Cooperative analysis and detection method for various pesticides in different plant tissues and application

Abstract

The invention discloses a collaborative analysis and detection method for various pesticides in different plant tissues and application. The method comprises the following steps: firstly, mixing crushed rice with an extraction solvent, sequentially carrying out ultrasonic extraction and purification, and determining four pesticides such as neonicotinoids, organophosphorus, pyrethroids and organochlorine by using a liquid chromatograph-mass spectrometer. The researched and developed method system has the advantages of short pretreatment process, few purification steps, simultaneous quantitative analysis of various pesticides and the like. The method has high accuracy and reproducibility.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide detection, and particularly relates to a method and application for co - analysis and detection of multiple types of pesticides in different plant tissues. Background Art

[0002] When detecting pesticide residues in plant tissues, the physical and chemical properties and matrix complexity of different organs pose significant challenges to the analysis results. For example, the waxy layer on the leaf surface, the high sugar or oil content in fruits, and the soil adsorbents on the roots can all interfere with the pesticide extraction efficiency. The fat - soluble components in leaves and peels are easily combined with organophosphorus or pyrethroid pesticides, making it difficult for traditional solvent extraction methods to completely release the target substances; while cellulose, lignin, and secondary metabolites (such as phenolic acids and alkaloids) in root and stem tissues may mask trace pesticides through adsorption or coprecipitation, reducing the detection sensitivity. In addition, endogenous substances in plant tissues (such as chlorophyll and carotenoids) are prone to matrix effects in chromatographic analysis, resulting in target peak deviation or signal suppression. Especially in multi - residue detection, the recovery rate differences of different pesticides in different matrices can reach more than 30%, and complex purification steps (such as combined with dispersive solid - phase extraction) are required to eliminate interference, but the optimization method needs to be verified separately for each tissue type, significantly increasing the detection cost and cycle.

[0003] Pesticide residues in agricultural products are related to national health. There are many deficiencies in the existing agricultural product analysis method system: on the one hand, the existing analysis method system has strong specificity, and the analysis and detection method processes for different types of pesticides in different agricultural products are different; on the other hand, in the pesticide residue pretreatment process represented by activated carbon treatment, there are disadvantages such as high solvent consumption, cumbersome purification steps, and long processing time to varying degrees. The lack of a highly universal agricultural product pesticide residue analysis method system limits the efficiency of agricultural product pesticide residue analysis and increases the difficulty of ensuring the safety of agricultural products. Developing a rapid and efficient agricultural product pesticide residue analysis and detection method system can provide technical support for relevant detection agencies and ensure the flow of safe agricultural products to the table. Summary of the Invention

[0004] The purpose of the present invention is to overcome the disadvantages and deficiencies of the prior art and provide a method for co - analysis and detection of multiple types of pesticides in different plant tissues.

[0005] Another purpose of the present invention is to provide the application of the above - mentioned method.

[0006] The purpose of the present invention is achieved by the following technical solutions:

[0007] A method for co - analysis and detection of multiple types of pesticides in different plant tissues, comprising the following steps:

[0008] (1) Cold-dry and pulverize the sample, add the mixed solvent, vortex and oscillate, perform ultrasonic extraction, filter to obtain the filtrate, add the solvent to the filter residue, vortex and oscillate, perform ultrasonic extraction, and filter 2 to 4 times. After combining the filtrates, obtain the extract.

[0009] (2) After concentrating the extract, add carbon black, vortex, centrifuge, and take the supernatant. Then add the mixed solvent to the lower-layer carbon black, repeat vortexing and centrifuging to obtain the second supernatant. Combine the two supernatants, concentrate, displace, and make up the volume to obtain the sample for analysis.

[0010] (3) Use a liquid chromatography-mass spectrometry (LC-MS) instrument to detect the sample for analysis.

[0011] The sample described in step (1) is plant tissue; preferably leaf or root tissue.

[0012] The mixed solvent is a mixed solvent of acetonitrile:dichloromethane = 1:1; preferably a mixed solvent of acetonitrile:dichloromethane = 1:1 containing 1 vol% ammonia water.

[0013] The addition amount of the mixed solvent described in step (1) to the sample is in the ratio of 10 - 50 mL:1 g; preferably 20 mL:1 g.

[0014] The conditions for vortex oscillation described in step (1) are vortex oscillation at 2000 - 3000 rpm for 1 - 5 min.

[0015] The conditions for ultrasonic extraction described in step (1) are ultrasonic extraction at 80 - 120 Hz for 10 - 30 min.

[0016] The concentration in step (2) is concentrated to 10 - 20% of the original volume.

[0017] The addition amount of carbon black described in step (2) to the mass of the sample is in the ratio of 1:5 - 20.

[0018] The conditions for vortex in step (2) are vortex oscillation at 2000 - 3000 rpm for 1 - 5 min.

[0019] The conditions for centrifugation in step (2) are centrifugation at 3000 - 5000 rpm for 5 - 20 min.

[0020] The addition amount of the mixed solvent described in step (2) to the sample is in the ratio of 5 - 25 mL:1 g; preferably 20 mL:1 g.

[0021] The displacement in step (2) is performed using methanol.

[0022] The detection parameters of the liquid chromatography-mass spectrometry instrument described in step (3) are as follows:

[0023] The carrier gas is high-purity argon (99.999%); the injection volume is 2 μL, and pulsed splitless injection is used; the chromatographic column is Acclaim TM RSLC 120 C18 2.2 μm (2.1×100 mm); the mobile phases are ultrapure water containing 0.2% formic acid (A) and methanol containing 2 mM ammonium formate (B); the ion source is an ESI source, and the sheath gas and auxiliary gas flow rates are 40 and 8 Arb respectively, spray voltage: 3500 V; the ion transfer tube temperature is 300 °C, and the heater temperature is 350 °C.

[0024] The target substances detected by using the liquid chromatography-mass spectrometry in step (3) include at least one of Dinoterufan, Thiachloprid-amide, Thiamethoxam, Imidacloprid, Clothianidin, Imidaclothiz, N-desmethyl-thiamethoxam, Acetamiprid, Acetamiprid-N-desmethyl, Sulfoxaflor, Imidachloprid-olefin, Thiacloprid, Dimethoate, CIAT, Prometon, Bromacil, Carbaryl, Ametryn, Atrazine, Diuron, Trifluralin, TCPy, Azoxystrobin, Boscalid, Pronamide, Chlorothalonil, Malathion, Metolachlor, Desulfinylfipronil, Fipronil, Glyphosate, Diazinon, Profenofos, Chlorpyrifos, Pendimethalin, Fenpropathrin, Permethrin, Bifenthrin.

[0025] Application of the above method for co-analysis and detection of multiple pesticides in different plant tissues in detecting pesticide residues in plant samples.

[0026] The present invention has the following advantages and effects compared with the prior art:

[0027] The present invention provides a pretreatment method for extracting pesticides from plant tissues. First, the rice powder is mixed with an extraction solvent, followed by ultrasonic extraction and purification in sequence. Then, the components can be determined using a liquid chromatography-mass spectrometry instrument, which can shorten the pretreatment time and improve the detection speed. This method adopts an optimized pretreatment technique, which can efficiently extract pesticide residues from plant samples and ensure the high accuracy and reliability of the detection results. Description of the Drawings

[0028] Figure 1 It is the average percentage graph of the recovery rate of the target pesticide in the rice leaves after being treated in Example 1 with added standard.

[0029] Figure 2 It is the average percentage graph of the recovery rate of the target pesticide in the rice root tissues after being treated in Example 2 with added standard.

[0030] Figure 3 It is the average percentage graph of the recovery rate of the target pesticide in the rice samples after being treated in Comparative Example 1 with added standard.

[0031] Figure 4 It is the average percentage graph of the recovery rate of the target pesticide in the rice samples after being treated in Comparative Example 2 with added standard.

[0032] Figure 5 It is the average percentage graph of the recovery rate of the target pesticide in the rice samples after being treated in Comparative Example 3 with added standard.

[0033] Figure 6 It is the average percentage graph of the recovery rate of the target pesticide in the rice samples after being treated in Comparative Example 4 with added standard.

[0034] Figure 7 It is the average percentage graph of the recovery rate of the target pesticide in the rice samples after being treated in Comparative Example 5 with added standard.

[0035] Figure 8 It is the average percentage graph of the recovery rate of the target pesticide in the rice samples after being treated in Comparative Example 6 with added standard.

[0036] Figure 9 It is the average percentage graph of the recovery rate of the target pesticide in the rice samples after being treated in Comparative Example 7 with added standard.

[0037] Figure 10 It is the average percentage graph of the recovery rate of the target pesticide in the rice samples after being treated in Comparative Example 8 with added standard.

[0038] Figure 11 It is the average percentage graph of the recovery rate of the target pesticide in the rice samples after being treated in Comparative Example 9 with added standard.

[0039] Figure 12 It is the average percentage graph of the recovery rate of the target pesticide in the rice samples after being treated in Comparative Example 10 with added standard.

[0040] Figure 13 Total ion current chromatograms of the pesticides in the examples and standard samples. Specific embodiments

[0041] The present invention will be further described in detail below in conjunction with the examples and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0042] If the specific test conditions are not indicated in the following embodiments, they are usually in accordance with the conventional test conditions or the test conditions recommended by the reagent company. The materials, reagents, etc. used, unless otherwise specified, are all reagents and materials obtained through commercial channels.

[0043] Example 1

[0044] (1) The rice leaf tissues were separated from the plants, freeze-dried, crushed by a crusher, and packed into aluminum foil paper. 0.5 g of the freeze-dried rice sample was weighed into a 50 mL centrifuge tube, and 50 ng each of the pesticide standard and the recovery indicator dissolved in methanol were added and allowed to stand for 24 h (the specific standards and recovery indicators added are shown in Table 1). Then, 10 mL of acetonitrile:dichloromethane = 1:1 (v:v, containing 1 vol% ammonia water) was added, vortexed at 2500 rpm for 1 min, sonicated at 100 Hz for 20 min, and then filtered to obtain the filtrate. The filtrate was transferred to a clean centrifuge tube, and then the mixed solvent, vortexing, and ultrasonic extraction processes were repeated in the remaining sample 3 times, and the extraction liquids were combined.

[0045] (2) The extraction liquid was concentrated to 5 mL with a gentle nitrogen stream, 0.05 g of graphitized carbon black was added, vortexed at 2500 rpm for 3 min with a vortex mixer, centrifuged at 4000 rpm for 10 min with a centrifuge, and the supernatant was transferred to a clean concentration tube; then, 5 mL of acetonitrile:dichloromethane = 1:1 (v:v, containing 1 vol% ammonia water) was added to the extraction liquid, and the above vortexing and centrifuging steps were repeated once, and then the supernatant was taken. The two supernatants were mixed, concentrated to less than 0.5 mL with a gentle nitrogen stream, and replaced with methanol 2 times, and fixed to 0.5 mL, and d 3 -Dinotefuran, d 4 -Imidacloprid, d 10 -Chlorpyrifos, d 14 -Trifluralin was used as the internal standard and detected by machine.

[0046] (3) A liquid chromatography-mass spectrometry instrument was used to detect the plant samples

[0047] Analysis was carried out using a Thermo TSQ QUANTIS series liquid chromatograph (LC) and a Thermo TSQ QUANTIS mass spectrometer (MS). The specific parameters are as follows:

[0048] The carrier gas is high-purity argon (99.999%); the injection volume is 2 μL, and pulsed splitless injection is used; the chromatographic column selected is Acclaim TM RSLC 120 C18 2.2 μm (2.1×100 mm). The mobile phases are ultrapure water containing 0.2% formic acid (A) and methanol containing 2 mM ammonium formate (B). The ion source is an ESI source, and the sheath gas and auxiliary gas flow rates are 40 and 8 Arb respectively, and the spray voltage is 3500 V. The temperature of the ion transfer tube is 300 °C, and the heater temperature is 350 °C.

[0049] The experimental results are as Figure 1 shown. The recovery rates of all pesticides measured by this method in the leaves range from 50.82±0.88% to 128.07±4.31%, meeting the extraction requirements.

[0050] Table 1 CAS numbers, quantitative and qualitative ions of target pesticides

[0051]

[0052] Example 2

[0053] (1) The rice root tissue was separated from the plant, freeze-dried and then crushed with a crusher and packed into aluminum foil. Accurately weigh 0.3 g of the rice sample into a 50 mL centrifuge tube, add 50 ng each of the pesticide standard and the recovery indicator dissolved in methanol, let stand for 24 h, then add 10 mL of acetonitrile:dichloromethane = 1:1 (v:v, containing 1% ammonia water), vortex at 2500 rpm for 1 min, ultrasonic at 100 Hz for 20 min, and then transfer the extract to a clean centrifuge tube. This process was repeated 3 times, and the extracts were combined.

[0054] (2) The extract was concentrated to 5 mL with a gentle nitrogen stream, then 0.03 g of graphitized carbon black was added, vortexed at 2500 rpm for 3 min, centrifuged at 4000 rpm for 10 min, and the purified liquid was transferred to a clean concentration tube. Add 5 mL of acetonitrile:dichloromethane = 1:1 (v:v, containing 1 vol% ammonia water) and repeat once. The purified extract was concentrated to 0.5 mL with a gentle nitrogen stream and replaced with methanol twice, made up to 0.5 mL, and added with d 3 -Dinotefuran, d 4 -Imidacloprid, d 10 -Chlorpyrifos, d 14 -Trifluralin as the internal standard and analyzed by machine.

[0055] (3) Detect plant samples using the liquid chromatography - mass spectrometry parameters in step (3) of Example 1.

[0056] The experimental results are as Figure 2 shown. The recovery rate range of all pesticides in the roots measured by this method is 59.90 ± 7.47% - 112.04 ± 8.93%.

[0057] Comparative Example 1

[0058] The experimental steps refer to Example 1, with the difference that the extraction solvent used in step (1) is replaced by acetonitrile and QuEChERS extraction reagent kit 60105 - 335 - B (4 g anhydrous magnesium sulfate, 1 g sodium chloride, 1 g sodium citrate, 0.5 g disodium hydrogen citrate), and the graphitized carbon black powder used in step (2) is replaced by QuEChERS purification reagent kit 60105 - 203 - B (150 mg anhydrous magnesium sulfate, 50 mg PSA). The recovery rate range of all pesticides in the leaves measured by this method is 17.24 ± 3.16% - 116.09 ± 15.17%.

[0059] Comparative Example 2

[0060] The experimental steps refer to Example 1, with the difference that the extraction solvent used in step (1) is replaced by acetonitrile and QuEChERS extraction reagent kit 60105 - 333 - B (6 g anhydrous magnesium sulfate, 1.5 g sodium peroxyacetate), and the graphitized carbon black powder used in step (2) is replaced by QuEChERS purification reagent kit 60105 - 203 - B. The recovery rate range of all pesticides in the leaves measured by this method is 11.34 ± 0.13% - 257.44 ± 4.44%.

[0061] Comparative Example 3

[0062] The experimental steps refer to Example 1, with the difference that the extraction solvent used in step (1) is replaced by dichloromethane. The recovery rate range of all pesticides in the leaves measured by this method is 0.00 ± 0.00% - 250.00 ± 38.62%.

[0063] Comparative Example 4

[0064] The experimental steps refer to Example 1, with the difference that the extraction solvent used in step (1) is replaced by acetonitrile. The recovery rate range of all pesticides in the leaves measured by this method is 0.00 ± 0.00% - 141.49 ± 14.52%.

[0065] Comparative Example 5

[0066] The experimental procedure was referred to Example 1, with the difference that the extraction solvent used in step (1) was replaced by acetonitrile containing 1% acetic acid. The recovery rate range of all pesticides in the leaves measured by this method was 0.00±0.00% to 328.70±46.96%.

[0067] Comparative Example 6

[0068] The experimental procedure was referred to Example 1, with the difference that the extraction solvent used in step (1) was replaced by acetonitrile:dichloromethane = 1:1 (v:v). The recovery rate range of all pesticides in the leaves measured by this method was 20.40±0.80% to 395.56±3.33%.

[0069] Comparative Example 7

[0070] The experimental procedure was referred to Example 1, with the difference that the extraction solvent used in step (1) was replaced by ammonia acetonitrile with pH = 9. The recovery rate range of all pesticides in the leaves measured by this method was 31.37±8.71% to 199.92±13.07%.

[0071] Comparative Example 8

[0072] The experimental procedure was referred to Example 1, with the difference that the extraction solvent used in step (1) was replaced by acetonitrile:dichloromethane = 1:1 (v:v, containing 3 vol% ammonia). The recovery rate range of all pesticides in the leaves measured by this method was 34.47±2.81% to 226.87±9.43%.

[0073] Comparative Example 9

[0074] The experimental procedure was referred to Example 1, with the difference that the pesticide standards added in step (1) were 10 ng and 200 ng, and the extraction solvent used was still acetonitrile:dichloromethane = 1:1 (v:v, containing 1% ammonia). The recovery rate range of pesticides in all leaves measured by this method was 56.67±1.56% to 140.52±12.78%, 51.79±0.46% to 132.72±9.76%.

[0075] Comparative Example 10

[0076] The experimental procedure was referred to Example 2, with the difference that the pesticide standards added in step (1) were 10 ng and 200 ng, and the extraction solvent used was still acetonitrile:dichloromethane = 1:1 (v:v, containing 1% ammonia). The recovery rate range of pesticides in all roots measured by this method was 50.82±0.88% to 128.07±4.31%, 54.42±2.35% to 124.46±12.42%.

[0077] By comparing the recovery rates of Example 1 with Comparative Examples 1-8, it can be found that the recovery rate of Example 1 is 50.82±0.88% to 128.07±4.31%, which is higher than that of the comparative examples and the range is more concentrated, meeting the experimental requirements. By comparing the recovery rates of Experimental Example 1 and Comparative Example 9, it can be found that the recovery rate of extracting pesticides with different concentrations in leaves by this method meets the experimental requirements. By comparing the recovery rates of Example 2 and Comparative Example 10, it can be found that the recovery rate of extracting pesticides with different concentrations in roots by this method meets the experimental requirements.

[0078] The present invention simplifies the pretreatment steps before detection, has a simple treatment method and uses a small volume of organic solvent. In the actual application process, it can realize the detection of multiple types of pesticides in different plant parts.

[0079] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A collaborative analysis and detection method for multiple types of pesticides in different plant tissues, characterized in that The steps include: (1) The sample is freeze-dried and crushed, a mixed solvent is added, vortexed, ultrasonically extracted, and filtered to obtain a filtrate. The remaining sample is subjected to the steps of adding solvent, vortexing, ultrasonically extracting, and filtering 2 to 4 times, and the filtrates are combined to obtain an extract; (2) After the extract is concentrated, carbon black is added, vortexed, centrifuged, and the supernatant is taken. The mixed solvent is then added to the lower layer of carbon black, and the vortexing and centrifugation are repeated to obtain a second supernatant. The two supernatants are combined, concentrated, replaced, and fixed to obtain a sample for the machine; (3) Use liquid chromatography-mass spectrometry to detect the samples on the machine.

2. The method according to claim 1, characterized in that: The mixed solvent is a mixed solvent of acetonitrile containing 1 vol% ammonia water and dichloromethane in a ratio of 1:

1.

3. The method according to claim 1, characterized in that: The ratio of the amount of the mixed solvent added in step (1) to the sample is 10-50 mL: 1 g; The sample in step (1) is plant tissue.

4. The method according to claim 1, characterized in that: The vortex oscillation condition of step (1) is 2000-3000 rpm vortex oscillation for 1-5 min; The ultrasonic extraction conditions in step (1) are 80-120 Hz ultrasonic extraction for 10-30 min.

5. The method according to claim 1, characterized in that: The concentration in step (2) is concentrated to 10-20% of the original volume; The mass ratio of the amount of carbon black added in step (2) to the sample is 1:5 to 20.

6. The method according to claim 1, characterized in that: The vortexing condition in step (2) is 2000-3000 rpm vortexing oscillation for 1-5 min. The centrifugation condition in step (2) is 3000-5000 rpm for 5-20 min.

7. The method according to claim 1, characterized in that: The ratio of the amount of the mixed solvent added in step (2) to the sample is 5-25 mL: 1 g; The replacement in step (2) is carried out using methanol.

8. The method according to claim 1, characterized in that: The detection parameters of the liquid chromatography-mass spectrometry instrument described in step (3) are as follows: The carrier gas was high-purity argon; the injection volume was 2 μL, pulsed splitless injection; the chromatographic column was Acclaim TM RSLC120C18 2.2μm The mobile phases were ultrapure water containing 0.2% formic acid and methanol containing 2 mM ammonium formate; the ion source was ESI source, the sheath gas and auxiliary gas flow rates were 40 and 8 Arb respectively, the spray voltage was 3500 V; the ion transfer tube temperature was 300°C, and the heater temperature was 350°C.

9. The method according to claim 1, characterized in that: The target substance detected by liquid chromatography-mass spectrometry in step (3) includes at least one of Dinoterufan, Thiachloprid-amide, Thiamethoxam, Imidacloprid, Clothianidin, Imidaclothiz, N-desmethyl-thiamethoxam, Acetamiprid, Acetamiprid-N-desmethyl, Sulfoxaflor, Imidachloprid-olefin, Thiacloprid, Dimethoate, CIAT, Prometon, Bromacil, Carbaryl, Ametryn, Atrazine, Diuron, Trifluralin, TCPy, Azoxystrobin, Boscalid, Pronamide, Chlorothalonil, Malathion, Metolachlor, Desulfinylfipronil, Fipronil, Glyphosate, Diazinon, Profenofos, Chlorpyrifos, Pendimethalin, Fenpropathrin, Permethrin, and Bifenthrin.

10. Use of the collaborative analysis and detection method for multiple types of pesticides in different plant tissues as claimed in any one of claims 1 to 9 in detecting pesticide residues in plant samples.

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