Method for extracting neonicotinoid insecticide and / or metabolite thereof from biological sample and application of neonicotinoid insecticide and / or metabolite thereof

Abstract

The invention discloses a method for extracting neonicotinoid insecticides and / or metabolites thereof from a biological sample and application of the neonicotinoid insecticides and / or metabolites thereof. The method realizes extraction and detection of as many as 10 neonicotinoid insecticides and metabolites thereof in a liquid biological sample and a solid biological sample, reduces errors caused by target object loss in the extraction and determination process, performs quantitative analysis by combining an internal standard method and an ultra-high performance liquid chromatography, improves the accuracy of a detection result, and reduces the detection cost. A new technical means is provided for monitoring residues of the neonicotinoid insecticide and metabolites thereof in organisms, the use of the neonicotinoid insecticide can be more normatively and strictly controlled, and the safety risk is reduced.

Description

Technical Field

[0001] The present invention relates to the field of pesticide detection, and in particular to a method for extracting neonicotinoid insecticides and / or their metabolites from biological samples and applications thereof. Background Art

[0002] Neonicotinoids have a unique neurotoxic mode of action, which targets nicotinic acetylcholine receptors on insect nerve cell membranes, leading to nerve signal transmission disorders and ultimately insect death. Neonicotinoids are widely used due to their broad-spectrum insecticidal activity, low toxicity to mammals, high efficacy and selectivity, and have gradually replaced older insecticides such as organophosphates and carbamates.

[0003] Studies have shown that exposure to neonicotinoids in the uterus of pregnant mice can lead to a "mother-to-child transmission" effect and may cause lasting damage to the behavior and brain function of their offspring. It can be seen that exposure to neonicotinoids may be harmful to mammals, such as reproductive toxicity, neurotoxicity, hepatotoxicity, genotoxicity, and endocrine disruption. Residues of neonicotinoid pesticides have been detected in water, soil, indoor dust, food, and even multiple human specimens. Regular monitoring of neonicotinoid pesticides and their metabolites in the human body will help to strictly control neonicotinoid pesticides and reduce residual risks.

[0004] At present, there are some methods for detecting neonicotinoid pesticides in liquid biological samples (such as CN113009057A and CN115901992A), but these methods are not suitable for detecting solid biological samples. The prior art (CN118393045A) has disclosed a method for detecting neonicotinoids and their metabolites in hair or nails, but the detected targets are relatively few. Summary of the invention

[0005] In order to solve the problem in the prior art that there is a lack of methods that can detect neonicotinoid pesticides and their metabolites in both liquid biological samples and solid biological samples, and to expand the detection range of target objects in solid biological samples, the present invention provides a method and application for extracting neonicotinoid pesticides and their metabolites from biological samples.

[0006] An object of the present invention is to provide a method for extracting neonicotinoid insecticides and / or their metabolites from biological samples.

[0007] Another object of the present invention is to provide application of the method in detecting neonicotinoid insecticides and / or their metabolites.

[0008] Another object of the present invention is to provide a method for detecting neonicotinoid insecticides and / or their metabolites.

[0009] In order to achieve the above object, the present invention is implemented by the following scheme:

[0010] The present invention first extracts neonicotinoid insecticides and their metabolites by liquid-liquid extraction using a mixed solvent of acetonitrile and dichloromethane, then adds formic acid to ionize them for subsequent column filtration, and then removes impurities by solid phase extraction, thereby reducing the interference of impurities and the influence of the matrix, obtaining an extract with higher purity, and greatly increasing the sensitivity of detection. Chromatography and mass spectrometry have the advantages of high sensitivity, high accuracy, high precision and good reproducibility. The present invention further performs sample detection by liquid chromatography tandem mass spectrometry to determine the content of neonicotinoid insecticides and their metabolites in a variety of biological samples.

[0011] A method for extracting neonicotinoid insecticides and / or their metabolites from a biological sample, wherein the neonicotinoid insecticides and / or their metabolites include at least one of acetamiprid, N-demethylacetamiprid, imidacloprid, 5-hydroxyimidacloprid, thiamethoxam, thiacloprid, thiacloprid amide, clothianidin, chlorothiazide and sulfoxaflor;

[0012] The method comprises the following steps: thoroughly mixing the biological sample and the extraction solvent, separating the layers, collecting the top layer solution to obtain a supernatant; mixing the supernatant with a 1-3% v / v formic acid aqueous solution to obtain a primary extraction product; performing solid phase extraction on the primary extraction product to obtain an extract containing the neonicotinoid insecticide and / or its metabolites;

[0013] The extraction solvent includes acetonitrile and dichloromethane, and the volume ratio of the acetonitrile to the dichloromethane is (0.5-2):1.

[0014] Preferably, the biological sample is a solid biological sample or a liquid biological sample.

[0015] More preferably, the solid biological sample comprises a stool sample.

[0016] Further preferably, the stool sample is a stool sample of an infant under 1 year old.

[0017] Further preferably, the biological sample is a solid biological sample, which is first dried and crushed, and then fully mixed with the extraction solvent.

[0018] More preferably, the method for drying the solid biological sample comprises freeze-drying.

[0019] More preferably, the method for comminuting the solid biological sample comprises grinding.

[0020] More preferably, the liquid biological sample comprises a urine sample.

[0021] Preferably, the volume ratio of the acetonitrile to the dichloromethane is 2:1.

[0022] Preferably, the supernatant is mixed with a 2% v / v formic acid aqueous solution.

[0023] Preferably, the biological sample and the extraction solvent are fully mixed, separated into layers, the top solution is collected to obtain supernatant 1, and the remaining solution is collected to obtain residual liquid; the residual liquid is fully mixed with the extraction solvent, separated into layers, and the top solution is collected to obtain supernatant 2; supernatant 1 and supernatant 2 are combined to obtain a mixed supernatant; the mixed supernatant is mixed with a 1-3% v / v formic acid aqueous solution to obtain a primary extraction product; the primary extraction product is subjected to solid phase extraction to obtain an extract containing the neonicotinoid insecticide and / or its metabolites. In this scheme, a total of 2 liquid-liquid extractions are performed.

[0024] Preferably, the biological sample and the extraction solvent are fully mixed, separated into layers, the top solution is collected to obtain supernatant 1, and the remaining solution is collected to obtain residual solution 1; the residual solution 1 is fully mixed with the extraction solvent, separated into layers, the top solution is collected to obtain supernatant 2, and the remaining solution is collected to obtain residual solution 2; the residual solution 2 is fully mixed with the extraction solvent, separated into layers, and the top solution is collected to obtain supernatant 3; supernatants 1 to 3 are combined to obtain a mixed supernatant; the mixed supernatant is mixed with a 1 to 3% v / v formic acid aqueous solution to obtain a primary extraction product; the primary extraction product is subjected to solid phase extraction to obtain an extract containing the neonicotinoid insecticide and / or its metabolites. In this scheme, a total of 3 liquid-liquid extractions are performed.

[0025] Preferably, the method of thoroughly mixing the biological sample and the extraction solvent comprises vortexing and / or ultrasound.

[0026] More preferably, the vortexing time is 0.5 to 2 minutes.

[0027] More preferably, the vortexing time is 1 min.

[0028] More preferably, the ultrasonic conditions include: frequency 30-50 KHz, power 80-120 W, time 10-20 min.

[0029] Further preferably, the ultrasonic conditions include: frequency 40KHz, power 100W, time 15min.

[0030] Preferably, the method of stratification comprises centrifugation.

[0031] More preferably, the centrifugal conditions include: a rotation speed of 4500-5500 rpm and a time of 5-15 min.

[0032] Further preferably, the centrifugal conditions include: rotation speed 5000 rpm, time 10 min.

[0033] Preferably, the mass volume ratio of the biological sample to the extraction solvent is 25 mg:7.5 mL.

[0034] Preferably, the volume ratio of the supernatant to the aqueous formic acid solution is 1:(4-8).

[0035] More preferably, the volume ratio of the supernatant to the aqueous formic acid solution is 1:6.

[0036] Preferably, the solid phase extraction comprises the following steps: adding the initial extraction product to an activated solid phase extraction column, washing with a column washing liquid, drying, eluting with methanol, and collecting the eluate; the column washing liquid comprises methanol, formic acid and water, and the volume ratio of methanol, formic acid and water is 10:2:(80-100).

[0037] More preferably, the volume ratio of methanol, formic acid and water is 10:2:88.

[0038] Preferably, the biological sample includes any one or more of body fluids, feces or hair.

[0039] The application of any of the methods described in detecting neonicotinoid insecticides and / or their metabolites should also be within the scope of protection of the present invention.

[0040] A method for detecting neonicotinoid insecticides and / or their metabolites, comprising the following steps: extracting a sample to be tested using the method for extracting neonicotinoid insecticides and / or their metabolites from a biological sample to obtain an extract, and then detecting the extract using ultra-high performance liquid chromatography;

[0041] The method comprises the following steps: fully mixing the sample to be tested and the extraction solvent, separating the layers, collecting the top layer solution to obtain a supernatant; mixing the supernatant with a 1-3% v / v formic acid aqueous solution to obtain a primary extraction product; performing solid phase extraction on the primary extraction product to obtain an extract containing the neonicotinoid insecticide and / or its metabolites; the extraction solvent comprises acetonitrile and dichloromethane, and the volume ratio of the acetonitrile to the dichloromethane is (0.5-2):1;

[0042] The neonicotinoid insecticide and / or its metabolites include at least one of acetamiprid, N-demethylacetamiprid, imidacloprid, 5-hydroxyimidacloprid, thiamethoxam, thiacloprid, thiacloprid amide, clothianidin, chlorothiazolin and sulfoxaflor.

[0043] Preferably, the biological sample is a solid biological sample or a liquid biological sample.

[0044] More preferably, the solid biological sample comprises a stool sample.

[0045] Further preferably, the stool sample is a stool sample of an infant under 1 year old.

[0046] Further preferably, the biological sample is a solid biological sample, which is first dried and crushed, and then fully mixed with the extraction solvent.

[0047] More preferably, the method for drying the solid biological sample comprises freeze-drying.

[0048] More preferably, the method for comminuting the solid biological sample comprises grinding.

[0049] More preferably, the liquid biological sample comprises a urine sample.

[0050] Preferably, the biological sample and the extraction solvent are fully mixed, separated into layers, the top solution is collected to obtain supernatant 1, and the remaining solution is collected to obtain residual liquid; the residual liquid is fully mixed with the extraction solvent, separated into layers, and the top solution is collected to obtain supernatant 2; supernatant 1 and supernatant 2 are combined to obtain a mixed supernatant; the mixed supernatant is mixed with a 1-3% v / v formic acid aqueous solution to obtain a primary extraction product; the primary extraction product is subjected to solid phase extraction to obtain an extract containing the neonicotinoid insecticide and / or its metabolites. In this scheme, a total of 2 liquid-liquid extractions are performed.

[0051] Preferably, the biological sample and the extraction solvent are fully mixed, separated into layers, the top solution is collected to obtain supernatant 1, and the remaining solution is collected to obtain residual solution 1; the residual solution 1 is fully mixed with the extraction solvent, separated into layers, the top solution is collected to obtain supernatant 2, and the remaining solution is collected to obtain residual solution 2; the residual solution 2 is fully mixed with the extraction solvent, separated into layers, and the top solution is collected to obtain supernatant 3; supernatants 1 to 3 are combined to obtain a mixed supernatant; the mixed supernatant is mixed with a 1 to 3% v / v formic acid aqueous solution to obtain a primary extraction product; the primary extraction product is subjected to solid phase extraction to obtain an extract containing the neonicotinoid insecticide and / or its metabolites. In this scheme, a total of 3 liquid-liquid extractions are performed.

[0052] Preferably, the method of thoroughly mixing the biological sample and the extraction solvent comprises vortexing and / or ultrasound.

[0053] More preferably, the vortexing time is 0.5 to 2 minutes.

[0054] More preferably, the vortexing time is 1 min.

[0055] More preferably, the ultrasonic conditions include: frequency 30-50 KHz, power 80-120 W, time 10-20 min.

[0056] Further preferably, the ultrasonic conditions include: frequency 40KHz, power 100W, time 15min.

[0057] Preferably, the method of stratification comprises centrifugation.

[0058] More preferably, the centrifugal conditions include: a rotation speed of 4500-5500 rpm and a time of 5-15 min.

[0059] Further preferably, the centrifugal conditions include: rotation speed 5000 rpm, time 10 min.

[0060] Preferably, the internal standard is thoroughly mixed with the sample to be tested and the extraction solvent, and the layers are separated to collect the top layer of the solution.

[0061] More preferably, the internal standard comprises isotope-labeled acetamiprid, isotope-labeled N-demethylacetamiprid, isotope-labeled imidacloprid, isotope-labeled 5-hydroxyimidacloprid, isotope-labeled thiamethoxam, isotope-labeled thiacloprid, isotope-labeled thiamethoxam amide, isotope-labeled clothianidin, isotope-labeled chlorpyrifos and isotope-labeled fluphenazine.

[0062] Further preferably, the isotope is a stable isotope.

[0063] Still more preferably, the stable isotopes include carbon, deuterium or nitrogen.

[0064] Still further preferably, the internal standard for detecting acetamiprid is 13 C6-acetamiprid, the internal standard for detecting N-demethylacetamiprid is 13 C2- 15 N-demethylacetamiprid, the internal standard for detecting imidacloprid is D4-imidacloprid, and the internal standard for detecting 5-hydroxyimidacloprid is 13 C- 15 N2-5-hydroxyimidacloprid, the internal standard for detecting thiamethoxam is 13 C4-15 N -Thiamethoxam, the internal standard for detecting thiamethoxam is 13 C6-thiacloprid, the internal standard for detecting thiacloprid amide is 13 C6-thiamethoxam, the internal standard for detecting clothianidin is 13 C4- 15 N-thiamethoxam, the internal standard for detecting chlorothiazide is D4-chlorothiazide, and the internal standard for detecting sulfoxaflor is D3- 13 C2- 15 N2-sulfoxaflor.

[0065] Preferably, the chromatographic conditions of the ultra-high performance liquid chromatography include: using a C18 chromatographic column; using 0.1% v / v formic acid aqueous solution as mobile phase A, using acetonitrile as mobile phase B, and in the gradient elution program, the volume percentage of mobile phase B changes as follows: 0-0.6 min, mobile phase B is 95%; 0.6-4.5 min, mobile phase B is 95%-1%; 4.5-5.5 min, mobile phase B is 1%; 5.5-6 min, mobile phase B is 1%-95%; 6-8 min, mobile phase B is 95%.

[0066] More preferably, the C18 chromatographic column is a Betasil C18 column with a length of 100 mm and an inner diameter of 2.1 mm and a filler particle diameter of 5 μm.

[0067] More preferably, the column temperature of the C18 chromatographic column is 38-40°C.

[0068] More preferably, the column temperature of the C18 chromatographic column is 40°C.

[0069] More preferably, the chromatographic conditions of the ultra-high performance liquid chromatography also include: an injection volume of 4 to 5 μL; a flow rate of 0.4 to 0.5 mL / min.

[0070] Further preferably, the chromatographic conditions of the ultra-high performance liquid chromatography also include: injection volume of 5 μL; flow rate of 0.5 mL / min.

[0071] Preferably, the detection conditions of the mass spectrometry include: using an electrospray ionization source, the scanning modes are positive ion mode and negative ion mode, the detection mode is multiple ion reaction detection mode, the dryer temperature is 275°C, the drying gas flow rate is 10L / min, the nebulizer pressure is 55psi, the sheath gas temperature is 350°C, the sheath gas flow rate is 11L / min, and the capillary voltage is 3500V.

[0072] Compared with the prior art, the present invention has the following beneficial effects:

[0073] The present invention realizes the extraction and detection of up to 10 neonicotinoid insecticides and their metabolites in liquid biological samples and solid biological samples, reduces the error caused by the loss of target objects during the extraction and determination process, combines the internal standard method and ultra-high performance liquid chromatography for quantitative analysis, and improves the accuracy of the detection results. It provides a new technical means for monitoring the residues of neonicotinoid insecticides and their metabolites in organisms, helps to standardize and strictly control the use of neonicotinoid insecticides, and reduce safety risks. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 The chromatograms are of 50 ng / mL standard solutions of 10 neonicotinoid insecticides and their metabolites. DETAILED DESCRIPTION

[0075] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0076] The instruments and equipment used in the following examples include: ultra-high performance liquid chromatograph, model: Agilent t1290infinityⅡHPLC system, manufacturer: Agilent; triple quadrupole mass spectrometer, model: Agilent 6470B, manufacturer: Agilent; chromatographic column, model: Betasil C18 column, 100×2.1mm, 5μm, manufacturer: Thermo Fisher Scientific; pipette, model 10 / 20 / 100 / 200 / 1000 / 5000μL:, manufacturer: Eppendorf; ultrasonic cleaning machine, model: KQ-700TDE, manufacturer: Kunshan Ultrasonics Corporation; electronic analytical balance, model: XSR204, manufacturer: Mettler Toledo; pure water system, model: Milli-Q Integral system, manufacturer: Millipore; centrifuge, model: Centrifuge 5804, manufacturer: Eppendorf; vacuum freeze dryer, model: FD-1A-50, manufacturer: Boyikang; solid phase extraction device, manufacturer: Merck; nitrogen blowdown apparatus, manufacturer: Organomation Ass ociates Inc.

[0077] The reagents and materials used in the following examples include: N-dm-ACE (purity 97.6%), IMZ (purity 98.0%), CLO (purity 98.6%), THX (purity 99.6%), THI (purity 99.2%), IMI (purity 98.7%), ACE (purity 99.6%), TA (purity 100%), SUF (purity 98.0%), 5-OH-IMI (Liquid), 13 C2- 15 NN-dm-ACE (Liquid), D4-IMZ (purity 97.2%), 13 C4

[0078] - 15 N-CLO(Liquid), 13 C4- 15 N-THX(Liquid),13 C6-THI (Liquid), D4-IMI (purity 99.0%), 13 C6-ACE(Liquid), 13 C6-TA(Liquid), D3- 13 C2- 15 N2-SUF(Liquid), 13 C- 15 N2-5-OH-IMI (Liquid) was purchased from CATO Research Chemicals Inc., Eugene, OR, USA; methanol (LC-MS), acetonitrile (LC-MS), and formic acid (LC-MS) were purchased from Fisher; and dichloromethane (analytical grade) was purchased from Komiou.

[0079] Example 1 Method for detecting 10 neonicotinoid insecticides and their metabolites in a sample

[0080] Table 1 Information on 10 neonicotinoid insecticides, their metabolites and internal standards

[0081]

[0082] The method provided in this embodiment detects 10 neonicotinoid insecticides and their metabolites as shown in Table 1, and the specific steps are as follows:

[0083] 1. Solvent preparation

[0084] (1) Mixed internal standard solution

[0085] The 10 isotope-labeled internal standards shown in Table 1 were dissolved in methanol at a final concentration of 500 ng / mL, respectively, and mixed evenly to obtain a mixed internal standard solution with a concentration of 50 ng / mL.

[0086] (2) Extraction solvent

[0087] Acetonitrile and dichloromethane were mixed in a volume ratio of 2:1 to obtain an extraction solvent.

[0088] (3) Column washing liquid

[0089] Methanol, formic acid and water were mixed in a volume ratio of 10:2:88 to obtain a column washing solution.

[0090] (4) Reconstitution solution

[0091] Methanol and water were mixed at a volume ratio of 1:3, and formic acid was added to a final concentration of 0.1% v / v to obtain a complex solution.

[0092] (5) Mixed standard solution

[0093] The 10 target standards shown in Table 1 were dissolved in methanol to obtain a mixed standard stock solution, wherein the final concentration of each target standard was 10 μg / mL.

[0094] The mixed standard stock solution was then gradiently 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, 2 ng / mL, 5 ng / mL, 10 ng / mL, 20 ng / mL and 50 ng / mL.

[0095] 2. Sample pretreatment

[0096] (1) Liquid-Liquid Extraction

[0097] Accurately weigh 25 mg of liquid sample or ground solid sample and put it into a 10 mL polypropylene centrifuge tube, add 50 μL of mixed internal standard solution, then add 2.5 mL of extraction solvent, vortex mix for 1 min, then ice bath ultrasonic (100 W, 40 kHz) for 15 min, then centrifuge at 5000 rpm / min for 10 min, collect the top layer of clear liquid and put it into a new centrifuge tube, record it as supernatant 1; add 2.5 mL of extraction solvent to the original centrifuge tube again, repeat the above vortex, ultrasonic and centrifugal process, repeat twice in total, obtain supernatant 2 and supernatant 3 in turn, that is, perform liquid-liquid extraction 3 times in total; combine supernatants 1 to 3, gently blow nitrogen at 25°C, concentrate to a volume of 0.5 mL, then add 3 mL of 2% v / v formic acid aqueous solution, mix well, and obtain the primary extraction product.

[0098] (2) Solid Phase Extraction

[0099] A solid phase extraction column (Bond Elut Plexa (60 mg / 3 mL, Agilent)) was activated by 3 mL of methanol and 3 mL of water, and then the initial extraction product was passed through the solid phase extraction column and washed with 2 mL of column washing liquid. It was vacuum dried under negative pressure conditions (10 MPa) for 1 min to remove the influence of the solvent in the column, and finally eluted with 3 mL of methanol. The eluate was collected and evaporated to dryness in mild nitrogen at 25°C to obtain the extracted product.

[0100] (3) Redissolution

[0101] The extracted product was dissolved in 250 μL of re-solution, vortexed for 1 min, and then filtered through a 0.22 μm PTFE filter membrane into a brown injection vial to obtain the sample for subsequent testing.

[0102] 3. Ultra-high performance liquid chromatography detection

[0103] The detection conditions are as follows: the chromatographic column is Betasil C18 column (length 100×inner diameter 2.1mm, filler particle diameter 5μm), the column temperature is set at 40°C, the injection volume is 5μL; the mobile phase A is 0.1% v / v formic acid aqueous solution, the mobile phase B is acetonitrile, the flow rate is 0.5mL / min, and the gradient elution program is shown in Table 2.

[0104] Table 2 Gradient elution program

[0105]

[0106] According to the above method, chromatographic detection was performed, and when the final concentration of each compound standard in the mixed standard solution was 50 ng / mL, the following was obtained: Figure 1 As shown in the chromatogram, it can be seen that the 10 neonicotinoid insecticides and their metabolites have clear characteristic peaks.

[0107] 4. Mass spectrometry analysis

[0108] The mass spectrometry conditions were as follows: an electrospray ionization source (ESI) was used, the scanning modes were positive ion mode and negative ion mode, the detection mode was multiple ion reaction detection mode, the dryer temperature was 275°C, the drying gas flow rate was 10 L / min, the nebulizer pressure was 55 psi, the sheath gas temperature was 350°C, the sheath gas flow rate was 11 L / min, the capillary voltage was 3500 V, and the mass spectrometry parameters of each target are shown in Table 3.

[0109] Table 3 Mass spectrometry parameters of 10 neonicotinoid insecticides and their metabolites and internal standards

[0110]

[0111]

[0112] 4. Results Analysis

[0113] (1) Establishment of standard curve

[0114] The internal standard method was used to quantitatively analyze 10 neonicotinoid insecticides and their metabolites. The specific steps were as follows: 12 mixed standard solutions of different concentrations were taken for ultra-high performance liquid chromatography detection and mass spectrometry analysis. The ratio of the chromatographic peak area of ​​each target at different concentrations to the chromatographic peak area of ​​the corresponding internal standard was used as the standard area ratio. The standard curve of each target was drawn with concentration as the horizontal axis and area ratio as the vertical axis, as shown in Table 4.

[0115] Table 4 Standard curves of 10 neonicotinoid insecticides and their metabolites

[0116]

[0117] (2) Analysis of the content of each target substance in the sample

[0118] 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 and substituted into the standard curve to calculate the content of each compound in the sample.

[0119] Example 2 Detection of 10 neonicotinoid pesticides and their metabolites in fecal samples

[0120] 1. Matrix Effect

[0121] (1) Stool sample processing and testing

[0122] Considering that the feces matrix may affect the quantitative analysis of the analyte, this example uses the post-extraction addition method to evaluate the matrix effect (ME) of feces samples. The specific steps are as follows:

[0123] The fecal sample was freeze-dried for 3 days, ground, and then processed according to the method of "sample pretreatment" in Example 1 of the present invention, with the only difference that no mixed internal standard solution was added in the step of "liquid-liquid extraction", and the sample obtained was the blank matrix.

[0124] To evaluate the spiked matrix response, 2.5 ng of standard substance and internal standard were spiked into 250 μL of matrix blank sample, and then the detection and analysis were performed according to the method of Example 1 of the present invention to obtain the spiked matrix response (A). Another 250 μL of blank matrix was taken and then the detection and analysis were performed according to the method of Example 1 of the present invention to obtain the blank matrix response (B). The response of the same concentration of the substance in the pure solvent was recorded as (C). The analysis was repeated three times, and the matrix effect (ME) of the target compound was calculated by the following formula:

[0125]

[0126] Among them, A represents the spike matrix effect, B represents the blank matrix effect, and C represents the response of the analyte at the same concentration in pure solvent.

[0127] The limit of detection (LOD) of the target compound was determined by the ratio of the chromatographic peak signal intensity (S) to the noise intensity (N) (S / N=3).

[0128] (2) Test results

[0129] Table 5 Limits of detection (LOD), blank matrix responses and ME of 10 neonicotinoid insecticides and their metabolites

[0130]

[0131] As shown in Table 5, the matrix effects of the 10 NEOs to be tested in fecal samples ranged from 6.4% ± 3.9 to 76.2 ± 2.8. This indicates that SUF has weak matrix inhibition, while other NEOs have obvious matrix inhibition. Correction by the internal standard method can eliminate the influence of the matrix effect of most compounds, meet the analysis requirements, and can accurately quantify even when the matrix effect is large.

[0132] 2. Spike recovery

[0133] (1) Preparation of spiked samples

[0134] This example uses feces of children under 1 year old to conduct methodological evaluation. The feces samples of children were freeze-dried for 3 days, ground, and 20 mg of feces samples were added to the mixed standard solution of the test substance 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.

[0135] (2) Detection

[0136] The spiked samples prepared in this example were used as liquid samples to be tested, and the contents of each neonicotinoid insecticide and its metabolites therein were tested according to the method of Example 1 of the present invention.

[0137] (3) Test results

[0138] Table 6 Spiked recoveries and relative deviations of 10 neonicotinoid insecticides and their metabolites

[0139]

[0140] As shown in Table 6, the spiked recoveries of 10 neonicotinoid insecticides and their metabolites reached 63±33% to 106±14%, indicating that the spiked recoveries and relative deviations of 10 neonicotinoid insecticides and their metabolites in our method were within the acceptable range (70% to 120%).

[0141] 3. The influence of the ratio of extraction reagents on the test results

[0142] A mixed standard solution of 10 target standards with a final concentration of 10 ng / mL was used as the test sample, and the detection was performed according to the method of Example 1 of the present invention, except that the volume ratio of acetonitrile and dichloromethane in the extraction solvent was 2:1, 1:1 or 1:2. The spike recovery rate was calculated.

[0143] The average recovery and relative deviation of each target and concentration are shown in Table 7. Compared with the other two ratios of extraction reagents, the instrument peak area is the largest when the volume ratio of acetonitrile and dichloromethane is 2:1. Therefore, in the range of recovery rate of 70% to 120%, the 2:1 acetonitrile dichloromethane mixed solution is confirmed as the optimal extraction reagent ratio with better instrument peak area.

[0144] Table 7 Spiked recoveries and peak areas of 10 neonicotinoid insecticides and their metabolites detected by different extraction reagents

[0145]

[0146]

[0147] 4. Impact of solid phase extraction cartridges on test results

[0148] A mixed standard solution of 10 target standards with a final concentration of 10 ng / mL was used as the test sample, and the detection was performed according to the method of Example 1 of the present invention, except that the model of the solid phase extraction cartridge was Bond ElutPlexa (60 mg / 3 mL, Agilent), Poly-Sery HLB Pro SPE Cartridge (200 mg / 6 mL, Anpel) or Oasis HLB 3cc (60 mg / 3 mL, Waters). The spike recovery rate was calculated.

[0149] As shown in Table 8, the instrument peak area was the highest when the solid phase extraction cartridge was Bond Elut Plexa (60 mg / 3 mL, Agilent) compared to the other two solid phase extraction cartridges. Therefore, in the range of recovery rate of 70% to 120%, Bond Elut Plexa (60 mg / 3 mL, Agilent) was confirmed as the best solid phase extraction cartridge with a higher instrument peak area.

[0150] Table 8 Spiked recoveries and peak areas of 10 neonicotinoid insecticides and their metabolites detected by different solid phase extraction columns

[0151]

[0152] 5. The influence of liquid-liquid extraction times on the test results

[0153] A mixed standard solution of 10 target standards with a final concentration of 10 ng / mL was used as a test sample, and detection was performed according to the method of Example 1 of the present invention, except that: liquid-liquid extraction was performed once, twice or three times in total.

[0154] As shown in Table 9, the peak areas of most neonicotinoids measured by different liquid-liquid extraction times were not significantly different, and the recovery rates were all in the range of 70% to 120%. Considering the saving of operation and saving of samples and reagents, 1 time can be used as the optimal liquid-liquid extraction time.

[0155] Table 9 Spiked recoveries and peak areas of 10 neonicotinoid insecticides and their metabolites detected by different liquid-liquid extraction times

[0156]

[0157] 6. Testing of actual fecal samples

[0158] 171 stool samples from donors were collected, freeze-dried for 3 days, and ground. The contents of 10 neonicotinoid pesticides and their metabolites in the stool samples were detected using the method in Example 1 of the present invention. The detection results are shown in Table 10. Due to the limitations of the instrument and the fact that stool samples may not contain all 10 neonicotinoid pesticides and their metabolites, a total of 8 neonicotinoid pesticides and their metabolites were actually detected in the 171 stool samples, among which IMI had the highest detection rate, indicating that the method in Example 1 of the present invention can detect the presence and content of neonicotinoid pesticides and their metabolites in stool samples.

[0159] Table 10 Detection of 10 neonicotinoid pesticides and their metabolites in 171 fecal samples

[0160]

[0161] Example 3 Detection of 10 neonicotinoid pesticides and their metabolites in urine samples

[0162] 1. Preparation of spiked samples

[0163] The mixed standard solution of the analyte and the mixed internal standard solution were added to the urine samples respectively to prepare spiked samples with final concentrations of 1.0 ng / mL (low concentration), 5.0 ng / mL (medium concentration) and 10.0 ng / mL (high concentration) of each standard, with 3 samples for each concentration.

[0164] 2. Detection

[0165] The contents of 10 neonicotinoid insecticides and their metabolites in the spiked samples of this example were detected by referring to the method of Example 1 of the present invention, and the spiked recovery rates were calculated.

[0166] 3. Test results

[0167] As shown in Table 11, the test results show that the recovery rates of most compounds in urine samples meet 70% to 120%, which indicates that the detection method provided by the present invention is also applicable to the determination of the contents of 10 neonicotinoid pesticides and metabolites in urine matrix.

[0168] Table 11 Recovery and relative deviation of 10 neonicotinoid pesticides and their metabolites in urine samples

[0169]

[0170] Example 4 Detection of 10 neonicotinoid pesticides and their metabolites in hair samples

[0171] 1. Preparation of spiked samples

[0172] After grinding the hair sample, take 20 mg of the hair sample and add the mixed standard solution of the analyte and the mixed internal standard solution respectively to prepare three gradient spiked samples with standard substance concentrations of 1.0 ng / mL, 5.0 ng / mL and 10.0 ng / mL, low, medium and high. The internal standard concentration of all gradient samples is 10 ng / mL, and there are 3 samples for each concentration.

[0173] 2. Detection

[0174] The contents of 10 neonicotinoid insecticides and their metabolites in the spiked samples of this example were detected by referring to the method of Example 1 of the present invention, and the spiked recovery rates were calculated.

[0175] 3. Test results

[0176] As shown in Table 12, the test results show that the recovery rate of most compounds in the hair samples is in the range of 70% to 120%. This indicates that the detection method provided by the present invention is also applicable to the determination of the contents of 10 neonicotinoid pesticides and metabolites in hair matrix.

[0177] Table 12 Recovery and relative deviation of 10 neonicotinoid pesticides and their metabolites in hair samples

[0178]

[0179] Comparison of Example 5 with existing neonicotinoid insecticide detection methods

[0180] 1. Preparation of spiked samples

[0181] Referring to the method in Example 2 of the present invention, a spiked sample was prepared using fecal samples of infants under 1 year old.

[0182] 2. Pretreatment of spiked samples

[0183] (1) In the prior art "CN113009057A", the method of "pretreatment of urine samples" was used to treat the spiked infant feces sample. The specific steps are as follows: 200 mg of infant feces was placed in a 10 mL PP tube, 50 μL of the mixed internal standard solution with a concentration of 50 ng / mL in Example 1 of the present invention was added, and then 20 μL of the prepared β-glucuronidase application solution was added, and the mixture was placed in a 37°C constant temperature oscillator, 200 r / min, and incubated for 12 h. Activate the Oasis HLB solid phase extraction column with 2 mL of methanol and 2 mL of water in turn, load the sample onto the column, and elute the analyte with 2 mL of dichloromethane-acetonitrile (7:3, v / v). After the eluate was dried by a gentle nitrogen flow, 1 mL of dichloromethane-acetonitrile (7:3, v / v) was added to dissolve the residue. After centrifugation at 13000 r / min for 10 min, the supernatant was taken. After drying under a gentle nitrogen stream, the solution was re-dissolved with 250 μL of methanol-formic acid water (methanol and 0.15% formic acid aqueous solution were mixed in a volume ratio of 1:9), and finally filtered through a 0.22 μm filter membrane for later use.

[0184] (2) Referring to the prior art "CN115901992A" "A method for detecting neonicotinoid pesticides and their metabolites in liquid milk", the infant feces spiked sample was treated, and the specific steps were as follows: 500 mg of infant feces was placed in a 10 mL PP tube, and the mixed internal standard solution with a concentration of 50 ng / mL in Example 1 of the present invention was added. Then 2.5 mL of acetonitrile was added, and the target compound was extracted by ultrasound for 10 min. 0.4 mL of saturated sodium chloride solution was added, and the upper organic phase was taken after centrifugation at 4000 rpm / min for 5 min. After drying with a gentle nitrogen flow, it was re-dissolved with 500 μL of acetonitrile. Finally, it was filtered through a 0.22 μm filter membrane and set aside.

[0185] 3. Detection

[0186] The spiked samples obtained by the two prior art pretreatment methods in the previous step were detected by referring to the method of Example 1 of the present invention, and the spiked recovery rate was calculated.

[0187] 4. Test results

[0188] (1) Compared with the detection method provided by the present invention, the method of the prior art "CN113009057A" requires a larger sample amount. As shown in Table 13, the test results show that after the infant feces are treated with the method of the prior art "CN113009057A", the spiked recovery rates of 10 neonicotinoid pesticides and their metabolites are in the range of 5% to 253%, and the recovery rate of one compound is lower than 15%, which cannot reach the recognized recovery standard (70% to 120%), and under the same batch of sample detection, the peak area of ​​the target object of the instrument is much lower than that of the method of Example 1 of the present invention. Therefore, the method of the prior art cannot be used for the detection of the content of 10 neonicotinoid pesticides and their metabolites in feces.

[0189] Table 13 Comparison of the recovery rates and peak areas of 10 neonicotinoid pesticides and their metabolites in fecal samples using different methods (spike recovery rate (%) ± (relative deviation, n = 3))

[0190]

[0191] (2) Compared with the detection method provided by the present invention, the prior art "CN115901992A" requires a larger sample amount. As shown in Table 14, the test results show that after the infant feces are treated with the method of the prior art "CN115901992A", the spiked recovery rate of 10 neonicotinoid pesticides and their metabolites is in the range of 74% to 91%. Under the same batch of sample detection, the target peak area of ​​the instrument is much lower than that of the method of Example 1 of the present invention. Therefore, the method provided by the present invention is significantly better than the prior art in detecting the content of 10 neonicotinoid pesticides and their metabolites in feces.

[0192] Table 14 Comparison of the recovery rates and peak areas of 10 neonicotinoid pesticides and their metabolites in fecal samples using different methods (spike recovery rate (%) ± (relative deviation, n = 3))

[0193]

[0194] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A method for extracting neonicotinoid pesticides and / or their metabolites from biological samples, characterized in that: The neonicotinoid insecticide and / or its metabolite comprises at least one of acetamiprid, N-demethylacetamiprid, imidacloprid, 5-hydroxyimidacloprid, thiamethoxam, thiacloprid, thiacloprid amide, clothianidin, chlorothiazolin and sulfoxaflor; The method comprises the following steps: thoroughly mixing the biological sample and the extraction solvent, separating the layers, collecting the top layer solution to obtain a supernatant; mixing the supernatant with a 1-3% v / v formic acid aqueous solution to obtain a primary extraction product; performing solid phase extraction on the primary extraction product to obtain an extract containing the neonicotinoid insecticide and / or its metabolites; The extraction solvent includes acetonitrile and dichloromethane, and the volume ratio of the acetonitrile to the dichloromethane is (0.5-2):

1.

2. The method according to claim 1, characterized in that The mass volume ratio of the biological sample and the extraction solvent is 25 mg: (2.5-7.5) mL.

3. The method according to claim 1, characterized in that The volume ratio of the supernatant to the formic acid aqueous solution is 1:(4-8).

4. The method according to claim 1, characterized in that: The solid phase extraction comprises the following steps: Add the initial extraction product to an activated solid phase extraction column, wash it with a column washing liquid, dry it, elute it with methanol, and collect the eluate; The column washing liquid comprises methanol, formic acid and water, and the volume ratio of the methanol, formic acid and water is 10:2:(80-100).

5. The method according to claim 1, characterized in that The biological sample is a solid biological sample or a liquid biological sample.

6. Use of the method according to any one of claims 1 to 5 in detecting neonicotinoid insecticides and / or their metabolites.

7. A method for detecting neonicotinoid insecticides and / or their metabolites, characterized in that: The method comprises the following steps: extracting the sample to be tested by the method described in claim 1 to obtain an extract, and then detecting the extract by ultra-high performance liquid chromatography.

8. The method according to claim 7, characterized in that In the method of claim 1, the internal standard is thoroughly mixed with the sample to be tested and the extraction solvent, and the layers are separated, and the top layer of the solution is collected.

9. The method according to claim 8, characterized in that The internal standard includes isotope-labeled acetamiprid, isotope-labeled N-demethylacetamiprid, isotope-labeled imidacloprid, isotope-labeled 5-hydroxyimidacloprid, isotope-labeled thiamethoxam, isotope-labeled thiacloprid, isotope-labeled thiamethoxam amide, isotope-labeled clothianidin, isotope-labeled chlorothiazide and isotope-labeled sulfoxaflor nitrile.

10. The method according to claim 7, characterized in that The chromatographic conditions of the ultra-high performance liquid chromatography include: using a C18 chromatographic column; using 0.1% v / v formic acid aqueous solution as mobile phase A, using acetonitrile as mobile phase B, and in the gradient elution program, the volume percentage of mobile phase B changes as follows: 0-0.6 min, mobile phase B is 95%; 0.6-4.5 min, mobile phase B is 95%-1%; 4.5-5.5 min, mobile phase B is 1%; 5.5-6 min, mobile phase B is 1%-95%; 6-8 min, mobile phase B is 95%.

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