CN115684448A

CN115684448A - Method for detecting residual quantity of fluopicoline and metabolites thereof in plant source product

Info

Publication number: CN115684448A
Application number: CN202211136773.3A
Authority: CN
Inventors: 荣杰峰; 林金俗; 曾秋霞; 黄伙水; 杨希; 邹强
Original assignee: Quanzhou Customs Comprehensive Technical Service Center
Current assignee: Quanzhou Customs Comprehensive Technical Service Center
Priority date: 2022-09-19
Filing date: 2022-09-19
Publication date: 2023-02-03

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

Method for detecting residual quantity of fluopicoline and metabolites thereof in plant source product

Technical Field

The application relates to the field of chemical analysis, in particular to a method for detecting residual quantity of fluopicoline and metabolites thereof in plant-derived products.

Background

Fluopyrone (Bicyclopyrone) is a novel 4-hydroxyphenylpyruvate dioxygenase (4-HPPD) inhibitor herbicide which is developed in the recent years and is mainly used in crop fields such as corn, beet and grains. 4-HPPD is a heme-iron (II) -independent dioxygenase. The function of the fluroxypyr is to block the function of 4-HPPD, thereby inhibiting the biosynthesis of carotenoids, causing the emergence of albino symptoms in the meristems of plants, and finally causing the death thereof. The flurtamone is a broad-spectrum herbicide, has very excellent selectivity, is mainly used for preventing and killing broad-leaved weeds and partial gramineous weeds in crop fields such as corn, sugarcane, grains (such as wheat and barley), has higher control effect on the broad-leaved weeds of large seeds such as ragweed, xanthium and the like, and has better control effect on glyphosate-resistant weeds. Fluroxypyr is normally metabolized in plants and animals to 2- (2-methoxyethoxymethyl) -6- (trifluoromethyl) pyridine-3-carboxylic acid (SYN 503780) and 2- (2-hydroxyethoxymethyl) -6- (trifluoromethyl) pyridine-3-carboxylic acid (CSCD 686480). The application of the fluopicoline has great flexibility, can be used from before sowing to after emergence, can also well play a role under different environmental conditions and different planting modes, and the fluopicoline is listed in 2015, is registered and listed in a plurality of countries such as the United states, canada, argentina, uyghur, australia and the like, and is widely applied based on the excellent characteristics, wherein the residual limit value of the fluopicoline in plant-derived products such as barley, wheat, field corn, sugarcane and the like is set in Australia and the United states, the limit value is as low as 0.02mg/kg, the residual limit value of the fluopicoline is revised in the United states environmental protection agency in 2022, the residual limit values in 12 products such as banana, onion, strawberry, watermelon and the like are newly increased, the limit value is as low as 0.01mg/kg, and the national food safety standard GB 2763-2021 sets the residual limit values in barley, wheat, corn, fresh corn, wheat germ and sugarcane, and the residual limit values are all in the sum of the fluopicoline and the CD 780 SYN and the CD 686480.

Research aiming at the flurtamone mainly focuses on the aspects of weeding activity, environmental behavior, safety evaluation and the like, and related reports on a flurtamone residue analysis method at home and abroad are few. The Zhang Qiang and the like adopt high performance liquid chromatography to establish a high performance liquid chromatography analysis method of 44% fluroxypyr-meptyl-sodium missible oil, and the analysis method of fluroxypyr in plant-derived products is not reported. At present, the related technologies do not report how to realize qualitative screening and quantitative analysis of residual quantity of fluopicoline and metabolites thereof in plant-derived products.

Disclosure of Invention

In order to solve the problems, the application provides a method for detecting the residual quantity of the fluroxypyr and the metabolites thereof in the plant source product.

The application provides a method for detecting residual quantity of fluopicoline and metabolites thereof in plant-derived products, which comprises the following steps:

extracting an extracting solution from a plant source product;

removing impurities from the extracting solution;

and performing mass spectrometry on the extracting solution after impurity removal and purification to obtain the residual quantity of the fluroxypyr and metabolites thereof in the plant source product.

Optionally, in some embodiments of the present application, the mass spectrometry comprises: and analyzing the extracting solution by adopting ultra-high performance liquid chromatography-quadrupole orbital hydrazine high-resolution mass spectrometry, wherein the adopted chromatographic column is a silica gel matrix chromatographic column, the adopted mobile phase is an aqueous solution containing formic acid with the volume percentage concentration of 0.2-0.25%, and gradient elution separation is adopted.

Optionally, in some embodiments of the present application, the silica gel matrix chromatography column is a Waters Acquity UPLC HSS T3 model 2.1mm × 100mm, and has a particle size of 1.8 μm.

Alternatively, in some embodiments of the present application, the conditions of the chromatography column are flow rate: 0.4mL/min-0.5mL/min; sample injection amount: 2 to 3 μ L.

Optionally, in some embodiments of the present application, the working conditions of the hplc-quadrupole orbitrap high-resolution mass spectrometry are as follows: the ionization mode is HESI; the spraying voltage is 3000V-4000V; the temperature of the capillary tube is 300-400 ℃; the temperature of the ion transmission tube is 300-400 ℃; the collection mode is as follows: the method is carried out in a parallel reaction monitoring mode, and the polarity mode is positive ions; sheath gas: 30arb-40arb; auxiliary gas: 5arb-10arb; scanning parameters of mass spectrum: the secondary scanning resolution is 17500dpi to 35000dpi; the secondary mass spectral collision energy is a normalized collision energy, which is 20%,40%, or 60%.

Optionally, in some embodiments of the present application, the extracting the extract from the plant-derived product comprises:

pretreating a plant source product to obtain a plant source sample;

mixing the plant source sample, a salting-out agent and an organic solvent, and extracting to obtain an extracting solution.

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

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

Optionally, in some embodiments herein, the mass ratio of the sample of plant origin to the 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).

Optionally, in some embodiments of the present application, the impurity removal purification employs a dispersive solid-phase extraction, in which the extraction solution, the graphitized carbon black, the octadecyl bonded silica gel, and the anhydrous magnesium sulfate are mixed, centrifuged, and a supernatant is obtained, and filtered to obtain the impurity-removed and purified extraction solution.

The application has the following beneficial effects:

the method for detecting the residue of the fluopicoline and the metabolin thereof in the plant-derived product is innovatively established, and the qualitative screening and synchronous quantification of the fluopicoline and the metabolin thereof are realized for the first time; the method is simple to operate, high in sensitivity and good in accuracy, is suitable for detecting the fluroxypyr and the metabolin thereof in the plant-derived products, and can provide technical support for risk monitoring of the fluroxypyr and the metabolin thereof in the plant-derived products.

Drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the description below are only some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.

FIG. 1 is a secondary mass spectrometric quantum ion (324.07730) extraction ion chromatogram of fluroxypyr on a Waters Acquity UPLC HSST3 chromatographic column;

FIG. 2 shows fluopicoline in Thermosyncronis C ₁₈ Determining quantum ion (324.07730) extraction ion chromatogram map by secondary mass spectrum on chromatographic column;

FIG. 3 is a secondary mass spectrometric quantum ion (324.07730) extraction ion chromatogram of fluroxypyr on a ThermoAccucoreaQ chromatographic column;

FIG. 4 is an extracted ion chromatogram of a secondary mass spectrum quanta ion of flurtamone (1 ng/mL);

FIG. 5 is a secondary mass spectrometry quantitative daughter ion extraction ion flow chromatogram of metabolite SYN503780 (1 ng/mL);

FIG. 6 is a secondary mass spectrometric quantitation of ion extraction ion current chromatogram for metabolite CSCD686480 (1 ng/mL);

FIG. 7 is a secondary mass spectrum of fluroxypyr (1 ng/mL) obtained in PRM mode;

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

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

FIG. 10 is a schematic diagram showing the effect of different extraction solvents on the extraction recovery of fluroxypyr and its metabolites SYN503780, CSCD 686480;

FIG. 11 is a graph showing the effect of different amounts of extraction solvents on the recovery of extraction of fluroxypyr and its metabolites SYN503780, CSCD 686480;

FIG. 12 is a schematic diagram showing the effect of different salting-out agents on the recovery of fluroxypyr and its metabolites SYN503780, CSCD686480 in a sample;

FIG. 13 is a graph showing the effect of different amounts of adsorbents on the recovery of extraction of the fluroxypyr metabolites SYN503780 and CSCD 686480;

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

Detailed Description

The technical solutions in the embodiments of the present application will be described clearly and completely with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only some embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

The embodiment of the application provides a method for detecting the residual quantity of fluopicoline and metabolites thereof in plant-derived products, which can be, but are not limited to, wheat, barley, wheat germ, corn, green Chinese onion, banana, sugarcane, onion, strawberry and watermelon. The metabolites of fluopicoline in plants are 2- (2-methoxyethoxymethyl) -6- (trifluoromethyl) pyridine-3-carboxylic acid (SYN 503780) and 2- (2-hydroxyethoxymethyl) -6- (trifluoromethyl) pyridine-3-carboxylic acid (CSCD 686480), and the structural formulas of fluopicoline and its metabolites (SYN 503780) and (CSCD 686480) are as follows:

the detection method comprises the following steps:

extracting an extracting solution from a plant source product, wherein the extracting solution may contain a target substance flurtamone to be detected and/or a metabolite of flurtamone. In one embodiment, the extract is extracted from the plant-derived product using a solvent method.

And removing impurities and purifying the extracting solution. In one embodiment, the extraction solution is purified from impurities by dispersed solid phase extraction.

And performing mass spectrometry on the purified extracting solution to obtain the residual quantity of the fluroxypyr and the metabolites thereof in the plant source product. In some embodiments, the mass spectrometry comprises: and (3) analyzing the extracting solution by adopting ultra-high performance liquid chromatography-quadrupole orbital hydrazine high-resolution mass spectrometry, wherein the adopted chromatographic column is a silica gel matrix chromatographic column, the adopted mobile phase is an aqueous solution containing formic acid with the volume percentage concentration of 0.2-0.25%, and gradient elution separation is adopted. In other embodiments, the mobile phase may be an aqueous solution having a concentration of 0.2%, 0.21%, 0.22%, 0.23%, 0.24%, or 0.25% formic acid by volume. Experiments show that the peak tailing phenomenon of the flurbiprofen and metabolites SYN503780 and CSCD686480 thereof is obviously improved after formic acid is added into a mobile phase system, and the experiments also prove that the peak shapes of the flurbiprofen and the metabolites SYN503780 and CSCD686480 thereof can reach a better state when the volume percentage concentration of the formic acid in the aqueous phase reaches 0.2%.

Ultra-high performance liquid chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrometry (UPLC-Q-Orbitrap HRMS) organically combines a quadrupole with high selectivity with an orbit trap with high resolution and high sensitivity, can carry out rapid screening and result confirmation of a target object and a non-target object, and compared with a low-resolution triple quadrupole mass spectrometry which uses a multi-reaction monitoring mode (MRM) to carry out quantitative analysis, the UPLC-Q-Orbitrap HRMS does not need to optimize the daughter ions of the target compound and related mass spectrometry parameters one by one, simultaneously overcomes the defects of limited number and false positive misjudgment of the traditional triple quadrupole mass spectrometry, and is widely applied in the food detection industry. In the existing research, the high resolution mass spectrometry technology is mainly used for quantitative analysis of target substances in a Full scan mode (FS), which cannot achieve better instrumental sensitivity than a triple quadrupole multiple reaction monitoring mode (MRM). In a Parallel Reaction Monitoring (PRM) mode applying a quadrupole/electrostatic field orbitrap high-resolution mass spectrometry (Q active), the sensitivity of an instrument method is greatly improved compared with an FS mode, and the method can be compared with an MRM mode of a triple quadrupole.

The sensitivity of the detection method provided by the embodiment meets the requirement of residue limit of related international and domestic regulations, and can make up for the blank that no detection method exists for fluopicoline and metabolites thereof in plant-derived products; has important effects on the evaluation and management of the influence of the compound on human health and environmental safety and the prevention of trade input risks.

In some embodiments of the present application, the silica gel matrix chromatography column is a Waters Acquity UPLC HSS T3 model 2.1mm x 100mm specification with a particle size of 1.8 μm. The Waters Acquity UPLC HSS T3 chromatographic column is a high-strength silica gel matrix chromatographic column based on a full-porous silica gel matrix bonding phase, and the retention performance of the chromatographic column is superior to that of the chromatographic column of the common C ₁₈ The column 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 chromatography column are flow rate: 0.4mL/min-0.5mL/min; sample introduction amount: 2 to 3 μ L. In a preferred embodiment, the flow rate of the column is 0.4mL/min and the sample size is 2. Mu.L.

In some embodiments of the present application, the working conditions of the hplc-quadrupole orbitrap high resolution mass spectrometry are as follows: the ionization mode is HESI; the spraying voltage is 3000V-4000V; the temperature of the capillary tube is 300-400 ℃; the temperature of the ion transmission tube is 300-400 ℃; the collection mode is as follows: the method is carried out in a parallel reaction monitoring mode, and the polarity mode is positive ions; sheath gas: 30arb-40arb; auxiliary gas: 5arb-10arb; scanning parameters of mass spectrum: the secondary scanning resolution is 17500dpi to 35000dpi; the secondary mass spectral collision energy is normalized collision energy, which is 20%,40%, or 60%.

In a specific example, the working conditions of the ultra performance liquid chromatography-quadrupole orbitrap high resolution mass spectrometry are as follows: the ionization mode is HESI; the spraying voltage is 3500V; the temperature of the capillary tube is 350 ℃; the temperature of the ion transmission tube is 350 ℃; the collection mode is as follows: the method is carried out in a parallel reaction monitoring mode, and the polarity mode is positive ions; sheath gas: 35arb; auxiliary gas: 10arb; scanning parameters of mass spectrum: the secondary scanning resolution is 17500dpi; the secondary mass spectral collision energy is normalized collision energy, which is 20%,40%, or 60%.

In some embodiments of the present application, extracting an extraction solution from a plant-derived product comprises:

and (3) pretreating the plant source product to obtain a plant source sample. In some embodiments, pre-treating the plant source product refers to: samples of grains of plant origin such as rice, wheat, corn (dried) and the like are crushed by a crusher and all pass through a standard screen of 425 μm. The vegetable and fruit samples are crushed by a knife grinder at 5000r/min and fully mixed.

Mixing the plant source sample, salting-out agent and organic solvent, and extracting to obtain extractive solution. In a specific example, 5g (accurate to 0.01 g) of prepared plant source sample is weighed in a 50mL polypropylene centrifuge tube, grain samples are added with 10mL of water in advance, vortex and uniform mixing are carried out, and standing is carried out for 30min; 5g of sodium chloride, 25mL of 0.4% formic acid acetonitrile solution and 1 ceramic homogeneous proton are added, vortex oscillation extraction is carried out for 5min, and centrifugation is carried out for 3min at 5000 r/min. Aspirate 1.5mL of supernatant into a 2mL polypropylene centrifuge tube.

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

In some embodiments of the present application, the organic solvent is 0.4% to 0.6% acetonitrile formate by volume. The adoption of 0.4-0.6% of acetonitrile formate is beneficial to improving the recovery rate of fluopicoline and the metabolites SYN503780 and CSCD686480 of the fluopicoline, thereby being beneficial to improving the extraction efficiency. In a preferred embodiment, the organic solvent is 0.4% acetonitrile formate by volume.

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

in some embodiments of the application, the impurity removal and purification is performed by adopting dispersed solid phase extraction, namely mixing the extracting solution, graphitized carbon black, octadecyl bonded silica gel and anhydrous magnesium sulfate, centrifuging, taking supernatant, and filtering to obtain the extracting solution after impurity removal and purification. The commonly used QuEChERS purifying reagents in plant-derived samples comprise GCB and C ₁₈ PSA and NH ₂ Etc. wherein PSA and NH ₂ The action mechanisms of the compounds are similar, the compounds have weak anion exchange capacity, and organic acids, polar pigments, fatty acids, saccharides and other components capable of forming hydrogen bonds in a sample can be effectively removed through the action of the hydrogen bonds and the compounds; c ₁₈ Removing nonpolar compounds such as volatile oil, terpenes, and lipids; GCB has extremely high adsorption capacity on polar and nonpolar organic interferents in a sample, and has obvious effect on removing pigments in plants; the anhydrous magnesium sulfate can remove water in the sample solution. The experiment investigates GCB, PSA and C ₁₈ And MgSO 2 ₄ The recovery rate of the three purifying reagents after adsorbing the 5 mu g/L fluroxypyr standard solution. The result shows that the recovery rates of the fluroxypyr and the metabolite SYN503780 and the metabolite CSCD686480 thereof after PSA purification are all lower than 80 percent, and the recovery rate is reduced more along with the increase of the dosage; GCB, C ₁₈ 、MgSO ₄ The average adsorption recovery rate of the three substances is between 90 and 110 percent.

In other embodiments, a QuEChERS (Quick, easy, cheap, effective, rugged, safe) method is adopted, an acetonitrile formate solution is used as an extraction solvent, salting out is carried out, an extracting solution is subjected to dispersed solid phase extraction and purification, a method for detecting residue of fluopicoline and metabolites thereof in plant-derived products is established based on a PRM mode of ultra-high performance liquid chromatography-quadrupole/electrostatic field orbital trap high resolution mass spectrum, a secondary mass spectrum database of fluopicoline and metabolites thereof is established, and qualitative screening and synchronous quantification of fluopicoline and metabolites thereof are realized. The sensitivity of the method meets the requirement of residual limit of relevant laws and regulations in China, and can make up for the blank that no detection method is available for the fluopicoline and the metabolin thereof in plant-derived products in China; has important effects on the evaluation and management of the influence of the compound on human health and environmental safety and the prevention of trade input risks.

In order to make the above implementation details and operations of the present invention clearly understood by those skilled in the art, and to make the performance of the method for detecting the residual amount of fluopicoline and its metabolites in plant-derived products of the present invention remarkably appear, the above technical solution is exemplified by experimental examples below.

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 are analytically pure and purchased from chemical reagents of national drug group, inc.; acetonitrile and methanol were both chromatographically pure and purchased from TEDIA corporation, usa. Graphitized carbon black (GCB, 40-120 μm), ethylenediamine-N-propylsilanized silica gel (PSA, 40-60 μm), and octadecyl-bonded silica gel (C) ₁₈ 40-60 μm) were purchased from Shanghai Ana spectral laboratory science and technology, inc. And (3) microporous filter membrane: 0.22 μm, organic phase type; ceramic proton homogeneity: 2cm (long) × 1cm (outer diameter). Fluroxyprione (C) ₁₉ H ₂₀ F ₃ NO ₅ 100. Mu.g/mL, bepure corporation). Fluroxyprione metabolite SYN503780:2- (2-methoxyethoxymethyl) -6- (trifluoromethyl) pyridine-3-carboxylic acid (C) ₁₁ H ₁₂ F ₃ NO ₄ 100. Mu.g/mL, bepure corporation); flurtamone metabolite CSCD686480:2- (2-Hydroxyethoxymethyl) -6- (trifluoromethyl) pyridine-3-carboxylic acid (C) ₁₀ H ₁₀ F ₃ NO ₄ Purity 97.0%, dr.

Q-exact quadrupole-electrostatic field orbitrap high resolution mass spectrometry system (Thermo Scientific, USA); ultiMate3000 rapid high performance liquid chromatography (Thermo Scientific, usa); milli-Q water purifier (Millipore, USA); a pulverizer (Shanghai Jiading grain and oil instruments, inc.); GM200 knife grinder (leys, germany); vortex oscillators (Talboys corporation, usa); centrifuge (Hunan instruments laboratory Instrument development Co., ltd., hunan province).

Preparing a standard solution:

an appropriate amount of CSCD686480 was weighed and dissolved in methanol to prepare a 1000mg/L standard stock solution. Respectively sucking a proper amount of fluopicoline, SYN503780 and CSCD686480 standard stock solutions, and diluting the raw materials into a mixed standard intermediate solution of 0.002mg/L of fluopicoline and 0.1mg/L of fluopicoline metabolites SYN503780 and CSCD686480 with methanol; and diluting the mixed standard intermediate solution by using methanol and a blank sample extracting solution to obtain a series of standard working solutions and a matrix matching standard working solution, wherein the concentration of the fluopicoline is 0.002 mu g/L, 0.004 mu g/L, 0.02 mu g/L, 0.04 mu g/L and 0.2 mu g/L, the concentration of fluopicoline metabolites SYN503780 and CSCD686480 is 0.1 mu g/L, 0.2 mu g/L, 1 mu g/L, 2 mu g/L and 10 mu g/L, and the working solutions are ready to use.

Sample pretreatment:

after being crushed by a crusher, grain samples such as rice, wheat, corn (dry) and the like can pass through a standard mesh screen with the diameter of 425 mu m, vegetable and fruit samples are crushed by a knife grinder with the speed of 5000r/min and are fully mixed. Weighing 5g (accurate to 0.01 g) of prepared sample in a 50mL polypropylene centrifuge tube, adding 10mL of water into grain sample in advance, uniformly mixing in a vortex manner, and standing for 30min; 5g of sodium chloride, 25mL of 0.4% formic acid acetonitrile solution and 1 ceramic homogeneous proton are added, vortex oscillation is carried out to extract for 5min, and centrifugation is carried out for 3min at 5000 r/min. Pipette 1.5mL of supernatant into a 2mL polypropylene centrifuge tube, add 5mg of GCB and 5mg of C ₁₈ And 50mg MgSO ₄ Vortex mixing for 1min, centrifuging at 12000r/min for 3min, collecting supernatant, and filtering with 0.22 μm organic filter membrane for determination.

The instrument conditions were as follows:

a chromatographic column: a Waters Acquity UPLC HSS T3 liquid chromatography column; flow rate: 0.4mL/min; sample introduction amount: 2 mu L of the solution; the mobile phase A is 0.2% formic acid water, and the mobile phase B is methanol; gradient elution procedure 0-2min,10% B;2-2.5min,10% -90% by weight B;2.5-5min,90% by weight B;5-5.1min,90% -10% by weight B;5.1-7min,10% by weight B.

HESI ionization mode; the spraying voltage is 3500V; the capillary temperature is 350 ℃; ion transfer tube temperature: 350 ℃; the collection mode is as follows: parallel Reaction Monitoring (PRM) mode, positive ion mode; sheath gas (N2): 35arb; auxiliary gas (N2): 10arb; secondary scanning resolution: 17500; secondary mass spectral collision energy is Normalized Collision Energy (NCE): 20%,40% and 60%.

Under the mass spectrum conditions, mass spectrum parameters of the fluroxypyr and the metabolin thereof obtained by optimization are shown in table 1.

TABLE 1

Experimental example 2 optimization of apparatus conditions

Regarding the optimal selection of the chromatography column:

this example compares 5. Mu.g/L fluroxypyr and metabolite standards on a Waters Acquity UPLC HSST3 column (2.1 mm 100mm 1.8 μm), thermoSyncrons C ₁₈ The peak widths and response values on the columns (2.1 mm. Times.100 mm. Times.1.9 μm) and on the ThermoAccucoreaQ column (2.1 mm. Times.150 mm. Times.2.6 μm) were good for both fluroxypyr metabolites SYN503780 and CSCD686480 on the three columns; however, for flurtamone, the compound is shown in ThermoAccucoreaQ C ₁₈ Relatively serious peak broadening and tailing appear on the chromatographic column, compared with ThermoSyncronis C ₁₈ The chromatographic column can obtain better peak shape and higher response on a Waters Acquity UPLC HSS T3 chromatographic column, the Waters Acquity UPLCHSS T3 chromatographic column is a high-strength silica gel matrix chromatographic column based on a full-porous silica gel matrix bonding phase, and the retention performance of the chromatographic column is superior to that of a common C ₁₈ And the column can obtain better peak shape and response. Thus, a Waters Acquity UPLC HSST3 chromatography column is a more preferred chromatography column. With specific reference to fig. 1-3, fig. 1 is a two-stage mass spectrometric quantum ion (324.07730) extraction ion chromatogram of fluroxypyr on a Waters Acquity UPLC HSST3 chromatographic column; FIG. 2 shows fluopicoline in Thermosyncronis C ₁₈ Secondary mass spectrometry quantum ion (324.07730) extraction ion chromatograms on chromatographic columns; fig. 3 is a secondary mass spectrometric quantum ion (324.07730) extraction ion chromatogram of fluroxypyr on a thermoaccucoreeaq chromatographic column.

Experimental example 3 optimization of apparatus conditions

Regarding the optimal selection of the mobile phase:

experimental examples the effect of different mobile phase systems (acetonitrile-water, acetonitrile-0.2% formic acid water, methanol-0.1% formic acid water, methanol-0.2% formic acid water and methanol-5 mmol/L ammonium acetate water (containing 0.1% formic acid)) on the chromatographic peak shape and signal response was examined. Results show that the peak types of the flurbiprofen and metabolites SYN503780 and CSCD686480 thereof in a mobile phase system (acetonitrile-water and methanol-water) without formic acid both have forward extending and tailing phenomena, the tailing phenomenon of the peak after formic acid is added in the mobile phase system is obviously improved, the peak shapes of the three substances can reach a better state when the formic acid content in the water phase reaches 0.2%, and the response and the peak shapes of the three substances are not obviously influenced by the addition of ammonium acetate in the water phase. The experimental result shows that both acetonitrile-0.2% formic acid water and methanol-0.2% formic acid water can be used as mobile phases, and the methanol-0.2% formic acid water is finally selected as the mobile phase because the system pressure is lower when the methanol is used as the mobile phase.

Experimental example 4 optimization of apparatus conditions

Optimized selection of mass spectrometry conditions:

the PRM mode of UPLC-Q-Orbitrap HRMS is utilized to detect the flurbicolone and metabolite mixed standard solution thereof, and only the molecular ion peak [ M + H ] of each compound is required to be provided] ⁺ The theoretically exact mass number, and the collision energy may be set to 20%,40%, or 60% using the Normalized Collision Energy (NCE). The PRM acquisition mode firstly utilizes the selective detection capability of a quadrupole mass analyzer to selectively detect parent ion information, namely a molecular ion peak [ M + H ] of a target compound in a primary mass spectrum] ⁺ Subsequently fragmenting the parent ions in a collision cell; and finally, detecting all fragment information of the selected parent ions in the secondary mass spectrum by using a high-resolution mass analyzer with high mass precision. The PRM technology is very similar to the MRM technology, the difference is that the two-stage mass spectrum detection mode is different, the PRM technology is that all the daughter ions are detected by a high-resolution mass analyzer after the fragmentation of the mother ions, compared with the MRM technology, the background noise can be further reduced by monitoring the accurate mass number of the daughter ions, and the interference of the ion noise is far smaller than that of the MRM technology, so that the sensitivity and the accuracy of the instrument are improved. Meanwhile, the PRM technology is simpler and more convenient to use, and accurate parent ion-daughter ion searching and related parameters optimization in advance are not needed. Perflufenim and its metabolites SYN503780 and CSCD6864 using PRM mode80, and the results are shown in fig. 4-9. FIG. 4 is an extracted ion chromatogram of a secondary mass spectrum quanta ion of flurtamone (1 ng/mL); FIG. 5 is a secondary mass spectrometry quantitative daughter ion extraction ion flow chromatogram of metabolite SYN503780 (1 ng/mL); FIG. 6 is a secondary mass spectrometric quantitive ion extraction ion current chromatogram of metabolite CSCD686480 (1 ng/mL); FIG. 7 is a secondary mass spectrum of fluroxypyr (1 ng/mL) obtained in PRM mode; FIG. 8 is a secondary mass spectrum of the metabolite SYN503780 (1 ng/mL) obtained in PRM mode; FIG. 9 is a secondary mass spectrum of the metabolite CSCD686480 (1 ng/mL) obtained in PRM mode.

Experimental example 5 optimization of pretreatment method

Regarding the optimized selection of the extraction solvent:

for the plant-derived sample, acetone, ethyl acetate, methanol, acetonitrile can be used as an extraction solvent. Methanol is excluded as the extraction solvent because the methanol extract cannot be separated from the aqueous phase by salting out. Experiment 0.4% acetone formate, 0.4% ethyl acetate formate, acetonitrile, 0.1% acetonitrile formate, 0.2% acetonitrile formate and 0.4% acetonitrile formate were selected as extraction solvents, and the extraction effects of different solvents were examined with the average recovery rate (added standard amount 5 μ g/kg) of flurtamone and its metabolites SYN503780 and CSCD 686480. For acetonitrile and acetonitrile formate solutions, wheat samples added with 5 mu g/kg of fluroxypyr and metabolites SYN503780 and CSCD686480 thereof are processed according to the sample pretreatment method of the experimental example 1 and then are measured on a machine; each experiment is measured in parallel for 3 times, and a solvent standard curve is adopted for calibration and quantification; 0.4% acetone formate and 0.4% ethyl formate were treated by the pretreatment of the sample according to Experimental example 1, and 1mL of each extract was subjected to nitrogen blowing at 40 ℃ and then redissolved in acetonitrile, and then loaded onto a machine. As can be seen from fig. 10, when 0.4% acetone formate was used as the extraction solvent, the recovery rate of the metabolite CSCD686480 was low; when 0.4% ethyl formate acetate is used as an extraction solvent, the recovery rate of a metabolite SYN545910 and a metabolite CSCD686480 is low; when acetonitrile is used as an extraction solvent, the recovery rates of the fluroxypyr and metabolites SYN503780 and CSCD686480 thereof are increased along with the increase of the content of formic acid, and the recovery rates of the three substances are better and the extraction efficiency is higher when 0.4% formic acid acetonitrile is extracted. Therefore, the pH value of the extraction solvent has a large influence on the extraction efficiency, and the research shows that 0.4% of acetonitrile formate is selected as the extraction solvent, so that the extraction efficiency is improved.

Experimental example 6 optimization of pretreatment method

Optimization of the amount of acetonitrile used as an extraction solvent:

wheat is used as an experimental sample, 10mL, 25mL, 40mL and 50mL are selected for experiment optimization of acetonitrile dosage, and the final recovery rate is used as a research basis. Referring to fig. 11, the results show that when the extraction solvent is 10mL, the recovery rate of the metabolite is relatively low due to the influence of the matrix effect, and when the amount of acetonitrile is 25mL, the recovery rate can meet the requirement of analytical determination, and when the amount of acetonitrile continues to increase, the change of the recovery rate is not obvious, and the detection limit of the method is increased. Therefore, the most preferable extraction solvent acetonitrile dosage is 25mL, the operation is convenient, and the consumable cost is reduced.

Experimental example 7 optimization of pretreatment method

Regarding the optimized selection of salting-out agent:

the salting-out agent mainly removes redundant water in a sample, so that the solubility of pesticide components in an organic phase is increased, and the extraction efficiency is improved. The experiment tests the effect of 3 common salting-out agents, namely sodium chloride, anhydrous sodium sulfate, and anhydrous magnesium sulfate + sodium chloride (3 + 1) mixed salting-out agents on the extraction effect of 3 pesticide components in the sample, and the result is shown in FIG. 12. As can be seen from the figure, when the salting-out agent is anhydrous sodium sulfate, anhydrous magnesium sulfate and sodium chloride (3 + 1), the extraction recovery rates of the three components are all lower than the extraction recovery rate when sodium chloride is used as the salting-out agent, thus demonstrating that the salting-out effect is favorable for improving the recovery rate of the extracting solution when the salting-out agent is sodium chloride, and the sodium chloride is low in price and reduces the cost.

Experimental example 8 optimization of pretreatment method

Regarding the optimal selection of the purification reagents:

the commonly used QuEChERS purifying reagents in plant-derived samples comprise GCB and C ₁₈ PSA and NH ₂ Etc. wherein PSA and NH ₂ Has weak anion exchange capacity and acts with the compound through hydrogen bondsOrganic acid, polar pigment, fatty acid, saccharide and other components capable of forming hydrogen bonds in the sample can be effectively removed; c ₁₈ Removing nonpolar compounds such as volatile oil, terpenes, and lipids; GCB has extremely high adsorption capacity on polar and nonpolar organic interferents in a sample, and has obvious effect on removing pigments in plants; the anhydrous magnesium sulfate can remove the water in the sample liquid. The experiment investigates GCB, PSA and C ₁₈ And MgSO ₄ The recovery rate of the 3 purifying reagents after adsorbing the 5 mu g/L fluroxypyr standard solution. The results show that the recovery rates of the three substances after PSA purification are all lower than 80 percent, and the recovery rates are reduced more along with the increase of the dosage; GCB, C ₁₈ 、MgSO ₄ The average adsorption recovery rate of the three substances is between 90 and 110 percent, thereby showing that GCB and C are selected ₁₈ 、MgSO ₄ As a purifying reagent, the method is favorable for improving the recovery rate of the extracting solution.

Experimental example 9 optimization of pretreatment method

Determination of the combined amount of the decontaminating agents:

when a plant-derived sample is purified by dispersed solid phase extraction, two or more purification reagents are usually used to remove interfering impurities such as lipids and saccharides from the sample. Experiment examined 3 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 ) The purification effect of the standard addition extracting solution of 5 mu g/kg on the wheat blank sample is shown in figure 13, and the results show that the recovery rates of 3 groups of purification reagents are all between 90 and 110 percent and can meet the experimental requirements; from the actual purifying effect, along with GCB and C ₁₈ The recovery rate of the flurtamone and metabolites SYN503780 and CSCD686480 thereof is slightly changed due to the increase of the using amount, and the combination I can meet the requirement of most samples on pigment removal. Thus, the purification reagent combination is selected to be 5mg GCB +5mg C ₁₈ +50mg MgSO ₄ It is favorable for improving the purification effect and reducing the comprehensive cost.

Experimental example 10 Effect of matrix Effect

The UPLC-Q-Orbitrap HRMS has stronger anti-interference capability, but the Matrix effect still exists, and the plant-derived samples are numerous, and the Matrix is complex, so the Matrix effect needs to be evaluated, and the Matrix effect (Matrix effects, ME) mainly generates influence on the ionization process of a target object by components other than the target object in the sample and the sample Matrix eluted from the target object in a high-resolution mass spectrum system, thereby causing ionization inhibition or enhancement. Experiment A blank matrix solution is prepared according to the sample pretreatment method in the experimental example 1, and a standard working solution and a matrix standard working solution are prepared according to the standard solution preparation method in the experimental example and are measured on a machine. The matrix effect was calculated as follows: ME = [ (slope of substrate matching calibration curve/slope of pure solvent standard curve) -1] × 100%, as shown in fig. 14, the results indicate that the substrate effect of fluroxypyr and its metabolites in rice, wheat, corn, sugarcane, banana, welsh onion, broccoli and watermelon is between 1% and 20%, the substrate effect is less than 20%, and the substrate effect of fluroxypyr and its metabolites in these 6 substrates is weak substrate effect without compensation for substrate effect.

Experimental example 11 methodological evaluation

Regarding the linear relationship and the quantitative lower limit:

the fluroxypyr and metabolin series standard working solution and the substrate matching standard working solution are prepared according to the standard solution preparation method of the experimental example 1, sample injection detection is carried out according to the instrument condition of the experimental example 1, the mass concentration (X, mu g/L) is used as a horizontal coordinate, and the peak area is used as a vertical coordinate (Y) to draw a standard working curve. Adding a proper amount of standard solution into the blank sample solution, performing machine measurement, and determining a lower limit of quantitation (LOQ) by using a 10-fold signal-to-noise ratio (S/N = 10), wherein the linear regression equation, the correlation coefficient (r), the linear range and the quantitative limit of the fluroxypyr and the metabolites thereof are shown in a table 2, and the table 2 is the regression equation, the correlation coefficient, the linear range and the quantitative limit of the fluroxypyr and the metabolites thereof.

TABLE 2

In table 2, Y: peak area of qualitative ion; x: mass concentration,. Mu.g/L.

Experimental example 12 recovery and relative standard deviation

Low, medium and high 3 level spiking recovery experiments were performed on wheat, corn, green onion, banana and sugarcane blank samples, respectively, with 6 replicates of each spiking concentration determination, with recovery and relative standard deviation (RSD, n = 6) as shown in table 3. The average recovery rate of the fluopicoline and the metabolites SYN503780 and CSCD686480 of the fluopicoline at 3 standard adding levels is 84.6-118.5%, and the RSD is 2.6-7.4%, so that the method for detecting the residual quantity of the fluopicoline and the metabolites of the fluopicoline in the plant source product has good accuracy and precision.

TABLE 3

Actual sample detection

The method is adopted to detect 5 market purchase samples of wheat, corn, green Chinese onion, banana and sugarcane respectively, qualitatively screen the fluroxypyr and metabolites thereof in the samples by adopting accurate mass number and retention time, and detect fluroxypyr residues in an entry corn seed by combining with secondary characteristic fragment ions for confirmation.

The method for analyzing and detecting the residual quantity of the flurtamone and the metabolites thereof in the plant-derived products by using the ultra-high performance liquid chromatography-quadrupole electrostatic orbitrap high-resolution mass spectrometry is established in the above embodiments of the application, under the optimized experimental conditions, the linear relationship of the flurtamone in the range of 0.002-0.2 mu g/L, the metabolite SYN503780 and the metabolite CSCD686480 in the range of 0.1-10 mu g/L is good, and the correlation coefficients are all larger than 0.995. The average recovery rate of the blank sample at the low, medium and high 3 levels by adding the standard is 84.6-118.5%, the RSD is 2.6-7.4%, the quantification limit of the fluroxypyr is 0.01 mu g/kg, and the quantification limit of the metabolites SYN503780 and CSCD686480 is 0.5 mu g/kg. The method is high in sensitivity, simple, rapid and accurate to operate, and can meet the detection requirement of residual quantity of the fluroxypyr and metabolites thereof in plant-derived products.

The most suitable pretreatment method and apparatus conditions are determined by optimizing the apparatus conditions, extraction solvent, purification mode and other conditions. The matrix effect of the samples is inspected, and the matrix effect of the fluroxypyr and the metabolites thereof in 8 samples is less than 20%, the fluroxypyr and the metabolites thereof are weak matrix effects, and no compensation measures need to be taken. Under the optimized experimental condition, the linear relation of the flurtamone in the range of 0.002-0.2 mu g/L, the metabolite SYN503780 and the metabolite CSCD686480 in the range of 0.1-10 mu g/L is good, and the correlation coefficients are all larger than 0.995. The average recovery of the blank samples at the low, medium and high 3-spiking levels ranged from 84.6% to 118.5%, the relative standard deviation (n = 6) ranged from 2.6% to 7.4%, the quantitation limit for fluroxypyr was 0.01 μ g/kg, and the quantitation limits for metabolite SYN503780 and metabolite CSCD686480 were 0.5 μ g/kg. The method is high in sensitivity, simple, rapid and accurate to operate, and can meet the detection requirement of residual quantity of the fluroxypyr and metabolites thereof in plant-derived products. The method is adopted to carry out determination on 25 samples, and the fluroxypyr residue is detected in one imported corn sample. The method is simple to operate, high in sensitivity and good in accuracy, is suitable for detecting the fluopicoline and the metabolites thereof in the plant-derived products, and can provide technical support for risk monitoring of the fluopicoline and the metabolites thereof in the plant-derived products.

The method for detecting the residual quantity of the fluroxypyr and the metabolites thereof in the plant-derived products, which is provided by the present application, is described in detail above, and the principle and the implementation manner of the present application are explained in the present application by using specific examples, and the description of the above examples is only used for helping to understand the method and the core idea of the present application; meanwhile, for those skilled in the art, according to the idea of the present application, the specific implementation manner and the application scope may be changed, and in summary, the content of the present specification should not be construed as a limitation to the present application.

Claims

1. A method for detecting residual quantity of fluopicoline and metabolites thereof in plant-derived products is characterized by comprising the following steps:

extracting an extracting solution from a plant source product;

removing impurities from the extracting solution and purifying;

and performing mass spectrometry on the extract after impurity removal and purification to obtain the residual quantity of the fluroxypyr and metabolites thereof in the plant source product.

2. The method for detecting the residual amount of fluopicoline and its metabolites in plant-derived products according to claim 1, wherein said mass spectrometric analysis comprises: and analyzing the extracting solution by adopting ultra-high performance liquid chromatography-quadrupole orbital hydrazine high-resolution mass spectrometry, wherein the adopted chromatographic column is a silica gel matrix chromatographic column, the adopted mobile phase is an aqueous solution containing formic acid with the volume percentage concentration of 0.2-0.25%, and gradient elution separation is adopted.

3. The method for detecting the residual amount of fluopicoline and its metabolites in plant-derived products as claimed in claim 2, wherein said silica gel matrix chromatographic column is a WatersAcquisty UPLC HSS T3 type chromatographic column, having a specification of 2.1mm x 100mm and a particle size of 1.8 μm.

4. The method for detecting the residual amount of fluopicoline and its metabolites in plant-derived products according to claim 2, wherein the conditions of the chromatographic column are flow rate: 0.4mL/min-0.5mL/min; sample introduction amount: 2 to 3 μ L.

5. The method for detecting the residual quantity of fluopicoline and metabolites thereof in plant-derived products according to claim 2, wherein the operating conditions of the ultra-high performance liquid chromatography-quadrupole orbitrap high-resolution mass spectrometry are as follows:

the ionization mode is HESI;

the spraying voltage is 3000V-4000V;

the temperature of the capillary tube is 300-400 ℃;

the temperature of the ion transmission tube is 300-400 ℃;

the collection mode is as follows: the method is carried out in a parallel reaction monitoring mode, and the polarity mode is positive ions;

sheath gas: 30arb-40arb;

auxiliary gas: 5arb-10arb;

scanning parameters of mass spectrum:

the secondary scanning resolution is 17500dpi to 35000dpi;

the secondary mass spectral collision energy is a normalized collision energy of 20%,40%, or 60%.

6. The method for detecting the residual amount of fluopicoline and its metabolites in plant-derived products according to claim 1, wherein said extracting the extract from the plant-derived products comprises:

pretreating a plant source product to obtain a plant source sample;

mixing the plant source sample, a salting-out agent and an organic solvent, and extracting to obtain the extracting solution.

7. The method for detecting the residual amount of fluopicoline and its metabolites in plant-derived products according to claim 6, wherein said organic solvent is any one of acetone formate, ethyl acetate formate, acetonitrile or acetonitrile formate; and/or

The salting-out agent is any one or a mixture of sodium chloride, anhydrous sodium sulfate and anhydrous magnesium sulfate.

8. The method for detecting the residual quantity of fluopicoline and its metabolites in plant-derived products according to claim 6, wherein the organic solvent is 0.4-0.6% by volume of acetonitrile formate.

9. The method for detecting the residual quantity of fluopicoline and metabolites thereof in plant-derived products according to claim 6, wherein the mass ratio of the plant-derived sample to the 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 detecting the residual quantity of the fluroxypyr and the metabolites thereof in the plant-derived products according to claim 1, wherein the impurity removal purification is performed by adopting dispersive solid-phase extraction, wherein the extraction solution, the graphitized carbon black, the octadecyl bonded silica gel and the anhydrous magnesium sulfate are mixed, and after centrifugation, the supernatant is taken and filtered to obtain the extraction solution after impurity removal and purification.