Method for determining the content of neonicotinoid insecticides and their metabolites in animal samples

Abstract

The present disclosure provides a method for determining the content of neonicotinoid insecticides and their metabolites in an animal sample, comprising: after extracting the animal sample with a solvent, adding QuEChERs salt to protect the neonicotinoid insecticides and their conversion products in the solvent and removing moisture to obtain an extract; adding a dispersive solid phase extraction filler to the extract, purifying the extract, concentrating and constant volume to obtain a test solution; detecting the test solution by high performance liquid chromatography-triple quadrupole mass spectrometry to obtain the content of neonicotinoid insecticides and their metabolites in the animal sample.

Description

Technical Field

[0001] This disclosure relates to the technical field of pesticide detection, and more particularly to a method for determining the content of neonicotinoid pesticides and their metabolites in animal samples. Background Technology

[0002] Neonicotinoids (NEOs) are a class of synthetic insecticides derived from natural nicotine. They are characterized by high efficiency, low toxicity, and broad spectrum of activity, and are considered the fourth generation of insecticides after organophosphates, carbamates, and pyrethroids. In recent years, neonicotinoids have been widely used, not only in agricultural pest control but also in aquaculture and the control and treatment of diseases and pests in pets and livestock.

[0003] Because neonicotinoid insecticides have good water solubility and novel modes of action, in addition to common seed treatment, foliar spraying and soil irrigation are also common application methods, making them easily migrated into natural water bodies with rainwater.

[0004] Currently, the detection and analysis of neonicotinoid pesticides mainly focus on water and sediment. However, some neonicotinoid pesticides discharged from agriculture enter water bodies and pose a threat to aquatic organisms. Furthermore, pesticides absorbed by crops can further affect terrestrial organisms, undergoing transformation and metabolism within them. Therefore, it is necessary to develop a method that can simultaneously determine neonicotinoid pesticides and their metabolites in animal samples. Summary of the Invention

[0005] In view of the above-mentioned technical problems, the main objective of this disclosure is to provide a method for determining the content of neonicotinoid insecticides and their metabolites in animal samples, so as to at least partially solve the aforementioned technical problems.

[0006] To solve the above-mentioned technical problems, the technical solution provided in this disclosure is as follows:

[0007] As one aspect of this disclosure, a method is provided for determining the content of neonicotinoid insecticides and their metabolites in animal samples, comprising:

[0008] After extracting animal samples with solvent, QuEChERs salt packs were added to protect neonicotinoid insecticides and their transformation products in the solvent and to remove water, resulting in an extract.

[0009] Dispersed solid-phase extraction packing material was added to the extract, the extract was purified, and then concentrated to a fixed volume to obtain the solution to be tested.

[0010] The content of neonicotinoid insecticides and their metabolites in animal samples was obtained by detecting the test solution using high performance liquid chromatography-triple quadrupole mass spectrometry.

[0011] In one embodiment, the solvent used for extraction is an acetonitrile-acetic acid mixture, comprising acetonitrile containing 1% acetic acid;

[0012] QuEChERs salt packs are buffered mixtures of inorganic salts, including the Agilent EN 15662 Extraction Salt Kit;

[0013] Dispersed solid phase extraction packings are adsorbents and purifiers capable of removing lipids and pigments, including a mixture of N-propylethylenediamine (PSA) and octadecyl bonded silica gel (C-18).

[0014] In one embodiment, the ratio of N-propylethylenediamine (PSA) to octadecyl bonded silica gel (C-18) in the N-propylethylenediamine (PSA) to octadecyl bonded silica gel (C-18) mixture is 1:1.

[0015] In one embodiment, the high performance liquid chromatography-triple quadrupole mass spectrometry method uses an electrospray ionization source in both positive and negative ion modes.

[0016] In one embodiment, chromatographic separation was performed using a Waters Atlantis T3 liquid chromatography column with gradient elution using a binary mobile phase.

[0017] In one embodiment, the binary mobile phase includes mobile phase A and mobile phase B;

[0018] Mobile phase A is a mixed aqueous solution of formic acid and ammonium acetate, comprising a mixed aqueous solution of formic acid and 5 mM ammonium acetate with a volume percentage of 0.01%;

[0019] Mobile phase B is methanol.

[0020] In one embodiment, the amount of animal sample added to the solvent was 90-110 mg / mL;

[0021] The amount of dispersed solid-phase extraction packing added to the extract is 180~220 mg / mL.

[0022] In one embodiment, the animal sample is a solid or semi-solid sample, including any one of animal muscle, animal tissues or organs.

[0023] In one embodiment, the neonicotinoid insecticide includes any one or more of the following: fipronil, flonicamid, imidacloprid, acetamiprid, thiamethoxam, thiamethoxam, acetamiprid, flupyrflurane, and thiamethoxam.

[0024] Metabolites include any one or more of denitrified imidacloprid hydrochloride, amide thiamethoxam, and demethyl acetamiprid.

[0025] As another aspect of this disclosure, an application is provided for the above-described method for determining the content of neonicotinoid insecticides and their metabolites in animal samples in the detection of ecological environment quality and food quality.

[0026] Based on the above technical solution, the method for determining the content of neonicotinoid insecticides and their metabolites in animal samples provided in this disclosure has at least the following beneficial effects:

[0027] Solvent extraction of animal samples followed by the addition of QuEChERS salt packs can enrich neonicotinoid insecticides and their metabolites in the animal samples, protect the neonicotinoid insecticides and their transformation products in the extract, remove water from the extract, promote the separation of the organic and aqueous phases, and facilitate the separation of the extract. Dispersed solid-phase extraction packing is used to adsorb and purify impurities in the extract, removing lipids and pigments from the animal samples and simplifying the processing steps. Combined with high performance liquid chromatography, the analysis rate is improved and the analysis time is shortened. Triple quadrupole mass spectrometry is used to effectively reduce background interference. This method has high selectivity, sensitivity, and anti-interference ability, effectively lowers the method detection limit, and can achieve rapid and accurate identification and quantification of multiple neonicotinoid insecticides and their metabolites in the test animal samples. Attached Figure Description

[0028] Figure 1 This is a schematic diagram illustrating the method for determining the content of neonicotinoid insecticides and their metabolites in animal samples according to embodiments of this disclosure;

[0029] Figure 2 This is a chromatographic separation diagram of neonicotinoid insecticides and their metabolites in animal samples in Example 2 of this disclosure.

[0030] Figure 3 This is a chromatographic separation diagram of neonicotinoid insecticides and their metabolites in animal samples from Example 3 of this disclosure. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this disclosure clearer, the following detailed description is provided in conjunction with specific embodiments and the accompanying drawings.

[0032] Currently, some studies have demonstrated the presence of neonicotinoid insecticides to varying degrees in different tissues of many animals, such as wild boars, seagulls, and domesticated cattle and sheep. Due to increasing resistance, the types of neonicotinoid insecticides are constantly expanding. Neonicotinoid insecticides are highly susceptible to transformation and metabolism in the environment and organisms. Researchers have focused primarily on the neonicotinoids themselves, neglecting the detection and analysis of their metabolites. Studies on animal samples with complex compositions and difficult purification are also scarce. Animal samples often contain large amounts of impurities such as lipids, proteins, and pigments that severely interfere with analysis. Pretreatment methods for such samples are often complex, time-consuming, and rarely achieve satisfactory impurity removal. Therefore, developing a method for simultaneously detecting multiple neonicotinoid insecticides and their transformation products in animal samples is undoubtedly a pressing problem for researchers.

[0033] In the process of developing the technical solution of this disclosure, it was found that the commonly used techniques for detecting neonicotinoid insecticides are liquid chromatography (LC) with a diode array detector (DAD), gas chromatography (GC), and liquid chromatography-tandem mass spectrometry (LC-MS / MS). Among these, LC-DAD is limited in its application to trace samples in complex matrices and has relatively poor sensitivity; gas chromatography is not suitable for the detection and analysis of neonicotinoid insecticides and their metabolites (such as acetamiprid) with high boiling points and low volatility, and has low universality; compared with gas chromatography, liquid chromatography is more suitable for analyzing neonicotinoid insecticide components that are non-volatile, thermally unstable, and highly polar. However, the number of target compounds that can be detected by LC-MS / MS is currently limited, mostly containing only 5-8 traditional neonicotinoid insecticide types, and it is difficult to detect metabolites and novel neonicotinoid insecticides in animal samples.

[0034] In view of the technical problems existing in related technologies, this disclosure provides a method for determining the content of neonicotinoid insecticides and their metabolites in animal samples. The method utilizes QuEChERs technology and dispersive solid-phase extraction to extract, enrich, and purify neonicotinoid insecticides and their metabolites, simplifying the cumbersome sample pretreatment steps. Combined with high performance liquid chromatography-tandem triple quadrupole mass spectrometry, the method can simultaneously and rapidly determine the content of neonicotinoid insecticides and their metabolites, which has high sensitivity and anti-interference ability, effectively improves the analysis rate and test accuracy, and accurately quantifies trace compounds, thereby reducing the method detection limit.

[0035] Specifically, as one aspect of this disclosure, a method for determining the content of neonicotinoid insecticides and their metabolites in animal samples is provided, wherein the water used in the process is LC-MS grade ultrapure water. Figure 1 This is a schematic diagram of a method for determining the content of neonicotinoid insecticides and their metabolites in animal samples according to an embodiment of this disclosure, including steps S101 to S103:

[0036] In step S101, the animal sample is activated with water and then extracted in a solvent. QuEChERs salt packs are then added to remove water from the solvent and protect the neonicotinoid insecticides and their metabolites in the solvent, thereby enriching the target compound to obtain the extract.

[0037] In step S102, a dispersed solid-phase extraction packing is added to the extract to remove impurities from the extract, purify neonicotinoid insecticides and their metabolites, and after purification, the extract is concentrated and diluted to obtain the test solution.

[0038] In step S103, the test solution is detected by high performance liquid chromatography-triple quadrupole mass spectrometry, and reaction monitoring scanning mode is selected as the data acquisition method. At the same time, the content of neonicotinoid insecticides and their metabolites in animal samples is obtained.

[0039] According to embodiments of this disclosure, a method for determining the content of neonicotinoid insecticides and their metabolites in animal samples is described below in conjunction with... Figure 1 A detailed explanation may be provided, which may include:

[0040] In step S101, after soaking the animal sample in water and ultrasonically activating it, a solvent is added and thoroughly vortexed for mixing. Then, QuEChERs salt packs and ceramic homogenizers are added and thoroughly shaken to protect the neonicotinoid insecticides and their metabolites in the animal sample. This process extracts and enriches them in the extract while removing water from the extract and promoting the separation of the organic and aqueous phases. The upper organic layer is then extracted by centrifugation to obtain the extract, which is rich in neonicotinoid insecticides and their transformation products. Further, if necessary, the animal sample can be pretreated using freeze-drying technology: after freeze-drying the collected animal sample to remove water, it is then thoroughly pulverized into powder using a grinder or mortar to obtain a homogenized animal sample. The amount of water used to activate the animal sample is replenished according to the sample's dry weight, approximately 10 times the sample's dry weight, with a fluctuation range not exceeding 10%, i.e., 10 ± 1 g of water is added for every 1 g of dried animal sample. If the water content of the fresh animal sample is ≥90%, there is no need to add water again. If too much water is added, it will affect the enrichment of the target compound by the QuEChERS salt pack and will not promote the salting-out process.

[0041] In step S102, the extract is transferred to a centrifuge tube, a dispersed solid-phase extraction packing is added to the extract and shaken thoroughly to remove impurities from the extract. After purification, the purified extract is concentrated and brought to a fixed volume, then transferred to a vial to obtain the solution to be tested. This purification method is simple and convenient to operate.

[0042] In step S103, high performance liquid chromatography-tandem triple quadrupole mass spectrometry is used to determine the neonicotinoid insecticides and their metabolites in the test solution. The temperature is kept constant at 35℃, which can significantly reduce interference and improve the detection sensitivity, and at the same time obtain the content of neonicotinoid insecticides and their metabolites.

[0043] According to embodiments of this disclosure, high-performance liquid chromatography (HPLC) can improve the analysis rate and shorten the analysis time, while triple quadrupole mass spectrometry (TQMS) has high sensitivity and anti-interference capabilities, effectively reducing background interference. Electrospray ionization (ESI) sources are selected in both positive and negative ion modes. High-purity argon and high-purity nitrogen are used for the collision gas and nebulizing gas, respectively. The negative ion ionization mode is used to determine the content of flonicamid, while the positive ion mode is used to determine the content of other neonicotinoid insecticides and their metabolites. Reaction monitoring scanning mode is selected as the data acquisition method.

[0044] According to embodiments of this disclosure, the selected solvent is an extraction solvent used to extract animal samples. It can be an organic solvent that is immiscible with water after salting out and has high polarity to maintain the state of the target compound. The solvent can be an acetonitrile-acetic acid mixture, including acetonitrile containing 1% acetic acid. The selected solvent should be miscible with water and able to separate from the aqueous phase after the addition of inorganic salts. For highly polar neonicotinoid insecticides, acetonitrile can exhibit strong selectivity and can be separated from the solubilizing aqueous phase through salting out. Compared to other solvents, it has higher extraction efficiency and stability, making it the primary extraction solvent in the pretreatment process. Adding 1% acetic acid to acetonitrile as an analytical preservative allows for the simultaneous extraction and enrichment of neonicotinoid insecticides and their metabolites in a single step. This further protects the target compound from being fully extracted, improves pretreatment efficiency, avoids the cumulative error of multiple experiments, and thus ensures the accuracy of subsequent detection.

[0045] According to embodiments of this disclosure, the selected QuEChERs salt pack is an inorganic salt mixture with buffering effect and good water absorption, which enables the extract to separate from the aqueous phase after salting out. The extract treated with acetonitrile separates from the aqueous phase through salting out, which is beneficial for the purification of the target compound. The QuEChERs salt pack can be the Agilent EN 15662 extraction salt kit, which contains magnesium sulfate, sodium chloride, sodium citrate, and sodium bicitrate sesquihydrate, and has a buffer salt system. Among them, the inorganic salts magnesium sulfate and sodium chloride have good water absorption, and sodium citrate and sodium bicitrate sesquihydrate can form a pH buffer system, which can maintain the molecular form of pH-sensitive neonicotinoid insecticides and improve their distribution efficiency in the organic phase.

[0046] According to embodiments of this disclosure, the dispersed solid-phase extraction packing material is selected from adsorbents and purifiers capable of removing biological components such as lipids, sugars, and pigments, including a mixture of N-propylethylenediamine (PSA) and octadecyl bonded silica gel (C-18), wherein the ratio of N-propylethylenediamine (PSA) to octadecyl bonded silica gel (C-18) is 1:1. In practical use, a weight ratio error within ±10% is acceptable, and it can be used to adsorb and remove a large number of impurities in the extract. N-propylethylenediamine can form hydrogen bonds with polar matrix components through the weak ion exchange of amine groups, thereby adsorbing and eliminating sugars, some pigments, and fatty acids in the sample matrix; the octadecyl bonded silica gel adsorbent has a strong adsorption effect on non-polar compounds and can be used to remove non-polar compounds in polar solutions and remove oils from the extract. For biological samples with complex compositions and rich in lipids, sugars, and pigments, the combination packing material of N-propylethylenediamine (PSA) and octadecyl bonded silica (C-18) can effectively adsorb interfering substances in biological samples that affect subsequent detection. At the same time, it can purify neonicotinoid insecticides and their metabolites. The operation is simple and quick, making it a relatively ideal packing material for dispersion solid phase extraction.

[0047] According to embodiments of this disclosure, a Waters Atlantis T3 liquid chromatography column with an inner diameter of 2.1 mm × length of 150 mm and a particle size of 3 μm is used for chromatographic separation during detection. Alternatively, a reversed-phase column equivalent to the Waters Atlantis T3 can be selected. Because the metabolites of neonicotinoid insecticides are highly polar and weakly basic, the interaction between the long alkyl chains of the stationary phase of a traditional C18 column and the strongly polar groups is weak, making chromatographic retention difficult. Furthermore, the residual silanol groups in the C18 column easily adsorb basic compounds, causing peak tailing and adversely affecting the detection of neonicotinoid insecticide metabolites using LC-MS / MS. However, the WatersAtlantis T3 liquid chromatography column selected in this disclosure is a reversed-phase C18 column. Compared with the trimethylsilane (TMS) end-capping of traditional C18 columns, its end-capping process can provide superior retention of polar compounds and water compatibility, while also improving peak shape and stability. It can provide excellent performance over a wider pH range and effectively separate different types of neonicotinoid insecticides and their metabolites.

[0048] According to embodiments of this disclosure, a binary mobile phase is used for gradient elution, wherein the binary mobile phase includes mobile phase A and mobile phase B; mobile phase A is a mixed aqueous solution of formic acid and ammonium acetate, comprising a mixed aqueous solution of 0.01% formic acid and 5mM ammonium acetate by volume; mobile phase B is methanol (LC-MS grade methanol). The flow rate in the binary mobile phase gradient elution can be set to 0.5 mL / min, the injection volume is 10 μL, and the mobile phase gradient elution program can be adjusted according to actual needs.

[0049] According to the embodiments of this disclosure, the amount of animal sample added to the solvent is 90~110 mg / mL, for example, 90 mg / mL, 95 mg / mL, 100 mg / mL, 110 mg / mL, etc.; the amount of solvent directly determines whether the internal standard concentration in the animal sample extract is accurate, and has a significant impact on the final determination result. The amount error should not exceed ±1%.

[0050] The amount of dispersive solid phase extraction (DSPE) packing added to the extract is 180~220 mg / mL, for example, it can also be 180 mg / mL, 190 mg / mL, 200 mg / mL, 210 mg / mL, etc. Adding too much dispersive solid phase extraction packing will cause waste and may even adsorb the target compound neonicotinoid insecticide and its metabolites. Adding too little will not be able to adsorb impurities cleanly and will affect the test results.

[0051] According to embodiments of this disclosure, the animal sample is a solid or semi-solid sample, including any one of animal muscle, animal tissues and organs. The animal sample can be a fresh animal sample or an animal sample that has been pre-freeze-dried and dehydrated, including animal muscle and some tissues and organs, such as solid or semi-solid animal samples such as heart, liver, lungs, brain, stomach, kidneys, skin, and blood.

[0052] According to embodiments of this disclosure, the neonicotinoid insecticides include any one or more of the following: fipronil, flonicamid, imidacloprid, acetamiprid, thiamethoxam, thiamethoxam, acetamiprid, flupyrflurane, and thiamethoxam.

[0053] The metabolites of neonicotinoid insecticides include any one or more of denitrifying imidacloprid hydrochloride, amide thiamethoxam, and demethyl acetamiprid.

[0054] As another aspect of this disclosure, a method for determining the content of neonicotinoid insecticides and their metabolites in animal samples is provided for the application in detecting the quality of the ecological environment.

[0055] To make the objectives, technical solutions, and advantages of this disclosure clearer, the technical solutions and principles of this disclosure are further illustrated below with reference to specific embodiments and accompanying drawings. It should be noted that the specific embodiments described below are merely illustrative examples, and the scope of protection of this disclosure is not limited thereto.

[0056] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available. Methods not specifically described in the examples are conventional and can be performed according to the techniques or conditions described in the literature or the product instructions.

[0057] Example 1

[0058] Nine neonicotinic insecticide standards were prepared: dinotefuran (DIN), flonicamid (SUL), imidacloprid (IMI), acetamiprid (ACE), thiamethoxam (CLO), thiamethoxam (TCP), nitenpyram (NIT), flupyrflufenone (FLU), and thiamethoxam (TMX); three neonicotinic insecticide metabolite standards: denitrified imidacloprid hydrochloride (Des-IMI-hyCl), amide thiamethoxam (TCP-amide), and demethyl acetamiprid (IM-2-1); and ten isotopically labeled standards (IMI-d4, ACE-...). 13 C, NIT- 13 C 15 N, TCP- 13 C, TMX- 13 C, CLO- 13 C, DIN-13 C, SUL- 13 C 15 N、IM-2-1- 13 C. TCP-amide- 13 C 15 The N) were diluted with methanol to prepare mixed standard stock solutions with a concentration of 1 mg / L. Mixed standard working solutions of 0.1~100 μg / L were prepared using various stock solutions. Specifically, these included DIN, NIT, TMX, CLO, IMI, ACE, TCP, SUL, FLU, IM-2-1, des-IMI-hyCl, and TCP-amide. The concentrations of each compound were 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 25 ng / mL, 50 ng / mL, and 100 ng / mL. The concentration of the isotope-labeled standard in the mixed standard working solutions was 20 μg / L.

[0059] The test solution was tested using high performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-MS / MS):

[0060] The high-performance liquid chromatograph (HPLC) was a Dionex Ultimate 3000 HPLC system. Chromatographic separation was performed using a Waters Atlantis T3 column (2.1 mm × 150 mm, 3 μm) at a column temperature of 35 °C. Mobile phase A consisted of a mixed aqueous solution of 0.01% formic acid and 5 mM ammonium acetate (v / v), and mobile phase B consisted of methanol. Gradient elution was performed using a binary mobile phase at a flow rate of 0.5 mL / min. The injection volume was 10 μL. The gradient elution program was set as follows: 0% mobile phase B for 0-1 minute; 100% mobile phase B for 4 minutes; 100% mobile phase B for 2 minutes; and 10% mobile phase B for 10 minutes.

[0061] The triple quadrupole mass spectrometer used was a Thermo Fisher Scientific TSQ Quantiva triple quadrupole mass spectrometer. The ion source was an electrospray ionization source, with high-purity argon as the collision gas and high-purity nitrogen as the nebulizer gas. Reaction monitoring scan (SRM) was selected as the data acquisition method. In positive ion mode, the spray voltage was 3.5 kV, the ion transfer tube and spray cone temperature were set to 350 °C, and the sheath gas pressure was 45 arb; the auxiliary gas pressure was 20 arb. In negative ion mode, the spray voltage was 2.8 kV, the ion transfer tube and spray cone temperature were set to 350 °C, the sheath gas pressure was 45 arb, and the auxiliary gas pressure was 10 arb.

[0062] When testing a solution containing neonicotinoid insecticides and their metabolites, the parameters of the instrument testing program should be adjusted according to Table 1. When testing neonicotinoid insecticides and their metabolites with isotopic internal standards, the parameters of the instrument testing program should be adjusted according to Table 2. Within the selected retention time, there may be a retention time drift of ±0.05 min. If it exceeds this range, the test should be repeated.

[0063] Table 1. LC-MS / MS parameters for determining neonicotinoid insecticides and their metabolites.

[0064]

[0065] Table 2. LC-MS / MS parameters for determining the isotopic internal standard neonicotinoid insecticides and their metabolites.

[0066]

[0067] Mixed standards at various concentration gradients were analyzed, and internal standard curves were plotted with the ratio of selected ion pair peak areas of the target analyte and its corresponding isotopic internal standard as the ordinate and the corresponding target analyte concentration as the abscissa. The correlation coefficients (R0) of the standard curves for nine neonicotinoid insecticides and three neonicotinoid insecticide metabolites were analyzed after testing. 2 The range of 0.9986-0.9996 indicates a good linear relationship in the test curve and a high degree of agreement with the original data, demonstrating the high feasibility of the method. For different types of neonicotinoid insecticides and their metabolites, the instrument detection limit obtained by this method is 0.07-6.75 ng / L, and the quantitation limit is 0.36-19.48 ng / L, effectively reducing the method detection limit.

[0068] Example 2

[0069] After thoroughly dehydrating and drying the red shrimp muscle using a vacuum freeze dryer, it was then pulverized and homogenized into a powder using a grinder. 1 g of homogenized red shrimp muscle was taken, and 200 μL of a mixed standard of neonicotinoid insecticides and their metabolites (IMI-d4, ACE-) was added to the homogenized red shrimp muscle. 13 C, NIT- 13 C 15 N, TCP- 13 C, TMX- 13 C, CLO- 13 C, DIN- 13 C, SUL- 13 C 15 N、IM-2-1- 13 C. TCP-amide- 13 C 15(N), where the concentration of each isotope mixed standard is 0.1 mg / L.

[0070] Take 1 g of homogenized red shrimp muscle and add 200 μL of a mixed standard of neonicotinoid insecticides and their metabolites (DIN, NIT, TMX, CLO, IMI, ACE, TCP, SUL, FLU, IM-2-1, des-IMI-hyCl, TCP-amide), where the concentration of each substance in the mixed standard is 0.1 mg / L; then add 200 μL of a mixed isotopic standard of neonicotinoid insecticides and their metabolites (IMI-d4, ACE- 13 C, NIT- 13 C 15 N, TCP- 13 C, TMX- 13 C, CLO- 13 C, DIN- 13 C, SUL- 13 C 15 N、IM-2-1- 13 C. TCP-amide- 13 C 15 (N), where the concentration of each isotope mixed standard is 0.1 mg / L.

[0071] Red shrimp muscle samples containing mixed standards and isotope mixed standards were soaked in 10 mL of ultrapure water and sonicated for 10 min to moisten the samples. Then, 10 mL of acetonitrile solution containing 1% acetic acid was added to extract and enrich neonicotinoid insecticides and their metabolites from the samples. After mixing thoroughly, the samples were sonicated for 15 min. After treatment, the samples were salted out using an Agilent EN 15662 extraction salt kit. After the organic and aqueous phases separated, the upper organic phase was collected to obtain the extract. Impurities in the extract were removed by adsorption using a 1:1 mixture of N-propylethylenediamine (PSA) and octadecyl bonded silica gel (C-18). The extract was then transferred to a clean collection bottle, and the eluent was blown to near dryness using high-purity nitrogen at a suitable flow rate. The volume was then adjusted to 0.5 mL with methanol and transferred to a vial to obtain the test solution, which was stored at -20°C for later analysis.

[0072] The test solution was analyzed using a high-performance liquid chromatography-tandem triple quadrupole mass spectrometer. The instrument parameters were adjusted according to Tables 1 and 2 in Example 1, such as… Figure 2 The results show chromatograms of 9 neonicotinoid insecticides, 3 neonicotinoid insecticide metabolites, and 10 isotopic internal standard compounds. Figure 2This is a chromatographic separation diagram of neonicotinoid insecticides and their metabolites in animal samples from Example 2 of this disclosure. It can be seen that the method provided in this disclosure can simultaneously determine multiple neonicotinoid insecticides and their metabolites, with both high detection limits and high detection accuracy. The concentrations of compounds in the muscle of red shrimp were calculated using the internal standard method. The occurrence of neonicotinoid insecticides and their metabolites in the muscle of red shrimp were as follows: C(DIN) = 0.051 ng / g(ww), C(NIT) = 0.117 ng / g(ww), C(TMX) = 0.034 ng / g(ww), C(IMI) = 0.031 ng / g(ww), C(ACE) = 0.040 ng / g(ww), C(SUL) = 0.026 ng / g(ww), C(IM-2-1) = 0.138 ng / g(ww). The recoveries of neonicotinoid insecticides and their metabolites in the muscle of red shrimp were between 74.62% and 103.7%.

[0073] Example 3

[0074] The same test method as in Example 2 was used, except that the red shrimp muscle was replaced with silver pomfret muscle after homogenization. 1 g of homogenized silver pomfret muscle was taken and 200 μL of a mixed standard of neonicotinoid insecticides and their metabolites (IMI-d4, ACE-) was added. 13 C, NIT- 13 C 15 N, TCP- 13 C, TMX- 13 C, CLO- 13 C, DIN- 13 C, SUL- 13 C 15 N、IM-2-1- 13 C. TCP-amide- 13 C 15 (N), where the concentration of each isotope mixed standard is 0.1 mg / L.

[0075] Take another 1 g of homogenized silver pomfret muscle and add 20 μL of a mixed standard of neonicotinoid insecticides and their metabolites (DIN, NIT, TMX, CLO, IMI, ACE, TCP, SUL, FLU, IM-2-1, des-IMI-hyCl, TCP-amide), and 200 μL of a mixed standard of isotopes of neonicotinoid insecticides and their metabolites (IMI-d4, ACE- 13 C, NIT- 13 C 15 N, TCP- 13 C, TMX- 13 C, CLO-13 C, DIN- 13 C, SUL- 13 C 15 N、IM-2-1- 13 C. TCP-amide- 13 C 15 N), wherein the concentration of each substance in the mixed standard and the concentration of the mixed standard of various isotopes are both 0.1 mg / L.

[0076] Figure 3 This is a chromatogram showing the separation of neonicotinoid insecticides and their metabolites in animal samples from Example 3 of this disclosure. The concentrations of compounds in silver pomfret muscle were calculated using the internal standard method, and the final concentrations of neonicotinoid insecticides and their metabolites in silver pomfret muscle were as follows: C(DIN) = 0.100 ng / g (ww), C(ACE) = 0.009 ng / g (ww). The recoveries of neonicotinoid insecticides and their metabolites in silver pomfret muscle were between 76.28% and 96.76%.

[0077] The method for determining the content of neonicotinoid insecticides and their metabolites in animal samples, as provided in this disclosure, removes water from the extract using a solvent and a QuEChERS extraction salt pack, thus protecting the extraction and enrichment of neonicotinoid insecticides and their metabolites in the animal sample. The extract is then purified using a dispersed solid-phase extraction packing material. Combined with high-performance liquid chromatography-tandem triple quadrupole mass spectrometry, this method can simultaneously and rapidly determine multiple neonicotinoid insecticides and their metabolites in animal samples. By optimizing the test parameters, high selectivity and high sensitivity for target compounds can be obtained, enabling rapid identification and accurate quantification of trace neonicotinoid insecticides and their metabolites in animal samples.

[0078] The specific embodiments described above further illustrate the purpose, technical solutions, and beneficial effects of this disclosure. It should be understood that the above descriptions are merely specific embodiments of this disclosure and are not intended to limit this disclosure. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this disclosure should be included within the protection scope of this disclosure.

Claims

1. A method for determining the content of thiamethoxam and its metabolite amide thiamethoxam in animal samples, comprising: After soaking the animal samples in water and ultrasonically wetting and activating them, a solvent was added and thoroughly vortexed and mixed. Then, QuEChERs salt packs and ceramic homogenants were added and thoroughly shaken to protect the thiamethoxam and its metabolite amide thiamethoxam in the animal samples. While extracting and enriching them into the extract, the water in the extract was removed, and the organic phase and aqueous phase were separated into layers. The upper organic solution was then extracted by centrifugation to obtain the extract. The QuEChERs salt pack is a buffered inorganic salt mixture, including the Agilent EN 15662 extraction salt kit, which contains magnesium sulfate, sodium chloride, sodium citrate, and sodium bicarbonate sesquihydrate. Sodium citrate and sodium bicarbonate sesquihydrate can form a pH buffer system, allowing pH-sensitive thiamethoxam and its metabolite thiamethoxam to maintain their molecular form and improving their partition efficiency in the organic phase. A dispersed solid-phase extraction packing material is added to the extract, wherein the dispersed solid-phase extraction packing material is an adsorbent and purifying agent capable of removing lipids, sugars and pigments, comprising a mixture of N-propylethylenediamine and octadecyl bonded silica gel in a ratio of 1:

1. After purifying the extract, the extract is concentrated and brought to a fixed volume to obtain the solution to be tested. The test solution was detected by high performance liquid chromatography-triple quadrupole mass spectrometry, and chromatographic separation was performed using a Waters Atlantis T3 liquid chromatography column with gradient elution using a binary mobile phase to obtain the content of thiamethoxam and its metabolite amide thiamethoxam in the animal sample; the animal sample was red shrimp muscle or silver pomfret muscle. The binary mobile phase includes mobile phase A and mobile phase B; The mobile phase A is a mixed aqueous solution of formic acid and ammonium acetate, comprising a mixed aqueous solution of formic acid and 5 mM ammonium acetate at a volume percentage of 0.01%; the mobile phase B is methanol. The mobile phase gradient elution program is set as follows: mobile phase B is 0% within 0-1 minute; mobile phase B is increased to 100% within 4 minutes; mobile phase B is maintained at 100% for 2 minutes; and mobile phase B is decreased to 10% within 10 minutes.

2. The method according to claim 1, wherein, The solvent is an acetonitrile-acetic acid mixed solution, including acetonitrile containing 1% acetic acid.

3. The method according to claim 1, wherein, The high-performance liquid chromatography-triple quadrupole mass spectrometry method uses an electrospray ionization source in both positive and negative ion modes.

4. The method according to claim 1, wherein, The amount of the animal sample added to the solvent was 90-110 mg / mL; The amount of the dispersed solid-phase extraction packing added to the extraction solution is 180~220 mg / mL.

5. The application of the method as described in any one of claims 1 to 4 in detecting ecological environment quality and food quality.

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