CN107478737B - Method for detecting residual quantity of 80 pesticides in white spirit - Google Patents
Method for detecting residual quantity of 80 pesticides in white spirit Download PDFInfo
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- CN107478737B CN107478737B CN201710585796.5A CN201710585796A CN107478737B CN 107478737 B CN107478737 B CN 107478737B CN 201710585796 A CN201710585796 A CN 201710585796A CN 107478737 B CN107478737 B CN 107478737B
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- G—PHYSICS
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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
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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
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Abstract
The invention discloses a rapid detection method for simultaneously determining 80 pesticide residues in white spirit by adopting ultra-high performance liquid chromatography-quadrupole/electrostatic field orbital trap high resolution mass spectrometry, which is characterized in that 80 pesticides comprise organophosphorus, carbamate, herbicide, bactericide and pesticide, 5 pesticides in total are used, a white spirit sample is subjected to nitrogen blowing to remove ethanol, acetonitrile is subjected to constant volume, supernatant is obtained after freezing and centrifugation, a microporous filter membrane is used for filtration, and filtrate is collected and enters the ultra-high performance liquid chromatography-quadrupole/electrostatic field orbital trap high resolution mass spectrometry for analysis and determination. Compared with the traditional detection method, the method has the advantages of wide coverage of pesticide species, simple and convenient pretreatment method, less used organic solvent and high detection sensitivity.
Description
Technical Field
The invention belongs to the technical field of pesticide residue analysis and detection, and particularly relates to a method for simultaneously determining 80 pesticide residues in white spirit by using ultra-high performance liquid chromatography-quadrupole/electrostatic field orbital trap high-resolution mass spectrometry.
Background
Pesticide residues (Pesticide residues) are a general term for trace amounts of Pesticide pathogens, toxic metabolites, degradants and impurities that are not decomposed and remain in organisms, harvest, soil, water bodies, atmosphere within a period of time after the Pesticide is used. Agricultural chemicals such as herbicides, plant growth regulators, insecticides, acaricides and bactericides, which are inevitably used in the course of planting of food crops, may eventually remain in the food. The liquor is prepared from starchy grains such as sorghum and wheat as main raw materials by fermenting, distilling, blending and storing. During distillation of the brewing raw materials containing the pesticides, the pesticides dissolved in alcohol may be distilled out together, and finally remain in the white spirit, which has potential threat to human health.
The control of pesticide residues in food requires a rapid, accurate and effective analysis method, high sensitivity and good selectivity. Obviously, the method for analyzing the pesticide residues can be used for completely analyzing various pesticides with different types, different polarities and different chemical properties by one-time sample injection, so that the analysis time is shortened, and the detection efficiency is improved. In various pesticide residue analyses, the most used method, chromatography-mass spectrometry, involves separation by GC or LC and qualitative and quantitative detection by mass spectrometry.
GC-MS or LC-MS methods are mostly adopted for detecting pesticide residues in white spirit, LC-MS and GC-MS are respectively adopted by Rong and the like to analyze 5 organophosphorus pesticides and 8 organochlorine pesticides in the white spirit, the method is high in sensitivity, but the pretreatment is complex, the time consumption is long, and the pesticide residue detection type is single each time. Because the white spirit is a complex beverage with high ethanol content, the pretreatment is more complex by further purification or liquid-liquid extraction method on the basis of removing ethanol, and the use amount of organic solvent is larger.
Disclosure of Invention
In view of the fact that the limit standard and the detection standard of various pesticide residues in white spirit and the defects of various pesticide residue technologies in the existing white spirit are not available at home and abroad, the invention aims to overcome the defects of the prior art, simplify the sample pretreatment method and shorten the sample analysis time, thereby providing the ultra-high performance liquid chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrometry method for simultaneously analyzing 5 types of 80 pesticides including organic phosphorus, carbamates, herbicides, bactericides and insecticides in white spirit by one injection, and the method has the advantages of high accuracy, good reproducibility and wider application range for simultaneously detecting various pesticide residues.
In order to achieve the purpose of the invention, the inventor conducts extensive experimental research and finally obtains the following technical scheme for detecting various pesticide residues in the white spirit, and the scheme specifically comprises the following steps:
(1) and sampling: if 1.0mL of white spirit sample is moved into a 2mL graduated centrifuge tube;
(2) nitrogen blowing: blowing nitrogen below 25 ℃ until all ethanol is removed;
(3) and constant volume: the volume of the sample blown with the nitrogen is determined to be 1.0mL by mass spectrum pure acetonitrile;
(4) and uniformly mixing: uniformly mixing the sample with the constant volume on a vortex oscillation instrument at 2000-6000 rpm for 2-5 min;
(5) and centrifuging: centrifuging the uniformly mixed sample at 4 ℃ at 6000-12000 rpm for 5-20 min; as a preferred embodiment, 10000rpm, 10min of centrifugation;
(6) and filtering: taking the centrifugal supernatant, filtering with a 0.22 μm needle head type filter head, and collecting the filtrate in a liquid phase vial;
(7) and ultra-high performance liquid chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrometry.
Wherein, in the step (7), the liquid phase condition is as follows: the chromatographic column adopts an Agilent Eclipse-XDB-C18 chromatographic column with the thickness of 2.1 multiplied by 100mM and 3.5 mu m, acetonitrile is taken as a mobile phase A, 5mM ammonium acetate water solution (containing 0.1% (V/V) formic acid) is taken as a mobile phase B, gradient elution is carried out, wherein the temperature of the column is 30-35 ℃, the flow rate is 0.2-0.3 mL/min, and the sample injection volume is 5-10 mu L; preferably, the column temperature is 30 ℃, the flow rate is 0.3mL/min, and the injection volume is 10. mu.L.
As a preferred embodiment, the conditions of gradient elution are as follows 1:
TABLE 1 mobile phase Linear elution conditions
| Time/minute | A/％ | B/％ |
| 0.0 | 20 | 80 |
| 1.0 | 20 | 80 |
| 10.0 | 85 | 15 |
| 17.0 | 100 | 0 |
| 20.0 | 100 | 0 |
| 21.0 | 20 | 80 |
In the step (7), the mass spectrum conditions are as follows: electrospray ion source with heating function (HESI), sheath gas: 30 arb; auxiliary gas: 10 arb; back blowing: 5 arb; ionization voltage: 3.5 kv; ion transfer tube temperature: 280 ℃; ion source temperature: 150 ℃; and (3) positive ion mode monitoring, wherein the positive ion mode monitoring is simultaneously measured by adopting two modes of full ms and target ms2 simultaneous scanning, the scanning range is 50-650m/z, the resolution of full ms scanning is 35000, and the resolution of target ms2 scanning mode is 17500.
In addition, the method also comprises the preparation of a standard curve, and the specific operation steps are as follows:
s1, preparing a standard stock solution: the pesticide is distinguished according to organophosphorus, carbamate, herbicide, bactericide and pesticide, 0.010g of various pesticide standard products are respectively and accurately weighed, the similar pesticide is placed in the same 10.0mL volumetric flask, dissolved by acetone and subjected to constant volume, and 80 pesticide mixed standard stock solution with the concentration of 1000 mug/mL is obtained;
s2, preparing a mixed standard solution: respectively sucking 1.0mL of the 5 pesticide mixed standard stock solutions in the step (8) and placing the 5 pesticide mixed standard stock solutions in a 10.0mL volumetric flask, diluting the acetonitrile to a constant volume to obtain 80 pesticide standard mixed stock solutions with the concentration of 100 mu g/mL, diluting the 80 pesticide standard mixed stock solutions step by using 50% (V/V) acetonitrile aqueous solution, and finally preparing 80 mixed pesticide standard working solutions of 5.0ng/mL, 10.0ng/mL, 20.0ng/mL, 50.0ng/mL, 100.0ng/mL and 200.0ng/mL, wherein the standard working solutions are stored at 0-4 ℃ in a dark place and can be used for 3 months;
s3, measuring by an instrument: sucking prepared mixed standard working solutions with different concentrations, filtering the mixed standard working solutions with a 0.22 mu m filter membrane, and then entering an ultra high performance liquid chromatography-quadrupole/electrostatic field orbit trap high resolution mass spectrometry for determination;
s4, calculation of pesticide residues: and (3) carrying out quantitative analysis on the pesticide target by adopting an external standard method, namely carrying out regression analysis on the corresponding concentration by using the peak area of the pesticide quantitative ion to obtain a standard curve.
And after performing ultra-high performance liquid chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrometry on the sample to be detected, substituting the peak area of the detected quantitative ion of the pesticide into the standard curve to obtain the content of the pesticide in the sample to be detected.
In the present invention, the pesticide package contains albendazole, fenthion sulfone, fenthion sulfoxide, benzothiazole, benalaxyl, fenclofos, fenthion sulfone, imidacloprid, fluazifop-p-butyl, profenofos, propoxur, fenamiphos, oryzalin, dichlorphos, dichlorvos, phos, fenamiphos, acetamiprid, chlorfenvinphos, chlorpyrifos, carbendazim, oxadixyl, famoxadone, diazinon, phos, phoxim, heptenophos, oryzophos, methamidophos, phorate, alachlor, mefenphos, chlorpyrifos-methyl, pirimiphos-methyl, carbaryl, metalaxyl, myclobutanil, quizalofop, monocrotophos, pirimicarb, carbofuran, phoxim, spirotetramat, malathion, imicarb, imazamidothion, methiocarb-methyl, triazophos, triazoxide, triaz, One or more of insecticidal urea, phenthoate, oxamyl, bitertanol, diphenoxylate, metoclopramide, terbufos sulphoxide, aldicarb sulphoxide, dimethomorph, aphidethoprophos, omethoate, acephate sulphoxide, ethyl parathion, ethyl glutathion, metolachlor, iprobenfos, clomazone, iprodione, indoxacarb and synergistic ether, and 80 or more pesticide residues can be detected simultaneously.
The qualitative and quantitative ions for the 80 pesticides are shown in table 2 of example 1.
The detection limit of the method is that the matrix standard preparation working solution of pesticides with different concentrations enters an ultra high performance liquid chromatography-quadrupole/electrostatic field orbit trap high resolution mass spectrum for detection, and the detection Limit (LOD) is realized by 3 times of signal to noise ratio (S/N is 3) technology, and is between 0.034ng/mL and 0.089 ng/mL.
The repeatability and the standard adding recovery rate of the method are as follows: 80 kinds of pesticide standard solutions are added into a blank sample, the blank sample is pretreated respectively and then enters into an ultra high performance liquid chromatography-quadrupole/electrostatic field orbit trap high resolution mass spectrometry for detection and analysis, and the recovery rate is calculated according to the addition amount and the measured value, and the result is shown in table 2 in the embodiment. As can be seen from Table 2, the recovery rates of the 80 pesticides are between 87.1% and 116.1%, and the average Relative Standard Deviation (RSD) is between 1.3% and 15.9%, which shows that the method has high recovery rate and good repeatability.
The method adopts two steps of impurity removal, wherein the first step is freezing centrifugation impurity removal, and the second step is filtration membrane impurity removal. In the prior art, the ultra-high performance liquid chromatography-quadrupole/electrostatic field orbit trap high resolution mass spectrometry generally only adopts filtration for impurity removal, and the invention also arranges freezing centrifugation for impurity removal, so as to primarily remove large-particle impurities in a sample, and also remove partial fat-soluble substances and macromolecular substances in the sample, thereby playing a role in quickly purifying the sample. And the quality to be analyzed cannot be lost by the two-step impurity removal because the impurity removal is carried out after the constant volume.
Particularly, the invention can simultaneously measure 80 or more pesticide residues, and the minimum detection limit is between 0.034ng/mL and 0.089 ng/mL. In the existing pesticide residue limit standard, most of pesticide limits are higher than 10ng/mL, the detection limit of the invention is obviously lower than that of the pesticide residues in the prior art, the sensitivity is high, and the pesticide residues in white spirit and trace amount thereof can be detected. The improvement of the sensitivity is not only benefited from the improvement of the detection performance of the instrument, but also benefited from the simple pretreatment method of the invention, only alcohol removal and filtration are carried out, steps such as purification and the like are not needed, the pesticide residue loss in the sample is small, and the detection limit is obviously reduced.
In addition, the invention has the following advantages:
(1) according to the method, ethanol is removed by nitrogen blowing in the pretreatment process of the sample, the temperature in the whole process is controlled below room temperature, the problem of easy decomposition characteristic of the thermally unstable pesticide is solved, and the recovery rate of the sample is improved.
(2) According to the invention, acetonitrile is adopted for constant volume in the pretreatment process of the sample, so that the solubility of the pesticide is improved, and the stability and the ionization efficiency are improved in the spray ionization process.
(3) The invention adopts a high-speed freezing and centrifuging mode, removes partial fat-soluble substances and macromolecular substances in the sample and plays a role in quickly purifying the sample.
(4) The invention adopts pretreatment modes such as nitrogen-blowing alcohol removal, high-speed freezing centrifugal purification and the like, and the pretreatment of the sample is simple, convenient and quick, uses less organic solvent, and is economic and environment-friendly.
(5) The invention adopts the ultra-high performance liquid chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrometry with high sensitivity, high resolution and strong anti-interference capability for detection, thereby having the advantages of accurate operation, high sensitivity and good repeatability.
Drawings
FIG. 1 is a TIC chart of 80 pesticide standards in example 1 of the invention.
FIG. 2 is a TIC chart of white spirit blank standard.
FIG. 3 is a TIC chart of a sample subjected to high-speed freeze centrifugation in example 2 of the present invention.
FIG. 4 is a graph of TIC of a sample subjected to filtration only in example 2 of the present invention.
FIG. 5 is a graph showing the effect of formic acid on the response of the pesticide in the apparatus of example 3 of the present invention.
FIG. 6 is a graph showing the effect of ammonium acetate on the pesticide peak profile in example 3 of the present invention; wherein A is an extraction diagram of methomyl added with 5mM ammonium acetate ions, B is an extraction diagram of methomyl without added with 5mM ammonium acetate ions, C is an extraction diagram of chlordimeform added with 5mM ammonium acetate ions, and D is an extraction diagram of chlordimeform without added with 5mM ammonium acetate ions.
FIG. 7 is a diagram of thiamethoxam ion extraction with different gradient treatments of the mobile phase in example 4 of the present invention; wherein A is a thiamethoxam ion extraction diagram when the initial concentration of the acetonitrile phase is 0%, and B is a thiamethoxam ion extraction diagram when the initial concentration of the acetonitrile phase is 20%; c is the thiamethoxam ion extraction diagram when the initial concentration of the acetonitrile phase is 50%.
Detailed Description
The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others of the concepts fall within the scope of the invention.
1. Instruments and reagents:
pesticide standard substance: albendazole, fenthion sulfone, fenthion sulfoxide, benzothiazole, benalaxyl, fenclofos, fenthion sulfone, imidacloprid, fluazifop-P, profenofos, propoxur, fenamiphos, oryzalin, dichlorvos, fenamiphos, acetamiprid, chlorfenvinphos, chlorpyrifos, carbendazim, oxadixyl, famoxadone, diazinon, fenphos, fluvophos, heptenophos, orythion, methamidophos, phorate, alachlor, profenofos, chlorpyrifos-methyl, pirimiphos-methyl, carbaryl, metalaxyl, myclobutanil, quizalofop-p, monocrotophos, pirimicarb, carbofuran, dimethoate, phosphamidon, malathion, azoxystrobin, methiocarb-methyl, methiocarb-ethyl, triazophos, triafola, triazoxide, Bifenacet, metoclopramide, terbufos sulfoxide, terbufos sulfone, aldicarb sulfoxide, dimethomorph, aphidethoprophos, omethoate, ethiprole, ethyl parathion, ethyl glutathion, metolachlor, iprobenfos, clomazone, iprodione, indoxacarb and synergistic ether, wherein acetonitrile and methanol are LC-MS grade reagents, and the distilled water meets the requirements of first-grade water in GB/T6682.
U.S. Thermo Fisher ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry; a LabnetVtexMixer VX200 oscillator, usa; german Sigma high speed refrigerated centrifuge.
Example 1 detection of residual amounts of 80 pesticides in white spirit sample A
1. Sample pretreatment:
accurately transferring 1.0mL of Chinese liquor sample and 2.0mL of PV tube, placing the PV tube containing the sample in a nitrogen blowing instrument, removing ethanol by nitrogen blowing, diluting to 1.0mL with acetonitrile, vortex oscillating for 2min, centrifuging at 4 deg.C and 12000rpm for 10min, collecting supernatant, filtering with 0.22 μm needle head type filter head, and collecting filtrate in liquid phase vial.
2. Preparing a standard working solution: the pesticide is distinguished according to organophosphorus, carbamate, herbicide, bactericide and pesticide, 0.010g of various pesticide standard products are respectively and accurately weighed, the similar pesticide is placed in the same 10.0mL volumetric flask, dissolved by acetone and subjected to constant volume, and 80 pesticide mixed standard stock solution with the concentration of 1000 mug/mL is obtained; respectively sucking 1.0mL of 5-class pesticide mixed standard stock solution in the mixed standard stock solution, placing the mixed standard stock solution in a 10.0mL volumetric flask, diluting with acetonitrile to a constant volume to obtain 80 pesticide standard mixed stock solutions 1 with the concentration of 100 mu g/mL, then diluting with 50% (V/V) acetonitrile step by step, finally preparing 80 mixed pesticide standard working solutions of 5.0ng/mL, 10.0ng/mL, 20.0ng/mL, 50.0ng/mL, 100.0ng/mL and 200.0ng/mL, storing the standard working solutions at 0-4 ℃ in a dark place, and storing the standard working solutions for 3 months;
3. measuring by an instrument: the prepared mixed standard working solution with different concentrations is sucked, filtered by a 0.22 mu m needle head type filter head, and the filtrate is collected in a liquid phase vial. Introducing the liquid phase small bottle filled with the standard working solution and the sample into an ultra-high performance liquid chromatography-quadrupole/electrostatic field orbital trap high resolution mass spectrometry for detection and analysis (the TIC images of 80 pesticide standard products are shown in figure 1); in addition, the invention also measures the blank white spirit without the standard substance, and the TIC graph is shown in figure 2.
4. And (3) calculating pesticide residues: and (3) carrying out quantitative analysis on the pesticide target by adopting an external standard method, namely carrying out regression analysis on the corresponding concentration by using the peak area of the pesticide quantitative ion to obtain a standard curve, measuring the treated sample, measuring the peak area of the detected pesticide quantitative ion, substituting the peak area into the standard curve, and solving the content of the pesticide in the sample.
5. When the ultra-high performance liquid chromatography-quadrupole/electrostatic field orbit trap high resolution mass spectrometry is used for measuring, the liquid phase conditions are as follows: the column was 2.1X 100mM, 3.5 μm Agilent Eclipse-XDB-C18 column, acetonitrile as mobile phase A, 5mM ammonium acetate solution (containing 0.1% (V/V) formic acid) as mobile phase B, and gradient elution was performed under the conditions shown in Table 1.
TABLE 1 mobile phase Linear elution conditions
| Time/minute | A/％ | B/％ |
| 0.0 | 20 | 80 |
| 1.0 | 20 | 80 |
| 10.0 | 85 | 15 |
| 17.0 | 100 | 0 |
| 20.0 | 100 | 0 |
| 21.0 | 20 | 80 |
Wherein the column temperature is 30 ℃, the flow rate is 0.3mL/min, and the sample injection volume is 10 muL.
The mass spectrum conditions of the mass spectrum are as follows: electrospray ion source with heating function (HESI), sheath gas: 30 arb; auxiliary gas: 10 arb; back blowing: 5 arb; ionization voltage: 3.5 kv; ion transfer tube temperature: ion source temperature: 150 ℃;
6. the data acquisition mode is as follows: positive ion mode monitoring, full ms and target ms are adopted2The scanning range is 50-650m/z, the resolution of full ms scanning is 35000, and the target ms is measured simultaneously2The scan mode resolution is 17500.
The TIC chart of 80 pesticide standard products is shown in figure 1, and the mass spectrum parameters are shown in the following table 2; the TIC chart of the white spirit blank standard substance is shown in figure 2, and the detection result of the white spirit sample is shown in table 3.
As can be seen from the following table 2, the recovery rate of the 80 pesticides of the invention is between 87.1% and 116.1%, and the average Relative Standard Deviation (RSD) is between 1.3% and 15.9%, which shows that the recovery rate of the method of the invention is high, and the repeatability is good. More particularly, the lowest detection line of 80 pesticides is between 0.034ng/mL and 0.089ng/mL, and compared with the existing pesticide residue limit standard in which most of the pesticide limits are higher than 10ng/mL, the method has high sensitivity and can detect the pesticide residues in the white spirit and trace amounts thereof.
Table 280 mass spectrum parameters and methodology parameters for the pesticides (n ═ 6)
TABLE 2
TABLE 3 detection results of the pesticide targets in sample A
Note: "-" indicates no detection.
Example 2 Effect of different modes of impurity removal on the Effect of analysis
In the prior art, the impurity removal of the pretreatment generally only adopts filtration or centrifugation, and the more steps are taken into consideration, the more the sample loss is, and the workload is increased. The experiment of the invention finds out the influence of the impurity removal mode on the analysis effect of 80 pesticides in the invention, and the specific steps are as follows:
accurately transferring 1.0mL of white spirit sample and 2.0mL of PV tube, placing the PV tube containing the sample in a nitrogen blowing instrument to remove ethanol by nitrogen blowing, diluting to 1.0mL by acetonitrile, carrying out vortex oscillation for 5min, respectively removing impurities by high-speed freezing centrifugation, filtering and removing impurities by a 0.22-micrometer needle head type filter head, purifying by three modes of high-speed freezing centrifugation and filtering and removing impurities, keeping the other parameters and modes unchanged, and placing the obtained sample in a liquid phase small bottle. The rest of the procedure was referred to example 1.
Experimental results and discussion:
the results are shown in FIGS. 1, 3 and 4, FIG. 3 is a TIC graph of a sample subjected to high-speed freeze centrifugation only, and FIG. 4 is a TIC graph of a sample subjected to filtration treatment only. As can be seen from the comparison between FIG. 3 and FIG. 4, the TIC graph response of the sample after freezing and centrifugation is lower than that of the sample after filtration, the interference peak of the sample after filtration is obviously more than that of the sample after freezing and centrifugation, some alcohol-soluble substances can be separated out to form micro-particles after the sample is subjected to alcohol removal and redissolution, and when the sample enters an instrument for detection, impurities are easily removed by filtration without a filter membrane, so that a liquid phase pipeline and a liquid spray needle are easily blocked, and therefore, the sample must be subjected to filtration before entering the instrument for analysis. The high-speed freezing centrifugation can remove part of alcohol-soluble substances in the redissolved solution, reduce the background value of the sample after the sample enters an instrument for analysis, and finally, the invention selects the mode of filtering and removing impurities after the high-speed freezing centrifugation for purification, and the TIC chart obtained by processing the sample is shown in figure 1.
Example 3 Effect of different flows on the Effect of analysis
In order to analyze the influence of different flows on the analysis result of the invention, the invention respectively adopts 4 groups of mobile phases of methanol-water, acetonitrile-formic acid water (containing 0.1 percent (V/V) formic acid) and acetonitrile-5 mM ammonium acetate water solution (containing 0.1 percent (V/V) formic acid) to carry out gradient elution comparison tests. The remaining experimental procedures and conditions were as in example 1.
Experimental results and discussion:
taking analysis time required by complete separation of 80 pesticides as a reference basis, respectively investigating gradient elution conditions of two mobile phases of methanol-water and acetonitrile-water, wherein when the methanol-water is used as the mobile phase, the complete separation of the 80 pesticides requires 35 minutes, and when the acetonitrile-water solution is used as the mobile phase, the complete separation of the 80 pesticides only requires 20 minutes, so that the acetonitrile-water is adopted as the mobile phase for further optimization.
The effect of the addition of formic acid and the absence of formic acid on the response of the pesticide in the instrument is comparatively analyzed by taking the peak area of the pesticide as a reference, and the result is shown in FIG. 5 (the response of metalaxyl, malathion, triadimenol, bromacil and diphenoxylate are taken as examples because the pesticide of the invention has too many types). As is clear from fig. 5, since the ionization of the agricultural chemical is promoted and the strength of the agricultural chemical in the apparatus is increased by adding formic acid to the mobile phase, the formic acid concentration is 0.1% (V/V) in consideration of the tolerance of the apparatus and the column by adding a certain amount of formic acid solution to the mobile phase.
In addition, the present invention also compares the effect of adding 5mM ammonium acetate in the mobile phase on the peak shape of the compound, i.e., comparing the effect of two groups of mobile phase of acetonitrile-formic acid (containing 0.1% (V/V) formic acid) and acetonitrile-5 mM ammonium acetate (containing 0.1% (V/V) formic acid) on the peak shape of the pesticide in the apparatus, and the result is shown in FIG. 6. After the ammonium acetate is added (fig. 6A and 6C), the peak patterns of the methomyl and the chlordimeform are better than those of the methomyl and the chlordimeform which are not added (fig. 6B and 6D), which shows that the peak patterns of the compounds can be optimized to a certain extent by proper amount of ammonium acetate in the mobile phase, and the corresponding strength effect of the compounds is improved. Therefore, acetonitrile-5 mM ammonium acetate aqueous solution (containing 0.1% (V/V) formic acid) is finally selected as the mobile phase for gradient elution in the invention.
Example 4 Effect of different gradients of the Mobile phase on the Effect of the analysis
In order to analyze the influence of different gradients of the mobile phase on the analysis effect, the invention adopts acetonitrile-5 mM ammonium acetate aqueous solution (containing 0.1% (V/V) formic acid) as the mobile phase, and explores the influence of different gradient analysis on the analysis effect. The remaining experimental procedures and conditions were as in example 1.
Experimental results and discussion:
the initial concentrations of the acetonitrile phases were set at 0%, 20%, 50%, respectively, and the results showed that, in the case of thiamethoxam, the compound retained 3min before had a severe solvent effect with splitting of the peaks (fig. 7A); when the concentration of acetonitrile is 50%, the strong polar substance in the sample matrix flows out fast along with the mobile phase, the compound before the retention time of 3min has stronger matrix effect (figure 7C), and when the initial concentration of acetonitrile is 20%, the effect is moderate (figure 7B).
In an elution gradient, 85% acetonitrile is taken as a gradient boundary, 45 pesticides peak in an acetonitrile 20% -85% interval, 34 pesticides peak in an acetonitrile 85% -100% interval, in order to achieve better separation degree of all compounds, shorten analysis time and obtain higher analysis efficiency, the gradient elution time is analyzed, target compounds are completely separated in the acetonitrile 20% -85% interval and the acetonitrile 85% -100% interval, enough scanning points are used for qualitative and quantitative determination as reference basis, and the results are shown in tables 4 and 5. As can be seen from the table, in the interval of acetonitrile concentration 20% -85%, the optimum elution time is 8-10min, and further experiments verify that when the elution time is 9min, all compounds can meet the requirements of qualitative and quantitative determination; in the interval of 85% -100% of acetonitrile concentration, the optimal elution time is 7-9min, and further experiments prove that when the elution time is 8min, all compounds can meet the requirements of qualitative and quantitative determination. The gradient elution of the final mobile phase is shown in table 1.
TABLE 4 selection of acetonitrile concentrations 20% -85% elution time
TABLE 5 selection of acetonitrile concentrations 85% -100% elution time
Claims (6)
1. A method for detecting pesticide residue in white spirit is characterized in that:
the specific operation steps are as follows:
(1) sampling;
(2) nitrogen blowing;
(3) and constant volume: the volume of the sample blown by nitrogen is fixed to 1.0mL by acetonitrile;
(4) and uniformly mixing: uniformly mixing the sample with constant volume in a vortex at 2000-6000 rpm for 2-5 min;
(5) and centrifuging: the blended sample is at 4°C, centrifuging at 6000-12000 rpm for 5-20 min;
(6) and filtering: filtering the centrifuged supernatant with 0.22 μm filter membrane;
(7) ultra-high performance liquid chromatography-quadrupole/electrostatic field orbit trap high-resolution mass spectrometry; the liquid phase conditions were: the chromatographic column adopts 2.1 × 100mM, 3.5 μm Agilent Eclipse-XDB-C18 chromatographic column, acetonitrile as mobile phase A, 5mM ammonium acetate solution containing 0.1% (V/V) formic acid as mobile phase B, and gradient elution is carried out, wherein the column temperature is 30-35%°C, the flow rate is 0.2-0.3 mL/min, and the sample injection volume is 5-10 mu L;
the conditions for gradient elution were:
The pesticide is albendazole, fenthion sulfone, fenthion sulfoxide, benzothiazole, benalaxyl, fenclofos, fenthion sulfone, imidacloprid, fluazifop-p-butyl, profenofos, propoxur, fenamiphos, oryzalin, dichlorvos, fenamiphos, acetamiprid, chlorfenvinphos, chlorpyrifos, carbendazim, oxadixyl, famoxadone, diazinon, fenphos, fluvophos, heptenophos, valethion, methamidophos, phorate, alachlor, profos, methamphos, chlorpyrifos-methyl, pirimiphos-methyl, carbaryl, metalaxyl, myclobutanil, quizalofop-p, monocarb, pirimicarb, carbofuran, dimethoate, phosphamidon, malathion, pyraclostrobin, azoxystrobin, methiocarb-methyl, methiocarb, methidathion, triazophos, triazoxide, Oxamyl, bitertanol, metamifop, metosulam, terbufos sulphoxide, aldicarb sulphoxide, dimethomorph, pirimiphos, omethoate, ethoprophos, iprobenfos, isofenoxanil, clomazone, indoxacarb and diethofecan.
2. The method of claim 1, wherein: the nitrogen blowing condition in the step (2) is 25°Nitrogen below C is blown to ethanol and is completely removed.
3. The method of claim 1, wherein: the centrifugal condition in the step (5) is 4°C，10000 rpm、10 min。
4. The method of claim 1, wherein: the column temperature was 30 ℃, the flow rate was 0.3mL/min, and the injection volume was 10. mu.L.
5. The method of claim 1, wherein: the mass spectrum conditions measured by the instrument in the step (7) are as follows: electrospray ion source with heating function (HESI), sheath gas: 30 arb; auxiliary gas: 10 arb; back blowing: 5 arb; ionization voltage: 3.5 kv; ion transfer tube temperature: 280°C; ion source temperature: 150°C; is justAnd (3) ion mode monitoring, wherein the ion mode monitoring is simultaneously measured by adopting two modes of full ms and target ms2 simultaneous scanning, the scanning range is 50-650m/z, the resolution of full ms scanning is 35000, and the resolution of target ms2 scanning mode is 17500.
6. The method according to claim 1, further comprising the preparation of a standard curve, the specific operating steps being as follows:
s1, preparing a standard stock solution: taking acetone as a solvent, and respectively distinguishing according to organophosphorus, carbamate, herbicide, bactericide and pesticide to prepare 5 standard stock solutions, wherein the concentration of each pesticide is 1.00 mg/mL;
s2, preparing a mixed standard solution: mixing the 5 standard stock solutions obtained in the step S1, preparing a mixed standard solution with each pesticide concentration of 0.01 mg/mL by using acetonitrile, and then diluting the mixed standard solution into a gradient solution by using an acetonitrile aqueous solution with the volume ratio of 50%;
s3, measuring by an instrument: sucking prepared mixed standard working solutions with different concentrations, filtering the mixed standard working solutions with a 0.22 mu m filter membrane, and then entering an ultra high performance liquid chromatography-quadrupole/electrostatic field orbit trap high resolution mass spectrometry for determination;
s4, calculation of pesticide residues: and (3) carrying out quantitative analysis on the pesticide target by adopting an external standard method, namely carrying out regression analysis on the corresponding concentration by using the peak area of the pesticide quantitative ion to obtain a standard curve.
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