be tested containing zearalenone and / or zearalenone derivatives is mixed with the internal standard and the extract, and then subjected to oscillation extraction. Anhydrous magnesium sulfate and anhydrous sodium acetate are then added, and the mixture is oscillated and mixed before centrifugation. The first supernatant is mixed with octadecylsilane and purified using the QuEChERS cleanup method. The purified second supernatant is taken, nitrogen-purged, and then redissolved with a redissolving solution. The sample is filtered and then detected by liquid chromatography-tandem mass spectrometry. The extract and the redissolving solution are acetonitrile aqueous solutions.

2. The detection method according to claim 1, wherein The internal standard is [ 13 C 18 ]-ZEN internal standard.

3. The detection method according to claim 1, wherein The extracting solution is a 70% to 90% (v / v) acetonitrile aqueous solution; and / or the reconstitution solution is a 2% to 8% (v / v) acetonitrile aqueous solution.

4. The detection method according to claim 1, wherein The mass volume ratio of the sample to be tested to the extract is 1 g: (3-5) mL; and / or, the mass ratio of anhydrous magnesium sulfate to anhydrous sodium acetate is (3-5):1; And / or, the volume mass ratio of the first supernatant to octadecylsilane is 1 mL: (40-60) mg.

5. The detection method according to claim 1, wherein The chromatographic column for liquid chromatography is C 18 The chromatographic column has the specifications of 100×2.1 mm and 2.6 μm.

6. The detection method according to claim 5, characterized in that The mobile phase of liquid chromatography includes mobile phase A and mobile phase B. Mobile phase A is 0.05%~0.15% (v / v) formic acid aqueous solution, and mobile phase B is acetonitrile.

7. The detection method according to claim 6, characterized in that The gradient elution conditions for liquid chromatography were as follows:

8. The detection method according to claim 7, characterized in that The column temperature of the liquid chromatography is 35°C to 40°C; and / or the flow rate is 0.2 to 0.4 mL / min.

9. The detection method according to claim 1, wherein The mass spectrometry scanning mode was negative ion scanning, and the detection method was multiple reaction monitoring; And / or, the ionization mode of the mass spectrometer is electrospray ionization, the spray voltage is: 4200~4800V, the nebulization temperature is: 520℃~580℃; the curtain gas is: 32~38 L / h, the nebulizer gas is: 45~55 mL / min, and the auxiliary gas is: 45~55 mL / min.

10. The detection method according to claim 1, characterized in that The sample to be tested includes: at least one of: zearalenone, α-zearalanol, β-zearalanol, α-zearalenol, β-zearalenol, zearalenone, α-zearalenol-4-glucoside, β-zearalenol-4-glucoside, zearalenone-4-glucoside, and zearalenone-4-sulfate ammonium salt.

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