CN114646704B - Mass spectrum detection method for residual quantity of pyrazophos in animal-derived food - Google Patents

Abstract

The invention discloses a mass spectrum detection method of residual quantity of fenpyrad in animal-derived food, which comprises the following steps: s1, preparing a standard working solution: sucking a proper amount of 100mg/L standard stock solution, preparing 1mg/L standard intermediate solution with acetone, and preserving at 0-4deg.C in dark place; sucking a proper amount of 1mg/L of the standard intermediate solution, and preparing a standard working solution with the concentration of 0.5 mug/L, 2 mug/L, 5 mug/L, 10 mug/L and 25 mug/L by using acetone, wherein the standard working solution is ready to use; s2, extracting the haloxyfop-R-methyl residue in the sample by acetonitrile, and removing impurities by ethylenediamine-N-propyl silanized silica gel, octadecyl bonded silica gel and anhydrous magnesium sulfate. According to the invention, by optimizing chromatographic conditions and investigating the influence of extraction solvent types, purifying agents and matrix effects on recovery rate, a QuEChERS-gas chromatography triple quadrupole mass spectrometry detection method of the halofop-butyl is established, so that a detection technical guarantee is provided for halofop-butyl residues in animal-derived products.

Description

Mass spectrum detection method for residual quantity of pyrazophos in animal-derived food

Technical Field

The invention relates to the technical field of chemical detection, in particular to a mass spectrum detection method for the residual quantity of halofop-butyl in animal-derived food.

Background

The fenpyrazamine is a novel broad-spectrum and high-activity pre-emergence soil treatment herbicide developed by Japanese combinatorial chemistry company. The herbicide belongs to isoxazole herbicides, and is a potential inhibitor for biosynthesis of Very Long Chain Fatty Acids (VLCFAs) in plants. The application crops of the fenpyrazamine are relatively wide, and the fenpyrazamine can be safely used for crops such as corn, cotton, peanut, wheat, sunflower, sorghum and the like; can effectively prevent and remove gramineous weeds such as green bristlegrass, crabgrass, barnyard grass and the like, broadleaf weeds such as amaranthus, stramonium, eggplant and abutilon and the like. Penoxsulam is considered to be the best agent against drug-resistant weeds such as ryegrass in countries such as australia. The haloxyfop-R-methyl has the excellent characteristics of wide weed killing spectrum, high activity, low dosage, good safety and the like, so that the haloxyfop-R-methyl is more and more widely paid attention to, and is registered in countries such as Australia, america, canada and the like in crop fields such as wheat, corn, soybean and cotton for sealing and weeding, wherein the Australian formulates residual limits of plant source products such as corn, cereal, bean seeds, soybean (dry) and sunflower seeds and residual limits of animal source foods such as poultry meat, poultry edible offal, meat (mammal), edible offal (mammal), milk and eggs, and the residual limits of animal source foods are as low as 0.002mg/kg.

Researches on the fenpyr mainly focus on the aspects of weeding activity, environmental behavior and safety evaluation, and few relevant reports are made at home and abroad on a residual analysis method of the fenpyr. In the prior art, a method for detecting the residue of the halofop-butyl in soil and water is established by using a high performance liquid chromatography, but an analysis method of the halofop-butyl in animal-derived foods such as meat and the like has not been reported yet. Therefore, the method for detecting and analyzing the residual of the fenpyrad in the animal-derived food can make up for the blank that the project does not have a detection method in the animal-derived food in China; the method has an extremely important role in evaluating and managing the influence of the compound on human health and environmental safety and preventing the risk of trade input and output.

Therefore, we propose a mass spectrum detection method for the residual quantity of the fenpyrad in the animal-derived food so as to solve the problems.

Disclosure of Invention

The invention aims to provide a mass spectrum detection method for the residual quantity of haloxyfop-R-methyl in animal-derived food, and aims to solve the problems, and the method is used for establishing a QuEChERS-gas chromatography triple quadrupole mass spectrum detection method for the haloxyfop-methyl by optimizing chromatographic conditions and examining the influence of the type of an extraction solvent, a purifying agent and the matrix effect on the recovery rate so as to provide a detection technical guarantee for the residual quantity of the haloxyfop-methyl in animal-derived products.

In order to achieve the above purpose, the present invention provides the following technical solutions: a mass spectrum detection method for the residual quantity of halofop-butyl in animal-derived food comprises the following steps:

s1, preparing a standard working solution: sucking a proper amount of 100mg/L standard stock solution, preparing 1mg/L standard intermediate solution with acetone, and preserving at 0-4deg.C in dark place; sucking a proper amount of 1mg/L of the standard intermediate solution, and preparing a standard working solution with the concentration of 0.5 mug/L, 2 mug/L, 5 mug/L, 10 mug/L and 25 mug/L by using acetone, wherein the standard working solution is ready to use;

s2, extracting the haloxyfop-R-methyl residue in the sample by acetonitrile, and removing impurities by ethylenediamine-N-propyl silanized silica gel, octadecyl bonded silica gel and anhydrous magnesium sulfate;

s3, centrifuging, taking supernatant nitrogen, blowing to dryness, adding acetone for redissolving and filtering, detecting by a gas chromatograph-triple quadrupole mass spectrometer, and quantifying by an external standard method.

Preferably, in step S2, the extracting of the residual haloxyfop-methyl with acetonitrile in the sample comprises the steps of:

s20, cutting the sample into small blocks, and crushing the small blocks into slurry samples by using a knife grinder at 5000 r/min;

s21, weighing 5g of the prepared sample into a 50mL polypropylene centrifuge tube, adding 2g of sodium chloride, 10mL of acetonitrile and a ceramic proton, extracting for 5min by vortex oscillation, and centrifuging for 3min at 4000 r/min;

s22, accurately sucking 1.5mL of supernatant into a 2mL polypropylene centrifuge tube, and adding 50mgPSA and 50mgC ₁₈ And 50mg anhydrous magnesium sulfate;

s23, vortex mixing 1min, centrifuging 12000r/min for 3min, accurately sucking 1mL of supernatant, blowing nitrogen at 60 ℃ to dryness, adding 1mL of acetone for redissolution, and then passing through a 0.22 mu m organic filter membrane.

Preferably, in the step S3, helium gas with the purity more than or equal to 99.999 percent is introduced, and a constant flow mode is adopted, wherein the flow rate is 1.0mL/min.

Preferably, in step S3, the gas chromatograph-triple quadrupole mass spectrometer detects conditions: after the temperature of the column box is raised to 70 ℃ for 0min, the temperature is raised to 230 ℃ at 20 ℃/min for 0min, and finally the temperature is raised to 310 ℃ at 30 ℃/min for 1min.

Preferably, in step S3, the gas chromatograph-triple quadrupole mass spectrometer sample injection conditions: the temperature of the sample inlet is 250-300 ℃, the sample injection amount is 0.5-1 mu L, and the sample injection mode adopts non-split sample injection.

Preferably, in step S3, the electron bombardment source: 70eV, ion source temperature: 230 ℃, transmission line temperature: solvent retardation at 280 ℃): 3min.

Preferably, in step S3, the multiple reaction monitoring mode: a pair of quantitative ions, a pair of qualitative ions, a retention time thereof, a pair of quantitative ions, a pair of qualitative ions, and a collision energy are selected.

Compared with the prior art, the invention has the beneficial effects that:

the invention establishes a detection method for determining the residual quantity of the halofop-butyl in animal-derived food by using a gas chromatography triple quadrupole mass spectrometry for the first time, and under the optimized experimental condition, the target compound has good linear relation within the range of 0.0005-0.025mg/L, and the correlation coefficient is more than 0.99. The average recovery rate of the blank sample at the low, medium and high 3 adding levels is 85.8% -105.5%, the relative standard deviation (n=6) is 3.1% -6.8%, the quantitative limit of the method is 0.001mg/kg, the method has high sensitivity, simple operation, rapidness, accuracy and low cost, and the method can meet the detection requirement of residual fenpyrad in animal-derived foods.

Drawings

FIG. 1 is a total ion flow diagram of the halofop-butyl in a mass spectrometry detection method of the residual quantity of the halofop-butyl in animal-derived food;

FIG. 2 is an MRM chromatogram of extracted ions of the halofop-butyl in the mass spectrum detection method of the residual quantity of the halofop-butyl in the animal-derived food;

FIG. 3 is a graph showing the relationship between the type of the extraction solution and the average recovery rate of the mass spectrometry method for detecting the residual quantity of the penoxsulam in the animal-derived food;

FIG. 4 shows the effect of different adsorbents of a mass spectrometry detection method for residual quantity of fenpyrad in animal-derived food on extraction recovery rate of labeled blank samples;

FIG. 5 shows the ME values of matrix effect of halofop-butyl in different samples of a mass spectrum method for detecting the residual amount of halofop-butyl in animal-derived food.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

1. Standard solution preparation

Standard working solution: sucking a proper amount of 100mg/L standard stock solution, preparing 1mg/L standard intermediate solution by using acetone, and preserving at 0-4 ℃ in a dark place for 1 month in the effective period; a proper amount of standard intermediate liquid of 1mg/L is sucked, and acetone is used for preparing standard working solutions with the concentration of 0.5 mug/L, 2 mug/L, 5 mug/L, 10 mug/L and 25 mug/L, and the standard working solutions are prepared for use.

2. QuEChERS pretreatment

Cutting pork, pork liver, chicken liver and other samples into small pieces, crushing into slurry by a knife grinder at 5000r/min, taking 10 eggs, removing shells, stirring uniformly, and taking about 200g of milk sample, stirring uniformly. Weighing 5g (accurate to 0.01 g) of the prepared sample into a 50mL polypropylene centrifuge tube, adding 2g of sodium chloride, 10mL of acetonitrile and a ceramic uniform proton, and carrying out vortex oscillation extraction for 5min; centrifuging at 4000r/min for 3min. Accurately sucking 1.5mL of supernatant into a 2mL polypropylene centrifuge tube, adding 50mgPSA and 50mgC ₁₈ And 50mg anhydrous magnesium sulfate. Vortex mixing 1min,12000r/min centrifuging for 3min, accurately sucking 1mL supernatant, blowing nitrogen at 60deg.C to dry, adding 1mL acetone, redissolving, and passing through 0.22 μm organic filter membrane for determination.

3. Instrument conditions

DB-17MS capillary chromatographic column (30 m x 0.25mm x 0.25 μm) column or equivalent; column box temperature rise program: maintaining at 70deg.C for 0min, and heating to 230deg.C at 20deg.C/min for 0min; finally, heating to 310 ℃ at 30 ℃/min, and keeping for 1min; carrier gas: helium with purity more than or equal to 99.999%, constant flow mode and flow rate of 1.0mL/min; sample inlet temperature: 280 ℃; sample injection amount: 1 μl; sample injection mode: sample introduction without diversion; electron bombardment source: 70eV; ion source temperature: 230 ℃; transmission line temperature: 280 ℃; solvent delay: 3min; multiple reaction monitoring mode: selecting a pair of quantitative ions, a pair of qualitative ions, a retention time thereof, a pair of quantitative ions, a pair of qualitative ions, and collision energy, see table 1;

TABLE 1 retention time, quantitative ion pair, qualitative ion pair, collision energy, regression equation, correlation coefficient, linear range, and quantitative limit for metaxazole

MS1 full scan is carried out by selecting 5mg/L of metazachlor standard solution, the peak response of molecular ions is found to be lower, and ions 228.6 and 178.6 with higher response are selected as parent ions in the experiment; and scanning the corresponding parent ions by product ions, and selecting two groups with the best signals as MRM quantitative and qualitative ion pairs. Total ion flow diagram of 0.01mg/L of pyrazophos-ethyl acetone solution and MRM (MRM) chromatogram of pyrazophos-ethyl extraction ion are shown in figures 1 and 2.

Instrument conditions, selection of instruments

The research aims at establishing a detection method for detecting and confirming the haloxyfop-methyl, and the gas chromatograph and the liquid chromatograph are abandoned to be used when the detection instrument is selected, and the triple quadrupole liquid chromatograph and the triple quadrupole gas chromatograph with stronger specific capacity are only used. Firstly, carrying out parent ion scanning and child ion scanning on the halofop-butyl according to a triple quadrupole rod liquid mass spectrometer method development program, and finding that the parent ion 391.2 of the halofop-butyl has better response, but the peak type analysis after optimization generally has poorer response, and the sensitivity is poorer and can not meet the requirements of related regulations; therefore, the triple quadrupole rod liquid chromatograph is abandoned to be used for detecting the haloxyfop-methyl, and the triple quadrupole rod gas chromatograph can obtain good peak, the method has high sensitivity, and finally, the determination of the residual quantity of the fenpyrad in the animal-derived food by using a triple quadrupole gas chromatograph-mass spectrometer is determined.

Mass spectrometry condition optimization

MS1 full scan is carried out by selecting 5mg/L of metazachlor standard solution, the peak response of molecular ions is found to be lower, and ions 228.6 and 178.6 with higher response are selected as parent ions in the experiment; and scanning the corresponding parent ions by product ions, and selecting two groups with the best signals as MRM quantitative and qualitative ion pairs. Total ion flow diagram of 0.01mg/L of pyrazophos-ethyl acetone solution and MRM (MRM) chromatogram of pyrazophos-ethyl extraction ion are shown in figures 1 and 2.

Optimization of pretreatment method, extraction mode and selection of extraction solvent

For animal-derived samples, impurities such as proteins and fats are the main interfering substances, most pretreatment processes need to add protein precipitants to remove part of proteins, and acetonitrile or acidic acetonitrile is used as an extractant to achieve the effects of precipitating proteins and eliminating emulsions. Because the experiment finally uses a triple quadrupole gas chromatograph as final detection equipment, acetonitrile is used as an extraction solvent, acetonitrile nitrogen is blown to dryness, and then is re-dissolved by solvents such as n-hexane and the like, and then is detected by a machine. Experiments compare the differences in the extraction effect of acetone, ethyl acetate, n-hexane, acetonitrile and 0.1% acetonitrile formate as extraction solvents. For acetonitrile and 0.1% acetonitrile formate, chicken, pig liver, pork, chicken liver, milk and egg samples added with 0.01mg/kg of pyrazophos are processed and put on the machine according to the experimental method of S2; extracting, purifying and centrifuging acetone, ethyl acetate and normal hexane solvent according to an experimental method of S2, and taking supernatant to pass through a 0.22 mu m organic filter membrane for direct measurement without nitrogen blowing and redissolution operation; all experiments were calibrated using a solvent standard curve. Each experiment was run in parallel 3 times to give a graph of extraction solution type versus average recovery as shown in figure 3. The results show that when n-hexane is used as an extraction solvent, the extraction recovery rate is low in various matrixes, and when ethyl acetate is used as the extraction solvent, the recovery rate in eggs is low, but the matrix effect is obvious in other matrixes, and the recovery rate is high; the extraction recovery rate is higher in all matrixes when the acetone is used as an extraction solvent, and the matrix effect is obvious. The recovery rate of acetonitrile and 0.1% acetonitrile formate is good, and the extraction efficiency is high; from the viewpoint of convenience in operation, acetonitrile is finally selected as an extraction solvent, as shown in fig. 3.

Selection of decontamination reagent

The QuEChERS purifying agent commonly used in animal-derived food has C ₁₈ PSA and NH ₂ Adsorbents, etc., wherein ethylenediamine-N-propyl silanized silica gel (PSA) and NH ₂ The adsorption mechanism of the adsorbent is similar, and the adsorbent has weak anion exchange capacity, and can effectively remove organic acid, polar pigment, fatty acid, saccharides and other components capable of forming hydrogen bonds in a sample through the action of the hydrogen bonds and the compounds; octadecylsilane chemically bonded silica (C) ₁₈ ) Removing nonpolar compounds such as volatile oil, terpenes, and lipids, and removing anhydrous magnesium sulfateRemoving water in the sample liquid. Experiments and surveys PSA, C ₁₈ And anhydrous MgSO ₄ Recovery rate of three substances after the standard solution of the metazopyr is adsorbed. 100mg of different adsorbents are weighed into a 1.5mL bullet-head centrifuge tube, 1mL of mixed standard solution of 0.05mg/L is added, vortex oscillation is carried out for 2min, centrifugation is carried out for 2min at 12000r/min, and supernatant is filtered by an organic filter membrane of 0.22 mu m and then is detected by a machine. Three adsorbents are arranged in parallel, the average recovery rate is calculated, and experimental results show that PSA and C ₁₈ Anhydrous MgSO ₄ The average absorption recovery rate of the metazopyr is 95-105%; it shows that none of the three substances can cause adsorption to the fenpyrad.

Determination of the amount of adsorbent combination

When the QuEChERS method is used for purifying animal-derived samples, two or more adsorption purifying agents are usually used for better removing impurities such as lipids and saccharides in the samples, which interfere with the analysis of instruments. Experiment three groups of adsorbents (I: 20mgPSA+20 mgC) ₁₈ +50mg anhydrous MgSO4, II: 50mgPSA+50mgC ₁₈ +50mg anhydrous MgSO4, III: 100mgpsa+100mgc ₁₈ +50mg anhydrous MgSO 4) and 0.02mg/kg standard recovery experiments are carried out on blank samples of chicken, pork liver, milk and egg, and the purification effect of different adsorbent combinations is examined, and the experimental results are shown in figure 4. Experimental results show that the three groups of compounded adsorbents can meet the requirement of experimental recovery rate, and the recovery rate range is between 90 and 110 percent; from the actual purification effect, with PSA and C ₁₈ The dosage is increased, the impurity peak is reduced, and the base line is more stable. Comprehensively considering purification effect, cost, convenience and interference impurities and baseline during instrument analysis, selecting 50mgPSA+50mgC ₁₈ +50mg anhydrous MgSO4 was the kind and amount of the adsorbent purifier for the final experiment, as shown in FIG. 4.

Influence of matrix Effect

The Matrix Effects (ME) refer to the co-extract of the sample of the substance to be detected, which enhances or inhibits the detection response of the substance to be detected, thereby affecting the accuracy and precision of the detection result. The matrix effect is mainly represented as a "matrix-induced chromatographic response enhancement phenomenon", i.e., a matrix enhancement effect, in a gas phase system. The sensitivity of the method and the accuracy of the method are reduced when the matrix effect is greatly influenced, and errors are brought to the measurement, so that the Matrix Effect (ME) needs to be evaluated in the process of developing and confirming the method. The matrix effect is related to the type of matrix and the content of matrix interferents, the characteristics of analytes, chromatographic separation conditions, ion source designs of different instruments, and the like, and can be quantitatively evaluated according to the following formula: matrix effect (ME,%) = [ (matrix matching standard curve slope/pure solvent standard curve slope) -1] ×100. The ME <20% is a weak matrix effect, negligible without taking compensation measures; 20% or less and 50% or less of the ME is a medium matrix effect, 50% or more of the ME is a strong matrix effect, and measures are taken to compensate the matrix effect. Test blank substrate solution is prepared according to the pretreatment method of S2, standard working solution is prepared according to the method of S1, and the standard working solution of the substrate is measured on a machine. The matrix effects in different matrixes are calculated according to the formula, as shown in fig. 5, and as can be seen from fig. 5, the matrix effects of the fenpyr-diethyl in the 6 matrixes are weak matrix effects, and the matrix effects do not need to be compensated.

Methodological verification, using linear range, correlation coefficient and method detection limit

Preparing standard working solutions with the concentration of 0.5 mug/L, 2 mug/L, 5 mug/L, 10 mug/L, 20 mug/L and 25 mug/L of a series of halofop-butyl matrixes by using a blank matrix solution, detecting according to S3 sample injection, drawing a standard working curve of the matrixes by using the concentration of the halofop-butyl (X, ng/mL) as an abscissa and using the peak area of the halofop-butyl as an ordinate, so as to obtain a linear equation and a correlation coefficient; the quantitative Limit (LOQ) was determined at S/n=10 after adding an appropriate amount of standard solution to the blank sample solution and the relevant data are shown in table 1.

Recovery rate

Standard addition and recovery experiments of different concentrations are respectively carried out on pork, pork liver, chicken liver, milk and egg blank samples, the standard addition and recovery experiments cover quantitative limits, main MRL values are measured for 6 times in parallel at each standard addition level, and experimental data of the addition concentration and recovery rate of the fenpyrad in the animal source samples are shown in Table 2.

Table 2 labeled recovery and relative standard deviation of metazachlor in different samples (n=6)

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And (3) detecting an actual sample: the method is used for detecting 5 laboratory daily detection samples of pork, pork liver, chicken liver, milk and eggs, and the detection results are all undetected.

The invention establishes a detection method for determining the residual quantity of the halofop-butyl in animal-derived food by using a gas chromatography triple quadrupole mass spectrometry for the first time, and under the optimized experimental condition, the target compound has good linear relation within the range of 0.0005-0.025mg/L, and the correlation coefficient is more than 0.99. The average recovery rate of the blank sample at the low, medium and high 3 adding levels is 85.8% -105.5%, the relative standard deviation (n=6) is 3.1% -6.8%, the quantitative limit of the method is 0.001mg/kg, the method has high sensitivity, simple operation, rapidness, accuracy and low cost, and the method can meet the detection requirement of residual fenpyrad in animal-derived foods.

Although the present invention has been described with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements and changes may be made without departing from the spirit and principles of the present invention.

Claims (2)

1. The mass spectrum detection method for the residual quantity of the halofop-butyl in the animal-derived food is characterized by comprising the following steps of:

s1, preparing a standard working solution: sucking a proper amount of 100mg/L standard stock solution, preparing 1mg/L standard intermediate solution with acetone, and preserving at 0-4deg.C in dark place; sucking a proper amount of 1mg/L of the standard intermediate solution, and preparing a standard working solution with the concentration of 0.5 mug/L, 2 mug/L, 5 mug/L, 10 mug/L and 25 mug/L by using acetone, wherein the standard working solution is ready to use;

s2, extracting the haloxyfop-R-methyl residue in a sample by using acetonitrile, removing impurities by using ethylenediamine-N-propyl silanized silica gel, octadecyl bonded silica gel and anhydrous magnesium sulfate, centrifuging, taking supernatant, blowing nitrogen to dryness, adding acetone, redissolving and filtering;

s3, detecting by a gas chromatograph-triple quadrupole mass spectrometer, and quantifying by an external standard method:

DB-17MS capillary chromatographic column: 30m 0.25mm 0.25 μm; column box temperature rise program: maintaining at 70deg.C for 0min, and heating to 230deg.C at 20deg.C/min for 0min; finally, heating to 310 ℃ at 30 ℃/min, and keeping for 1min; carrier gas: helium with purity more than or equal to 99.999%, constant flow mode and flow rate of 1.0mL/min; sample inlet temperature: 280 ℃; sample injection amount: 1 μl; sample injection mode: sample introduction without diversion; electron bombardment source: 70eV; ion source temperature: 230 ℃; transmission line temperature: 280 ℃; solvent delay: 3min; multiple reaction monitoring mode: retention time 8.67min, quantitative ion pair: 228.6 and 179.1, a collision energy of 15eV, a qualitative ion pair 178.6 and 159.0, a collision energy of 15eV;

the animal-derived food includes: pork, pork liver, chicken liver, egg and milk.

2. The mass spectrometry detection method for residual quantity of pyribenzoxim in animal-derived food according to claim 1, characterized by comprising the following steps in step S2:

s20, cutting the sample into small blocks, and crushing the small blocks into slurry samples by using a knife grinder at 5000 r/min;

s21, weighing 5g of the prepared sample into a 50mL polypropylene centrifuge tube, adding 2g of sodium chloride, 10mL of acetonitrile and a ceramic proton, extracting for 5min by vortex oscillation, and centrifuging for 3min at 4000 r/min;

s22, accurately sucking 1.5mL of supernatant into a 2mL polypropylene centrifuge tube, and adding 50mgPSA and 50mgC ₁₈ And 50mg anhydrous magnesium sulfate;

s23, vortex mixing 1min, centrifuging 12000r/min for 3min, accurately sucking 1mL of supernatant, blowing nitrogen at 60 ℃ to dryness, adding 1mL of acetone for redissolution, and then passing through a 0.22 mu m organic filter membrane.

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