CN112485349A - Method for determining residues of five benzimidazoles pesticides in cereal grains - Google Patents
Method for determining residues of five benzimidazoles pesticides in cereal grains Download PDFInfo
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Abstract
The invention belongs to the technical field of agricultural product detection, and discloses a method for determining the residual quantity of five benzimidazole pesticides in cereal grains, which comprises the steps of soaking the cereal grains, coarsely grinding the cereal grains, and secondarily grinding the particles obtained by coarse grinding by a semi-dry method; adding water into the secondarily ground particles, and uniformly stirring to obtain a mixed solution; extracting the mixed solution by using acetonitrile to obtain extract liquor; connecting the small solid-phase extraction column to a solid-phase extraction device for purifying the extraction liquid; presetting conditions of a chromatographic column, a mobile phase and a mass spectrum, and determining carbendazim, probenazole, benomyl, probenazole and fuberidazole by adopting a liquid chromatography-tandem mass spectrometry method; obtaining a regression curve and carrying out quantitative judgment. The method uses the liquid chromatography-tandem mass spectrometry to determine the benzimidazoles, can realize the respective determination of the contents of the carbendazim, the probenazole, the benomyl, the probenazole and the fuberidazole, and is more convenient to determine and operate.
Description
Technical Field
The invention belongs to the technical field of agricultural product detection, and particularly relates to a method for determining the residual quantity of five benzimidazole pesticides in cereal grains.
Background
At present: in agricultural production, organic bactericides with active benzimidazole ring as a parent body are used for sterilization, the compounds all show systemic bactericidal activity, and representative compounds of the organic bactericides comprise five compounds of carbendazim, probenazole, benomyl, probenazole and fuberidazole, and the representative compounds are metabolized and activated in a plant body or nearby the plant body to become methyl benzimidazole-2-carbamate, so that the bactericidal activity is shown. The grain is the main source for providing energy for human body, and the intake amount is huge. However, when the benzimidazole bactericide is sprayed on cereal crops, residues are generated in the cereal crops, and the residues can harm human health through a food chain. In order to realize food safety, the benzimidazole compound in the cereal grains is detected, but the existing method for detecting the benzimidazole compound in the cereal grains cannot realize simultaneous detection of a plurality of compounds, needs to carry out detection for a plurality of times and has complex operation.
Through the above analysis, the problems and defects of the prior art are as follows: the existing method for detecting benzimidazoles in cereal grains cannot realize simultaneous detection of multiple compounds, needs to carry out detection for multiple times, and is complex to operate.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a method for measuring the residual quantity of five benzimidazole pesticides in cereal grains.
The invention is realized in such a way that a method for measuring the residual quantity of five benzimidazole pesticides in cereal grains comprises the following steps:
step one, soaking cereal grains at room temperature for 15-20h, wiping surface water by using filter paper, carrying out coarse grinding, and carrying out secondary grinding on particles obtained by the coarse grinding by adopting a semi-dry method;
placing the secondarily ground particles in a beaker, adding water, and uniformly stirring to obtain a mixed solution; extracting the mixed solution by using acetonitrile to obtain extract liquor;
connecting the small solid-phase extraction column to a solid-phase extraction device for purifying the extraction liquid;
step four, presetting conditions of a chromatographic column, a mobile phase and a mass spectrum, and determining carbendazim, probenazole, benomyl, probenazole and fuberidazole by adopting a liquid chromatography-tandem mass spectrometry method; the mass spectrum parameters were as follows:
table 15 mass spectrometric parameters of benzimidazole fungicides
Taking a proper amount of five benzimidazole pesticides, and preparing 1.0-1.5 mL of standard reference solution containing 40-48 micrograms of organic chloride reference substance by using petroleum ether;
step six, precisely measuring mixed standard reference solution, preparing a solution containing 5 mug, 10 mug, 50 mug, 100 mug, 250 mug and 500 mug in each 1L by petroleum ether, injecting sample according to conditions of chromatographic column, mobile phase and mass spectrum, and repeating for 3 times;
step seven, taking the average value of 3 times to draw a standard curve, taking the concentration of the sample as a vertical coordinate and the peak area as a horizontal coordinate, performing regression calculation, determining a linear range, and obtaining a corresponding coefficient with good linear relation in the concentration range;
eighthly, determining the specific category of the pesticide based on the peak-out time and the qualitative and quantitative particle pair determined in the fourth step, and respectively substituting the peak areas into the standard curve in the seventh step to obtain the residual quantity of the five benzimidazoles pesticides in the sample;
and step nine, calculating the five benzimidazole pesticide residues in the sample in the cereal grain based on the five benzimidazole pesticide residues in the sample.
Further, in the step one, the specific process adopted for soaking the cereal grains in the sample at the room temperature is as follows:
stirring grain samples, and removing impurities on grains; after the removal is finished, carrying out vibration screening on the grain samples;
after the vibration screening is finished, putting corresponding enzyme and hydrated cereal grains into the soaking appliance;
soaking grains in the soaking device at indoor temperature for 15-20 hr, and observing the grain soaking condition;
further, in the first step, the semidry method comprises the following steps:
(1) placing the granules obtained by coarse grinding into a beaker, adding distilled water and fully stirring;
(2) stirring, standing at room temperature for 2h to allow water to be fully immersed in the particles;
(3) grinding particles by using a grinder, detecting the particle size by using a particle size detection device arranged on the grinder, and transmitting the detected particle size data to a grinder controller;
(4) the pulverizer controller analyzes the particle size data in real time, judges whether the fine requirement is met, and controls the pulverizer to reduce the pulverizing gap and continue grinding when the fine requirement is not met;
(5) when the relevant particle size reaches the fine requirement, the ground powder is collected.
Further, in the second step, the process of stirring the secondarily ground particles in the beaker is as follows:
placing the secondarily ground particles in a beaker, and adding a proper amount of water according to a certain proportion;
placing the beaker below a stirrer, slowly starting the stirrer, and stirring for 25-40 min;
the state of the particles being stirred was observed, and accelerated stirring was performed until the particles were mixed.
Further, in the second step, the step of extracting the mixed solution is as follows:
1) adding acetonitrile into the mixed solution, and putting the mixed solution added with the acetonitrile into a constant-temperature oscillator for uniform oscillation;
2) adding sodium chloride solution into the oscillated mixed solution, shaking for 10min, mixing uniformly, salting out, standing and layering;
3) filtering the supernatant, and centrifuging the supernatant by using a centrifuge;
4) the upper acetonitrile extract was obtained and filtered with a filtration membrane.
Further, in step 1), the oscillating includes: the oscillation temperature is 35 ℃; the oscillation speed was 150 r/min.
Further, in the step 2), the salting-out temperature is 30-38 ℃.
Further, in step 3), the centrifuging includes: the centrifugation speed is 3000r/min, and the centrifugation time is 15 min.
Further, in the third step, the purification of the extraction liquid specifically includes:
(1) the solid phase extraction column is activated by methanol and water in sequence in advance;
(2) pouring the extract into a funnel, filtering by using filter paper, and allowing the filtrate to flow through a small column;
(3) after the extract liquid is drained naturally, the small column is cleaned by acetonitrile aqueous solution;
(4) after the cleaning solution naturally flows out, blowing out residual solution in the small column by using a suction ball;
(5) connecting a centrifugal pipe at the outlet of the column, leaching the small column with methanol, and blowing out residual liquid in the small column with a suction ball after the methanol naturally flows out.
Further, in the fourth step, the determination of carbendazim, probenazole, benomyl, probenazole and fuberidazole by using the liquid chromatography-tandem mass spectrometry method comprises the following steps:
the measuring and heating method comprises the following steps: the initial temperature is maintained at 65 deg.C for 1min, and raised to 200 deg.C at 20 deg.C per minute, then raised to 220 deg.C at 2 deg.C per minute, then raised to 260 deg.C at 5 deg.C per minute, and finally raised to 280 deg.C at 10 deg.C/min, and maintained for 15 min.
Further, the presetting of the chromatographic column, the mobile phase and the mass spectrum condition comprises the following steps:
(1) selecting a ZORBAX eclipse XDS-C18 column;
(2) using 10mmol/L ammonium acetate aqueous solution (0.1% formic acid) -methanol solution as mobile phase of carbendazim, probenazole, benomyl, probenazole and fuberidazole;
(3) selecting an ionization mode as electrospray ionization; the ion spray voltage is 4 kV; the atomizing gas is nitrogen, the drying gas is nitrogen, and the collision gas is argon.
(4) Multiple reaction monitoring was performed.
Further, in the sixth step, the temperature of the petroleum ether is 60-90 ℃.
Further, in the seventh step, the linear range of the concentration of the standard curve is 1 mug/kg-5000 mug/kg.
By combining all the technical schemes, the invention has the advantages and positive effects that: according to the invention, the acetonitrile is used for extracting the benzimidazole pesticide in the cereal grains, so that the separation is convenient, and the higher extraction efficiency can be kept; the benzimidazole content can be respectively measured by using the liquid chromatography-tandem mass spectrometry, so that the contents of carbendazim, probenazole, benomyl, probenazole and fuberidazole can be measured, and the measurement operation is more convenient.
The method can effectively detect the residual quantity of the 5 benzimidazole bactericides in the peanuts and the soybeans, has the characteristics of sensitivity, stability and reliability, and provides an effective way for monitoring the quality safety of the peanuts and the soybeans in China.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.
Fig. 1 is a flow chart of a method for determining the residual quantity of five benzimidazoles pesticides in cereal grains according to an embodiment of the present invention.
Fig. 2 is a flow chart illustrating a secondary grinding process using a semi-dry method according to an embodiment of the present invention.
FIG. 3 is a flow chart of the extraction of mixed liquid according to the embodiment of the present invention.
FIG. 4 is a flow chart of the purification of the extraction liquid provided by the embodiment of the present invention.
FIG. 5 is a flow chart of the preset conditions of the chromatographic column, the mobile phase and the mass spectrum according to the embodiment of the invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Aiming at the problems in the prior art, the invention provides a method for measuring the residual quantity of five benzimidazole pesticides in cereal grains, which is described in detail below with reference to the accompanying drawings.
As shown in fig. 1, the method for determining the residual amounts of five benzimidazoles pesticides in cereal grains provided by the embodiment of the present invention comprises the following steps:
s101, soaking cereal grains at room temperature for 15-20h, wiping surface water by using filter paper, carrying out coarse grinding, and carrying out secondary grinding on particles obtained by the coarse grinding by adopting a semi-dry method;
s102, placing the secondarily ground particles in a beaker, adding water, and uniformly stirring to obtain a mixed solution; extracting the mixed solution by using acetonitrile to obtain extract liquor;
s103, connecting the small solid-phase extraction column to a solid-phase extraction device for purifying extraction liquid;
s104, presetting conditions of a chromatographic column, a mobile phase and a mass spectrum, and measuring carbendazim, probenazole, benomyl, probenazole and fuberidazole by adopting a liquid chromatography-tandem mass spectrometry method;
and S105, obtaining a concentration-peak area regression curve for quantitative judgment.
As shown in fig. 2, the semidry method according to the embodiment of the present invention includes the following steps:
s201, placing the particles obtained by coarse grinding into a beaker, adding distilled water, and fully stirring;
s202, stirring, standing at room temperature for 2 hours to enable water to be fully immersed into the particles;
s203, grinding the particles by using a grinder, detecting the particle size by using a particle size detection device arranged on the grinder, and transmitting the detected particle size data to a grinder controller;
s204, the pulverizer controller analyzes the particle size data in real time, judges whether the fine requirement is met, and controls the pulverizer to reduce the pulverizing gap and continue grinding when the fine requirement is not met;
and S205, collecting the grinding powder when the relevant particle size reaches the fine requirement.
As shown in fig. 3, the steps of performing mixed liquid extraction according to the embodiment of the present invention are as follows:
s301, adding acetonitrile into the mixed solution, and putting the mixed solution added with acetonitrile into a constant-temperature oscillator for uniform-speed oscillation;
s302, adding a sodium chloride solution into the oscillated mixed solution, shaking for 10min, uniformly mixing, salting out, standing and layering;
s303, filtering the supernatant, and centrifuging the supernatant by using a centrifuge;
and S304, obtaining an upper acetonitrile extracting solution, and filtering by using a filtering membrane.
In step S301, the oscillation provided by the embodiment of the present invention includes: the oscillation temperature is 35 ℃; the oscillation speed was 150 r/min.
In step S302, the salting-out temperature provided by the embodiment of the present invention is 30 to 38 ℃.
In step S303, the centrifugation provided in the embodiment of the present invention includes: the centrifugation speed is 3000r/min, and the centrifugation time is 15 min.
As shown in fig. 4, the purification of the extraction liquid provided by the embodiment of the present invention specifically includes:
s401, activating a solid-phase extraction column by using methanol and water in sequence in advance;
s402, pouring the extract into a funnel, filtering by using filter paper, and allowing the filtrate to flow through a small column;
s403, after the extract liquid naturally drains, cleaning the small column with acetonitrile aqueous solution;
s404, after the cleaning liquid naturally flows out, blowing out residual liquid in the small column by using a suction ball;
s405, connecting a centrifugal pipe at the outlet of the column, leaching the small column with methanol, and blowing out residual liquid in the small column with a suction ball after the methanol naturally flows out.
As shown in fig. 5, the presetting of the chromatographic column, the mobile phase and the mass spectrum conditions provided by the embodiment of the invention includes:
s501, selecting a ZORBAX eclipse XDS-C18 column;
s502, using 10mmol/L ammonium acetate water solution (0.1% formic acid) -methanol solution as a mobile phase of carbendazim, probenazole, benomyl, probenazole and fuberidazole;
s503, selecting an ionization mode to be electrospray ionization; the ion spray voltage is 4 kV; the atomizing gas is nitrogen, the drying gas is nitrogen, and the collision gas is argon.
And S504, carrying out multi-reaction monitoring.
The process of stirring the twice-ground particles in the beaker provided by the embodiment of the invention is as follows:
placing the secondarily ground particles in a beaker, and adding a proper amount of water according to a certain proportion;
placing the beaker below a stirrer, slowly starting the stirrer, and stirring for 25-40 min;
the state of the particles being stirred was observed, and accelerated stirring was performed until the particles were mixed.
The grain sample room temperature soaking process provided by the embodiment of the invention comprises the following steps:
stirring grain samples, and removing impurities on grains; after the removal is finished, carrying out vibration screening on the grain samples;
after the vibration screening is finished, putting corresponding enzyme and hydrated cereal grains into the soaking appliance;
and (3) placing the soaking appliance at indoor temperature, soaking the grains for 15-20h, and observing the grain soaking condition in the soaking process.
The quantitative judgment provided by the embodiment of the invention comprises the following steps:
performing linear regression on the standard curve to determine a linear range, and obtaining a corresponding coefficient with good linear relation in a concentration range;
and comparing the concentrations of the five benzimidazole pesticides in the cereal grains to obtain a judgment result.
The linear regression of the standard curve provided by the embodiment of the invention comprises the following steps:
firstly, taking a proper amount of five benzimidazole pesticides, and preparing 1.0-1.5 mL of standard reference solution containing 40-48 micrograms of organic chloride reference substance by using petroleum ether;
secondly, precisely measuring a mixed standard control solution, preparing a solution containing 5 mug, 10 mug, 50 mug, 100 mug, 250 mug and 500 mug in each 1L by petroleum ether, and performing sample injection analysis by liquid chromatography-tandem mass spectrometry for 3 times;
and finally, taking the average value for 3 times to draw a standard curve, and performing regression calculation by taking the concentration of the sample as a vertical coordinate and the peak area as a horizontal coordinate.
The temperature of the petroleum ether provided by the embodiment of the invention is 60-90 ℃.
The residual liquid in the pillars was blown off.
In step S04, the determination of carbendazim, probenazole, benomyl, probenazole and fuberidazole by liquid chromatography-tandem mass spectrometry provided in the embodiment of the present invention includes:
the measuring and heating method comprises the following steps: the initial temperature is maintained at 65 deg.C for 1min, raised to 200 deg.C at 20 deg.C/min, raised to 220 deg.C at 2 deg.C/min, raised to 260 deg.C at 5 deg.C/min, and raised to 280 deg.C at 10 deg.C/min, and maintained for 15 min.
The linear range of the concentration of the standard curve provided by the embodiment of the invention is 1 mug/kg-5000 mug/kg.
The technical solution of the present invention is further described with reference to the following specific examples.
Example 1:
step one, soaking cereal grains in a sample at room temperature for 15 hours, wiping surface water by using filter paper, carrying out coarse grinding, and carrying out secondary grinding on particles obtained by the coarse grinding by adopting a semi-dry method;
the semidry method comprises the following steps of:
(1) placing the granules obtained by coarse grinding into a beaker, adding distilled water and fully stirring;
(2) stirring, standing at room temperature for 2h to allow water to be fully immersed in the particles;
(3) grinding particles by using a grinder, detecting the particle size by using a particle size detection device arranged on the grinder, and transmitting the detected particle size data to a grinder controller;
(4) the pulverizer controller analyzes the particle size data in real time, judges whether the fine requirement is met, and controls the pulverizer to reduce the pulverizing gap and continue grinding when the fine requirement is not met;
(5) collecting the ground powder when the relevant particle size meets the fine requirement;
placing the secondarily ground particles in a beaker, adding water, and uniformly stirring to obtain a mixed solution; extracting the mixed solution by using acetonitrile to obtain extract liquor;
connecting the small solid-phase extraction column to a solid-phase extraction device for purifying the extraction liquid;
step four, presetting conditions of a chromatographic column, a mobile phase and a mass spectrum, and determining carbendazim, probenazole, benomyl, probenazole and fuberidazole by adopting a liquid chromatography-tandem mass spectrometry method;
taking a proper amount of five benzimidazole pesticides, and preparing 1.0mL of standard reference solution containing 40 mu g of organic chloride reference substance by using petroleum ether;
step six, precisely measuring mixed standard reference solution, preparing a solution containing 5 mug, 10 mug, 50 mug, 100 mug, 250 mug and 500 mug in each 1L by petroleum ether, injecting sample according to conditions of chromatographic column, mobile phase and mass spectrum, and repeating for 3 times;
step seven, taking the average value of 3 times to draw a standard curve, taking the concentration of the sample as a vertical coordinate and the peak area as a horizontal coordinate, performing regression calculation, determining a linear range, and obtaining a corresponding coefficient with good linear relation in the concentration range;
eighthly, determining the specific category of the pesticide based on the peak-out time and the qualitative and quantitative ion pairs determined in the fourth step, and respectively substituting the peak areas into the standard curve in the seventh step to obtain the residual quantity of the five benzimidazoles pesticides in the sample;
and step nine, calculating the five benzimidazole pesticide residues in the sample in the cereal grain based on the five benzimidazole pesticide residues in the sample.
Example 2:
step one, soaking cereal grains at room temperature for 18h, wiping surface water by using filter paper, carrying out coarse grinding, and carrying out secondary grinding on particles obtained by the coarse grinding by adopting a semi-dry method;
the semidry method comprises the following steps of:
(1) placing the granules obtained by coarse grinding into a beaker, adding distilled water and fully stirring;
(2) stirring, standing at room temperature for 2h to allow water to be fully immersed in the particles;
(3) grinding particles by using a grinder, detecting the particle size by using a particle size detection device arranged on the grinder, and transmitting the detected particle size data to a grinder controller;
(4) the pulverizer controller analyzes the particle size data in real time, judges whether the fine requirement is met, and controls the pulverizer to reduce the pulverizing gap and continue grinding when the fine requirement is not met;
(5) collecting the ground powder when the relevant particle size meets the fine requirement;
placing the secondarily ground particles in a beaker, adding water, and uniformly stirring to obtain a mixed solution; extracting the mixed solution by using acetonitrile to obtain extract liquor;
connecting the small solid-phase extraction column to a solid-phase extraction device for purifying the extraction liquid;
step four, presetting conditions of a chromatographic column, a mobile phase and a mass spectrum, and determining carbendazim, probenazole, benomyl, probenazole and fuberidazole by adopting a liquid chromatography-tandem mass spectrometry method;
taking a proper amount of five benzimidazole pesticides, and preparing 1.2mL of standard reference solution containing about 44 mu g of organic chloride reference substance by using petroleum ether;
step six, precisely measuring mixed standard reference solution, preparing a solution containing 5 mug, 10 mug, 50 mug, 100 mug, 250 mug and 500 mug in each 1L by petroleum ether, injecting sample according to conditions of chromatographic column, mobile phase and mass spectrum, and repeating for 3 times;
step seven, taking the average value of 3 times to draw a standard curve, taking the concentration of the sample as a vertical coordinate and the peak area as a horizontal coordinate, performing regression calculation, determining a linear range, and obtaining a corresponding coefficient with good linear relation in the concentration range;
eighthly, determining the specific category of the pesticide based on the peak-out time and the qualitative and quantitative ion pairs determined in the fourth step, and respectively substituting the peak areas into the standard curve in the seventh step to obtain the residual quantity of the five benzimidazoles pesticides in the sample;
and step nine, calculating the five benzimidazole pesticide residues in the sample in the cereal grain based on the five benzimidazole pesticide residues in the sample.
Example 3:
step one, soaking cereal grains at room temperature for 20 hours, wiping surface water by using filter paper, carrying out coarse grinding, and carrying out secondary grinding on particles obtained by the coarse grinding by adopting a semi-dry method;
the semidry method comprises the following steps of:
(1) placing the granules obtained by coarse grinding into a beaker, adding distilled water and fully stirring;
(2) stirring, standing at room temperature for 2h to allow water to be fully immersed in the particles;
(3) grinding particles by using a grinder, detecting the particle size by using a particle size detection device arranged on the grinder, and transmitting the detected particle size data to a grinder controller;
(4) the pulverizer controller analyzes the particle size data in real time, judges whether the fine requirement is met, and controls the pulverizer to reduce the pulverizing gap and continue grinding when the fine requirement is not met;
(5) collecting the ground powder when the relevant particle size meets the fine requirement;
placing the secondarily ground particles in a beaker, adding water, and uniformly stirring to obtain a mixed solution; extracting the mixed solution by using acetonitrile to obtain extract liquor;
connecting the small solid-phase extraction column to a solid-phase extraction device for purifying the extraction liquid;
step four, presetting conditions of a chromatographic column, a mobile phase and a mass spectrum, and determining carbendazim, probenazole, benomyl, probenazole and fuberidazole by adopting a liquid chromatography-tandem mass spectrometry method;
taking a proper amount of five benzimidazole pesticides, and preparing 1.5mL of standard reference solution containing about 48 mu g of organic chloride reference substance by using petroleum ether;
step six, precisely measuring mixed standard reference solution, preparing a solution containing 5 mug, 10 mug, 50 mug, 100 mug, 250 mug and 500 mug in each 1L by petroleum ether, injecting sample according to conditions of chromatographic column, mobile phase and mass spectrum, and repeating for 3 times;
step seven, taking the average value of 3 times to draw a standard curve, taking the concentration of the sample as a vertical coordinate and the peak area as a horizontal coordinate, performing regression calculation, determining a linear range, and obtaining a corresponding coefficient with good linear relation in the concentration range;
eighthly, determining the specific category of the pesticide based on the peak-out time and the qualitative and quantitative ion pairs determined in the fourth step, and respectively substituting the peak areas into the standard curve in the seventh step to obtain the residual quantity of the five benzimidazoles pesticides in the sample;
and step nine, calculating the five benzimidazole pesticide residues in the sample in the cereal grain based on the five benzimidazole pesticide residues in the sample.
The technical effects of the present invention will be described in detail with reference to experiments.
1. Experimental part
1.1 instruments, reagents and materials
1260-6460 liquid chromatography-tandem mass spectrometer (Agilent, USA); electronic balances (Mettler Toledo, switzerland); KQ-500DV ultrasonic cleaning machine (Kunshan ultrasonic instruments Co., Ltd.); ultra pure water machines (Millipore, USA); a pH meter (Mettler Toledo, Switzerland); centrifuge (HITACHI); rotary evaporator (BUCHI, switzerland); nitrogen blowing apparatus (organization corporation); MS 3basic vortex mixer (German IKA company)
Acetonitrile (chromatographically pure, Merck, germany), methanol (chromatographically pure, pharmaceutical group), formic acid (chromatographically pure); disodium hydrogen phosphate, sodium chloride, sodium hydroxide (analytically pure, mijo, Tianjin); ammonium acetate (analytically pure, national drug group); anhydrous sodium sulfate (analytically pure, burned at 650 deg.C for 4h before use, stored in a desiccator, and cooled for use); ENVI-Carb/LC-NH2Small column (Supelco, 500mg/6 mL);
1.2 preparation of Standard solution
And (3) standard substance: carbendazim, probenazole, benomyl, probenazole and fuberidazole (all in the department of agriculture, 100 mg/L).
Preparation of a standard solution: respectively taking the contents of carbendazim, probenazole, benomyl, probenazole and fuberidazole, preparing standard solutions with the mass concentration of 10mg/L by using methanol, and shaking uniformly for later use.
1.3 sample pretreatment
1.3.1 preparation of samples
And crushing the sample by a crusher, sieving by a 20-mesh sieve, and uniformly mixing for later use.
1.3.2 sample extraction
Accurately weighing 5g of sample (accurate to 0.01g) in a 50mL centrifuge tube, adding 5 mL-10 mL of water, uniformly mixing and soaking in a vortex manner, standing for 10min, and adding 10mL of 0.2mol/L Na2HPO4And (3) uniformly mixing the buffer solution and 3g of sodium chloride, adjusting the pH value to 7.0-8.5 by using 2mol/L NaOH solution, adding 20mL of acetonitrile, oscillating and extracting for 30min, and centrifuging for 5min at 4000r/min to be purified.
1.3.2 sample clarification
In ENVI-Carb/LC-NH2Adding 2cm high anhydrous sodium sulfate into the column, activating ENVI-Carb/LC-NH with 5mL formic acid-methanol (2: 98) before loading2Small column, when the liquid level reaches the top of sodium sulfate, quickly transferring 4.0mL of the extract to pass through the column, eluting with 10mL of formic acid-methanol (2: 98), collecting all the effluent liquid to a heart-shaped flask, rotating in a water bath at 40 ℃ to evaporate to dryness, adding 1.0mL of mobile phase to dissolve the residual liquidAnd filtering the residue with a 0.45 μm filter membrane in a sample injection bottle for determination by a liquid chromatography-tandem mass spectrometer.
1.4 apparatus conditions
1.4.1 chromatographic conditions
A chromatographic column: AgilentZORBAX Eclipse Plus C18Chromatography column (30 mm. times.100 mm 3.5 μm); mobile phase: 0.1% formic acid-10 mmol/L ammonium acetate (A) and methanol (B) gradient elution; elution procedure: the initial state A is 90%, the phase A is reduced to 20% in 0-7 min, the phase A is reduced to 0% in 1min after being kept for 2min, the phase A is kept for 4min, then the phase A is increased from 0% to 90% in 1min, and the initial state is recovered after being kept for 5 min; flow rate: 0.4 mL/min; sample introduction volume: 10 mu L of the solution; column temperature: at 40 ℃.
1.4.2 Mass Spectrometry conditions
An ion source: electrospray ion (Jet-ESI) source, scan mode: positive ion (ESI)+) And the detection mode is as follows: multiple Reaction Monitoring (MRM) mode, spray voltage 4kV, dry gas and atomization gas are nitrogen, dryer temperature 350 ℃, dry gas flow 12L/min, atomization gas pressure 40psi, cracking voltage 100V, other conditions are detailed in Table 1.
Table 15 mass spectrometric parameters of benzimidazole fungicides
2. Results and analysis
2.1 optimization of the extraction method
Acetonitrile as a solvent with good permeability has good solubility for most pesticides, so acetonitrile is used as an extraction solvent in the experiment. In the experimental process, after acetonitrile is added into peanut and soybean samples, the samples are easy to agglomerate in a solvent and are not beneficial to osmotic extraction of the solvent, and after 5-10 mL of water is added to fully wet and disperse the soybean samples, extraction and homogenate are carried out, so that the dispersing effect of the samples is better. The pH value of the sample solution has great influence on the extraction of the substance to be detected. The pH values of the sample solutions are respectively adjusted to 7.0, 8.0, 9.0, 10.0 and 12.0, and experiments show that only carbendazim and thiabendazole generate peaks when the pH value is 12.0; when the pH value is 10.0, the fuberin does not peak; when the pH values were 7.0, 8.0, and 9.0, peaks were observed in 5 samples, but the recovery rates of the samples differed. Further experiments showed that when the pH of the sample solution was adjusted to above 8.5, the recovery of fuberidazine was low. Comprehensively considering the recovery rate of each substance to be detected, and finally determining the pH value of the extraction sample solution to be 7.0-8.5.
2.2 selection of purification method
Considering that peanuts and soybeans contain a large amount of lipid substances, a solid-phase extraction and purification method for removing the lipid substances and other impurities by using a graphitized carbon-amino composite column is selected experimentally. The results of comparative investigation on the effects of methanol and formic acid-methanol (2: 98) as eluting solvents show that the recovery rates of carbendazim and fuberidazole are poor when methanol is used as the eluting solvent, and the recovery rates of 5 pesticides are satisfactory when formic acid-methanol (2: 98) is used as the eluting solvent.
2.3 selection of the Mobile phase
Experiments compared the elution effect of acetonitrile-water, methanol-water, 10mmol/L ammonium acetate (0.1% formic acid) -methanol solution as mobile phase. As a result, the 5 bactericides have poor peak shapes when acetonitrile-water and methanol-water are used as mobile phases; when 10mmol/L ammonium acetate (0.1% formic acid) -methanol solution was used as mobile phase, the peak shape was found to be symmetrical and the response value was high.
The chromatographic results of 2.45 benzimidazole bactericides in a Multiple Reaction Monitoring (MRM) mode are as follows:
carbendazim (carbendazim): 8.522 min; benomyl (benomyl): 8.437 min; thiabendazole (thiabendazole): 8.845 min; fuberin (thiophanate-methyl): 9.239 min; benomyl (thiophanate-ethyl): 10.096 min.
2.5 Linear Range and quantitative limits
Accurately weighing 5g (accurate to 0.01g) of sample, processing according to a method of '1.3.2', taking the obtained matrix solution as a solvent, and preparing series of standard solutions with mass concentrations of 0.5 mu g/L, 1.0 mu g/L, 2.0 mu g/L, 5.0 mu g/L, 10.0 mu g/L and 20.0 mu g/L. The measurement is carried out under the optimized condition, the mass concentration (X, mu g/L) is taken as the abscissa, the peak area (Y) is taken as the ordinate, and regression analysis is carried out to obtain the linear equation and the correlation coefficient of each substance to be measured (see table 2). The result shows that the linear relation of each substance to be detected is good within the mass concentration range of 0.5 mu g/L-20.0 mu g/L, and the correlation coefficient is not less than 0.9993.
TABLE 25 Linear equations and correlation coefficients for benzimidazole fungicides
| Name (R) | Linear equation of equations | Linear Range (μ g/L) | Coefficient of correlation (r) |
| Carbendazim | Y＝17078.8×X－887.1 | 0.5～20.0 | 0.99988470 |
| Benomyl | Y＝39091.4×X－1724.6 | 0.5～20.0 | 0.99987326 |
| Medicine for treating ear disease | Y＝6304.9×X－481.1 | 0.5～20.0 | 0.99979781 |
| Prothion carbendazim | Y＝14063.2×X－1500.7 | 0.5～20.0 | 0.99965670 |
| Thiabendazole | Y＝8006.8×X－120.6 | 0.5～20.0 | 0.99934462 |
And (3) performing a standard addition experiment on the negative samples of the peanuts and the soybeans, and calculating the signal to noise ratio (S/N) to 10 to obtain the quantitative limits of the carbendazim, the benomyl, the probenazole, the fuberidazole and the probenazole to be 1.0 mu g/kg.
2.6 recovery and precision of the Process
Adding 1.0 mu g/kg, 2.0 mu g/kg and 10.0 mu g/kg of carbendazim, benomyl, probenazole, fuberidazine and probenazole into recessive samples of peanuts and soybeans respectively, and measuring 6 times at each level, wherein the average recovery rate of each substance to be measured is 73.2-110.4%, and the Relative Standard Deviation (RSD) is 2.98-9.85% (see table 3).
Table 3 sample 5 benzimidazole fungicides with standard recovery and relative standard deviation (n ═ 6)
The invention establishes a liquid chromatography-tandem mass spectrometry method capable of simultaneously detecting 5 benzimidazole bactericides, such as carbendazim, benomyl, probenazole, fuberidazole and probenazole in peanuts and soybeans. The method can effectively detect the residual quantity of the 5 benzimidazole bactericides in the peanuts and the soybeans, has the characteristics of sensitivity, stability and reliability, and provides an effective way for monitoring the quality safety of the peanuts and the soybeans in China.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A method for measuring the residual quantity of five benzimidazole pesticides in cereal grains is characterized by comprising the following steps:
step one, soaking cereal grains at room temperature for 15-20h, wiping surface water by using filter paper, carrying out coarse grinding, and carrying out secondary grinding on particles obtained by the coarse grinding by adopting a semi-dry method;
the semidry method comprises the following steps of:
(1) placing the granules obtained by coarse grinding into a beaker, adding distilled water and fully stirring;
(2) stirring, standing at room temperature for 2h to allow water to be fully immersed in the particles;
(3) grinding particles by using a grinder, detecting the particle size by using a particle size detection device arranged on the grinder, and transmitting the detected particle size data to a grinder controller;
(4) the pulverizer controller analyzes the particle size data in real time, judges whether the fine requirement is met, and controls the pulverizer to reduce the pulverizing gap and continue grinding when the fine requirement is not met;
(5) collecting the ground powder when the relevant particle size meets the fine requirement;
placing the secondarily ground particles in a beaker, adding water, and uniformly stirring to obtain a mixed solution; extracting the mixed solution by using acetonitrile to obtain extract liquor;
connecting the small solid-phase extraction column to a solid-phase extraction device for purifying the extraction liquid;
step four, presetting conditions of a chromatographic column, a mobile phase and a mass spectrum, and determining carbendazim, probenazole, benomyl, probenazole and fuberidazole by adopting a liquid chromatography-tandem mass spectrometry method;
taking a proper amount of five benzimidazole pesticides, and preparing 1.0-1.5 mL of standard reference solution containing 40-48 micrograms of organic chloride reference substance by using petroleum ether;
accurately measuring mixed standard reference solution, preparing various solutions with different concentrations by using petroleum ether, and injecting samples according to chromatographic column, mobile phase and mass spectrum conditions;
step seven, taking the average value of 3 times to draw a standard curve, taking the concentration of the sample as a vertical coordinate and the peak area as a horizontal coordinate, performing regression calculation, determining a linear range, and obtaining a corresponding coefficient with good linear relation in the concentration range;
eighthly, determining the specific category of the pesticide based on the peak-out time and the qualitative and quantitative ion pairs determined in the fourth step, and respectively substituting the peak areas into the standard curve in the seventh step to obtain the residual quantity of the five benzimidazoles pesticides in the sample;
and step nine, calculating the five benzimidazole pesticide residues in the sample in the cereal grain based on the five benzimidazole pesticide residues in the sample.
2. The method for determining the residual quantity of five benzimidazoles pesticides in cereal grains according to claim 1, wherein in the second step, the step of extracting the mixed solution comprises the following steps:
1) adding acetonitrile into the mixed solution, and putting the mixed solution added with the acetonitrile into a constant-temperature oscillator for uniform oscillation;
2) adding sodium chloride solution into the oscillated mixed solution, shaking for 10min, mixing uniformly, salting out, standing and layering;
3) filtering the supernatant, and centrifuging the supernatant by using a centrifuge;
4) the upper acetonitrile extract was obtained and filtered with a filtration membrane.
3. The method for determining the residual amounts of five benzimidazoles pesticides in cereal grains according to claim 2, wherein in the step 1), the oscillating comprises: the oscillation temperature is 35 ℃; the oscillation speed was 150 r/min.
4. The method for measuring the residual quantity of five benzimidazole pesticides in cereal grains according to claim 2, wherein the salting-out temperature in the step 2) is 30-38 ℃.
5. The method for determining the residual amounts of five benzimidazoles pesticides in cereal grains according to claim 2, wherein in the step 3), the centrifuging comprises: the centrifugation speed is 3000r/min, and the centrifugation time is 15 min.
6. The method for determining the residual amounts of five benzimidazoles pesticides in cereal grains according to claim 1, wherein in step three, the purification of the extract specifically comprises:
(1) the solid phase extraction column is activated by methanol and water in sequence in advance;
(2) pouring the extract into a funnel, filtering by using filter paper, and allowing the filtrate to flow through a small column;
(3) after the extract liquid is drained naturally, the small column is cleaned by acetonitrile aqueous solution;
(4) after the cleaning solution naturally flows out, blowing out residual solution in the small column by using a suction ball;
(5) connecting a centrifugal pipe at the outlet of the column, leaching the small column with methanol, and blowing out residual liquid in the small column with a suction ball after the methanol naturally flows out.
7. The method for determining the residual amounts of five benzimidazoles pesticides in cereal grains according to claim 1, wherein in the fourth step, the determination of carbendazim, probenazole, benomyl, probenazole and fuberidazole by using a liquid chromatography-tandem mass spectrometry method comprises the following steps:
the measuring and heating method comprises the following steps: the initial temperature is maintained at 65 deg.C for 1min, and raised to 200 deg.C at 20 deg.C per minute, then raised to 220 deg.C at 2 deg.C per minute, then raised to 260 deg.C at 5 deg.C per minute, and finally raised to 280 deg.C at 10 deg.C/min, and maintained for 15 min.
8. The method for determining the residual quantity of five benzimidazoles pesticides in cereal grains according to claim 1, wherein the presetting of the conditions of the chromatographic column, the mobile phase and the mass spectrum comprises the following steps:
(1) selecting a ZORBAX eclipse XDS-C18 column;
(2) using 10mmol/L ammonium acetate aqueous solution (0.1% formic acid) -methanol solution as mobile phase of carbendazim, probenazole, benomyl, probenazole and fuberidazole;
(3) selecting an ionization mode as electrospray ionization; the ion spray voltage is 4 kV; the atomizing gas is nitrogen, the drying gas is nitrogen, and the collision gas is argon;
(4) multiple reaction monitoring was performed.
9. The method for determining the residual quantity of five benzimidazoles pesticides in cereal grains according to claim 1, wherein in the sixth step, the temperature of the petroleum ether is 60 ℃ to 90 ℃.
10. The method for determining the residual quantity of five benzimidazoles pesticides in cereal grains according to claim 1, wherein in the seventh step, the linear range of the concentration of the standard curve is 1 μ g/kg to 5000 μ g/kg.
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| WO2017216193A1 (en) * | 2016-06-14 | 2017-12-21 | Exosect Limited | Seed coatings comprising pesticide |
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