Method for detecting neonicotinoid pesticides in honey and application thereof

Abstract

The application discloses a method for detecting neonicotinoid pesticides in honey and application thereof, and belongs to the technical field of food safety. The method comprises the following steps: weighing 1g of a honey sample, adding 0.35mL of an EDTA-Mcilvaine buffer solution, and vortexing to dissolve; subsequently, 0.6mL of 1% formic acid acetonitrile (v / v) is added, and after vortexing, centrifugation and freezing treatment, the upper acetonitrile extraction solution is taken for analysis and determination. The liquid chromatography-tandem mass spectrometry method is used to determine seven kinds of neonicotinoid pesticides in honey, and the external standard method is used for quantification. The application utilizes the honey matrix to induce the sugar precipitation effect, and combines freezing treatment to realize one-step extraction, pre-concentration and purification of the target, has the advantages of simple pretreatment, economy, environmental protection, rapid detection, low matrix effect and high sensitivity, is suitable for high-throughput detection of a large number of honey samples, and provides a reliable analysis tool for food safety risk assessment.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of food safety detection, in particular to a method for detecting neonicotinoid pesticides in honey. BACKGROUND

[0002] Honey is a pure natural sweet food brewed by bees, and is favored by consumers due to its high nutritional value and medicinal value. As a major honey producing, consuming and exporting country in the world, China has achieved significant growth in honey production in recent decades. The quality and safety of honey have important influences on people's health and the sustainable development of the honey industry.

[0003] Neonicotinoid pesticides are a new type of insecticide containing a nicotine structure, and have the characteristics of high efficiency, low toxicity and wide spectrum. They are the most widely used insecticides in the world. However, the large-scale application of neonicotinoid pesticides may lead to the contact of pollinating insects such as bees with neonicotinoid pesticides through various channels, and the problem of neonicotinoid pesticide residues in honey has attracted more and more attention. A global survey published in Science in 2017 showed that 75% of honey contained at least one quantifiable neonicotinoid pesticide, and the pesticide existed in honey samples in all continents and regions of the world. The European Union has stipulated that the maximum residue limit of neonicotinoid pesticides in honey is 10-200 mu g / kg. Therefore, it is of great significance to develop and establish an accurate, rapid, sensitive and low-cost method for detecting neonicotinoid pesticides in honey.

[0004] The high sugar and complex matrix in honey can have a negative impact on mass spectrometric detection and chromatographic separation, which is a great challenge for analyzing pesticide residues in honey. At present, the sample preparation techniques used in the detection of neonicotinoid pesticides in honey mainly include dispersive solid-phase extraction, solid-phase extraction and QuEChERS method. However, these methods usually require extraction, purification and / or concentration steps, and are complicated to operate, time-consuming to process, large in organic reagent consumption and high in cost.

[0005] Therefore, it is necessary to establish a simple and cost-effective pretreatment method, and for this purpose, a method for detecting neonicotinoid pesticides in honey is provided. SUMMARY

[0006] The method provided by the application can realize one-step extraction, purification and pre-concentration of neonicotinoid pesticides in honey through matrix-induced sugar dialysis liquid-liquid extraction under the assistance of freezing, and rapid qualitative and quantitative determination of seven neonicotinoid pesticides can be obtained by combining liquid chromatography-tandem mass spectrometry detection, which has the characteristics of simple pretreatment, economic environmental protection, rapid detection, low matrix effect and high sensitivity.

[0007] To achieve the above object, the present application provides the following technical scheme: a method for detecting neonicotinoid pesticides in honey, comprising the following steps:

[0008] S1, sample dissolution: dissolving the honey sample in EDTA-Mcilvaine buffer solution;

[0009] S1.1: accurately weigh 1.0 g of honey sample in a 2 mL centrifuge tube;

[0010] S1.2: add 3.5 mL of EDTA-Mcilvaine buffer solution, vortex thoroughly until the honey is completely dissolved;

[0011] S2, extraction and separation: add an extraction solvent to the sample solution, perform vortex extraction and centrifugal separation, then perform freezing treatment, and take the upper extraction liquid;

[0012] S2.1: add 0.6 mL of formic acid acetonitrile (v / v) with a concentration range of 0.5% to 1.5% to the sample solution obtained in step S1 as an extraction solvent;

[0013] S2.2: vortex extraction for 1.0 to 2.0 minutes;

[0014] S2.3: centrifuge at a speed of 8000 to 12000 r / min for 3 to 7 minutes, and the centrifugation temperature ranges from 0 to 10°C;

[0015] S2.4: freeze the mixed solution after centrifugation at -40 to -30°C for 10 to 20 minutes;

[0016] S2.5: take the upper extraction liquid, filter through a filter membrane with a pore size range of 0.1 to 0.3 μm to obtain the sample to be tested;

[0017] S3, detection and quantification: using liquid chromatography-tandem mass spectrometry to detect and quantify neonicotinoid pesticides in the extraction liquid;

[0018] S3.1: using liquid chromatography-tandem mass spectrometry to detect the sample to be tested;

[0019] S3.2: establish a standard curve by preparing a standard working solution of the test substance;

[0020] S3.3: qualitatively determine the test substance according to the ion pair and retention time, and quantitatively determine the test substance by external standard method according to the peak area and standard curve.

[0021] Preferably, the neonicotinoid pesticides are 7 kinds, which are nitenpyram, thiamethoxam, clothianidin, imidacloprid, chlorocholine, acetamiprid and thiacloprid.

[0022] Preferably, in step S1.1, the honey sample is first melted and mixed at 40°C water bath before weighing.

[0023] Preferably, in step S2.1, the extraction solvent is 1% formic acid in acetonitrile (v / v).

[0024] In step S2.2, the vortex extraction time is 1.5 minutes; the vortex speed ranges from 1500 r / min to 3000 r / min.

[0025] Preferably, in step S2.3, the centrifugation condition is 10000 r / min for 5 minutes at 4°C; in step S2.4, the freezing temperature is -35°C for 15 minutes.

[0026] Preferably, in step S2.5, the filtration step uses a 0.22 μm filter membrane made of polytetrafluoroethylene.

[0027] Preferably, in step S3.1, the liquid chromatography condition is as follows:

[0028] An ACQUITY BEH C18 column with a 2.1 mm x 100 mm x 1.7 μm particle size is used.

[0029] The column temperature is 35°C.

[0030] The injection volume is 2 μL.

[0031] The mobile phase A is 0.05% (v / v) formic acid in water, and the mobile phase B is acetonitrile.

[0032] The flow rate of the mobile phase is 0.4 mL / min.

[0033] The gradient elution program is as follows:

[0034] 0-4.0 min, 5% B phase;

[0035] 4.0-6.0 min, 5% to 20% B phase;

[0036] 6.0-8.0 min, 20% to 40% B phase;

[0037] 8.0-9.0 min, 40% to 95% B phase;

[0038] 9.0-11.0 min, 95% B phase;

[0039] 11.0-13.5 min, 95% to 5% B phase;

[0040] 13.5-14.5 min, 5% B phase.

[0041] Preferably, in the step S3.1, the mass spectrometry conditions are as follows:

[0042] ESI ion source, positive ion scanning mode;

[0043] Multiple ion reaction monitoring (MRM) mode;

[0044] Ion source temperature 550℃;

[0045] Electrospray voltage 5000V;

[0046] Collision dissociation energy CAD is set to medium;

[0047] Gas 1 and gas 2 are both 50psi;

[0048] The curtain gas (CUR) is 35psi.

[0049] Preferably, in the step S3.2, the concentration of the standard working solution is in the range of 0.5-200 μg / L, and the preparation solvent is acetonitrile.

[0050] The application of a method for detecting neonicotinoid pesticides in honey is used to simultaneously detect seven neonicotinoid pesticides in honey, including nitenpyram, thiamethoxam, clothianidin, imidacloprid, chlorothalonil, acetamiprid and thiacloprid.

[0051] Compared with the prior art, the beneficial effects of the present application are as follows:

[0052] 1. The present application is based on the principle of sugar effusion effect, which uses monosaccharides in honey matrix to induce the two-phase separation of acetonitrile and water, without the need for additional induction of layered reagents, simplifying the operation steps and reducing the cost of reagents.

[0053] 2. Acetonitrile is used as an extraction solvent, which can effectively extract target pesticides, while retaining sugars and proteins in the water phase and inhibiting the extraction of beeswax, achieving the purification of target substances.

[0054] 3. Through freezing treatment, the two-phase separation of acetonitrile and water is further promoted, the recovery rate of target substances is improved, and the sugar content in the acetonitrile phase is almost completely removed, enhancing the purification effect.

[0055] 4. Using EDTA-Mcilvaine buffer solution to dissolve the sample can complex the divalent cations in honey, reduce matrix interference, and achieve pre-concentration of target substances through optimized conditions, improving detection sensitivity.

[0056] 5. The method of the present application can complete the extraction, purification and pre-concentration of neonicotinoid pesticides in honey in one step, can simultaneously detect seven neonicotinoid pesticides, has the characteristics of simple operation, economic and environmental protection, good purification effect, rapid detection and high sensitivity. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 Operation schematic diagram of the method for detecting neonicotinoid pesticides in honey according to the present application.

[0058] Figure 2 Extraction ion current chromatogram (XIC chromatogram) of 7 neonicotinoid pesticide standard working solutions (50 μg / L) determined by liquid chromatography-tandem mass spectrometry.

[0059] Figure 3 Total ion current chromatogram (TIC chromatogram) of sample spiked extract (50 μg / L).

[0060] Figure 4 Effect of extraction solvent volume on phase separation.

[0061] Figure 5 Effect of addition of formic acid in acetonitrile extraction solvent on recovery (n = 3).

[0062] Figure 6 Effect of freezing on sugar removal from extract.

[0063] Figure 7 Effect of freezing on recovery (n = 3);

[0064] Figure 8 Flowchart of the method for detecting neonicotinoid pesticides in honey according to the present application. DETAILED DESCRIPTION

[0065] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0066] Please refer to Figures 1 to 8 The present application provides a technical solution: a method for detecting neonicotinoid pesticides in honey, comprising the following steps:

[0067] S1. Sample dissolution

[0068] S1.1: Accurately weigh 1.0 g of honey sample into a 2 mL centrifuge tube. To ensure the uniformity of the honey sample, it is recommended to melt and mix the honey sample in a 40°C water bath before weighing.

[0069] S1.2: Add 3.5 mL of EDTA-Mcilvaine buffer solution to the centrifuge tube and vortex thoroughly (about 30 seconds) until the honey is completely dissolved to form a uniform sample solution.

[0070] Preparation of EDTA-Mcilvaine buffer solution:

[0071] Weigh 7.163 g of disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), dissolve in a suitable amount of deionized water, and dilute to 100 mL to obtain a 0.2 mol / L disodium hydrogen phosphate solution.

[0072] Weigh 2.101 g of citric acid monohydrate (C6H8O7·H2O), dissolve in a suitable amount of deionized water, and dilute to 100 mL to obtain a 0.1 mol / L citric acid solution.

[0073] Mix 100 mL of 0.1 mol / L citric acid solution with 62.5 mL of 0.2 mol / L disodium hydrogen phosphate solution, adjust the pH to 4.0 ± 0.05 with hydrochloric acid to obtain a Mcilvaine buffer solution.

[0074] Weigh 6.05 g of disodium ethylenediaminetetraacetate (Na2EDTA·2H2O) and add it to 162.5 mL of Mcilvaine buffer solution, shake to dissolve, and you get the EDTA-Mcilvaine buffer solution. This buffer solution is used to dissolve the honey sample and can effectively complex the divalent cations in the honey, reducing matrix interference.

[0075] S2. Extraction and separation

[0076] S2.1: Add 0.6 mL of 1% formic acid acetonitrile (v / v) as the extraction solvent to the sample solution obtained in step S1.

[0077] Preparation of 1% formic acid acetonitrile (v / v) extraction solution:

[0078] Take 1 mL of formic acid (HCOOH) and add 99 mL of acetonitrile (CH3CN) and mix well to obtain a 1% formic acid acetonitrile solution. This solution can effectively extract neonicotinoid pesticides from honey while reducing matrix effects.

[0079] Optimization: Based on the above, the present application investigates 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL and 0.8 mL in the selection of extraction solvent volume. As shown in Figure 4 When the addition volume is 0.3 mL and 0.4 mL, no two-phase separation of acetonitrile phase and water phase is observed, or the phase separation is not complete. Research shows that when the addition volume is 0.5 mL, the recovery rate and precision of the target substance are not satisfactory, and when the addition volume of the extraction solvent is in the range of 0.6 to 0.8 mL, there is no significant effect on the extraction recovery. In order to improve the sensitivity of the method and further reduce the amount of organic reagent, the present application determines 0.6 mL as the optimal extraction solvent volume.

[0080] On the basis of the foregoing, the present application investigates pure acetonitrile and 1% formic acid acetonitrile (v / v) in the selection of extraction solvent. The experimental results show (see Figure 5 ), 1% formic acid acetonitrile (v / v) as an extraction solvent can significantly improve the recovery rate of nitenpyram. Therefore, the present application selects 1% formic acid acetonitrile (v / v) as the extraction solvent;

[0081] S2.2: Perform vortex extraction, and the vortex time is 1.5 minutes. The vortex speed ranges from 1500 r / min to 3000 r / min, ensuring that the target pesticide is fully transferred to the acetonitrile phase.

[0082] S2.3: Centrifuge at a speed of 10000 r / min for 5 minutes, and the centrifugation temperature is 4℃. After centrifugation, the solution is divided into two layers, and the target pesticide is mainly distributed in the upper acetonitrile phase.

[0083] S2.4: Freeze the mixed solution after centrifugation at -35℃ for 15 minutes. Freezing treatment can further promote the separation of acetonitrile and water into two phases, reduce the solubility of sugar in acetonitrile, and enhance the purification effect.

[0084] Optimization: On the basis of the foregoing, the present application investigates the effect of freezing on purification and recovery rate, and the freezing temperature is -35℃ and the freezing time is 15 min. It is found that the freezing time exceeds 15 min, and ice crystals are easily produced in the acetonitrile phase. According to GB5009.8-2023 "National Food Safety Standard Determination of Fructose, Glucose, Sucrose, Maltose and Lactose in Food" Method 1 High Performance Liquid Chromatography, the concentration of sugar in the extraction solution before and after freezing is detected. As shown in Figure 7 , after freezing treatment, fructose and glucose in the extraction solution are not detected, indicating that freezing treatment has a further purification effect on sugar in the extraction solution. In addition, the results show Figure 6 ) that the recovery rate of each target after freezing is improved to a certain extent, which is related to the further separation of acetonitrile-water into two phases caused by freezing

[0085] S2.5: Take the upper extraction solution, filter it through a 0.22μm polytetrafluoroethylene (PTFE) filter membrane, and obtain a sample solution to be detected. The pore size of the filter membrane is selected to be 0.22μm, which can effectively remove impurities and avoid clogging the chromatographic column.

[0086] S3. Detection and quantification

[0087] S3.1: Use a liquid chromatograph-tandem mass spectrometer to detect the sample to be detected.

[0088] Chromatographic conditions:

[0089] ACQUITY UPLC® BEH C18 column, 2.1 mm x 100 mm x 1.7 μm, column temperature set at 35 °C; injection volume 2 μL; mobile phase A and B were 0.05% (v / v) formic acid in water and acetonitrile, respectively, at a flow rate of 0.4 mL / min. The gradient elution program was as follows:

[0090] 0-4.0 min, maintain 5% B phase;

[0091] 4.0-6.0 min, linearly increase B phase from 5% to 20%;

[0092] 6.0-8.0 min, linearly increase B phase from 20% to 40%;

[0093] 8.0-9.0 min, linearly increase B phase from 40% to 95%;

[0094] 9.0-11.0 min, maintain 95% B phase;

[0095] 11.0-13.5 min, linearly decrease B phase from 95% to 5%;

[0096] 13.5-14.5 min, maintain 5% B phase.

[0097] The total analysis time was 14.5 min. The gradient elution program could effectively separate the seven neonicotinoid pesticides, while ensuring a relatively short analysis time and improving the detection efficiency.

[0098] Mass spectrometry conditions:

[0099] Electrospray ionization (ESI) ion source was used for detection in positive ion scan mode. The multiple ion reaction monitoring (MRM) mode was selected for secondary mass spectrometry detection to improve the sensitivity and selectivity of detection. The optimized mass spectrometry conditions were as follows:

[0100] Ion source temperature: 550 °C;

[0101] Electrospray voltage: 5000 V;

[0102] Collisional dissociation energy (CAD): medium;

[0103] Ion source gas 1 and gas 2: both 50 psi;

[0104] Curtain gas (CUR): 35 psi.

[0105] The characteristic ions and parameters of each target were as shown in Table 1: ​

[0106]

[0107]

[0108] Note: Sub-ions with "*" are quantitative ions.

[0109] S3.2: Establish a standard curve by preparing a standard working solution of the analyte.

[0110] Preparation of standard solution: Take 1 mL of the mixed standard solution of 7 neonicotinoid pesticides (100 μg / mL, purchased from Tianjin Alta Technology Co., Ltd.), and dilute to 100.0 mL with acetonitrile to obtain a mixed standard stock solution of 1000 μg / L. Take an appropriate amount of the mixed standard stock solution, and dilute with acetonitrile step by step to prepare mixed standard sequence working solutions with concentrations of 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, 100.0, and 200.0 μg / L. These standard solutions are used to establish a standard curve to achieve external standard quantitative analysis.

[0111] S3.3: Qualitative analysis of the analyte according to ion pairs and retention time, and external standard quantitative analysis of the analyte according to peak area and standard curve.

[0112] Using the prepared mixed standard sequence working solution, analysis is performed according to the above chromatographic and mass spectrometric conditions. The standard curve is plotted with the target concentration as the abscissa and the mass spectrometric response area as the ordinate. According to the linear regression equation of the standard curve, the concentration of each target in the sample to be tested is calculated. The external standard method is used for quantitative analysis, and the content of each pesticide in the sample is obtained by comparing the peak area of the target in the sample with the standard curve.

[0113] Example 1: Detection of neonicotinoid pesticides in honey

[0114] 1. Overview of the example

[0115] This example aims to verify a method for detecting neonicotinoid pesticides in honey. The linear range, detection limit, quantification limit, recovery rate, and precision of the method are verified through experiments to prove its feasibility and reliability in actual detection.

[0116] 2. Experimental materials and equipment

[0117] Reagents:

[0118] EDTA-Mcilvaine buffer solution (preparation method is described above).

[0119] 1% formic acid acetonitrile (v / v) extract.

[0120] 7 kinds of neonicotinoid pesticide mixed standard solution (100 μg / mL, purchased from Tianjin Altatech Co., Ltd.).

[0121] Acetonitrile, formic acid and other reagents are chromatographically pure.

[0122] Equipment:

[0123] Liquid chromatography-tandem mass spectrometer (AB Sciex 6500+, USA).

[0124] Vortex mixer.

[0125] Centrifuge (maximum speed 12000 r / min).

[0126] Freezing equipment (-35℃).

[0127] 0.22 μm polytetrafluoroethylene (PTFE) filter membrane.

[0128] 3. Experimental steps

[0129] Sample preparation:

[0130] Accurately weigh 1.0 g of honey sample into a 2 mL centrifuge tube, melt and mix the sample in a 40℃ water bath.

[0131] Add 3.5 mL of EDTA-Mcilvaine buffer solution, vortex thoroughly (about 30 seconds) until the honey is completely dissolved.

[0132] Extraction and purification:

[0133] Add 0.6 mL of 1% formic acid acetonitrile extractant to the sample solution, vortex extract for 1.5 minutes (vortex speed 1500-3000 r / min).

[0134] Centrifuge at 10000 r / min for 5 minutes, centrifuge temperature is 4℃.

[0135] Freeze the mixed solution after centrifugation at -35℃ for 15 minutes.

[0136] Take the upper extract, filter through a 0.22 μm PTFE filter membrane to obtain the sample solution to be tested.

[0137] Detection conditions:

[0138] Liquid chromatography conditions:

[0139] Chromatographic column: ACQUITY BEH C18 chromatographic column with a size of 2.1 mm x 100 mm x 1.7 μm.

[0140] Column temperature: 35℃. ​

[0141] Injection volume: 2 pL.

[0142] Mobile phase A: 0.05% (v / v) formic acid in water.

[0143] Mobile phase B: Acetonitrile.

[0144] Flow rate: 0.4 mL / min.

[0145] Gradient elution program:

[0146] 0 - 4.0 min: 5% B phase.

[0147] 4.0 - 6.0 min: 5% to 20% B phase.

[0148] 6.0 - 8.0 min: 20% to 40% B phase.

[0149] 8.0 - 9.0 min: 40% to 95% B phase.

[0150] 9.0 - 11.0 min: 95% B phase.

[0151] 11.0 - 13.5 min: 95% to 5% B phase.

[0152] 13.5 - 14.5 min: 5% B phase.

[0153] Mass spectrometry conditions:

[0154] Ion source: ESI (positive ion mode).

[0155] Scan mode: Multiple ion reaction monitoring (MRM).

[0156] Ion source temperature: 550 °C.

[0157] Electrospray voltage: 5000 V.

[0158] Collisional activation energy (CAD): medium.

[0159] Gas 1 and gas 2: both 50 psi.

[0160] Curtain gas (CUR): 35 psi.

[0161] The characteristic ions and parameters for each target are shown in the preceding table.

[0162] Data analysis:

[0163] Prepare the mixed standard sequence working solutions at concentrations of 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, 100.0, and 200.0 pg / L.

[0164] The standard solution and the sample liquid to be tested were analyzed using a liquid chromatograph-tandem mass spectrometer. The target concentration was taken as the abscissa, and the mass spectrometric response area was taken as the ordinate. A standard curve was plotted.

[0165] According to the linear regression equation of the standard curve, the concentration of each target in the sample to be tested was calculated, and the external standard method was used for quantification.

[0166] 4. Results and verification

[0167] 1) Linear range and detection limit:

[0168] The concentration of each target standard sequence working solution was taken as the abscissa (X), and the mass spectrometric peak area was taken as the ordinate (Y). A standard curve was plotted (Table 2). The results showed that nitenpyram and thiamethoxam had good linearity in the range of 1-200 μg / L, thiamethoxam, imidacloprid, chlorocholine, acetamiprid and thiacloprid had good linearity in the range of 0.5-200 μg / L, and the correlation coefficient R 2 >0.9996.

[0169] The concentration of 3 times the signal-to-noise ratio (S / N) was defined as the detection limit (LOD), and the lowest spiked level with acceptable recovery (70%-120%) and precision (≤20%) was defined as the quantification limit (LOQ). The results showed that the detection limit of this method was 0.03-0.3 μg / kg, and the method quantification limit was 0.3-0.6 μg / kg.

[0170] Table 2: Linear range, linear equation, correlation coefficient, detection limit and quantification limit of 7 neonicotinoid pesticides

[0171]

[0172] 2) Recovery and precision:

[0173] Honey samples containing no nitenpyram, thiamethoxam, thiamethoxam, imidacloprid, chlorocholine, acetamiprid and thiacloprid were selected for spiked recovery experiments at three addition levels of one quantification limit concentration, 1.5 μg / kg and 6 μg / kg. The average recovery and relative standard deviation (RSD) were calculated, and the results are shown in Table 3. The results showed that the average recovery of the 7 neonicotinoid pesticides was between 80.3% and 116.2%, and the RSD was between 4.4% and 18.5%, indicating that the method had good accuracy and precision, and met the requirements of pesticide residue analysis.

[0174] Table 3: Recovery and relative standard deviation of 7 neonicotinoid pesticides

[0175]

[0176] 3) Matrix effect evaluation:

[0177] The method is used to extract blank matrix solution and prepare standard curve, and the matrix effect is evaluated by the ratio of the slope of matrix curve to the slope of solvent curve, and the calculation formula is: matrix effect (%) = (slope of matrix curve / slope of solvent curve-1) x 100%, and the absolute value of matrix effect is less than 20%, which indicates that the matrix effect is weak or negligible, and if the matrix effect value is greater than 20% or less than -20%, it indicates that the matrix effect is enhanced or the obvious inhibition effect. The matrix effects of 7 neonicotinoid pesticides in 4 kinds of honey (multifloral, pagoda tree, osmanthus and linden) are investigated, and the results (Table 4) show that the matrix effect value is between -3.8% and 12.6%, which indicates that the matrix effect is not obvious or negligible.

[0178] Table 4: Matrix effect value of 7 neonicotinoid pesticides

[0179]

[0180] 5. Conclusion

[0181] The embodiment verifies a method for detecting neonicotinoid pesticides in honey. The experimental results show that the method has a good linear range (0.5-200 μg / L), a low detection limit (0.03-0.3 μg / kg), a high recovery rate (80.3%-116.2%) and a low matrix effect (-3.8% to 12.6%), which can meet the detection requirements of neonicotinoid pesticides in honey, and provides a reliable technical means for food safety monitoring.

[0182] Although the embodiments of the present application have been shown and described, it can be understood by those skilled in the art that various changes, modifications, replacements and variations can be made to the embodiments without departing from the principles and spirits of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method of detecting neonicotinoid pesticides in honey, characterised in that: The method comprises the following steps: S1, sample dissolution: dissolving the honey sample in EDTA-Mcilvaine buffer solution; S1.1: accurately weigh 1.0 g of honey sample in a 2 mL centrifuge tube; S1.2: add 3.5 mL of EDTA-Mcilvaine buffer solution, and vortex thoroughly until the honey is completely dissolved; S2, extraction and separation: add an extraction solvent to the sample solution, perform vortex extraction and centrifugal separation, and then perform freezing treatment, and take the upper layer of the extraction liquid; S2.1: add 0.6 mL of formic acid acetonitrile with a concentration range of 0.5% to 1.5% by volume to the sample solution obtained in step S1 as an extraction solvent; S2.2: perform vortex extraction for 1.0 to 2.0 minutes; S2.3: centrifuge at a speed of 8000 to 12000 r / min for 3 to 7 minutes, and the centrifugation temperature ranges from 0°C to 10°C; S2.4: freeze the mixed solution after centrifugation at -40°C to -30°C for 10 to 20 minutes; S2.5: take the upper layer of the extraction liquid, filter through a filter membrane with a pore size range of 0.1 to 0.3 μm, and obtain the sample to be tested; S3, detection and quantification: detect and quantify the neonicotinoid pesticides in the extraction liquid by liquid chromatography-tandem mass spectrometry; S3.1: use a liquid chromatography-tandem mass spectrometer to detect the sample to be tested; S3.2: establish a standard curve by preparing a standard working solution of the test substance; S3.3: qualitatively analyze the test substance according to the ion pair and retention time, and quantitatively analyze the test substance by external standard method according to the peak area and standard curve; The neonicotinoid pesticides are 7 kinds, which are nitenpyram, thiamethoxam, clothianidin, imidacloprid, chlorothalonil, acetamiprid and thiacloprid; In step S3.1, the liquid chromatography conditions are as follows: Use an ACQUITY UPLC® BEH C18 chromatographic column with a size of 2.1 mm × 100 mm × 1.7 μm; The column temperature is 35°C; The injection volume is 2 μL; The mobile phase A is 0.05% formic acid aqueous solution, and the mobile phase B is acetonitrile; The flow rate of the mobile phase is 0.4 mL / min; The gradient elution program is as follows: 0-4.0 min, 5% B phase; 4.0-6.0 min, 5% to 20% B phase; 6.0-8.0 min, 20% to 40% B phase; 8.0-9.0 min, 40% to 95% B phase; 9.0-11.0 min, 95% B phase; 11.0-13.5 min, 95% to 5% B phase; 13.5-14.5 min, 5% B phase; In step S3.1, the mass spectrometry conditions are as follows: ESI ion source, positive ion scanning mode; Multiple ion reaction monitoring mode; Ion source temperature 550°C; Electrospray voltage 5000 V; Collision-induced dissociation energy CAD set to medium; Gas 1 and gas 2 are both 50 psi; Gas curtain gas is 35 psi.

2. A method of detecting neonicotinoid pesticides in honey according to claim 1, characterised in that: In step S1.1, the honey sample is first melted and mixed at 40℃ water bath before weighing.

3. A method of detecting neonicotinoid pesticides in honey according to claim 1, characterised in that: In step S2.1, the concentration of the extraction solvent is 1% formic acid acetonitrile by volume. In step S2.2, the vortex extraction time is 1.5 minutes, and the vortex speed ranges from 1500 r / min to 3000 r / min.

4. A method of detecting neonicotinoid pesticides in honey according to claim 1, characterized in that: In step S2.3, the centrifugation conditions are: speed 10000 r / min, time 5 minutes, and temperature 4℃; in step S2.4, the freezing temperature is -35℃, and the freezing time is 15 minutes.

5. A method of detecting neonicotinoid pesticides in honey according to claim 1, characterized in that: In step S2.5, the filter used in the filtration step is a 0.22 μm filter membrane made of polytetrafluoroethylene.

6. A method of detecting neonicotinoid pesticides in honey according to claim 1, characterised in that: In step S3.2, the concentration of the standard working solution ranges from 0.5 to 200 μg / L, and the preparation solvent is acetonitrile.

7. The use of a method for detecting neonicotinoid pesticides in honey, according to any one of claims 1 to 6, characterized in that: The method is used for simultaneous detection of 7 neonicotinoid pesticides in honey, including nitenpyram, thiamethoxam, clothianidin, imidacloprid, chlorothalonil, acetamiprid, and thiacloprid.

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