CN113514590A - Low-toxicity dispersive solid-phase extraction method for detecting pesticide and metabolite residues thereof in fruits - Google Patents
Low-toxicity dispersive solid-phase extraction method for detecting pesticide and metabolite residues thereof in fruits Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/062—Preparation extracting sample from raw material
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Abstract
The invention discloses a low-toxicity dispersion solid-phase extraction method for detecting pesticide and metabolite residues thereof in fruits. A low-toxicity dispersive solid-phase extraction method for detecting pesticide and metabolite residues thereof in fruits comprises the following steps: (1) sample extraction: weighing a homogenized fruit sample, adding ethanol, mixing with a homogenized matrix, adding ammonium sulfate, mixing uniformly to enable an organic phase and a water phase to be layered at room temperature, transferring a target extract into an upper-layer ethanol phase after centrifugation, and collecting a supernatant in a centrifugal tube; (2) sample purification: and adding a purifying agent into the obtained supernatant for purification to obtain a purified solution, and filtering the purified solution to obtain a sample solution to be detected after solid-phase extraction. The dispersive solid-phase extraction method provided by the invention can be used for detecting the neonicotinoid pesticides and metabolites thereof, has the advantages of no use of highly toxic organic solvents in the operation process, simple and convenient operation, and good sensitivity, accuracy and precision.
Description
Technical Field
The invention relates to the technical field of pesticide residue detection, in particular to a low-toxicity dispersion solid-phase extraction method for detecting pesticide and metabolite residues of the pesticide in fruits.
Background
Pesticide residue in the environment and food can threaten human health in various different ways, an effective analysis method is established to monitor pesticide residue in the environment and food samples, and important technical support can be provided for the supervision department to carry out pesticide residue risk assessment. At present, the dispersive solid-phase extraction technology is the most widely applied pretreatment method in the standard pesticide residue detection method, but with the development of the detection technology, the traditional methods need to be further improved in the aspects of environmental protection, operation efficiency, sensitivity and the like. The low-toxicity dispersed solid phase extraction technology can effectively reduce the dosage of high-toxicity organic solvent, and the use of high-efficiency adsorption material can also improve the purification efficiency of the method and meet the increasingly improved pesticide residue analysis requirement.
The dispersed solid phase extraction technology based on the modified carbon nano material is applied to the detection of pesticide residues. The carbon nano tube derivative has the obvious advantages of low density, high porosity, large specific surface area, chemical inertness and the like, is widely applied to the fields of catalysis, gas separation, adsorption separation and the like, and is a potential good purifying agent in the pesticide residue detection technology. Therefore, the use of different surface-modified multi-walled carbon nanotube materials as a pesticide residue purifying agent is an important development trend for the application and improvement of the dispersed solid-phase extraction technology.
In view of high cost performance and sustainable development, the application of low-toxicity and green organic solvents in pesticide residue analysis is receiving more and more attention. Ethanol is one of the most abundant low-toxicity organic solvents, and has good dissolving effect on different types of organic compounds. However, because of its good intersolubility with water, it is difficult to perform effective enrichment and separation of target substances in a high water content matrix, and thus there are few reports on the extraction of fruit pesticide residues. Therefore, the method for constructing the phase separation by the efficient and green strategy takes the ethanol as the extracting agent and is applied to the analysis and detection of the neonicotinoid insecticides, so that the method has wide application prospect in the aspect of pesticide residue analysis.
Disclosure of Invention
The invention solves the problems in the prior art, and aims to provide a low-toxicity dispersed solid-phase extraction method for detecting pesticide and metabolite residues in fruits.
In order to achieve the purpose, the invention adopts the technical scheme that: a low-toxicity dispersive solid-phase extraction method for detecting pesticide and metabolite residues thereof in fruits comprises the following steps:
(1) sample extraction: weighing a homogenized fruit sample, adding ethanol, mixing with a homogenized matrix, adding ammonium sulfate, mixing uniformly to enable an organic phase and a water phase to be layered at room temperature, transferring a target extract to an upper ethanol phase after centrifugation, and collecting supernatant in a centrifuge tube;
(2) sample purification: and adding a purifying agent into the obtained supernatant for purification to obtain a purified solution, and filtering the purified solution to obtain a sample solution to be detected after solid-phase extraction.
The filtration of the purified solution provided by the invention means that the purified solution passes through an organic filter membrane with the diameter of 0.22 mu m.
According to the sample pretreatment method provided by the invention, the extraction solvent is ethanol, and the ethanol phase and the water phase are separated by utilizing the ammonium sulfate with a specific proportion, so that the toxicity and the cost are lower compared with acetonitrile used in the traditional dispersed solid phase extraction, and the development trend of green analysis is met. The dispersive solid-phase extraction method provided by the invention can be used for detecting the neonicotinoid pesticides and metabolites thereof, has the advantages of no use of highly toxic organic solvents in the operation process, simple and convenient operation, and good sensitivity, accuracy and precision.
Preferably, the step (1) specifically comprises the following steps: weighing a homogenized fruit sample into a centrifuge tube, adding ethanol and ultrapure water, performing vortex extraction, wherein the adding ratio of the homogenized fruit sample to the ethanol is 1:2-4g/mL, the volume ratio of the ethanol to the ultrapure water is 2-4:1, adding ammonium sulfate, uniformly mixing, centrifuging, promoting the layering of an organic phase and a water phase, the mass ratio of the ammonium sulfate to the homogenized fruit sample is 2:5, transferring a target extract to an upper ethanol phase, and collecting a supernatant in the centrifuge tube.
Preferably, the addition ratio of the purifying agent and the supernatant in the step (2) is 0.05-0.20:1 g/mL.
Preferably, the purifying agent is selected from more than one of multi-walled carbon nanotubes, carboxylated multi-walled carbon nanotubes, hydroxylated multi-walled carbon nanotubes and aminated multi-walled carbon nanotubes. The purifying agent is a surface-modified multi-wall carbon nanotube, is a novel adsorbent material, and can selectively remove main pigment interference components in a matrix sample with high pigment content without causing the recovery loss of pesticides to be detected.
The invention also provides a method for detecting pesticide and metabolite residues in fruits, which comprises the following steps:
(1) obtaining a sample solution to be detected after solid-phase extraction according to the low-toxicity dispersion solid-phase extraction method for detecting pesticide and metabolite residues in the fruits;
(2) carrying out high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) on the solution to be detected to detect pesticide and metabolite residues in the fruits;
liquid chromatography conditions: a chromatographic column: c18, 100mm × 2.1mm, 3.0 μm; sample introduction amount: 10 mu L of the solution; flow rate: 0.4mL min-1(ii) a Column temperature: 35 ℃; methanol is used as a mobile phase A, 5mmol L-1Ammonium acetate and 20mmol L-1Taking the formic acid mixed aqueous solution as a mobile phase B for gradient elution, wherein the gradient elution procedure is as follows: 0.0-1.0 min/10% A, 1.0-4.0 min/10% -90% A, 4.0-8.4 min/90% A, 8.4-8.5 min/90% -10% A, 8.5-10.0 min/10% A;
the mass spectrum conditions are as follows: a spray ionization source; monitoring multiple reactions; scanning positive ions; the ionization temperature is 550 ℃; the spraying voltage is 5500V; atomizer pressure 55 psi.
Preferably, the pesticide and the metabolite thereof are selected from more than one of imidacloprid, acetamiprid, thiamethoxam and clothianidin, and the detection conditions are shown in the following table 1:
TABLE 1
The pesticide in the invention is neonicotinoid pesticide.
The invention also protects the application of the detection method in detecting pesticide and metabolite residues in fruits.
Preferably, the fruit is citrus or peach. The technology based on the dispersive solid-phase extraction and the high performance liquid chromatography-tandem mass spectrometry is applied to the analysis of the residues of the neonicotinoid pesticides and the metabolites thereof in the fruits, and the dosage of the high-toxicity organic solvent and the extraction cost in the detection process can be effectively reduced. The established method can be widely used for measuring the neonicotinoid pesticide residues in fruit samples such as oranges, peaches and the like.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the sample pretreatment method, the separation of the ethanol phase and the water phase is promoted by utilizing the ammonium sulfate with a specific proportion, so that the ethanol can be used as an extracting agent for the dispersed solid phase extraction, and compared with acetonitrile used in the traditional dispersed solid phase extraction, the method is lower in toxicity and cost, and meets the development trend of green analysis.
2. The purifying agent used for the dispersive solid-phase extraction is a surface-modified multi-wall carbon nano tube, is a novel adsorbent material, can selectively remove main pigment interference components in a matrix sample with high pigment content, and cannot cause the recovery loss of pesticides to be detected.
3. The sample is subjected to ethanol extraction and carboxylated multi-walled carbon nanotube purification by adopting a dispersed solid phase extraction pretreatment technology, the method meets the analysis requirements of neonicotinoid pesticides and metabolites thereof in oranges and peaches, the accuracy, precision and sensitivity meet the requirements, and the purification effect is good. Can be used for detecting the residue of neonicotinoid pesticides and metabolites thereof in fruits.
Drawings
FIG. 1 is a standard curve and linear correlation coefficient of residue of 4 kinds of pesticides and metabolites thereof.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof. The equipment and reagents used in the present invention are, unless otherwise specified, conventional commercial products in the art.
In the following examples, the detection apparatus for HPLC-MS is HPLC-MS (Shimadzu LC-20A XR-HPLC system, AB-SCIEX 4000Q-TRAP triple quadrupole mass spectrometer).
An Ultra AQ C18 column (100 mm. times.2.1 mm, 3.0 μm) in methanol (A), 5mmol L-1Ammonium acetate and 20mmol L-1The formic acid mixed aqueous solution (B) is used as a mobile phase and the flow rate is 0.4mL min-1. Separating by gradient elution under the following conditions: 0.0-1.0 min/10% A, 1.0-4.0 min/10% -90% A, 4.0-8.4 min/90% A, 8.4-8.5 min/90% -10% A, 8.5-10.0 min/10% A, the injection amount is 10 muL. The mass spectrometry was performed using an electrospray ionization (ESI) source, Multiple Reaction Monitoring (MRM) and positive mode, with an ionization temperature of 550 ℃, a spray voltage of 5500V, and an atomizer pressure of 55 psi. The pesticide is neonicotinoid pesticide, the pesticide and its metabolite are selected from more than one of imidacloprid, acetamiprid, thiamethoxam and clothianidin, and the detection conditions are shown in table 1.
Example 1
A method for detecting pesticide and metabolite residues in citrus comprises the following steps:
(1) homogenizing: the citrus fruits are cut and homogenized to obtain homogenate, and 5g of the homogenate is weighed and placed into a 50mL centrifuge tube.
(2) Extraction: adding 15mL of ethanol and 5mL of ultrapure water into a centrifugal tube for weighing the citrus homogenate, performing vortex extraction for 5min, adding 2g of ammonium sulfate, uniformly mixing, and centrifuging at 4000rpm for 5 min.
(3) Dispersed solid phase extraction and purification: 1mL of the supernatant was transferred to a 2mL centrifuge tube containing 5mg of carboxylated multiwalled carbon nanotubes. Centrifuging after vortex shaking for 1min, and filtering with 0.22 μm organic filter membrane to obtain citrus matrix extractive solution.
(4) Preparing 4 kinds of standard stock solutions of pesticide residues of imidacloprid, acetamiprid, thiamethoxam and clothianidin, respectively diluting the stock solutions with ethanol to form mixed standard solutions, wherein the mass concentration of the imidacloprid, the mass concentration of the acetamiprid, the mass concentration of the thiamethoxam and the mass concentration of the clothianidin are respectively 20mg/L and 20mg/L, then preparing the citrus matrix extracting solution treated in the steps (1) to (3) into standard solutions of 0.005mg/L, 0.01mg/L, 0.05mg/L, 0.1mg/L and 0.2mg/L respectively, carrying out HPLC-MS/MS sample injection analysis, measuring each concentration for 3 times, taking the concentration as abscissa x and the average of the peak areas as ordinate y, the standard curves and linear correlation coefficients for the 4 residues were obtained, as shown in FIG. 1.
(5) Adding the 4 pesticide standard solutions into the blank matrix sample to a concentration of 0.05mg/kg as a sample, performing HPLC-MS/MS sample injection analysis after the treatment of the steps (1) to (3), measuring the peak area of each residue, measuring each concentration for 3 times, comparing with the standard curve of the figure 1, and calculating the recovery rate and the coefficient of variation.
Comparative example 1
The same as example 1, except that: the extractant is acetonitrile.
Comparative example 2
The same as example 1, except that: the extractant is methanol.
Example 1, comparative example 1 and comparative example 2 test the extraction effect of different extraction solvents on three pesticides and high-toxicity metabolite residues in citrus, and mainly select the following 3 extraction solvents: acetonitrile, methanol and ethanol. After 2g of ammonium sulfate was added, acetonitrile and ethanol could be separated from water, the sample solution was supernatant, methanol could not be separated, the sample solution was a mixed solution of methanol and water, and the influence of different extraction solvents on the recovery rate of pesticide residue is shown in table 2 below.
TABLE 2
Table 2 shows that the extraction efficiency of acetonitrile and ethanol on pesticide residues is between 70% and 110%, the extraction requirement is met, and the ethanol is selected as the optimal extraction solvent in consideration of lower toxicity of the ethanol.
Example 1, comparative example 1 and comparative example 2 test the influence of different amounts of extractant on the recovery rate of pesticide residue, and the following 3 amounts of ethanol extractant were selected: 10mL, 15mL, 20 mL. After 2g of ammonium sulfate was added, 15mL and 20mL of ethanol could be separated from water, the sample solution was supernatant, 10mL of ethanol could not be separated, the sample solution was a mixed solution of ethanol and water, and the effect of different amounts of ethanol on the recovery rate of pesticide residue is shown in table 3 below.
TABLE 3
From table 3, it follows: the best recovery rate was selected for 15mL of ethanol.
Example 2
A method for detecting pesticide and metabolite residues in citrus comprises the following steps:
(1) homogenizing: the citrus fruits are cut and homogenized to obtain homogenate, and 5g of the homogenate is weighed and placed into a 50mL centrifuge tube.
(2) Extraction: adding 15mL of ethanol and 5mL of ultrapure water into a centrifugal tube for weighing the citrus homogenate, performing vortex extraction for 5min, adding 2g of ammonium sulfate, uniformly mixing, and centrifuging at 4000rpm for 5 min.
(3) Dispersed solid phase extraction and purification: 1mL of the supernatant was transferred to a 2mL centrifuge tube containing 5mg of carboxylated multiwalled carbon nanotubes. Vortex, shake for 1min, centrifuge, filter with 0.22 μm organic filter membrane into sample bottle, and detect by HPLC-MS/MS under the above conditions.
This example tests the effect of different purifiers on the effect of pesticide residue on the fruit matrix, and mainly selects the following 7 purifiers: PSA, GCB, C18, multi-wall carbon nanotubes, carboxylated multi-wall carbon nanotubes, hydroxylated multi-wall carbon nanotubes and aminated multi-wall carbon nanotubes. The effect of different types of scavengers on the recovery of pesticide residues is shown in table 4 below.
TABLE 4
As can be seen from Table 4, the carboxylated multi-wall carbon nanotubes were selected as the best decontaminant because the recovery of the pesticide residue after decontamination was minimally affected by the substrate.
Meanwhile, the embodiment tests the influence of different dosages of the carboxylated multi-wall carbon nanotube on the purification effect of the sample, and mainly selects the following 5 dosages: 0. 5mg, 10mg, 20mg, 30 mg. The effect of different amounts of carboxylated multi-wall carbon nanotubes on the adsorption and purification recovery rate of pesticide residues is shown in the following table 5.
TABLE 5
From table 5, it follows: when the carboxylated multi-wall carbon nano tube is 5mg, the recovery rate is higher, so that the 5mg carboxylated multi-wall carbon nano tube is selected as the purifying agent.
Example 3
A method for detecting pesticide and metabolite residues in fruits comprises the following steps:
(1) homogenizing: the orange/peach is cut and homogenized to obtain homogenate, and 5g of the homogenate is weighed and put into a 50mL centrifuge tube.
(2) Extraction: adding 15mL of ethanol and 5mL of ultrapure water into a centrifugal tube for weighing the fruit homogenate, performing vortex extraction for 5min, adding 2g of ammonium sulfate, uniformly mixing, and centrifuging at 4000rpm for 5 min.
(3) Dispersed solid phase extraction and purification: 1mL of the supernatant was transferred to a 2mL centrifuge tube containing 5mg of carboxylated multiwalled carbon nanotubes. Centrifuging after vortex shaking for 1min, and filtering with 0.22 μm organic filter membrane to obtain citrus/peach matrix extractive solution.
(4) Preparing 4 standard stock solutions of residues, diluting the stock solutions into mixed standard solutions of 20mg/L by using ethanol respectively, then preparing the citrus/peach matrix extracting solutions processed in the steps (1) to (3) into standard solutions of 0.005mg/L, 0.01mg/L, 0.05mg/L, 0.1mg/L and 0.2mg/L respectively, analyzing by HPLC-MS/MS sample injection, measuring each concentration for 3 times, and obtaining standard curves and linear correlation coefficients of the 4 residues by taking the concentration as a horizontal coordinate x and taking a peak area average value as a vertical coordinate y, as shown in figure 1.
(5) Adding 4 pesticide standard solutions into the blank fruit matrix sample to the concentrations of 0.02, 0.05 and 0.2mg/kg respectively to serve as added samples, carrying out HPLC-MS/MS sample injection analysis after the treatment according to the steps (1) to (3), measuring the peak area of each residue, measuring each concentration for 3 times, comparing with a standard curve, calculating the recovery rate and the variation coefficient, and verifying the accuracy, precision and sensitivity of the method. The data results are shown in table 6 below.
TABLE 6
Note that: RSD is the coefficient of variation.
As shown in Table 6, the detection method provided by the invention has better recovery results when used for detecting 4 neonicotinoid pesticides, the addition recovery rates of the citrus and the peach are respectively between 76% -82% and 78% -86%, the reproducibility is good, and the RSD in the citrus and the peach is respectively between 0.8% -6.8% and 1.7% -7.1%, which shows that the method can be used for well enriching and analyzing the target substances in the citrus and the peach.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (8)
1. A low-toxicity dispersive solid-phase extraction method for detecting pesticide and metabolite residues thereof in fruits is characterized by comprising the following steps:
(1) sample extraction: weighing a homogenized fruit sample, adding ethanol, mixing with a homogenized matrix, adding ammonium sulfate, mixing uniformly to enable an organic phase and a water phase to be layered at room temperature, transferring a target extract into an upper-layer ethanol phase after centrifugation, and collecting a supernatant in a centrifugal tube;
(2) sample purification: and adding a purifying agent into the obtained supernatant for purification to obtain a purified solution, and filtering the purified solution to obtain a sample solution to be detected after solid-phase extraction.
2. The low-toxicity dispersive solid-phase extraction method for detecting pesticide and metabolite residues in fruits according to claim 1, wherein the step (1) specifically comprises the following steps: weighing a homogenized fruit sample into a centrifuge tube, adding ethanol and ultrapure water, performing vortex extraction, wherein the adding ratio of the homogenized fruit sample to the ethanol is 1:2-4g/mL, the volume ratio of the ethanol to the ultrapure water is 2-4:1, adding ammonium sulfate, uniformly mixing, centrifuging, promoting the layering of an organic phase and a water phase, the mass ratio of the ammonium sulfate to the homogenized fruit sample is 2:5, transferring a target extract to an upper ethanol phase, and collecting a supernatant in the centrifuge tube.
3. The low-toxicity dispersive solid-phase extraction method for detecting pesticide and metabolite residues in fruits according to claim 1 or 2, wherein the addition ratio of the purifying agent and the supernatant in the step (2) is 0.05-0.20:1 g/mL.
4. The method for detecting the low toxicity dispersed solid phase extraction of pesticide and metabolite residues in fruits according to claim 1, wherein the purifying agent is selected from one or more of multi-walled carbon nanotubes, carboxylated multi-walled carbon nanotubes, hydroxylated multi-walled carbon nanotubes and aminated multi-walled carbon nanotubes.
5. A method for detecting pesticide and metabolite residues in fruits is characterized by comprising the following steps:
(1) the low-toxicity dispersive solid-phase extraction method for detecting pesticide and metabolite residues in fruits according to claim 1, so as to obtain a sample solution to be detected after solid-phase extraction;
(2) carrying out high performance liquid chromatography tandem mass spectrometry on the liquid to be detected to detect pesticide and metabolite residues in the fruits;
liquid chromatography conditions: a chromatographic column: c18, 100mm × 2.1mm, 3.0 μm; sample introduction amount: 10 mu L of the solution; flow rate: 0.4mL min-1(ii) a Column temperature: 35 ℃; methanol is used as a mobile phase A, 5mmol L-1Ammonium acetate and 20mmol L-1Taking the formic acid mixed aqueous solution as a mobile phase B for gradient elution, wherein the gradient elution procedure is as follows: 0.0-1.0 min/10% A, 1.0-4.0 min/10% -90% A, 4.0-8.4 min/90% A, 8.4-8.5 min/90% -10% A, 8.5-10.0 min/10% A;
the mass spectrum conditions are as follows: a spray ionization source; monitoring multiple reactions; scanning positive ions; the ionization temperature is 550 ℃; the spraying voltage is 5500V; atomizer pressure 55 psi.
6. The method for detecting pesticide and metabolite residues in fruits according to claim 5, wherein the pesticide and metabolite residues are selected from more than one of imidacloprid, acetamiprid, thiamethoxam and clothianidin, and the detection conditions are shown in the following table:
7. use of the method of claim 5 for detecting pesticide and metabolite residues in fruit.
8. The method of claim 7 for detecting pesticide and its metabolite residues in fruit, wherein the fruit is citrus or peach.
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