Pesticide residue detection method for fishes

Abstract

The invention provides a pesticide residue detection method for fishes, and belongs to the technical field of pesticide residue detection. The detection method comprises the following steps: adding MgSO4 solid powder into a fish meat sample, extracting a target compound by using an acetonitrile solution containing 2.5-3.5 v / v% of formic acid, and detecting by adopting an ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry to obtain a pesticide residue determination result, wherein the chromatographic conditions are as follows: an acetic acid aqueous solution with the concentration of 0.008-0.012 v / v% is taken as a mobile phase A, and acetonitrile is taken as a mobile phase B. According to the invention, the ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry is adopted, the detection conditions are optimized, and the residues of 31 compounds can be simultaneously detected from a fish sample. The detection method is good in accuracy and high in sensitivity, the detection limit ranges from 0.01 ng / mL to 0.05 ng / mL, and the requirement for detecting the pesticide residues in the fish can be met.

Description

Technical Field

[0001] The present invention relates to the technical field of pesticide residue detection, and in particular to a pesticide residue detection method for fish. Background Art

[0002] With the development of modern agriculture, various pesticides are widely used for pest control, with neonicotinoids, organophosphorus insecticides, pyrethroids, and phenoxy herbicides being the most common. These compounds are highly effective and fast-acting, but due to their stability and water solubility in the environment, some pesticides and their metabolites can enter aquatic environments through agricultural runoff and rainfall, causing water pollution. Studies have shown that pesticides are widely detected in aquatic environments, particularly in rivers and lakes near aquaculture areas and intensive agricultural regions. Residues of neonicotinoids, organophosphorus insecticides, pyrethroids, and phenoxy pesticides are prevalent. These pesticides can be absorbed and accumulated in aquatic organisms, particularly fish, which, due to their crucial role in the food chain, are a potential reservoir for pesticides and their metabolites.

[0003] Once pesticides and their metabolites enter fish, they can be transferred to humans through the food chain, posing a potential risk of chronic exposure. Long-term consumption of aquatic products containing pesticide residues can adversely affect the nervous, endocrine, and immune systems, and may even lead to chronic poisoning or cancer. Given that fish is a significant source of protein in the human diet, its food safety is receiving increasing attention.

[0004] Currently, the detection of pesticide residues and their metabolites in fish samples still faces significant technical challenges. Existing methods have limitations in extraction efficiency, method versatility, and the ability to remove sample matrix interference. For example: 1. Low extraction efficiency: Existing methods do not fully extract pesticides and their metabolites with large polarity differences, affecting the accuracy of quantitative analysis; 2. Complex and time-consuming operation: The multi-step pre-treatment process reduces detection efficiency and is not conducive to large-scale sample processing; 3. Poor adaptability: Some methods are only applicable to a certain type or a few types of pesticide substances and lack the ability to deal with complex types of pollution (such as the coexistence of insecticides and herbicides). Summary of the Invention

[0005] In view of this, the present invention provides a method for detecting pesticide residues in fish to solve the above problems.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for detecting pesticide residues in fish, comprising the following steps: (1) Drying the fish sample and grinding it to obtain fish powder; (2) Mix the fish powder with water until it becomes a meat paste, add MgSO4 solid powder and acetonitrile solution containing 2.5~3.5v / v% formic acid, perform vortex extraction, then sonicate and centrifuge, and collect the upper solution to obtain supernatant 1; (3) Repeat step (2), collect the upper solution to obtain supernatant 2, and combine supernatants 1 and 2 to obtain the extraction product; (4) Remove fat from the extracted product, filter it through a filter membrane, and detect it using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry to obtain the pesticide residue determination results; The chromatographic conditions were as follows: 0.008-0.012 v / v% acetic acid aqueous solution as mobile phase A, and acetonitrile as mobile phase B; The elution gradient program of the chromatography is: Time (min) Mobile phase A (v / v%) Mobile phase B (v / v%) 1 95 5 3.2 60 40 4 50 50 4.5 50 50 5 5 95 6.6 5 95 7.2 95 5 8 95 5 Preferably, the mass ratio of the fish sample to the MgSO4 solid powder is 1:0.9~1.1.

[0007] Preferably, the rotation speed of the vortex extraction is 2300-2700 rpm, and the vortex extraction time is 2.5-3.5 min.

[0008] Preferably, the chromatographic column is a Waters BEH C18 column, with a length of 100 mm, an inner diameter of 2.1 mm, and a filler particle diameter of 1.7 μm.

[0009] Preferably, the chromatographic conditions further include: column temperature set at 38-42°C, injection volume of 2-4 μL, and flow rate of 0.28-0.32 mL / min.

[0010] Preferably, the pesticide residues include dimethyl phosphate, dimethyl phosphate thioester, diethyl phosphate, diethyl dithiophosphate, diethyl tetrathiophosphate, p-nitrophenol, 3,5,6-trichloro-2-pyridinol, dimethyl dithiophosphate, 2-isopropyl-6-methyl-4-pyridinol, 3-phenoxybenzoic acid, 4-fluoro-3-phenoxybenzoic acid, trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid, cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, 6-chloronicotinic acid, sulfoxaflor, allyl imidacloprid, nitrosoimidacloprid, imidacloprid, thiacloprid, clothianidin, dinotefuran, flonicamid, demethylthiacloprid, thiacloprid amide, chlorothiazolin, thiamethoxam, acetamiprid, N-demethylacetamiprid and 5-hydroxyimidacloprid.

[0011] Preferably, a triple quadrupole tandem mass spectrometer is used for detection, an electrospray ionization source is used, the scanning modes are positive ion mode and negative ion mode, and the detection mode is multiple ion reaction detection mode; the ion source parameters are: ion source temperature is 530~570 ° C, ionization voltage is 4300~4700 V, spray gas is 53~57 psi, auxiliary heating gas is 53~57 psi, and curtain gas is 33~37 psi.

[0012] Preferably, the ultrasound frequency is 38-42 kHz, the power is 90-110 W, and the ultrasound time is 13-17 min.

[0013] Preferably, the mass volume ratio of the fish powder to the acetonitrile solution containing 2.5-3.5 v / v% formic acid is 0.1 g:1.8-2.2 mL.

[0014] Preferably, the pore size of the filter membrane is 0.20-0.25 μm.

[0015] By adopting the above technical solution, the present invention has the following beneficial effects: the detection method of the present invention comprises the following steps: adding MgSO4 solid powder to a fish sample, extracting the target compound with an acetonitrile solution containing 2.5-3.5 v / v% formic acid, and detecting the target compound using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry to obtain the pesticide residue determination result; wherein the chromatographic conditions are: using an aqueous acetic acid solution with a concentration of 0.008-0.012 v / v% as mobile phase A, and using acetonitrile as mobile phase B. The present invention uses ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry to optimize the detection conditions and can simultaneously detect residues of 31 compounds in fish samples. The detection method of the present invention also has good accuracy and high sensitivity, with a detection limit of 0.01-0.05 ng / mL, which can meet the requirements for detecting pesticide residues in fish. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the chromatogram of 10 ng / mL standard solutions of 31 pesticides and their metabolites in Example 4.

[0017] Figure 2 The chromatograms of 31 pesticides and their metabolites in Comparative Example 6 are shown.

[0018] Figure 3 The chromatogram of 31 pesticides and their metabolites in Comparative Example 7 is shown.

[0019] Figure 4 The chromatograms of 31 pesticides and their metabolites in Comparative Example 8 are shown.

[0020] Figure 5 The chromatograms of 31 pesticides and their metabolites in Comparative Example 9 are shown.

[0021] Figure 6 The chromatogram of 31 pesticides and their metabolites in Comparative Example 10. DETAILED DESCRIPTION

[0022] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] The models and brands of the instruments and equipment used in the examples of the present invention are as follows: Ultra-high performance liquid chromatography (UPLC), model: Shimadzu Nexera LC-40D ultra-high performance liquid chromatography system, manufacturer: Shimadzu Instruments (Suzhou) Co., Ltd. Triple quadrupole mass spectrometer, model: AB Sciex QTRAP 5500+, manufacturer: Shanghai SCIEX Aibo Caisi Analytical Instrument Trading Co., Ltd.; Chromatographic column, model: Waters BEH C18 column, 100 × 2.1 mm, 1.7 µm, manufacturer: Waters-Waters China official website; Pipette, model 10 / 20 / 100 / 200 / 1000 / 2500 μL, brand: Eppendorf; Ultrasonic cleaning machine, model: KQ-700TDE, brand: Kunshan Ultrasonics Corporation; Electronic analytical balance, model: XSR204, brand: Mettler Toledo; Pure water system, model: Milli-Q Integral system, brand: Millipore; Centrifuge, model: Centrifuge 5804, brand: Eppendorf; Vacuum freeze dryer, model: FD-1A-50, manufacturer: Boyikang (Beijing) Instrument Co., Ltd. Solid phase extraction device, brand: Merck; The nitrogen blowdown apparatus was purchased from the official website of Organomation Associates Inc.

[0024] Methanol (LC-MS), acetonitrile (LC-MS), and formic acid (LC-MS) used in the examples of the present invention were purchased from Thermo Fisher Scientific Inc.

[0025] Example 1. Method for detecting 31 pesticides and their metabolites in samples 1. Target compound and internal standard

[0026] The present invention provides a method for detecting multiple pesticide residues in fish samples, using 31 compounds shown in Table 1 as target compounds, specifically including: dimethyl phosphate, dimethyl phosphate thioester, diethyl phosphate, diethyl dithiophosphate, diethyl tetrathiophosphate, p-nitrophenol, 3,5,6-trichloro-2-pyridinol, dimethyl dithiophosphate, 2-isopropyl-6-methyl-4-pyridinol, 3-phenoxybenzoic acid, 4-fluoro-3-phenoxybenzoic acid, trans-3-(2,2-dichlorovinyl)-2,2- Dimethylcyclopropanecarboxylic acid, cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, 6-chloronicotinic acid, sulfoxaflor, allyl imidacloprid, nitrosoimidacloprid, imidacloprid, thiacloprid, clothianidin, dinotefuran, flonicamid, demethylthiacloprid, thiacloprid amide, chlorothiazolin, thiamethoxam, acetamiprid, N-demethylacetamiprid and 5-hydroxyimidacloprid; 27 isotopically labeled internal standards, including: D6-DMP, D 10 -DETP, D4-PNP, 13 C3-TCPY,D 10 -DMDTP, 13 C4-IMPY, 13 C6-3-PBA, D3- 13 C2-trans-DCCA, D3- 13 C2-cis-DCCA, D3-2,4-D, D2- 13 C- 15 N-2, 4, 5-T, 13 C6-6-CN, D3- 13 C2- 15 N2-SUF, 13 C- 15 N2-Of-IMI, 13 C3- 15 N2-NIT, D4-IMI, 13 C6-THI, 13 C4- 15 N-CLO, 13 C5-DIN, 18 O- 15 N-FLO, D4-N-DMT, 13 C6-TA, D4-IMZ, 13 C4- 15 N-THX, 13 C6-ACE, 13 C2- 15 NN-dm-ACE, 13 C- 15 N2-5-OH-IMI.

[0027] Table 1 Information of 31 compounds and internal standards

[0028] 2. Solution Preparation (1) Mixed internal standard solution The 27 isotope-labeled internal standards listed in Table 1 were dissolved in methanol at a final concentration of 500 ng / mL, mixed evenly, and diluted to obtain a mixed internal standard solution with a concentration of 10 ng / mL.

[0029] (2) Extraction solvent Formic acid was added to acetonitrile at a volume ratio of 3:100 and mixed to obtain an extraction solvent.

[0030] (3) Mixed standard solution The standards of the 31 target substances listed in Table 1 were dissolved in methanol to prepare mixed standard stock solutions, where the final concentration of each target substance standard was 10 µg / mL.

[0031] The mixed standard stock solution was then serially diluted with methanol to obtain mixed standard solutions with final concentrations of each compound standard of 0.01 ng / mL, 0.02 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL and 50 ng / mL.

[0032] 3. Sample Pretreatment (1) Sample preparation The fish meat was dissected and separated from the fish body to obtain the fish meat. The fish meat was cut into sections and dried in a vacuum freeze dryer for 48 hours to obtain freeze-dried fish meat samples. The freeze-dried fish meat samples were ground in a mortar and passed through a 100-mesh sieve to obtain fish meat powder.

[0033] (2) Liquid-liquid extraction Weigh 0.1 g of fish powder, moisten with 100 µL of ultrapure water, and vortex for 1 minute until a paste forms. Add 0.1 g of MgSO₄ solid powder and 2 mL of acetonitrile solution containing 3% formic acid at 2500 rpm / min, and vortex for 3 minutes. Ultrasonicate (frequency 40 kHz, power 100 W) for 15 minutes, adding ice to prevent overheating. Centrifuge for 10 minutes (4500 rpm) to separate the layers. Collect the upper layer to obtain Supernatant 1. Repeat the above extraction steps once, collecting the upper layer to obtain Supernatant 2. Combine Supernatants 1 and 2 to obtain 5 mL of mixed supernatant. Place the mixed supernatant under a gentle nitrogen atmosphere at 25°C and blow until nearly dry to obtain the extracted product.

[0034] (3) Freeze-drying after reconstitution Dissolve the extract in 500 µL of reconstitution solution (pure acetonitrile), vortex for 1 minute to mix, and freeze at -20°C for 12 hours to precipitate the fat. After precipitation, aspirate the supernatant and filter through a 0.22 µm PTFE filter into a brown injection vial to obtain the sample for subsequent analysis.

[0035] 4. Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry The detection conditions were as follows: the chromatographic column was a Waters BEH C18 column (length 100 × inner diameter 2.1 mm, filler particle diameter 1.7 µm), the column temperature was set at 40°C, and the injection volume was 3 µL; the mobile phase A was 0.01 v / v% acetic acid in water, and the mobile phase B was acetonitrile. The flow rate was 0.3 mL / min, and the gradient elution program was shown in Table 2.

[0036] Table 2 Gradient elution program

[0037] 5. Mass Spectrometry Analysis A triple quadrupole tandem mass spectrometer was used for detection. The mass spectrometry conditions were as follows: an electrospray ionization source (ESI) was used, the scan modes were positive ion mode and negative ion mode, and the detection mode was multiple ion reaction detection mode. The ion source parameters were: ion source temperature of 550 °C, ionization voltage of 4500 V, spray gas of 55 psi, auxiliary heating gas of 55 psi, curtain gas of 35 psi, and collision gas flow rate of medium intensity (9). The mass spectrometry parameters of each target compound are shown in Table 3.

[0038] Table 3 Mass spectrometric parameters of 31 pesticides, their metabolites, and internal standards

[0039] 4. Results Analysis (1) Establishment of standard curve The internal standard method was used for the quantitative analysis of 31 pesticides and their metabolites. The specific steps were as follows: 12 mixed standard solutions of different concentrations were taken for ultra-performance liquid chromatography detection and mass spectrometry analysis. The ratio of the chromatographic peak area of ​​each target substance at different concentrations to the chromatographic peak area of ​​the corresponding internal standard substance was used as the standard area ratio. The standard curves of each target substance were drawn with concentration as the horizontal axis and area ratio as the vertical axis, as shown in Table 4.

[0040] Table 4 Standard curves of 31 pesticides and their metabolites

[0041] The results showed that within the linear range of 0.01 ng / mL~50 ng / mL, the linear relationships of the 31 compounds were all greater than 0.99, indicating a good linear relationship.

[0042] (2) Analysis of the content of each target substance in the sample The ratio of the peak area of ​​each target compound measured in the sample to the chromatographic peak area of ​​the corresponding internal standard was used as the sample area ratio and substituted into the standard curve to calculate the content of each compound in the sample.

[0043] Example 2. Evaluation of the detection method in Example 1 using fish samples 1. Preparation of spiked samples This example used fish meat samples for methodological evaluation. Fish meat was dissected and separated from the fish body to obtain fish meat. The fish meat was cut into sections and dried in a vacuum freeze dryer for 48 hours to obtain freeze-dried fish meat samples. The freeze-dried fish meat samples were then ground in a mortar and passed through a 100-mesh sieve to obtain fish meat powder.

[0044] 0.1 g of fish sample was added with the mixed standard solution of the analyte and the mixed internal standard solution to prepare spiked samples with a final concentration of 1.0 ng / mL (low concentration) and 10.0 ng / mL (high concentration) for each standard, with 3 samples for each concentration.

[0045] 2. Detection The spiked sample prepared in this example was used as the liquid sample to be tested. The content of each pesticide and its metabolite therein was tested according to the method of Example 1 of the present invention. The test results are shown in Table 5.

[0046] Table 5 Spiked recoveries and relative deviations of 31 pesticides and their metabolites As shown in Table 5, the spiked recoveries for the 31 pesticides and their metabolites ranged from 70% to 113%. This indicates that the spiked recoveries and relative deviations for the 31 pesticides and their metabolites detected using the method of the present invention were within the acceptable range (70% to 120%). The detection method of Example 1 of the present invention can achieve simultaneous extraction and determination of 31 target compounds, is simple to operate, low-cost, and has good detection accuracy (spike recovery of 70% to 113%), high precision (RSD ≤ 4.5), and strong specificity, meeting recognized standard requirements.

[0047] Example 3 Detection of 31 pesticides and their metabolites in actual fish samples Fifty fish samples collected from a South China Sea expedition were freeze-dried for 48 hours, ground, and sieved. The samples were then tested for 31 pesticides and their metabolites using the method described in Example 1. The results are shown in Table 6. Due to instrument limitations and the fact that fish samples may not contain all 31 pesticides and their metabolites, a total of 28 pesticides and their metabolites were detected in the 50 fish samples, with thiamethoxam (THX) having the highest detection rate. The method described in Example 1 offers high precision, excellent stability, and a low detection limit (0.01–0.05 ng / mL), meeting the practical requirements for testing fish samples.

[0048] Table 6 Detection of 31 pesticides and their metabolites in 50 fish samples Note: The data of “detection rate” in the table are rounded off.

[0049] Example 4 A mixed standard working solution of 31 target compound standards with a final concentration of 10 ng / mL was used as the fish sample to be tested. 0.1 g of fish powder was weighed, moistened with 100 μL of water, and vortexed for 1 minute until the mixture became a paste. To 0.1 g of MgSO₄ solid powder, add 2 mL of acetonitrile solution containing 3% v / v formic acid. Vortex at 2500 rpm for 3 minutes, then sonicate (40 kHz, 100 W) for 15 minutes, adding ice to prevent overheating. Centrifuge at 4500 rpm for 10 minutes, separate the layers, and collect the upper layer to obtain Supernatant 1. Repeat the above extraction steps once, collecting the upper layer to obtain Supernatant 2. Combine Supernatants 1 and 2 to obtain 5 mL of a mixed supernatant. Place the mixed supernatant under a gentle nitrogen purge at 25°C and concentrate to near dryness to obtain the extract. This extract is then tested according to the method in Example 1.

[0050] Comparative Example 1 Different from Example 4, when extracting the target compound, the extraction was performed with a solvent having a volume ratio of acetonitrile and dichloromethane of 1:1, and MgSO4 solid powder was added.

[0051] Comparative Example 2 Different from Example 4, when extracting the target compound, the extraction was performed with a solvent of acetonitrile and dichloromethane in a volume ratio of 1:1, and no MgSO4 solid powder was added.

[0052] Comparative Example 3 Different from Example 4, when extracting the target compound, acetonitrile solvent containing 3% formic acid was used for extraction, and no MgSO4 solid powder was added.

[0053] Comparative Example 4 Different from Example 4, when extracting the target compound, the extraction was performed with a solvent having a volume ratio of 1:1 between n-hexane and dichloromethane, and MgSO4 solid powder was added.

[0054] Comparative Example 5 Different from Example 4, when extracting the target compound, the extraction was performed with a solvent having a volume ratio of 1:1 between n-hexane and dichloromethane, and no MgSO4 solid powder was added.

[0055] The extraction solvents for Example 4 and Comparative Examples 1 to 5 are shown in Table 7, and the spiked recoveries of the target compounds using different extraction solvents are shown in Table 8.

[0056] Table 7 Different extraction reagent combinations for 31 target compounds and their metabolites

[0057] Table 8 Spiked recovery of different extraction reagent combinations

[0058] As shown in Table 8, Example 4 achieved the best extraction results when using different extraction reagent combinations, successfully extracting 31 compounds with recoveries ranging from 80% to 118%, meeting the generally accepted recovery standard of 70% to 120%. A mixed solvent of acetonitrile and 3% formic acid, supplemented with MgSO₄, effectively extracted pesticides and selectively removed lipid interference in fish, offering the advantages of rapid extraction, low cost, and high recovery.

[0059] Comparative Example 6 Different from Example 4, in the step “ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry detection”, the mobile phase was changed to ultrapure water (mobile phase A) and methanol (mobile phase B).

[0060] Comparative Example 7 Different from Example 4, in the step “ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry detection”, the mobile phases were changed to ultrapure water (mobile phase A) and acetonitrile (mobile phase B).

[0061] Draw a chromatogram based on the test results. Figures 1-3 shown. Figure 1 It shows that Example 4 uses ultrapure water (mobile phase A) and acetonitrile (mobile phase B) added with 0.01% acetic acid for detection. All 31 target compounds have clear characteristic peaks, good peak shape, good response, obvious substance separation, and reasonable time arrangement. Figure 2 It shows that when ultrapure water (mobile phase A) and methanol (mobile phase B) of Comparative Example 6 are used for detection, most of the organophosphorus metabolites do not produce peaks, and the peaks of the substances are concentrated around 1 minute, and the substance separation effect is even worse. Figure 3 The results show that the detection of the 31 target compounds using ultrapure water (mobile phase A) and acetonitrile (mobile phase B) in Comparative Example 7 showed a decrease in the number of peak substances and a decrease in the response of the substances. Therefore, the ultrapure water (mobile phase A) and acetonitrile (mobile phase B) mobile phase system with 0.01% acetic acid added in Example 4 provided the best response and separation effect for each compound.

[0062] Comparative Example 8 Different from Example 4, in the step of “ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry detection”, the gradient elution program is different, see Table 9.

[0063] Comparative Example 9 Different from Example 4, in the step of “ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry detection”, the gradient elution program is different, see Table 9.

[0064] Comparative Example 10 Different from Example 4, in the step of “ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry detection”, the gradient elution program is different, see Table 9.

[0065] Draw a chromatogram based on the test results.

[0066] Table 9 Different gradient elution programs

[0067] The chromatograms of Example 4 and Comparative Examples 8 to 10 are shown in the table below: Figure 1 、 Figure 4 、 Figure 5 and Figure 6As shown, compared with Example 4, when the procedure of Comparative Example 8 was used for elution, the peak time of the substances was too concentrated; when the procedure of Comparative Example 9 was used for elution, no substance peaked in the last 4 minutes, and the time could still be optimized; when the procedure of Comparative Example 10 was used, the substances peaked in the 3-4 minute range, and the separation effect deteriorated. Therefore, the elution gradient of Example 4 had the best response and separation effect for each compound.

[0068] Example 5 Comparison with existing pesticide detection methods 1. Preparation of spiked samples Referring to the method in Example 2 of the present invention, a spiked sample was prepared by freeze-drying and grinding a fish sample.

[0069] 2. Pretreatment of spiked samples Spiked fish samples were processed using the method described in the prior art patent application CN 109298111 A, entitled "Method for Simultaneous Detection of Multiple Pesticide Residues in Fruits and Vegetables." The specific steps are as follows: 20 g of sample was weighed into a centrifuge tube, 7 g of sodium chloride and 20 mL of acetonitrile were added, and the mixture was vortexed and homogenized at 10,000 rpm for 3 minutes. The mixture was then centrifuged at 4,000 rpm for 5 minutes. 10 mL of the supernatant was removed and blown to near dryness with nitrogen at 40°C. The residue was then reconstituted with a methanol solution containing 2% dichloromethane. The reconstituted solution was loaded onto an activated solid-phase extraction column, and the effluent was collected. The collected effluent was blown to near dryness with nitrogen at 40°C, the column was rinsed with methanol, and then filtered through a 0.22 μm microporous membrane for analysis.

[0070] 3. Detection The spiked samples obtained by the two pretreatment methods in the previous step were detected by referring to the method of Example 1 of the present invention, and the spiked recovery rates were calculated. The test results are shown in Table 10.

[0071] Table 10 Comparison of the recovery rates of 31 pesticides and their metabolites in fish samples using different methods (spiked recovery (%) ± (relative deviation,%) n =3))

[0072] The results showed that, compared to the detection method provided by the present invention, the method disclosed in patent publication number "CN 109298111 A" requires a larger sample volume and requires subsequent solid-phase extraction and cleanup, resulting in higher costs. The results in Table 10 show that, after treating fish samples with the method disclosed in patent publication number "CN 109298111 A," the spiked recoveries for 31 pesticides ranged from 5% to 185%, with recoveries for four compounds falling below 15%, failing to meet the generally accepted recovery standard of 70% to 120% and failing to accurately quantify all 31 compounds simultaneously. Therefore, this prior art method cannot be used to detect the levels of 31 pesticides and their metabolites in fish samples.

[0073] It can be seen from the above embodiments that the present invention provides a method for detecting pesticide residues in fish. The method for detecting pesticide residues in fish provided by the present invention has strong resistance to matrix interference, high sensitivity, high stability, simple operation and low cost.

[0074] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for detecting pesticide residues in fish, characterized in that: The following steps are involved: (1) Drying the fish sample and grinding it to obtain fish powder; (2) Mix the fish powder with water until it becomes a meat paste, add MgSO4 solid powder and acetonitrile solution containing 2.5~3.5v / v% formic acid, perform vortex extraction, then sonicate and centrifuge, and collect the upper solution to obtain supernatant 1; (3) Repeat step (2), collect the upper solution to obtain supernatant 2, and combine supernatants 1 and 2 to obtain the extraction product; (4) Remove fat from the extracted product, filter it through a filter membrane, and detect it using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry to obtain the pesticide residue determination results; The chromatographic conditions were as follows: 0.008-0.012 v / v% acetic acid aqueous solution as mobile phase A, and acetonitrile as mobile phase B; The elution gradient program of the chromatography is:

2. The detection method according to claim 1, wherein The mass ratio of fish meat sample to MgSO4 solid powder is 1:0.9~1.

1.

3. The detection method according to claim 1, wherein The rotation speed of the vortex extraction is 2300-2700 rpm, and the time of the vortex extraction is 2.5-3.5 min.

4. The detection method according to claim 1, wherein The chromatographic column was a Waters BEH C18 column with a length of 100 mm, an inner diameter of 2.1 mm, and a filler particle diameter of 1.7 μm.

5. The detection method according to claim 1, wherein The chromatographic conditions also include: column temperature set at 38-42°C, injection volume of 2-4 μL, and flow rate of 0.28-0.32 mL / min.

6. The detection method according to claim 1, characterized in that The pesticide residues include dimethyl phosphate, dimethyl phosphate thioester, diethyl phosphate, diethyl dithiophosphate, diethyl tetrathiophosphate, p-nitrophenol, 3,5,6-trichloro-2-pyridinol, dimethyl dithiophosphate, 2-isopropyl-6-methyl-4-pyridinol, 3-phenoxybenzoic acid, 4-fluoro-3-phenoxybenzoic acid, trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid , cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, 6-chloronicotinic acid, flubendiamide, allyl imidacloprid, nitrosoimidacloprid, imidacloprid, thiacloprid, clothianidin, dinotefuran, flubendiamide, demethylthiacloprid, thiacloprid amide, chlorothiazolin, thiamethoxam, acetamiprid, N-demethylacetamiprid and 5-hydroxyimidacloprid.

7. The detection method according to claim 6, characterized in that A triple quadrupole tandem mass spectrometer was used for detection, with an electrospray ionization source, positive and negative scan modes, and multiple ion reaction detection. The ion source parameters were as follows: ion source temperature 530-570 °C, ionization voltage 4300-4700 V, spray gas 53-57 psi, auxiliary heating gas 53-57 psi, and curtain gas 33-37 psi.

8. The detection method according to claim 1, wherein The ultrasound frequency is 38-42Khz, the power is 90-110W, and the ultrasound time is 13-17 minutes.

9. The detection method according to claim 1, wherein The mass volume ratio of fish powder to acetonitrile solution containing 2.5~3.5v / v% formic acid is 0.1g:1.8~2.2mL.

10. The detection method according to claim 7, characterized in that: The pore size of the filter membrane is 0.20-0.25 μm.

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