Method for determining prochloraz and thiabendazole in plant-derived food

Abstract

The application discloses a method for determining prothioconazole and chlorothalonil in plant source food, which comprises the following steps: S1, preparing a sample to be measured, diluting or dissolving the sample with water, high-speed centrifugation, and taking supernatant; S2, purifying the sample to be measured, using a mixed cation exchange solid phase extraction purification column, washing the column with water, vacuum drying, and then eluting with methanol; S3, derivative of the sample to be measured, adding O-bistrimethylsilyl trifluoroacetamide + trimethylchlorosilane; S4, purification of the derivative, adding saturated sodium chloride solution, and taking the lower dichloromethane layer; S5, detecting the sample to be measured, feeding the derivative sample solution and standard working solution into a gas chromatography-tandem mass spectrometer, taking the concentration of the pesticide derivative in the standard solution as the abscissa, the peak area of the quantitative ion pair as the ordinate, drawing a working curve, and quantifying by an external standard method. The application can check the plant source food, improve the safety of the plant source food circulating on the market, and guarantee food safety standards.

Description

TECHNICAL FIELD

[0001] The present application relates to food detection, in particular a method for determining prothioconazole and clofencet in plant-derived food. BACKGROUND

[0002] Prothioconazole belongs to the organic heterocyclic fungicide, has strong inhibitory and killing effect on fungi and bacterial diseases, can effectively prevent and control bacterial diseases such as rice bacterial leaf blight and vegetable bacterial angular spot, and at present, only a small amount of literature reports the determination of prothioconazole in cucumber, tomato, apple, grape and citrus fruits by liquid chromatography-tandem mass spectrometry (LC-MS-MS). GB2763-2021 "National Food Safety Standard Maximum Residue Limits of Pesticides in Food" and GB2763.1-2022 "National Food Safety Standard Maximum Residue Limits of 112 Pesticides such as 2,4-D Butyric Acid Sodium Salt in Food" have made provisions for the residue of prothioconazole in cereals, vegetables, fruits, which is limited to 0.05-1mg / kg.

[0003] Clofencet is a systemic conductive benzoic acid herbicide, which is also a metabolic product of herbicide clofencet dimethyl in plant body, and is often used for controlling annual and perennial broadleaf weeds in cereals and gramineous crops. There is no literature report on the determination of clofencet residue. GB2763-2021 has made limit provisions for clofencet in cereals, vegetables, fruits, beverages, oils and oilseeds, medicinal plants, sugar crops, nuts, edible fungi, condiments, etc., all of which are 0.01mg / kg.

[0004] At present, there is no detection standard for the above two pesticides, and there is no literature report on fruit juice, vegetable juice, tea beverage, tea concentrate juice and instant tea, so it is necessary to establish a method for simultaneously determining the above two pesticides in plant-derived processed food, and to provide technical support for the quality and safety detection of related food. SUMMARY

[0005] In view of the deficiencies in the prior art, the purpose of the present application is to provide a method for determining prothioconazole and clofencet in plant-derived food, which can check plant-derived food, improve the safety of plant-derived food circulating on the market, and ensure food safety standards.

[0006] To achieve the above purpose, the present application provides the following technical scheme: a method for determining prothioconazole and clofencet in plant-derived food, characterized in that it comprises the following steps:

[0007] S1. Prepare the sample to be tested, dilute the fruit juice or vegetable juice or tea beverage or tea concentrate juice sample with water or dissolve the instant tea sample with water, then centrifuge at high speed, and take the supernatant for purification;

[0008] S2. Purify the sample to be tested, take the supernatant and purify it by using a mixed cation exchange solid phase extraction column through gravity, then wash the column with water, discard the effluent, vacuum dry, then elute with methanol, collect all the eluent in a calibrated centrifuge tube; place the eluent in a water bath, nitrogen blow to dry, then redissolve with dichloromethane;

[0009] S3. Derive the sample to be tested, take the above-mentioned dichloromethane redissolved material, add derivative reagent O-bistrimethylsilyl trifluoroacetamide + trimethylchlorosilane, derive at 70℃ for 30 minutes; take out and cool to room temperature, then purify;

[0010] S4. Purification of the derivative, add saturated sodium chloride solution to the derivative, vortex thoroughly, then stand at room temperature not higher than 25℃ for more than 30 minutes, then high-speed centrifuge, take the lower dichloromethane layer for gas chromatograph-mass spectrometer determination;

[0011] S5. Detect the sample to be tested, separately inject the sample to be tested after derivation and the standard working solution on the gas chromatograph-mass spectrometer, draw a working curve with the concentration of pesticide derivative in the standard solution as the abscissa and the peak area of the quantitative ion pair as the ordinate, and quantitatively determine by external standard method;

[0012] Prepare the standard solution before any of S1-S5, prepare the mixed standard solution of different concentrations by diluting the thiamethoxam and chlorothene stock solution with dichloromethane, and derive it as above S3 and S4.

[0013] As a further improvement of the present application, the solid phase extraction column filler is a polymer with styrene-based-divinylbenzene as the matrix and mixed cation exchange groups bonded.

[0014] As a further improvement of the present application, the solid phase extraction column is further activated with methanol and water in sequence before use.

[0015] As a further improvement of the present application, add saturated sodium chloride solution to the derivative product to remove excess derivative reagent.

[0016] As a further improvement of the present application, the parameters of the gas chromatograph-mass spectrometer are as follows:

[0017] Chromatographic conditions:

[0018] Chromatographic column: Agilent HP-5MS capillary column 30m*0.25mm*0.25μm; chromatographic column temperature program: 70℃ for 1min, increase to 220℃ at 25℃ / min and maintain for 1min, then increase to 300℃ at 15℃ / min and maintain for 6min; carrier gas: helium, purity 99.999%; carrier gas flow rate: 1.0mL / min; injection port temperature: 240℃; injection amount: 1μL; injection mode: splitless injection;

[0019] Mass spectrometry conditions:

[0020] Ion source temperature: 300℃; transfer line temperature: 280℃; collision gas: argon; scan type: multiple reaction monitoring mode; qualitative and quantitative ion pairs of two pesticide derivative products, collision energy are as follows

[0021]

[0022] The quantitative ion pairs are marked in the table.

[0023] The beneficial effects of the present application, the scheme can reduce the boiling point by the method of derivatization, improve the stability and detection sensitivity of analysis, can overcome the problem of high polarity and large boiling point of prothioconazole and chlorothion, which leads to the difficulty of direct analysis by gas chromatography. Through the scheme, it can provide support for related food safety supervision and detection, improve the safety of plant source food circulating in the market, and ensure food safety standards. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 MRM spectrum of prothioconazole derivative product.

[0025] Figure 2 MRM spectrum of chlorothion derivative product.

[0026] Figure 3 Secondary mass spectrum of prothioconazole derivative product and mass spectrum possible cracking pathway analysis.

[0027] Figure 4 Secondary mass spectrum of chlorothion derivative product and mass spectrum possible cracking pathway analysis. DETAILED DESCRIPTION

[0028] The present application will be further described in detail below with reference to the embodiments given in the accompanying drawings.

[0029] Referring to Figures 1-4 , as shown,

[0030] Example 1

[0031] The present embodiment provides a method for preparing a GC-MS-MS standard curve of prothioconazole and chlorothion.

[0032] (1) Preparation of standard solution

[0033] Take 0.1 mL of commercial prothioconazole and chlorothion stock solution (1000 μg / mL) in a 10 mL volumetric flask, and dilute to 10 mL with dichloromethane to prepare a 10 μg / mL mixed standard solution.

[0034] Take the appropriate amount of 10 μg / mL of mixed label use liquid, with dichloromethane in turn dilution preparation of 500, 200, 100, 50, 20, 10, 5 μg / L of serial concentration mixed label, for making standard curve.

[0035] (2) Derivatization and purification of derivative

[0036] Take the above prepared 10 μg / mL, 500 μg / L, 200 μg / L, 100 μg / L, 50 μg / L, 20 μg / L, 10 μg / L, 5 μg / L of mixed label solution 1 mL in 10 mL glass centrifuge tube with cover, add 100 μL of derivatization reagent O-bistrimethylsilyl trifluoroacetamide (BSTFA) + trimethylchlorosilane (TMCS) (99+1), put into electric heating drying box at 70℃ for 30 minutes.

[0037] Take out the centrifuge tube and cool to room temperature, then add 1 mL of saturated sodium chloride solution to the derivative, cover the cover and vortex thoroughly, then stand at room temperature not higher than 25℃ for 30 minutes or more, then centrifuge at high speed and take the lower dichloromethane layer solution for the next step of GC-MS-MS determination.

[0038] (3) Optimization of multiple reaction monitoring conditions

[0039] Take the derivative of the above 10 μg / mL mixed label use liquid for primary mass spectrometry full scan, scan mass number range 50-500u; according to the primary mass spectrometry full scan results, select one ion with relatively large relative abundance as the parent ion, the parent ions of thiabendazole derivative and chlorothion acid derivative are m / z 233.1 and m / z 433.0 respectively, carry out secondary mass spectrometry sub-ion full scan, then select two sub-ions with relatively large relative abundance, finally optimize the collision voltage of the selected two pairs of parent and daughter ion pairs. The secondary mass spectrometry sub-ion full scan graphs of two pesticide derivatives are shown in Figure 1 and Figure 2 , Figure 1 and Figure 2 The possible cleavage pathways of the two pesticide derivative sub-ions are also provided. The optimized multiple reaction monitoring conditions are shown in Table 1.

[0040] Table 1

[0041]

[0042] Among them, * is marked as quantitative ion pair.

[0043] (4) GC-MS-MS detection

[0044] The gas phase conditions are: chromatographic column Agilent HP-5MS capillary column 30 m x 0.25 mm x 0.25 μm; chromatographic column temperature program: 70°C for 1 min, 25°C / min to 220°C and keep for 1 min, 15°C / min to 300°C for 6 min; carrier gas: helium, purity 99.999%; carrier gas flow rate: 1.0 mL / min; injection port temperature: 240°C; injection volume: 1 μL; injection mode: splitless injection. The mass spectrometry conditions are: ion source temperature: 300°C; transfer line temperature: 280°C; collision gas: argon; scan type: multiple reaction monitoring mode, the conditions are shown in Table 1.

[0045] The derivatives of the mixed standard at the concentrations of 500, 200, 100, 50, 20, 10, 5 μg / L are analyzed in turn under the above instrument conditions, and the MRM typical spectra of the two pesticide derivatives at 100 μg / L are shown in Figure 3 and Figure 4 .

[0046] (5) Preparation of standard curve

[0047] The external standard curve is plotted with the response peak area of the quantitative ion pair of the two pesticide derivatives as the abscissa and the concentration as the ordinate, which is used for sample detection and quantification.

[0048] Based on the embodiment, first, the thiamethoxam and chlorothalonil are subjected to silanization derivatization by using the derivatization reagent O-bistrimethylsilyl trifluoroacetamide (BSTFA) + trimethylchlorosilane (TMCS) (99+1), through the derivatization, the polarity of the thiamethoxam and the chlorothalonil is effectively reduced, the produced derivative is stable and beneficial to gasification, and higher sensitivity can be obtained in the GC-MS-MS detection.

[0049] Second, after the completion of the derivatization reaction, in order to remove the excess derivatization reagent to avoid the pollution of the instrument, the derivative is washed with saturated sodium chloride solution, and white crystalline substances are precipitated after standing, which are removed by high-speed centrifugation, so that the excess derivatization reagent is effectively prevented from entering the instrument to pollute the liner, the chromatographic column and the ion source, the service life of the chromatographic column is greatly prolonged, and the frequency of instrument maintenance is reduced.

[0050] Example 2

[0051] The embodiment provides a pretreatment method of plant source samples such as fruit juice, vegetable juice, tea beverage, tea concentrate, instant tea and the like.

[0052] (1) Sample pretreatment

[0053] Each of 5 g of juice and vegetable juice samples was diluted with 5 mL of water in a 50 mL centrifuge tube, and then centrifuged at 9000 r / min, and the supernatant was purified. 5 g of tea beverage was diluted with 5 mL of water in a 50 mL centrifuge tube, and then purified. 2 g of tea concentrate was diluted with 8 mL of water in a 50 mL centrifuge tube, and then purified. 0.1 g of instant tea was dissolved in 10 mL of water in a 50 mL centrifuge tube, and then purified.

[0054] (2) Purification by solid phase extraction

[0055] A solid phase extraction column with a 150 mg / 6 mL size of a polymer material with a styrene-divinylbenzene matrix and a mixed cation exchange group was used, and was pre-activated with 6 mL of methanol and 6 mL of water. The pretreated sample solution to be purified was added to the column, and was naturally passed through the column by gravity. After the sample solution passed through the column, 6 mL of water was added to the column, and was vacuum-dried. Then, 1 mL of methanol was added to the column, and was passed through the column. After the methanol passed through the column, the effluent was discarded. Then, 6 mL of methanol was added to the column, and the effluent was collected in a 10 mL glass centrifuge tube. The collected effluent was blown dry with nitrogen in a 45°C water bath, and 1 mL of dichloromethane was added to the residue in the centrifuge tube to re-dissolve the residue, and was subjected to derivatization.

[0056] (3) Derivatization and purification of the derivative

[0057] The above 1 mL of dichloromethane re-dissolved material was subjected to derivatization and purification of the derivative according to (2) of the reference example 1.

[0058] Based on this example, first, the liquid sample (juice, vegetable juice, tea beverage, tea concentrate) was diluted by adding water, and the solid sample (instant tea) was dissolved, so that the thiamethoxam and prochloraz remaining in the sample when passing through the column could be better adsorbed.

[0059] Second, the column with a polymer material with a styrene-divinylbenzene matrix and a mixed cation exchange group was used, which could ensure good recovery of thiamethoxam and prochloraz while ensuring the purification effect.

[0060] Again, dichloromethane is used as a solvent in the derivatization process. On the one hand, dichloromethane can be used to re-dissolve the eluate of the solid phase extraction in the previous step after blowing dry, which can ensure that the two pesticides are completely re-dissolved and the recovery rate is ensured. On the other hand, using dichloromethane as a solvent, the derivative can obtain better sensitivity. The present application also compares the use of acetonitrile and n-hexane as solvents, respectively. The results show that the sensitivity of the derivative is greatly reduced when acetonitrile is used as a solvent. When n-hexane is used as a solvent, the sensitivity of the standard sample derivative is normal, but the residual solvent capacity is poor when the column-passed sample with added standard sample is blown dry with nitrogen. The target is wrapped by polar impurities, and n-hexane cannot dissolve the target, resulting in a low recovery rate. Dichloromethane can take both into account, so dichloromethane is preferably used as a solvent.

[0061] Example 3

[0062] The present embodiment provides a method for determining the added recovery content of benthival and chlorothion in plant source samples such as fruit juice, vegetable juice, tea beverage, tea concentrate, instant tea, etc.

[0063] Take negative samples of fruit juice, vegetable juice, tea beverage, tea concentrate, instant tea without benthival and chlorothion, add mixed standard samples for standard addition recovery test, add 3 concentrations, each concentration is 6 parallel, refer to the sample pretreatment in Example 2 to obtain the sample to be tested, and then refer to the instrument conditions in Example 1 for detection. The standard curve obtained in Example 1 is used for quantification, and the recovery rate and precision are calculated according to the amount of standard addition. The standard addition concentration, recovery rate and precision of various samples are shown in Table 2. The limit of quantification is determined by the signal-to-noise ratio of the derivative after standard addition in the sample, and the limit of quantification obtained in various samples is shown in Table 2.

[0064] Table 2: Recovery rate, precision (n=6) and limit of quantification

[0065]

[0066] It can be known from Table 2 that the sample pretreatment and instrument detection method provided by the application can quickly detect the thiabendazole and chlorophenoxy acid residues in fruit juice, vegetable juice, tea beverage, tea concentrate and instant tea, and the accuracy, precision and sensitivity of the method meet the requirements of pesticide residue detection. The quantitative limit of the two pesticides in fruit juice, vegetable juice, tea beverage and tea concentrate is less than 10 μg / kg. Instant tea is a granular solid beverage that is easy to dissolve in water without tea residue, which is processed through processes such as extraction, filtration, concentration and drying, and is made of fresh leaves as raw materials. The concentration multiple is large, and it is generally recommended to brew 1g of tea leaves with 200mL of boiled water (tea-water ratio 1:200). The quantitative limit of thiabendazole and chlorophenoxy acid in the solid particles of instant tea of the application is 50 μg / kg and 100 μg / kg, which is equivalent to 0.25 μg / L and 0.5 μg / L in tea soup, which is much lower than 10 μg / L. GB2763-2021 limits the amount of chlorophenoxy acid in beverage (including tea, fruit juice and vegetable juice) to 0.01 mg / kg, and does not make a limit provision for tea beverage and instant tea. GB2763-2021 does not make a limit provision for the five plant source products involved in the application. If the products without limit provision are executed according to the "uniform standard" of 0.01 mg / kg, the quantitative limit of the above-mentioned five plant source products in the application is also less than 0.01 mg / kg, which meets the detection requirements.

[0067] The above is only the preferred embodiment of the application, and the protection scope of the application is not limited to the above-mentioned embodiments. Any technical solution falling within the idea of the application shall fall within the protection scope of the application. It should be noted that for ordinary skilled persons in the art, some improvements and decorations without departing from the principles of the application shall also be considered as the protection scope of the application.

Claims

1. A method for determining thiabendazole and chlorothalonil in plant-derived food, characterized by, Comprising the following steps: ​ S1. Preparation of the sample to be tested, taking the juice or vegetable juice or tea beverage or tea concentrate sample with water dilution or taking the instant tea sample with water dissolution, high-speed centrifugation, and taking the supernatant for purification; S2. Purification of the sample to be tested, taking the filler as a polymer solid-phase extraction column with styrene-based-divinylbenzene as the matrix and bonded mixed cation exchange groups, pre-activating with methanol and water in turn; adding the pretreated sample solution to be purified to the column, passing the column naturally by gravity, and washing the column with water and vacuum drying after the whole sample solution passes through the column; Then add methanol to the column, and discard the effluent after the methanol passes through the column; Add methanol again for elution, and collect all the effluent; blow dry the collected effluent in a water bath with nitrogen, and redissolve the residue in the centrifuge tube with dichloromethane, and then derivatize; S3. Derivatization of the analyte, taking the above-mentioned dichloromethane redissolved material, adding derivatization reagent O-bistrimethylsilyl trifluoroacetamide + trimethylchlorosilane, and derivatizing at 70°C for 30 minutes; take out and cool to room temperature, and then purify; S4. Purification of the derivative, adding saturated sodium chloride solution to the derivative, vortexing thoroughly, and standing at room temperature not higher than 25°C for more than 30 minutes, and then taking the lower dichloromethane layer for gas chromatograph-tandem mass spectrometry determination after high-speed centrifugation; S5. Detection of the sample to be tested, respectively injecting the derivatized sample solution and the standard working solution into the gas chromatograph-tandem mass spectrometer, and drawing a working curve with the concentration of the pesticide derivative in the standard solution as the abscissa and the peak area of the quantitative ion pair as the ordinate, and quantifying by external standard method; Prepare the standard solution before any of steps S1-S5, and prepare the mixed standard solution of different concentrations by diluting the thiabendazole and chlorothene stock solution with dichloromethane, and the derivatization is the same as S3 and S4 above.

2. The method according to claim 1, wherein the plant-derived food is selected from the group consisting of tea, coffee, cocoa, and chocolate. The parameters of the gas chromatograph-tandem mass spectrometer are as follows: Chromatographic conditions: Chromatographic column: Agilent HP-5 MS capillary column 30 m*0.25 mm*0.25 μm; chromatographic column temperature program: 70°C for 1 min, increased to 220°C at 25°C / min and maintained for 1 min, and then increased to 300°C at 15°C / min and maintained for 6 min; Carrier gas: helium, purity 99.999%; carrier gas flow rate: 1.0 mL / min; injection port temperature: 240°C; injection volume: 1 μL; injection mode: splitless injection.

3. The method according to claim 2, wherein The parameters of the gas chromatograph-tandem mass spectrometer are as follows: Mass spectrometry conditions: Ion source temperature: 300°C; transfer line temperature: 280°C; collision gas: argon; scan type: multiple reaction monitoring mode; the qualitative and quantitative ion pairs of the two pesticide derivative products and the collision energies are as follows: The * mark in the table is the quantitative ion pair.

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