A method for detecting pesticide residues in fish

Abstract

This invention provides a method for detecting pesticide residues in fish, belonging to the field of pesticide residue detection technology. The detection method includes 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 it using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry (UHPLC-MS / MS) to obtain the pesticide residue determination results; wherein the chromatographic conditions are: using an aqueous solution of acetic acid with a concentration of 0.008~0.012 v / v% as mobile phase A, and acetonitrile as mobile phase B. This invention uses UHPLC-MS / MS to optimize the detection conditions, enabling the simultaneous detection of 31 compounds in fish samples. Furthermore, the detection method of this invention has good accuracy and high sensitivity, with a detection limit of 0.01~0.05 ng / mL, which meets the requirements for detecting pesticide residues in fish.

Description

Technical Field

[0001] This invention relates to the field of pesticide residue detection technology, and in particular to a method for detecting pesticide residues in fish. Background Technology

[0002] With the development of modern agriculture, various pesticides are widely used in pest and disease control, among which neonicotinoid insecticides, organophosphate insecticides, pyrethroid insecticides, and phenoxy herbicides are the most common. These compounds are characterized by high efficiency and rapid action, but due to their strong stability in the environment and high water solubility, some pesticides and their metabolites can enter the aquatic environment through agricultural runoff and rainfall, causing water pollution. Existing studies have shown that pesticides are widely detected in the aquatic environment, especially in rivers and lakes near aquaculture areas and intensive agricultural areas, where residues of neonicotinoids, organophosphates, pyrethroids, and phenoxy pesticides are prevalent. These pesticides can be absorbed and accumulated in aquatic organisms, especially fish, which, due to their important position in the food chain, are more likely to become enrichment carriers of pesticides and their metabolites.

[0003] Pesticides and their metabolites, once ingested by fish, can potentially be transferred to humans through the food chain, posing a risk of chronic exposure. Long-term consumption of aquatic products containing pesticide residues may adversely affect the nervous, endocrine, and immune systems, and may even lead to chronic poisoning or cancer. Given that fish is an important 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 interference from the sample matrix. For example:

[0005] 1. Low extraction efficiency: Existing methods do not completely extract pesticides and their metabolites with large polarity differences, affecting the accuracy of quantitative analysis;

[0006] 2. Complex and time-consuming operation: The multi-step pretreatment process reduces detection efficiency and is not conducive to large-scale sample processing;

[0007] 3. Poor adaptability: Some methods are only applicable to certain types or a few types of pesticides, and lack the ability to deal with complex types of pollution (such as the coexistence of insecticides and herbicides). Summary of the Invention

[0008] In view of this, the present invention provides a method for detecting pesticide residues in fish to solve the above problems.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0010] This invention provides a method for detecting pesticide residues in fish, comprising the following steps:

[0011] (1) The fish meat sample was dried and then ground to obtain fish meat powder;

[0012] (2) Mix fish meat powder with water until it becomes a paste, add MgSO4 solid powder and acetonitrile solution containing 2.5~3.5 v / v% formic acid, perform vortex extraction, then sonicate, centrifuge, and collect the upper layer solution to obtain supernatant 1;

[0013] (3) Repeat step (2) to collect the upper layer of solution to obtain supernatant 2. Combine supernatant 1 and 2 to obtain the extraction product;

[0014] (4) Remove the fat from the extract, filter it through a filter membrane, and detect it by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry to obtain the pesticide residue determination results;

[0015] The chromatographic conditions were as follows: acetic acid aqueous solution with a concentration of 0.008~0.012 v / v% was used as mobile phase A, and acetonitrile was used as mobile phase B.

[0016] The elution gradient program for chromatography is as follows:

[0017] 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

[0018] Preferably, the mass ratio of fish meat sample to MgSO4 solid powder is 1:0.9~1.1.

[0019] Preferably, the vortex extraction speed is 2300~2700 rpm and the vortex extraction time is 2.5~3.5 min.

[0020] 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 packing particle diameter of 1.7 µm.

[0021] Preferably, the chromatographic conditions further include: column temperature set at 38~42℃, injection volume at 2~4 μL, and flow rate at 0.28~0.32 mL / min.

[0022] Preferably, the pesticide residue includes dimethyl phosphate, dimethyl phosphate thioester, diethyl phosphate, diethyl dithiophosphate, diethyl tetrathiophosphate, p-nitrophenol, 3,5,6-trichloro-2-pyridinephenol, dimethyl dithiophosphate, 2-isopropyl-6-methyl-4-pyridinephenol, 3-phenoxybenzoic acid, 4-fluoro-3-phenoxybenzoic acid, and trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane. Carboxylic acids, cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid, 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, 6-chloronicotinic acid, flonicamid, allyl imidacloprid, nitrosopyram, imidacloprid, thiamethoxam, thiamethoxam, dinotefuran, flonicamid, demethylthiamethoxam, thiamethoxam, chlorothiamethoxam, thiamethoxam, acetamiprid, N-demethylacetamiprid and 5-hydroxyimidacloprid.

[0023] Preferably, a triple quadrupole tandem mass spectrometer is used for detection, employing an electrospray ionization source. The scanning modes are positive ion mode and negative ion mode, and the detection mode is multi-ion reaction detection mode. The ion source parameters are: ion source temperature 530~570 ℃, ionization voltage 4300~4700 V, spray gas 53~57 psi, auxiliary heating gas 53~57 psi, and curtain gas 33~37 psi.

[0024] Preferably, the frequency of the ultrasound is 38~42kHz, the power is 90~110W, and the ultrasound time is 13~17min.

[0025] Preferably, the mass-to-volume ratio of fish powder to acetonitrile solution containing 2.5-3.5 v / v% formic acid is 0.1 g: 1.8-2.2 mL.

[0026] Preferably, the pore size of the filter membrane is 0.20~0.25 µm.

[0027] By adopting the above technical solution, the present invention has the following beneficial effects: The detection method of the present invention includes the following steps: adding MgSO4 solid powder to fish meat samples, extracting the target compound with an acetonitrile solution containing 2.5~3.5 v / v% formic acid, and detecting it using ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry to obtain the pesticide residue determination results; wherein, the chromatographic conditions are: using an aqueous solution of acetic acid with a concentration of 0.008~0.012 v / v% as mobile phase A, and acetonitrile as mobile phase B. The present invention uses ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry, which optimizes the detection conditions and can simultaneously detect the residues of 31 compounds from fish samples. Furthermore, the detection method of the present invention 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. Attached Figure Description

[0028] Figure 1 The chromatogram is of a 10 ng / mL standard solution of 31 pesticides and their metabolites in Example 4.

[0029] Figure 2 The chromatograms are of 31 pesticides and their metabolites in Comparative Example 6.

[0030] Figure 3 The chromatograms are of 31 pesticides and their metabolites in Comparative Example 7.

[0031] Figure 4 The chromatograms are of 31 pesticides and their metabolites in Comparative Example 8.

[0032] Figure 5 The chromatograms are of 31 pesticides and their metabolites in Comparative Example 9.

[0033] Figure 6 The chromatograms are of 31 pesticides and their metabolites in Comparative Example 10. Detailed Implementation

[0034] The technical solutions provided by the present invention will be 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.

[0035] The model and brand information of the instruments and equipment used in the embodiments of this invention are as follows:

[0036] Ultra-high performance liquid chromatograph, model: Shimadzu Nexera LC-40D ultra-high performance liquid chromatography system, manufacturer: Shimadzu Instruments (Suzhou) Co., Ltd.;

[0037] Triple quadrupole mass spectrometer, model: AB Sciex QTRAP 5500+, manufacturer: Shanghai SCIEX Analytical Instruments Trading Co., Ltd.;

[0038] Chromatographic column, model: Waters BEH C18 column, 100 × 2.1 mm, 1.7 µm, manufacturer: Waters China official website;

[0039] Pipettes, models 10 / 20 / 100 / 200 / 1000 / 2500μL, brand: Eppendorf;

[0040] Ultrasonic cleaner, model: KQ-700TDE, brand: Kunshan Ultrasonics Corporation;

[0041] Electronic analytical balance, model: XSR204, brand: Mettler Toledo;

[0042] Pure water system, model: Milli-Q Integral system, brand: Millipore;

[0043] Centrifuge, model: Centrifuge 5804, brand: Eppendorf;

[0044] Vacuum freeze dryer, model: FD-1A-50, manufacturer: Boyikang (Beijing) Instrument Co., Ltd.;

[0045] Solid phase extraction device, brand: Merck;

[0046] The nitrogen blowing device was purchased from the website of Organomation Associates Inc.

[0047] The methanol (LC-MS), acetonitrile (LC-MS), and formic acid (LC-MS) used in the embodiments of this invention were purchased from Thermo Fisher Scientific.

[0048] Example 1. Method for detecting 31 pesticides and their metabolites in a sample.

[0049] 1. Target compound and internal standard

[0050] This invention provides a method for detecting multiple pesticide residues in fish samples, using 31 compounds as target compounds as shown in Table 1, specifically including: dimethyl phosphate, dimethyl phosphate thioester, diethyl phosphate, diethyl dithiophosphate, diethyl tetrathiophosphate, p-nitrophenol, 3,5,6-trichloro-2-pyridinephenol, dimethyl dithiophosphate, 2-isopropyl-6-methyl-4-pyridinephenol, 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, flonicamid, allyl imidacloprid, nitrosopyram, imidacloprid, thiamethoxam, thiamethoxam, dinotefuran, flonicamid, demethylthiamethoxam, thiamethoxam, chlorothiamethoxam, thiamethoxam, acetamiprid, N-demethylacetamiprid and 5-hydroxyimidacloprid; 27 isotopically labeled internal standards, specifically 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- 13C2-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.

[0051] Table 1 Information on 31 compounds and internal standards

[0052]

[0053]

[0054] 2. Solution preparation

[0055] (1) Mixed internal standard solution

[0056] The 27 isotope-labeled internal standards shown in Table 1 were dissolved in methanol at a final concentration of 500 ng / mL, mixed thoroughly, and diluted to obtain a mixed internal standard solution with a concentration of 10 ng / mL.

[0057] (2) Extraction solvent

[0058] Formic acid was added to acetonitrile at a volume ratio of 3:100 and mixed thoroughly to obtain the extraction solvent.

[0059] (3) Mixing standard solutions

[0060] The 31 target analytes shown in Table 1 were dissolved in methanol to obtain a mixed standard stock solution, wherein the final concentration of each target analyte standard was 10 µg / mL.

[0061] The mixed standard stock solution was then serially diluted with methanol to obtain mixed standard solutions with final concentrations 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, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL, and 50 ng / mL for each compound.

[0062] 3. Sample pretreatment

[0063] (1) Sample preparation

[0064] The fish meat was separated from the fish body through dissection. After being cut into sections, the fish meat was 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.

[0065] (2) Liquid-liquid extraction

[0066] Weigh 0.1 g of fish meat powder, add 100 µL of ultrapure water to moisten, and vortex for 1 min until it becomes a paste. Add 0.1 g of MgSO4 solid powder, add 2 mL of acetonitrile solution containing 3% formic acid, and vortex for 3 min at 2500 rpm. Sonicate (frequency 40 kHz, power 100 W) for 15 min, adding ice to prevent overheating. Centrifuge for 10 min (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 in a mild nitrogen atmosphere at 25°C until nearly dry to obtain the extraction product.

[0067] (3) Reconstitute and then freeze to remove fat.

[0068] The extraction product was dissolved in 500 µL of reconstituted solution (pure acetonitrile), vortexed for 1 min to mix, and then frozen at -20°C for 12 h to precipitate the fat. After precipitation, the supernatant was collected, filtered through a 0.22 µm PTFE membrane into a brown vial to obtain the sample for subsequent analysis.

[0069] 4. Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry

[0070] The detection conditions were as follows: the chromatographic column was a Waters BEH C18 column (length 100 × inner diameter 2.1 mm, packing particle diameter 1.7 µm), the column temperature was set at 40℃, and the injection volume was 3 μL; mobile phase A was 0.01 v / v% acetic acid aqueous solution, mobile phase B was acetonitrile, the flow rate was 0.3 mL / min, and the gradient elution program was shown in Table 2.

[0071] Table 2 Gradient elution program

[0072]

[0073] 5. Mass spectrometry analysis

[0074] The detection was performed using a triple quadrupole tandem mass spectrometer under the following conditions: electrospray ionization (ESI) source was used, with scanning modes of positive and negative ion modes, and detection mode of multi-ion reaction detection. Ion source parameters were: ion source temperature 550 °C, ionization voltage 4500 V, spray gas 55 psi, auxiliary heating gas 55 psi, curtain gas 35 psi, and collision gas flow rate at a moderate intensity (9). The mass spectrometric parameters of each target analyte are shown in Table 3.

[0075] Table 3. Mass spectrometry parameters of 31 pesticides, their metabolites, and internal standards.

[0076]

[0077]

[0078] 4. Results Analysis

[0079] (1) Establishment of the standard curve

[0080] The internal standard method was used to perform quantitative analysis of 31 pesticides and their metabolites. The specific steps are as follows: 12 mixed standard solutions of different concentrations were taken for detection by ultra-high performance liquid chromatography and mass spectrometry analysis. The ratio of the chromatographic peak area of ​​each target analyte to the chromatographic peak area of ​​the corresponding internal standard at different concentrations was used as the standard area ratio. The standard curves of each target analyte were plotted with concentration as the abscissa and area ratio as the ordinate, as shown in Table 4.

[0081] Table 4. Standard curves of 31 pesticides and their metabolites

[0082]

[0083]

[0084] The results showed that within the linear range of 0.01 ng / mL to 50 ng / mL, the linearity of all 31 compounds was greater than 0.99, indicating good linearity.

[0085] (2) Analysis of the content of each target substance in the sample

[0086] The ratio of the peak area of ​​each target compound measured in the sample to the chromatographic peak area of ​​the corresponding internal standard is used as the sample area ratio. This ratio is then substituted into the standard curve to calculate the content of each compound in the sample.

[0087] Example 2. Evaluation of the detection method in Example 1 using fish meat samples.

[0088] 1. Preparation of spiked samples

[0089] This embodiment utilizes fish meat samples for methodological evaluation. Fish meat was dissected and separated from the fish body. The fish meat was then cut into segments 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.

[0090] Take 0.1g of fish meat sample and add it to 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. Three samples are prepared for each concentration.

[0091] 2. Testing

[0092] The spiked sample prepared in this embodiment was used as the liquid sample to be tested. The content of each pesticide and its metabolites in the sample was detected according to the method of Embodiment 1 of the present invention. The test results are shown in Table 5.

[0093] Table 5. Spiked recoveries and relative deviations of 31 pesticides and their metabolites

[0094]

[0095] As shown in Table 5, the spiked recoveries of 31 pesticides and their metabolites ranged from 70% to 113%. This indicates that using the method of the present invention, the spiked recoveries and relative deviations of the 31 pesticides and their metabolites are 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 in cost, has good detection accuracy (spiking recoveries of 70% to 113%), high precision (RSD ≤ 4.5), and strong specificity, meeting the recognized standard requirements.

[0096] Example 3: Detection of 31 pesticides and their metabolites in actual fish meat samples.

[0097] Fifty fish meat samples were collected from fish caught by a South China Sea scientific expedition team. After freeze-drying for 48 hours, grinding, and sieving, the content of 31 pesticides and their metabolites in the fish meat samples was detected using the method described in Example 1 of this invention. The detection results are shown in Table 6. Due to instrument limitations and the fact that fish meat samples may not contain all 31 pesticides and metabolites, a total of 28 pesticides and their metabolites were actually detected in the 50 fish meat samples, with thiamethoxam (THX) showing the highest detection rate. The detection method described in Example 1 of this invention has high accuracy, good stability, and a low detection limit (0.01~0.05 ng / mL), which can meet the actual detection needs of fish meat samples.

[0098] Table 6. Detection of 31 pesticides and their metabolites in 50 fish meat samples.

[0099]

[0100] Note: The data for "detection rate" in the table are rounded off.

[0101] Example 4

[0102] A mixed standard working solution, with a final concentration of 10 ng / mL for each of the 31 target compounds, was used as the fish meat sample to be tested. 0.1 g of fish meat powder was weighed, moistened with 100 μL of water, and vortexed for 1 min until a paste-like consistency was achieved.

[0103] 0.1 g of MgSO4 solid powder was added to 2 mL of acetonitrile solution containing 3 v / v% formic acid. The mixture was vortexed at 2500 rpm for 3 min, followed by sonication (40 kHz, 100 W) for 15 min, with ice added to prevent overheating. The mixture was centrifuged at 4500 rpm for 10 min to separate the layers, and the upper layer was collected to obtain supernatant 1. This extraction step was repeated once, and the upper layer was collected to obtain supernatant 2. Supernatants 1 and 2 were combined to obtain 5 mL of mixed supernatant. The mixed supernatant was placed under a mild nitrogen atmosphere at 25°C and concentrated to near dryness to obtain the extract. The product was then analyzed according to the method in Example 1.

[0104] Comparative Example 1

[0105] Unlike Example 4, the target compound was extracted using a solvent with a volume ratio of acetonitrile and dichloromethane of 1:1, with the addition of MgSO4 solid powder.

[0106] Comparative Example 2

[0107] Unlike Example 4, the target compound was extracted using a solvent with a volume ratio of acetonitrile and dichloromethane of 1:1, without the addition of MgSO4 solid powder.

[0108] Comparative Example 3

[0109] Unlike Example 4, the target compound was extracted using acetonitrile solvent containing 3% formic acid, without the addition of MgSO4 solid powder.

[0110] Comparative Example 4

[0111] Unlike Example 4, the target compound was extracted using a solvent with a volume ratio of n-hexane to dichloromethane of 1:1, with the addition of MgSO4 solid powder.

[0112] Comparative Example 5

[0113] Unlike Example 4, the target compound was extracted using a solvent with a volume ratio of n-hexane to dichloromethane of 1:1, without the addition of MgSO4 solid powder.

[0114] The extraction solvents for Examples 4 and Comparative Examples 1-5 are shown in Table 7, and the spiked recoveries of the target compounds for different extraction solvents are shown in Table 8.

[0115] Table 7. Different extraction reagent combinations for 31 target compounds and their metabolites analytes

[0116]

[0117] Table 8 Spike recoveries of different extraction reagent combinations

[0118]

[0119]

[0120] As shown in Table 8, when using different combinations of extraction reagents, Example 4 showed the best extraction effect, successfully extracting 31 compounds with recoveries ranging from 80% to 118%, meeting the generally accepted recovery standard (70% to 120%). The combination of acetonitrile and 3% formic acid in a mixed solvent with MgSO4 in a fish-based biological matrix can efficiently extract pesticides and selectively remove lipid interference, offering advantages such as speed, low cost, and high recovery rate.

[0121] Comparative Example 6

[0122] Unlike Example 4, in the step "Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection", the mobile phase is changed to: ultrapure water (mobile phase A) and methanol (mobile phase B).

[0123] Comparative Example 7

[0124] Unlike Example 4, in the step "Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection", the mobile phase is changed to: ultrapure water (mobile phase A) and acetonitrile (mobile phase B).

[0125] A chromatogram was plotted based on the test results, as shown below. Figures 1-3 As shown. Figure 1 The results show that in Example 4, ultrapure water (mobile phase A) with 0.01% acetic acid added and acetonitrile (mobile phase B) were used for detection. All 31 target compounds had clear characteristic peaks, good peak shapes, good responses, obvious substance separation, and reasonable time arrangement. Figure 2 The results showed that when using ultrapure water (mobile phase A) and methanol (mobile phase B) in Comparative Example 6 for detection, most organophosphorus metabolites did not produce peaks, and the substances concentrated in the peaks around 1 minute, indicating poorer substance separation. Figure 3 The results showed that using ultrapure water (mobile phase A) and acetonitrile (mobile phase B) in Comparative Example 7 resulted in a decrease in the peak concentrations of the 31 target compounds, and a reduction in the response of the substances. Therefore, the ultrapure water (mobile phase A) and acetonitrile (mobile phase B) mobile phase system with added 0.01% acetic acid in Example 4 demonstrated the best response and separation performance for each compound.

[0126] Comparative Example 8

[0127] Unlike Example 4, the gradient elution procedure in the step “Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection” is different, as shown in Table 9.

[0128] Comparative Example 9

[0129] Unlike Example 4, the gradient elution procedure in the step “Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection” is different, as shown in Table 9.

[0130] Comparative Example 10

[0131] Unlike Example 4, the gradient elution procedure in the step “Ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry detection” is different, as shown in Table 9.

[0132] A chromatogram was plotted based on the test results.

[0133] Table 9 Different gradient elution procedures

[0134]

[0135]

[0136] The chromatograms of Example 4 and Comparative Examples 8-10 are shown in the following figures. Figure 1 , Figure 4 , Figure 5 and Figure 6As shown, compared with Example 4, when using the program of Comparative Example 8 for elution, the elution peak times were too concentrated; when using the program of Comparative Example 9 for elution, no substances eluted in the last 4 minutes, and the time could be further optimized; when using the program of Comparative Example 10, the substances concentrated in the 3-4 minute elution, and the separation effect deteriorated. Therefore, the elution gradient detection of each compound in Example 4 showed the best response and separation effect.

[0137] Example 5: Comparison with existing pesticide detection methods

[0138] 1. Preparation of spiked samples

[0139] Referring to the method in Example 2 of this invention, spiked samples were prepared by freeze-drying and grinding fish meat samples.

[0140] 2. Pretreatment of spiked samples

[0141] The fish spiked sample was processed according to the method described in the invention patent "Method for Simultaneous Detection of Multiple Pesticide Residues in Fruits and Vegetables" (publication number CN 109298111 A). The specific steps are as follows: Weigh 20g of sample into a centrifuge tube, add 7g of sodium chloride and 20mL of acetonitrile, vortex mix, and homogenize at 10000 rpm for 3min; then centrifuge at 4000 rpm for 5min. Take 10mL of supernatant, blow it nearly dry under nitrogen at 40℃, and redissolve the residue with a methanol solution containing 2% dichloromethane. Load the redissolved solution onto an activated solid-phase extraction column and collect the eluent; blow the collected eluent nearly dry under nitrogen at 40℃, rinse the solid-phase extraction column with methanol solution, and then filter it through a 0.22 μm microporous membrane for analysis.

[0142] 3. Testing

[0143] The spiked samples obtained by the two pretreatment methods in the previous step were tested according to the method of Example 1 of the present invention, and the spiked recovery rate was calculated. The test results are shown in Table 10.

[0144] Table 10 Comparison of recoveries of 31 pesticides and their metabolites in fish samples using different methods (spiked recovery rate (%) ± (relative deviation,%)) n = 3))

[0145]

[0146]

[0147] The results show that, compared with the detection method provided by this invention, the method in patent publication number "CN 109298111 A" requires a larger sample volume and necessitates extraction followed by solid-phase extraction purification, resulting in higher costs. The detection results in Table 10 indicate that after processing fish samples using the method in patent publication number "CN 109298111 A", the spiked recoveries of 31 pesticides ranged from 5% to 185%, with four compounds showing recoveries below 15%, failing to meet the generally accepted recovery standard (70%–120%) and thus unable to simultaneously and accurately quantify 31 compounds. Therefore, this prior art method cannot be used to detect the content of 31 pesticides and their metabolites in fish samples.

[0148] As can be seen from the above embodiments, 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.

[0149] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for detecting pesticide residues in fish, characterized in that, Includes the following steps: (1) The fish meat sample was dried and then ground to obtain fish meat powder; (2) Mix fish meat powder with water until it becomes a paste, add MgSO4 solid powder and acetonitrile solution containing 2.5~3.5 v / v% formic acid, perform vortex extraction, then sonicate, centrifuge, and collect the upper layer solution to obtain supernatant 1; (3) Repeat step (2) to collect the upper layer of solution to obtain supernatant 2. Combine supernatant 1 and 2 to obtain the extraction product; (4) Remove the fat from the extract, filter it through a filter membrane, and detect it by ultra-high performance liquid chromatography-triple quadrupole tandem mass spectrometry to obtain the pesticide residue determination results; The chromatographic conditions were as follows: acetic acid aqueous solution with a concentration of 0.008~0.012 v / v% was used as mobile phase A, and acetonitrile was used as mobile phase B. The elution gradient program for chromatography is as follows: ； The chromatographic column was a Waters BEH C18 column, with a length of 100 mm, an inner diameter of 2.1 mm, and a packing particle diameter of 1.7 µm. 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, and 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, flonicamid, allyl imidacloprid, nitrosopyram, imidacloprid, thiamethoxam, thiamethoxam, dinotefuran, flonicamid, demethylthiamethoxam, thiamethoxam, chlorothiamethoxam, thiamethoxam, acetamiprid, N-demethylacetamiprid and 5-hydroxyimidacloprid; The detection was performed using a triple quadrupole tandem mass spectrometer with an electrospray ionization source. The scanning modes were positive ion mode and negative ion mode, and the detection mode was multi-ion reaction detection mode. The ion source parameters were as follows: ion source temperature 530~570 ℃, ionization voltage 4300~4700 V, spray gas 53~57 psi, auxiliary heating gas 53~57 psi, and curtain gas 33~37 psi.

2. The detection method according to claim 1, characterized in that, The mass ratio of fish meat sample to MgSO4 solid powder was 1:0.9~1.

1.

3. The detection method according to claim 1, characterized in that, The vortex extraction speed is 2300~2700 rpm, and the vortex extraction time is 2.5~3.5 min.

4. The detection method according to claim 1, characterized in that, The chromatographic conditions also include: column temperature set at 38~42℃, injection volume at 2~4 μL, and flow rate at 0.28~0.32 mL / min.

5. The detection method according to claim 1, characterized in that, The ultrasound frequency is 38~42kHz, the power is 90~110W, and the ultrasound time is 13~17min.

6. The detection method according to claim 1, characterized in that, The mass-to-volume ratio of fish meat powder to acetonitrile solution containing 2.5-3.5 v / v% formic acid is 0.1 g : 1.8-2.2 mL.

7. The detection method according to claim 1, characterized in that, The pore size of the filter membrane is 0.20~0.25 µm.

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