CN115684448A - A method for detecting the residues of flumetrazone and its metabolites in plant-derived products - Google Patents

Abstract

The application discloses a method for detecting residual quantity of fluopicoline and metabolites thereof in plant source products, which comprises the following steps: extracting an extracting solution from a plant source product; removing impurities from the extract; and performing mass spectrometry on the purified extracting solution to obtain the residual quantity of the fluroxypyr and metabolites thereof in the plant source product. The application establishes a method for detecting the residue of the fluopicoline and the metabolites thereof in the plant-derived product, and realizes the qualitative screening and synchronous quantification of the fluopicoline and the metabolites thereof for the first time; the method is simple to operate, high in sensitivity and good in accuracy.

Description

A method for detecting the residues of flumetrazone and its metabolites in plant-derived products

technical field

The present application relates to the field of chemical analysis, in particular to a method for detecting the residues of flumetrazone and its metabolites in plant-derived products.

Background technique

Bicyclopyrone is a new type of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) inhibitor herbicide developed by Syngenta in recent years. It is mainly used in crop fields such as corn, sugar beet and grain. 4-HPPD is a heme iron(II)-independent dioxygenase. The function of flufenpyrone is to block the function of 4-HPPD, thereby inhibiting the biosynthesis of carotenoids, causing the albino symptoms of plant meristems, and eventually causing their death. Flupyroxydone is a broad-spectrum herbicide with excellent selectivity. It mainly controls broad-leaved weeds and some gramineous weeds in crop fields such as corn, sugarcane, and cereals (such as wheat and barley). Large-seeded broad-leaved weeds such as ragweed and cocklebur are more effective in controlling glyphosate-resistant weeds. Fluoxypyrone is often metabolized in plants and animals to 2-(2-methoxyethoxymethyl)-6-(trifluoromethyl)pyridine-3-carboxylic acid (SYN503780) and 2-(2-hydroxy Ethoxymethyl)-6-(trifluoromethyl)pyridine-3-carboxylic acid (CSCD686480). The application of flufenzazone is very flexible, and it can be used from before sowing to after emergence. In addition, it can also play a good role in different environmental conditions and different planting methods. Since 2015, flufenzazone It was launched in 2010, and has been registered and listed in the United States, Canada, Argentina, Uruguay, Australia and other countries. It is widely used based on its excellent characteristics. Among them, Australia and the United States have developed plant-derived products such as barley, wheat, field corn, and sugarcane. The residue limit value in the product, the limit value is as low as 0.02mg/kg. In 2022, the US Environmental Protection Agency revised the residue limit of flumetrazone, and added the residue limit in 12 products such as bananas, onions, strawberries, and watermelon , the limit value is as low as 0.01mg/kg. China's national food safety standard GB2763-2021 stipulates the residue limits in barley, wheat, corn, fresh corn, wheat germ and sugarcane. The sum of SYN503780 and CSCD686480.

The research on flufenzol mainly focuses on herbicidal activity, environmental behavior and safety evaluation, and there are few domestic and foreign reports on the residue analysis methods of flufenzol. Zhang Qiang et al established a high performance liquid chromatography analysis method for 44% diflufenidone·scented metolachlor EC by using high performance liquid chromatography, but there is no report on the analytical method of diflufenidone in plant-derived products. At present, there is no report in the related art on how to realize the qualitative screening and quantitative analysis of the residues of flufenazol and its metabolites in plant-derived products.

Contents of the invention

In order to solve the above problems, the present application provides a method for detecting the residues of flufenazol and its metabolites in plant-derived products.

The present application provides a method for detecting the residues of flufenazol and its metabolites in plant-derived products, which includes:

extracts from products of plant origin;

Purify the extract by removing impurities;

Perform mass spectrometry analysis on the extract after impurity removal and purification, and obtain the residual amount of flufenpyrone and its metabolites in the plant-derived product.

Optionally, in some embodiments of the present application, the mass spectrometry analysis includes: using ultra-high performance liquid chromatography-quadrupole orbital hydrazine high-resolution mass spectrometry to analyze the extract, wherein the chromatographic column used is silica gel The matrix chromatographic column adopts mobile phase as an aqueous solution containing formic acid with a concentration of 0.2%-0.25% by volume, and adopts gradient elution for separation.

Optionally, in some embodiments of the present application, the silica gel-based chromatographic column is a Waters Acquity UPLCHSS T3 chromatographic column with a size of 2.1 mm×100 mm and a particle size of 1.8 μm.

Optionally, in some embodiments of the present application, the conditions of the chromatographic column are flow rate: 0.4 mL/min-0.5 mL/min; injection volume: 2 μL-3 μL.

Optionally, in some embodiments of the present application, the working conditions of the ultra-high performance liquid chromatography-quadrupole orbital hydrazine high-resolution mass spectrometry: the ionization mode is HESI; the spray voltage is 3000V-4000V; the temperature of the capillary is 300°C-400°C; the temperature of the ion transfer tube is 300°C-400°C; the acquisition method: in parallel reaction monitoring mode, the polarity mode is positive ion; sheath gas: 30arb-40arb; auxiliary gas: 5arb-10arb; The scanning parameters of the mass spectrometer: the resolution of the secondary scanning is 17500dpi-35000dpi; the collision energy of the secondary mass spectrometry is the normalized collision energy, and the normalized collision energy is 20%, 40% or 60%.

Optionally, in some embodiments of the present application, the extraction of the extract from plant-derived products includes:

Pretreatment of plant-derived products to obtain plant-derived samples;

The plant source sample, the salting-out agent and the organic solvent are mixed and extracted to obtain an extract.

Optionally, in some embodiments of the present application, the organic solvent is any one of acetone formate, ethyl acetate formate, acetonitrile or acetonitrile formate; and/or, the salting-out agent is sodium chloride, Mix any one or more of anhydrous sodium sulfate or anhydrous magnesium sulfate.

Optionally, in some embodiments of the present application, the organic solvent is formic acid acetonitrile with a concentration of 0.4%-0.6% by volume.

Optionally, in some embodiments of the present application, the mass ratio of the plant source sample to the salting-out agent is (1-1.5):1; the mass ratio of the plant source sample to the organic solvent The volume ratio is 1:(4-5).

Optionally, in some embodiments of the present application, the impurity removal and purification adopts dispersive solid-phase extraction: the extract, graphitized carbon black, octadecyl bonded silica gel and anhydrous magnesium sulfate are mixed, and After centrifugation, the supernatant was taken, and filtered to obtain the extract after removal of impurities.

The application has the following beneficial effects:

This application has innovatively established a detection method for the residues of fenpyrazone and its metabolites in plant-derived products, and for the first time realized the qualitative screening and simultaneous quantification of fenpyrazone and its metabolites; the method of this application The operation is simple, the sensitivity is high and the accuracy is good, and it is suitable for the detection of flufenazol and its metabolites in plant-derived products, and can provide technical support for the risk monitoring of flufenzazone and its metabolites in plant-derived products.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings that need to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application. For those skilled in the art, other drawings can also be obtained based on these drawings without any creative effort.

Fig. 1 is the ion chromatogram extracted by the secondary mass spectrometry quantitative ion (324.07730) of flufenzazone on the Waters Acquity UPLC HSST3 chromatographic column;

Fig. 2 is the ion chromatogram extracted by the secondary mass spectrometry quantant ion (324.07730) of flufenzazone on the ThermoSyncronis C ₁₈ chromatographic column;

Fig. 3 is the ion chromatogram extracted by the secondary mass spectrometry quantant ion (324.07730) of flufenzazone on the ThermoAccucoreaQ chromatographic column;

Fig. 4 is the extracted ion chromatogram of the secondary mass spectrometry quantitative ion of flumetrazone (1ng/mL);

Figure 5 is the ion flow chromatogram of the secondary mass spectrometry quantitative ion extraction ion flow chromatogram of metabolite SYN503780 (1ng/mL);

Fig. 6 is the ion flow chromatogram of the secondary mass spectrometry quantitative ion extraction ion flow chromatogram of metabolite CSCD686480 (1ng/mL);

Fig. 7 is the secondary mass spectrogram of flumetrazone (1ng/mL) obtained in PRM mode;

Figure 8 is the secondary mass spectrum of the metabolite SYN503780 (1ng/mL) obtained in PRM mode;

Figure 9 is the secondary mass spectrum of the metabolite CSCD686480 (1ng/mL) obtained in PRM mode;

Figure 10 is a schematic diagram of the influence of different extraction solvents on the extraction recovery of flumetrazone and its metabolites SYN503780 and CSCD686480;

Figure 11 is a schematic diagram showing the influence of the amount of different extraction solvents on the extraction recovery of flumetrazone and its metabolites SYN503780 and CSCD686480;

Figure 12 is a schematic diagram of the influence of different salting-out agents on the recovery rate of flumetrazone and its metabolites SYN503780 and CSCD686480 in the sample;

Figure 13 is a schematic diagram showing the influence of the amount of different adsorbents on the recovery rate of the extraction of flufenpyrone metabolites SYN503780 and CSCD686480;

Figure 14 is a schematic diagram of the matrix effect of flumetrazone and its metabolites SYN503780 and CSCD686480 in different matrices.

Detailed ways

The technical solutions in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all of them. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without making creative efforts belong to the scope of protection of this application.

The embodiment of the present application provides a method for detecting the residues of flumetrazone and its metabolites in plant-derived products, and the involved plant-derived products may be, but not limited to, wheat, barley, wheat germ, corn, scallions, bananas, sugarcane , Onion, Strawberry, Watermelon. The metabolites of flufenpyrone in plants are 2-(2-methoxyethoxymethyl)-6-(trifluoromethyl)pyridine-3-carboxylic acid (SYN503780) and 2-(2-hydroxyethyl The structural formulas of oxymethyl)-6-(trifluoromethyl)pyridine-3-carboxylic acid (CSCD686480), flumetrazone and its metabolites (SYN503780) and (CSCD686480) are as follows:

Detection methods include:

The extract solution is extracted from the plant-derived product, and the extract solution may contain the target substance flumezazone and/or metabolites of flumezazone. In one embodiment, the solvent method is used to extract the extract from the product of plant origin.

Purify the extract. In one embodiment, dispersive solid phase extraction is used for purification to remove impurities in the extract.

Mass spectrometry analysis was carried out on the extracted solution after impurity removal and purification, and the residues of flufenazol and its metabolites in the plant-derived products were obtained. In some embodiments, the mass spectrometry analysis includes: using ultra-high performance liquid chromatography-quadrupole orbital hydrazine high-resolution mass spectrometry to analyze the extract, wherein the chromatographic column used is a silica gel matrix chromatographic column, and the mobile phase is The aqueous solution with the concentration of 0.2%-0.25% formic acid is separated by gradient elution. In some other embodiments, the mobile phase may be an aqueous solution with a volume percent concentration of 0.2%, 0.21%, 0.22%, 0.23%, 0.24% or 0.25% formic acid. Tests have shown that after adding formic acid to the mobile phase system, the peak tailing phenomenon of flumetrazone and its metabolites SYN503780 and CSCD686480 has been significantly improved. The test also proves that when the volume percent concentration of formic acid in the aqueous phase reaches 0.2% When , the peak shapes of flufenzazone and its metabolites SYN503780 and CSCD686480 can reach a better state.

Ultrahigh performance liquid chromatography-quadrupole/orbitrap high resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) combines highly selective quadrupole with high resolution , high-sensitivity orbitrap organic combination, can carry out rapid screening and result confirmation of target and non-target objects, compared with low-resolution triple quadrupole mass spectrometry using multiple reaction monitoring mode (MRM) for quantitative analysis, UPLC-Q -Orbitrap HRMS does not need to optimize the product ions of target compounds and related mass spectrometry parameters one by one, and it also makes up for the shortcomings of the traditional triple quadrupole mass spectrometry detection of limited number of compounds and false positives. It has been widely used in the food testing industry. In existing studies, the quantitative analysis of target objects using high-resolution mass spectrometry mainly uses the full scan mode (Full scan, FS), which cannot obtain better results compared with the triple quadrupole multiple reaction monitoring mode (MRM). sensitivity of the instrument method. In the parallel reaction monitoring (PRM) mode of quadrupole/electrostatic field orbitrap high-resolution mass spectrometry (Q Exactive), the sensitivity of the instrument method has been greatly improved compared with the FS mode, which can be compared with the triple quadrupole comparable to the MRM model of rods.

The sensitivity of the detection method provided in this example meets the residue limit requirements of relevant international and domestic laws and regulations, and can make up for the absence of a detection method for flufenazol and its metabolites in plant-derived products; and environmental security, preventing trade import risks plays an important role.

In some embodiments of the present application, the silica gel-based chromatographic column is a Waters Acquity UPLC HSS T3 chromatographic column with a specification of 2.1 mm×100 mm and a particle size of 1.8 μm. Waters Acquity UPLC HSS T3 chromatographic column is a high-strength silica-based chromatographic column based on a fully porous silica-based bonded phase. Its retention performance is better than that of ordinary C ₁₈ columns, and it can obtain better peak shape and response, thereby obtaining better peak shape and higher response.

In some embodiments of the present application, the conditions of the chromatographic column are: flow rate: 0.4mL/min-0.5mL/min; injection volume: 2μL-3μL. In a preferred embodiment, the flow rate of the chromatographic column is 0.4 mL/min, and the injection volume is 2 μL.

In some embodiments of the present application, the working conditions of ultra-high performance liquid chromatography-quadrupole orbital hydrazine high-resolution mass spectrometry: the ionization mode is HESI; the spray voltage is 3000V-4000V; the temperature of the capillary is 300°C-400°C; The temperature of the ion transfer tube is 300°C-400°C; the acquisition method: in the parallel reaction monitoring mode, the polarity mode is positive ion; the sheath gas: 30arb-40arb; the auxiliary gas: 5arb-10arb; the scanning parameters of the mass spectrometer: two The resolution of the primary scan is 17500dpi-35000dpi; the collision energy of the secondary mass spectrometer is the normalized collision energy, and the normalized collision energy is 20%, 40% or 60%.

In a specific example, the working conditions of ultra-high performance liquid chromatography-quadrupole orbital hydrazine high-resolution mass spectrometry are: the ionization mode is HESI; the spray voltage is 3500V; the temperature of the capillary is 350°C; the temperature of the ion transfer tube is 350°C ℃; acquisition method: in parallel reaction monitoring mode, polarity mode is positive ion; sheath gas: 35arb; auxiliary gas: 10arb; mass spectrometry scanning parameters: secondary scanning resolution is 17500dpi; secondary mass spectrometry collision energy is normalized Normalized collision energy, the normalized collision energy is 20%, 40% or 60%.

In some embodiments of the present application, extracting the extract from the plant source product comprises:

The plant-derived product is pretreated to obtain a plant-derived sample. In some embodiments, the pretreatment of plant-derived products refers to: crushing plant-derived products such as rice, wheat, corn (dry) and other grain samples with a pulverizer so that all of them can pass through a 425 μm standard mesh sieve. The vegetable and fruit samples were pulverized with a knife grinder at 5000r/min and thoroughly mixed.

The plant source sample, the salting-out agent and the organic solvent are mixed and extracted to obtain an extract. In a specific example, weigh 5g (accurate to 0.01g) of the prepared plant source sample in a 50mL polypropylene centrifuge tube, add 10mL of water to the grain sample in advance, vortex and mix, and let it stand for 30min; add 5g of chloride Sodium, 25mL 0.4% formic acid acetonitrile solution and 1 ceramic homogenizer, vortex extraction for 5min, centrifugation at 5000r/min for 3min. Pipette 1.5mL supernatant into a 2mL polypropylene centrifuge tube.

In some embodiments of the present application, the organic solvent is any one of acetone formate, ethyl acetate formate, acetonitrile or acetonitrile formate; and/or, the salting-out agent is sodium chloride, anhydrous sodium sulfate or anhydrous sulfuric acid Any one or more mixtures of magnesium.

In some embodiments of the present application, the organic solvent is formic acid acetonitrile with a concentration of 0.4%-0.6% by volume. The use of 0.4%-0.6% formic acid acetonitrile is beneficial to improving the recovery rate of flufenazol and its metabolite SYN503780 and metabolite CSCD686480, thereby improving the extraction efficiency. In a preferred embodiment, the organic solvent is formic acid acetonitrile with a concentration of 0.4% by volume.

In some embodiments of the present application, the mass ratio of the plant source sample to the salting-out agent is (1-1.5):1. In some other embodiments, the mass ratio of the plant source sample to the salting-out agent may also be 1:1, 1.2:1, 1.3:1, 1.4:1 or 1.5:1. The mass volume ratio of the plant source sample to the organic solvent is 1: (4-5). In some other embodiments, the mass volume ratio of the plant source sample to the organic solvent is 1:4, 1:4.5 or 1:5.

In some embodiments of the present application, dispersive solid-phase extraction is used for impurity removal and purification: the extract, graphitized carbon black, octadecyl bonded silica gel and anhydrous magnesium sulfate are mixed, and the supernatant is taken after centrifugation, After filtering, the extracted solution after impurity removal and purification is obtained. QuEChERS purification reagents commonly used in plant-derived samples include GCB, C ₁₈ , PSA, and NH ₂ . Among them, PSA and NH ₂ have similar mechanisms of action, and both have weak anion exchange capabilities. They interact with compounds through hydrogen bonds and can effectively remove Organic acids, polar pigments, fatty acids, sugars and other components that can form hydrogen bonds in the sample; C ₁₈ removes non-polar compounds such as volatile oils, terpenes, lipids, etc.; GCB is effective for polar and non-polar compounds in the sample Organic interfering substances have extremely high adsorption capacity, and have a remarkable effect on removing pigments in plants; anhydrous magnesium sulfate can remove water in sample liquid. The recovery rate of the 5 μg/L flupidalone standard solution after adsorption by three purification reagents of GCB, PSA, C ₁₈ and MgSO ₄ was investigated in the experiment. The results showed that after purification by PSA, the recoveries of flumetrazone and its metabolite SYN503780 and metabolite CSCD686480 were all lower than 80%, and the recoveries decreased more with the increase of dosage; GCB, C ₁₈ , MgSO ₄ The average adsorption recoveries of the three substances are all between 90% and 110%.

In some other embodiments, the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) method is adopted, and acetonitrile formic acid solution is used as the extraction solvent. After salting out, the extract is purified by dispersive solid-phase extraction. The PRM mode of phase chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry has established a detection method for the residues of flufenzadone and its metabolites in plant-derived products. Secondary mass spectrometry database to realize the qualitative screening and simultaneous quantification of flumetrazone and its metabolites. The sensitivity of this method meets the residue limit requirements of relevant international and domestic laws and regulations, and it can make up for the absence of a detection method for flumetrazone and its metabolites in plant-derived products in my country; It plays an important role in preventing trade import risks.

In order to make the above-mentioned implementation details and operations of the present invention clearly understood by those skilled in the art, and to reflect the significant progress of the method for detecting the residues of flufenazol and its metabolites in plant-derived products according to the embodiment of the present invention, the following The above technical solution is illustrated by an experimental example.

Experimental example 1

Selection of reagents and instruments:

Anhydrous magnesium sulfate (MgSO ₄ ), anhydrous sodium sulfate (Na ₂ SO ₄ ), sodium chloride (NaCl), n-hexane, acetone, and ethyl acetate were all analytically pure, purchased from Sinopharm Chemical Reagent Co., Ltd.; acetonitrile Both methanol and methanol were chromatographically pure and purchased from TEDIA, USA. Graphitized carbon black (GCB, 40-120 μm), ethylenediamine-N-propyl silylated silica gel (PSA, 40-60 μm) and octadecyl bonded silica gel (C ₁₈ , 40-60 μm) were purchased from Shanghai An Spectrum Experiment Technology Co., Ltd. Microporous membrane: 0.22μm, organic phase type; ceramic homogeneous: 2cm (length) × 1cm (outer diameter). Flupyrazone (C ₁₉ H ₂₀ F ₃ NO ₅ , 100 μg/mL, Bepure Company). Fluoxypyrone metabolite SYN503780: 2-(2-methoxyethoxymethyl)-6-(trifluoromethyl)pyridine-3-carboxylic acid (C ₁₁ H ₁₂ F ₃ NO ₄ , 100 μg/mL, Bepure Company); flumetrazone metabolite CSCD686480: 2-(2-hydroxyethoxymethyl)-6-(trifluoromethyl)pyridine-3-carboxylic acid (C ₁₀ H ₁₀ F ₃ NO ₄ , purity 97.0 %, Dr. Ehrenstorfer Company, Germany).

Q-Exactive quadrupole-electrostatic field orbitrap high-resolution mass spectrometry system (Thermo Scientific, USA); UltiMate3000 fast high-performance liquid chromatography system (Thermo Scientific, USA); Milli-Q pure water instrument (Millipore, USA); pulverizer (Shanghai) Jiading Grain and Oil Instrument Co., Ltd.); GM200 Knife Grinder (Leich, Germany); Vortex Oscillator (Talboys, USA); Centrifuge (Hunan Xiangyi Laboratory Instrument Development Co., Ltd.).

Standard solution preparation:

Weigh an appropriate amount of CSCD686480 and dissolve it in methanol to prepare a 1000mg/L standard stock solution. Take an appropriate amount of flumezazone, SYN503780 and CSCD686480 standard stock solutions, dilute with methanol to form a mixed standard intermediate solution of flumezazone 0.002mg/L, flumezazone metabolites SYN503780 and CSCD686480 0.1mg/L; Methanol and blank sample extract were diluted and mixed with the standard intermediate solution to obtain a series of standard working solutions and matrix-matched standard working solutions, in which the concentration of flumetrazone was 0.002 μg/L, 0.004 μg/L, 0.02 μg/L, 0.04 μg/L , 0.2μg/L, and the concentrations of the flumetrazone metabolites SYN503780 and CSCD686480 are 0.1μg/L, 0.2μg/L, 1μg/L, 2μg/L, 10μg/L, and the working solution is prepared and used immediately.

Sample pretreatment:

Rice, wheat, corn (dry) and other grain samples were pulverized with a pulverizer so that they could all pass through a 425 μm standard mesh sieve. Vegetable and fruit samples were pulverized with a knife mill at 5000r/min and fully mixed. Weigh 5g (accurate to 0.01g) of the prepared sample into a 50mL polypropylene centrifuge tube, add 10mL water to the grain sample in advance, vortex and mix, and let it stand for 30min; add 5g sodium chloride, 25mL 0.4% formic acid acetonitrile solution and 1 ceramic homogenizer, vortex extraction for 5 minutes, and centrifugation at 5000r/min for 3 minutes. Draw 1.5mL supernatant into a 2mL polypropylene centrifuge tube, add 5mg GCB, 5mg C ₁₈ and 50mg MgSO ₄ , vortex and mix for 1min, centrifuge at 12000r/min for 3min, take the supernatant and pass it through a 0.22μm organic filter membrane, to be determined .

Instrument Conditions:

Chromatographic column: Waters Acquity UPLC HSS T3 liquid chromatography column; flow rate: 0.4mL/min; injection volume: 2μL; mobile phase A is 0.2% formic acid water, mobile phase B is methanol; gradient elution program: 0-2min, 10% B; 2-2.5min, 10%-90%B; 2.5-5min, 90%B; 5-5.1min, 90%-10%B; 5.1-7min, 10%B.

HESI ionization mode; spray voltage 3500V; capillary temperature 350°C; ion transfer tube temperature: 350°C; acquisition mode: parallel reaction monitoring (PRM) mode, positive ion mode; sheath gas (N2): 35arb; auxiliary gas ( N2): 10 arb; secondary scanning resolution: 17500; normalized collision energy (NCE) of secondary mass spectrometry: 20%, 40%, 60%.

Under the above mass spectrometry conditions, the optimized mass spectrometry parameters of flumetrazone and its metabolites are shown in Table 1.

Table 1

Optimization of Experimental Example 2 Instrument Conditions

Optimal selection of chromatographic columns:

This experimental example compares the 5μg/L standard solution of flumetrazone and its metabolites on Waters Acquity UPLCHSST3 chromatographic column (2.1mm×100mm×1.8μm), ThermoSyncronis C ₁₈ chromatographic column (2.1mm×100mm×1.9μm) and ThermoAccucoreaQ The peak width and response value on the chromatographic column (2.1mm × 150mm × 2.6μm), for two kinds of flufenzazone metabolites SYN503780 and CSCD686480, the peak shape is all good on three kinds of chromatographic columns; On the ThermoAccucoreaQ C ₁₈ chromatographic column, there will be more serious peak broadening and tailing. Compared with the ThermoSyncronis C ₁₈ chromatographic column, better peak shape and higher response can be obtained on the Waters Acquity UPLC HSS T3 chromatographic column, Waters Acquity UPLC HSS T3 chromatographic column is a high-strength silica gel-based chromatographic column, based on a fully porous silica-based bonded phase, its retention performance is better than that of ordinary C ₁₈ columns, and better peak shape and response can be obtained. Therefore, the Waters Acquity UPLCHSST3 chromatographic column is a more preferred chromatographic column. For the specific results, please refer to Figure 1-Figure 3. Figure 1 is the ion chromatogram of the secondary mass spectrometry quantion ion (324.07730) of flumezazone on the WatersAcquity UPLC HSST3 chromatographic column; The secondary mass spectrometry quantant ion (324.07730) on the _C18 chromatographic column extracts the ion chromatogram; Fig. 3 is the secondary mass spectrometry quantant ion (324.07730) extracts the ion chromatogram of flumetrazone on the ThermoAccucoreaQ chromatographic column.

Optimization of Experimental Example 3 Instrument Conditions

Regarding the optimal choice of mobile phase:

The experimental examples have investigated different mobile phase systems (acetonitrile-water, acetonitrile-0.2% formic acid water, methanol-water, methyl alcohol-0.1% formic acid water, methanol-0.2% formic acid water and methyl alcohol-5mmol/L ammonium acetate water (containing 0.1% formic acid)) on the chromatographic peak shape and signal response. The results showed that the peak shapes of flumetrazone and its metabolites SYN503780 and CSCD686480 in the mobile phase system (acetonitrile-water and methanol-water) without formic acid all appeared fronting and tailing, and the mobile phase system added formic acid. The tailing phenomenon has been significantly improved. When the formic acid content in the water phase reaches 0.2%, the peak shapes of the three substances can reach a better state. Adding ammonium acetate in the water phase has no obvious effect on the response and peak shape of the three substances. The experimental results show that both acetonitrile-0.2% formic acid water and methanol-0.2% formic acid water can be used as the mobile phase. Since methanol is used as the mobile phase, the system pressure is small, so methanol-0.2% formic acid water is finally selected as the mobile phase.

Optimization of Experimental Example 4 Instrument Conditions

Optimal selection of mass spectrometry conditions:

Using the PRM mode of UPLC-Q-Orbitrap HRMS to detect the mixed standard solution of flumezazone and its metabolites, only the molecular ion peak [M+H] ⁺ theoretical accurate mass of each compound is required, and the collision energy is normalized The normalized collision energy (NCE) can be set to 20%, 40%, 60%. The PRM acquisition mode first uses the selective detection capability of the quadrupole mass analyzer to selectively detect the parent ion information of the target compound in the primary mass spectrometer, that is, the molecular ion peak [M+H] ⁺ , and then the parent ion is detected in the collision cell Fragmentation is carried out; finally, all fragment information of the selected parent ion is detected in the secondary mass spectrometer by using a high-resolution mass analyzer with high mass precision. PRM technology is very similar to MRM technology, the difference is the detection mode of secondary mass spectrometry. PRM technology detects all product ions through high-resolution mass analyzer after fragmentation of parent ion. The accurate mass number can further reduce the background noise, and the ion noise interference is much smaller than that of MRM technology, thus improving the sensitivity and accuracy of the instrument. At the same time, the PRM technology is also easier to use, and there is no need to find accurate precursor ions-product ions and optimize related parameters in advance. Use the PRM mode to detect flumezazone and its metabolites SYN503780 and CSCD686480. Please refer to Figure 4-Figure 9 for the results. Fig. 4 is the extracted ion chromatogram of the secondary mass spectrometry quantifier ion of flufenzazone (1ng/mL); Fig. 5 is the extracted ion flow chromatogram of the quadruple mass spectrometry quantifier ion of metabolite SYN503780 (1ng/mL); Fig. 6 is the secondary mass spectrometry quantitative ion extraction ion flow chromatogram of the metabolite CSCD686480 (1ng/mL); Figure 7 is the secondary mass spectrogram of flufenox (1ng/mL) obtained in PRM mode; Figure 8 is The secondary mass spectrum of metabolite SYN503780 (1ng/mL) obtained in PRM mode; Figure 9 is the secondary mass spectrum of metabolite CSCD686480 (1ng/mL) obtained in PRM mode.

Optimization of Experimental Example 5 Pretreatment Method

Optimal selection of extraction solvents:

For plant-derived samples, acetone, ethyl acetate, methanol, and acetonitrile can be used as extraction solvents. Methanol was excluded as the extraction solvent because the methanol extract could not be separated from the water phase even after salting out. In the experiment, 0.4% formic acid acetone, 0.4% formic acid ethyl acetate, acetonitrile, 0.1% formic acid acetonitrile, 0.2% formic acid acetonitrile and 0.4% formic acetonitrile were selected as the extraction solvents, and the average recovery rate of flumetrazone and its metabolites SYN503780 and CSCD686480 (Scaling amount 5μg/kg) to investigate the extraction effect of different solvents. For acetonitrile and formic acid acetonitrile solutions, the wheat samples added with 5 μg/kg flumetrazone and its metabolites SYN503780 and CSCD686480 were treated according to the sample pretreatment method of Experimental Example 1 and then tested on the machine; each experiment was measured in parallel 3 times, using solvent The standard curve was used for calibration and quantification; for 0.4% acetone formate and 0.4% ethyl acetate formate, after treatment according to the sample pretreatment method in Experimental Example 1, 1 mL of the extract was blown with nitrogen at 40°C, reconstituted with acetonitrile, and put on the machine. As can be seen from Figure 10, when 0.4% formic acid acetone is used as the extraction solvent, the recovery rate of metabolite CSCD686480 is low; Solvent, as the content of formic acid increases, the recovery rate of flumetrazone and its metabolites SYN503780 and CSCD686480 also increases, and the recovery rate of the three substances is better when extracting with 0.4% formic acid acetonitrile, and the extraction efficiency is higher . This shows that the pH value of the extraction solvent has a greater impact on the extraction efficiency, and the study concluded that choosing 0.4% formic acid acetonitrile as the extraction solvent is conducive to improving the extraction efficiency.

Optimization of Experimental Example 6 Pretreatment Method

About the optimization of extraction solvent acetonitrile consumption:

Taking wheat as the experimental sample, 10mL, 25mL, 40mL and 50mL were selected to optimize the amount of acetonitrile in the experiment, and the final recovery rate was used as the basis for investigation. Please refer to Figure 11. The results show that when the extraction solvent is 10mL, the recovery rate of metabolites is relatively low due to the influence of the matrix effect. When the amount of acetonitrile is 25mL, the recovery rate can meet the analysis and determination requirements. The rate change is not obvious, and it will lead to the increase of the detection limit of the method. This shows that the most preferable amount of extraction solvent acetonitrile is 25mL, the operation is more convenient, and the cost of consumables is reduced.

Optimization of Experimental Example 7 Pretreatment Method

Optimal selection of salting-out agent:

The function of the salting-out agent is mainly to remove excess water in the sample, increase the solubility of the pesticide components in the organic phase, and help improve the extraction efficiency. This experiment tested the effect of three commonly used salting-out agents, namely sodium chloride, anhydrous sodium sulfate, and anhydrous magnesium sulfate + sodium chloride (3+1) mixed salting-out agent on the extraction effect of three pesticide components in the sample. The results are shown in Figure 12. As can be seen from the figure, when the salting-out agent adopts anhydrous sodium sulfate and anhydrous magnesium sulfate+sodium chloride (3+1), the extraction recoveries of the three components are all lower than the extraction when sodium chloride is the salting-out agent The recovery rate shows that when the salting-out agent is sodium chloride, the salting-out effect is conducive to improving the recovery rate of the extract, while the price of sodium chloride is low and the cost is reduced.

Optimization of Experimental Example 8 Pretreatment Method

Optimal selection of purification reagents:

QuEChERS purification reagents commonly used in plant-derived samples include GCB, C ₁₈ , PSA, and NH ₂ . Among them, PSA and NH ₂ have similar mechanisms of action, and both have weak anion exchange capabilities. They interact with compounds through hydrogen bonds and can effectively remove Organic acids, polar pigments, fatty acids, sugars and other components that can form hydrogen bonds in the sample; C ₁₈ removes non-polar compounds such as volatile oils, terpenes, lipids, etc.; GCB is effective for polar and non-polar compounds in the sample Organic interfering substances have extremely high adsorption capacity, and have a remarkable effect on removing pigments in plants; anhydrous magnesium sulfate can remove water in sample liquid. The recovery rate of the 5 μg/L flupidalone standard solution after adsorption by three kinds of purification reagents, GCB, PSA, C ₁₈ and MgSO ₄ , was investigated in the experiment. The results show that the recovery rates of the three substances after PSA purification are _all lower than 80%, _and the recovery rates decrease more with the increase of the dosage; %, which shows that the selection of GCB, C ₁₈ , MgSO ₄ as purification reagents is beneficial to improve the recovery rate of the extract.

Optimization of Experimental Example 9 Pretreatment Method

Determination of the combined dosage of purification reagents:

When plant-derived samples are purified by dispersive solid-phase extraction, two or more purification reagents are usually used to better remove interfering impurities such as lipids and sugars in the sample. The experiment investigated three groups of purification reagents (I: 0mg GCB+0mg C ₁₈ +50mg MgSO ₄ , II: 5mg GCB+5mg C ₁₈ +50mg MgSO ₄ , III: 10mg GCB+10mg C ₁₈ +50mg MgSO ₄ , IV: 20mg GCB+20mg C ₁₈ +50mg MgSO ₄ ) on the purification effect of 5 μg/kg spiked extract of wheat blank sample, as shown in Figure 13, the results show that the recoveries of the three groups of purification reagents are all between 90-110%. Both can meet the experimental requirements; From the perspective of the actual purification effect, with the increase of the amount of GCB and C ₁₈ , the recovery rate of flumetrazone and its metabolites SYN503780 and CSCD686480 changes little, and the combination I can meet the requirements of most samples. Pigment removal. This shows that choosing the combination of purification reagents as 5mgGCB+5mg C ₁₈ +50mg MgSO ₄ is conducive to improving the purification effect and reducing the overall cost.

The influence of experimental example 10 matrix effect

UPLC-Q-Orbitrap HRMS has stronger anti-interference ability, but matrix effects still exist, and there are many plant-derived samples with complex matrices. Therefore, matrix effects need to be evaluated. Matrix effects (Matrix effects, ME) The mass spectrometry system is mainly composed of components other than the target in the sample, and the sample matrix co-eluted with the target affects the ionization process of the target, resulting in ionization inhibition or enhancement. In the experiment, a blank matrix solution was prepared according to the sample pretreatment method of Experimental Example 1, and a standard working solution and matrix standard working solution were prepared according to the standard solution preparation method of the Experimental Example. Calculate the matrix effect according to the following formula: ME=[(matrix matching calibration curve slope/pure solvent standard curve slope)-1]×100%, as shown in Figure 14, the results show that rice, wheat, corn, sugarcane, banana, green onion, The matrix effects of flumezazone and its metabolites in broccoli and watermelon were between 1% and 20%, and the matrix effects were all less than 20%. Shown as weak matrix effect, no need to compensate for matrix effect.

Experimental Example 11 Methodological Evaluation

Regarding the linear relationship and the lower limit of quantitation:

According to the preparation method of the standard solution in Experimental Example 1, the standard working solution of flumetrazone and its metabolite series and the matrix matching standard working solution were prepared, and the sample was injected and tested according to the instrument conditions in Experimental Example 1. The mass concentration (X, μg/L) was used as the horizontal Coordinates, draw the standard working curve with its peak area as the ordinate (Y). After adding an appropriate amount of standard solution to the blank sample solution, it was measured on the computer, and the lower limit of quantification (LOQ) was determined with 10 times the signal-to-noise ratio (S/N=10), the linear regression equation and correlation coefficient of flufenazol and its metabolites (r), linear range and limit of quantification are shown in Table 2, and Table 2 is the regression equation, correlation coefficient, linear range and limit of quantification of flumetrazone and its metabolites.

Table 2

In Table 2, Y: peak area of quantitative ion; X: mass concentration, μg/L.

Experimental example 12 rate of recovery and relative standard deviation

The standard addition recovery experiment of low, medium and high levels was carried out on wheat, corn, scallion, banana and sugarcane blank samples respectively, and each concentration of standard addition was measured 6 times in parallel, the recovery rate and relative standard deviation (RSD, n=6 ) see Table 3. The average recoveries of flufenpyrone and its metabolites SYN503780 and CSCD686480 at the three spiked levels were 84.6%-118.5%, and the RSDs were 2.6%-7.4%. The method for the residues of flufenazol and its metabolites has good accuracy and precision.

table 3

Actual sample testing

This method was used to detect 5 samples each of wheat, corn, scallions, bananas, and sugarcane purchased in the market, and the accurate mass and retention time were used to qualitatively screen the flupidalone and its metabolites in the samples, and combined with the secondary characteristics As confirmed by fragment ions, fluroxyfen residue was detected in an imported corn species.

The above-mentioned examples of the present application established an analysis and detection method for the determination of the residues of fenflufen and its metabolites in plant-derived products by ultra-high performance liquid chromatography-quadrupole electrostatic orbitrap high-resolution mass spectrometry. Under optimized experimental conditions, The linear relationship of flufenpyrone in the range of 0.002-0.2μg/L, metabolite SYN503780 and metabolite CSCD686480 was good in the range of 0.1-10μg/L, and the correlation coefficients were all greater than 0.995. The average recovery rate of the blank sample spiked at the low, medium and high levels was 84.6%-118.5%, the RSD was 2.6%-7.4%, the limit of quantification of flumetrazone was 0.01 μg/kg, and the metabolites SYN503780 and CSCD686480 were quantified The limit is 0.5μg/kg. The method has the advantages of high sensitivity, simple operation, rapidity and accuracy, and can meet the detection requirements of flufenazol and its metabolite residues in plant-derived products.

The most suitable pretreatment method and instrument conditions are determined by optimizing conditions such as instrument conditions, extraction solvents, and purification methods. The matrix effect of the samples was investigated, and it was found that the matrix effect of flumetrazone and its metabolites in the 8 samples were all less than 20%, which was a weak matrix effect, and no compensation measures were needed. Under the optimized experimental conditions, the linear relationship between flufenzazone 0.002-0.2μg/L, metabolite SYN503780 and metabolite CSCD686480 was good in the range of 0.1-10μg/L, and the correlation coefficients were all greater than 0.995. The average recovery rate of the blank sample at the low, medium and high 3 spiked levels was 84.6%-118.5%, the relative standard deviation (n=6) was 2.6%-7.4%, and the limit of quantitation of fenfluoxydone was 0.01 μg/ kg, the limit of quantification for metabolite SYN503780 and metabolite CSCD686480 was 0.5 μg/kg. The method has the advantages of high sensitivity, simple operation, rapidity and accuracy, and can meet the detection requirements of flufenazol and its metabolite residues in plant-derived products. The method was used to determine 25 samples, and the residue of flufenazol was detected in one imported corn sample. The method is simple to operate, high in sensitivity and good in accuracy, and is suitable for the detection of flufenzazone and its metabolites in plant-derived products. support.

The method for detecting the residues of flufenazol and its metabolites in plant-derived products provided by this application has been described in detail above. In this paper, specific examples have been used to illustrate the principles and implementation methods of this application. The above examples The description is only used to help understand the method of the present application and its core idea; at the same time, for those skilled in the art, according to the idea of the present application, there will be changes in the specific implementation and scope of application. In summary, The contents of this specification should not be understood as limiting the application.

Claims (10)

1. A method for detecting flumetrazone and its metabolite residues in plant-derived products, characterized in that it comprises: extracts from products of plant origin; Purify the extract by removing impurities; Perform mass spectrometry analysis on the extract after impurity removal and purification, and obtain the residual amount of flufenpyrone and its metabolites in the plant-derived product. 2. The method for detecting the residues of flumetrazone and its metabolites in plant-derived products according to claim 1, wherein the mass spectrometry analysis comprises: using ultra-high performance liquid chromatography-quadrupole orbital hydrazine high The extract is analyzed by resolution mass spectrometry, wherein the chromatographic column used is a silica gel-based chromatographic column, the mobile phase is an aqueous solution containing 0.2%-0.25% formic acid by volume, and gradient elution is used for separation. 3. The method according to claim 2 for detecting the residues of flumetrazone and its metabolites in plant-derived products, wherein the silica gel-based chromatographic column is a WatersAcquity UPLC HSS T3 type chromatographic column with a specification of 2.1 mm×100mm, the particle size is 1.8μm. 4. The method for detecting the residual amount of flumetrazone and its metabolites in plant-derived products according to claim 2, characterized in that the condition of the chromatographic column is flow rate: 0.4mL/min-0.5mL/min; Injection volume: 2μL-3μL. 5. The method for detecting the residual amount of flumetrazone and its metabolites in plant-derived products according to claim 2, characterized in that, the working conditions of the ultra-high performance liquid chromatography-quadrupole orbital hydrazine high-resolution mass spectrometry : The ionization method is HESI; The spray voltage is 3000V-4000V; The temperature of the capillary is 300°C-400°C; The temperature of the ion transfer tube is 300°C-400°C; Acquisition method: in the parallel reaction monitoring mode, the polarity mode is positive ion; Sheath gas: 30arb-40arb; Auxiliary gas: 5arb-10arb; Scan parameters for mass spectrometry: The secondary scanning resolution is 17500dpi-35000dpi; The collision energy of the secondary mass spectrometer is the normalized collision energy, and the normalized collision energy is 20%, 40% or 60%. 6. The method for detecting the residual amount of flumezazone and its metabolites in plant-derived products according to claim 1, wherein the extraction of the extract from plant-derived products comprises: Pretreatment of plant-derived products to obtain plant-derived samples; The plant source sample, salting-out agent and organic solvent are mixed and extracted to obtain the extract. 7. The method for detecting flumetrazone and its metabolite residues in plant-derived products according to claim 6, wherein the organic solvent is acetone formate, ethyl acetate formate, acetonitrile or acetonitrile formate either; and/or The salting-out agent is any one or a mixture of sodium chloride, anhydrous sodium sulfate or anhydrous magnesium sulfate. 8. The method for detecting the residues of flumetrazone and its metabolites in plant-derived products according to claim 6, characterized in that the organic solvent is formic acid acetonitrile with a concentration of 0.4%-0.6% by volume. 9. the method for flumetrazone and its metabolite residues in the detection plant source product according to claim 6, is characterized in that, the mass ratio of described plant source sample and described salting-out agent is (1- 1.5): 1; The mass volume ratio of the plant source sample to the organic solvent is 1:(4-5). 10. the method for flumetrazone and metabolite residue thereof in the detection plant source product according to claim 1, is characterized in that, described impurity removal purification adopts dispersive solid-phase extraction: described extracting solution, graphitization Carbon black, octadecyl bonded silica gel and anhydrous magnesium sulfate are mixed, the supernatant is obtained after centrifugation, and the extract after removal of impurities and purification is obtained after filtration.

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