Method for detecting neonicotinoid pesticide in honey and application

Abstract

The invention discloses a method for detecting neonicotinoid pesticides in honey and application, and belongs to the technical field of food safety. The method comprises the following steps: weighing 1 g of a honey sample, adding 0.35 mL of an EDTA-Mcilvaine buffer solution, and carrying out vortex dissolution; and then adding 0.6 mL of 1% formic acid acetonitrile (v / v), carrying out vortex treatment, centrifugation and freezing treatment, and sucking an upper acetonitrile extract for analysis and determination. The seven neonicotinoid pesticides in the honey are determined by liquid chromatography-tandem mass spectrometry, and are quantified by an external standard method. According to the method, one-step extraction, pre-concentration and purification of a target object are realized by utilizing the honey matrix induced saccharification effect and combining freezing treatment, and the method has the advantages of simple pretreatment, economy, environmental protection, rapidness in 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 invention relates to the technical field of food safety detection, and in particular to a method for detecting neonicotinoid pesticides in honey. Background Art

[0002] Honey is a pure natural sweet food made by bees. It is widely loved by consumers for its high nutritional value and medicinal value. As the world's major producer, consumer and exporter of honey, my country has achieved significant growth in honey production in recent decades. The quality and safety of honey has an important impact on people's health and the sustainable development of the honey industry.

[0003] Neonicotinoid pesticides are a new type of insecticide containing nicotine structure. They are highly effective, low toxic and broad-spectrum, and are currently the most widely used type of insecticide in the world. However, the large-scale use of neonicotinoid pesticides may lead to bees and other pollinating insects being exposed to neonicotinoid pesticides through various pathways. The problem of neonicotinoid pesticide residues in honey has attracted increasing attention. A global survey study published in Science in 2017 showed that 75% of honey contained at least one quantifiable neonicotinoid pesticide, which was present in honey samples from all continents and regions around the world. The European Union has set a maximum residue limit of 10 to 200 μg / kg for neonicotinoid pesticides in honey. 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 spectrometry detection and chromatographic separation, which is a huge challenge in analyzing pesticide residues in honey. At present, the sample preparation techniques used in the detection of neonicotinoid pesticides in honey are mainly dispersed solid phase extraction, solid phase extraction and QuEChERS. However, these methods usually require extraction, purification and / or concentration steps, which are cumbersome to operate, take a long time to process, use a large amount of organic reagents and are costly.

[0005] Therefore, it is necessary to establish a pretreatment method that is simple to operate and cost-effective. To this end, we proposed a method for detecting neonicotinoid pesticides in honey. Summary of the invention

[0006] The method provided by the present invention can achieve one-step extraction, purification and pre-concentration of neonicotinoid pesticides in honey by matrix-induced sugar liquid-liquid extraction under the assistance of freezing before pretreatment, and can obtain rapid qualitative and quantitative determination of 7 neonicotinoid pesticides by combining liquid chromatography-tandem mass spectrometry detection, which has the characteristics of simple pretreatment, economic and environmental protection, rapid detection, low matrix effect and high sensitivity.

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

[0008] S1. Sample dissolution: dissolve the honey sample in EDTA-Mcilvaine buffer solution;

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

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

[0011] S2, extraction and separation: adding an extraction solvent to the sample solution, performing vortex extraction and centrifugal separation, and then freezing the sample solution to obtain the upper extract;

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

[0013] S2.2: performing vortex extraction, the vortex time being 1.0 min to 2.0 min;

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

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

[0016] S2.5: Take the upper layer of extract and filter it through a filter membrane with a pore size range of 0.1 μm to 0.3 μm to obtain a sample to be tested;

[0017] S3. Detection and quantification: Liquid chromatography-tandem mass spectrometry was used to detect and quantify neonicotinoid pesticides in the extract;

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

[0019] S3.2: Establish a standard curve by preparing the standard working solution of the substance to be tested;

[0020] S3.3: The analytes are qualitatively analyzed based on ion pairs and retention times, and quantitatively analyzed by external standard method using peak areas and standard curves.

[0021] Preferably, there are 7 neonicotinoid pesticides, namely, nitenpyram, thiamethoxam, clothianidin, imidacloprid, chlorthiazolin, acetamiprid and thiacloprid.

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

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

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

[0025] Preferably, in S2.3, the centrifugation conditions are: rotation speed 10000 r / min, time 5 minutes, temperature 4°C; in step S2.4, the freezing temperature is -35°C, and the freezing time is 15 minutes.

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

[0027] Preferably, in step S3.1, the liquid chromatography conditions are:

[0028] Using the 2.1 mm × 100 mm × 1.7 μm ACQUITY BEH C18 column;

[0029] Column temperature: 35°C;

[0030] Injection volume 2 μL;

[0031] Mobile phase A was 0.05% (v / v) formic acid in water, and mobile phase B was acetonitrile;

[0032] Mobile phase flow rate: 0.4 mL / min;

[0033] The gradient elution program was:

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

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

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

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

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

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

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

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

[0042] The ESI ion source was used in positive ion scanning mode;

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

[0044] Ion source temperature 550°C;

[0045] Electrospray voltage: 5000 V;

[0046] The collision dissociation energy CAD is set to medium;

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

[0048] The curtain air (CUR) was 35 psi.

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

[0050] An application of a method for detecting neonicotinoid pesticides in honey is used to simultaneously detect 7 neonicotinoid pesticides in honey, including nitenpyram, thiamethoxam, clothianidin, imidacloprid, chlorthiazolin, acetamiprid and thiacloprid.

[0051] Compared with the prior art, the present invention has the following beneficial effects:

[0052] 1. The present invention is based on the principle of sugar precipitation effect and utilizes monosaccharides in the honey matrix to induce the two-phase separation of acetonitrile and water. No additional inducing stratification reagent is needed, which simplifies the operation steps and reduces the reagent cost.

[0053] 2. Using acetonitrile as the extraction solvent can effectively extract the target pesticide, while retaining sugar and protein in the aqueous phase and inhibiting the extraction of beeswax, thereby achieving the purification of the target.

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

[0055] 4. Use EDTA-Mcilvaine buffer solution to dissolve the sample, which can complex the divalent cations in honey, reduce matrix interference, and achieve pre-concentration of the target by optimizing conditions to improve detection sensitivity.

[0056] 5. The method of the present invention can complete the extraction, purification and pre-concentration of neonicotinoid pesticides in honey in one step, and can detect 7 neonicotinoid pesticides at the same time. It has the characteristics of simple operation, economy and environmental protection, good purification effect, rapid detection and high sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 This is a schematic diagram of the operation of a method for detecting neonicotinoid pesticides in honey according to the present invention.

[0058] Figure 2 This is the extracted ion current chromatogram (XIC chromatogram) of the standard working solution (50 μg / L) of 7 neonicotinoid pesticides determined by liquid chromatography-tandem mass spectrometry.

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

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

[0061] Figure 5 The effect of adding formic acid to the acetonitrile extraction solvent on the recovery rate (n=3).

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

[0063] Figure 7 is the effect of freezing on the recovery rate (n=3);

[0064] Figure 8 The present invention is a schematic flow chart of a method for detecting neonicotinoid pesticides in honey. DETAILED DESCRIPTION

[0065] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0066] See also Figures 1 to 8 The present invention 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 1.0g of honey sample into a 2mL centrifuge tube. To ensure the homogeneity of the honey sample, it is recommended to melt and mix the honey sample in a 40℃ 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 it in an appropriate amount of deionized water, and make up 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 it in an appropriate amount of deionized water, and make up 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 and 62.5 mL of 0.2 mol / L disodium hydrogen phosphate solution, and 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), add it to 162.5 mL of Mcilvaine buffer solution, shake to dissolve, and obtain EDTA-Mcilvaine buffer solution. This buffer solution is used to dissolve honey samples, which can effectively complex divalent cations in honey and reduce matrix interference.

[0075] S2. Extraction and separation

[0076] S2.1: Add 0.6 mL of 1% formic acid acetonitrile (v / v) as an 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), add 99 mL of acetonitrile (CH3CN), mix well, and obtain a 1% formic acid acetonitrile solution. This solution can be used as an extraction solvent to effectively extract neonicotinoid pesticides in honey while reducing the matrix effect.

[0079] Optimization description: Based on the above, the present invention examines 0.3mL, 0.4mL, 0.5mL, 0.6mL, 0.7mL and 0.8mL when selecting the volume of the extraction solvent. Figure 4 As shown, when the added volume is 0.3mL and 0.4mL, no separation of the acetonitrile phase and the aqueous phase is observed, or the phase separation is incomplete. Studies have found that when the added volume is 0.5mL, the recovery rate and precision of the target are not satisfactory, and when the amount of extraction solvent added is in the range of 0.6 to 0.8mL, there is no significant effect on the extraction recovery rate. In order to improve the sensitivity of the method and further reduce the amount of organic reagents used, the present invention determines 0.6mL as the optimal extraction solvent volume.

[0080] On the basis of the above, the present invention examines pure acetonitrile and 1% formic acid acetonitrile (v / v) when selecting the extraction solvent. The experimental results show (see Figure 5 ), 1% formic acid acetonitrile (v / v) as the extraction solvent can significantly improve the recovery rate of nitenpyram. Therefore, the present invention selects 1% formic acid acetonitrile (v / v) as the extraction solvent;

[0081] S2.2: Perform vortex extraction with a vortex time of 1.5 minutes and a vortex speed ranging from 1500r / min to 3000r / min to ensure that the target pesticide is fully transferred to the acetonitrile phase.

[0082] S2.3: Centrifuge at 10000r / min for 5 minutes at 4°C. 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°C for 15 minutes. Freezing can further promote the separation of acetonitrile and water phases, while reducing the solubility of sugars in acetonitrile and enhancing the purification effect.

[0084] Optimization description: Based on the above, the present invention investigates the effect of freezing on purification and recovery rate, with a freezing temperature of -35°C and a freezing time of 15 minutes. The study found that when the freezing time exceeds 15 minutes, ice crystals are easily generated in the acetonitrile phase. Referring to the first method of high performance liquid chromatography in GB5009.8-2023 "National Food Safety Standard for the Determination of Fructose, Glucose, Sucrose, Maltose and Lactose in Food", the concentration of sugars in the extract before and after freezing was detected. Figure 7 As shown in the figure, after freezing treatment, neither fructose nor glucose was detected in the extract, indicating that freezing treatment further purified the sugars in the extract. In addition, the results showed that ( Figure 6 ) After freezing, the recovery rates of each target compound were improved to a certain extent, which was related to the further separation of acetonitrile-water phases caused by freezing.

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

[0086] S3. Detection and Quantification

[0087] S3.1: Use liquid chromatography-tandem mass spectrometry to detect the sample to be tested.

[0088] Chromatographic conditions:

[0089] Using the 2.1 mm × 100 mm × 1.7 μm ACQUITY BEH C18 chromatographic column, column temperature set to 35°C; injection volume 2μL; 0.05% (v / v) formic acid aqueous solution and acetonitrile as mobile phase A and mobile phase B, respectively, mobile phase flow rate 0.4mL / min. Gradient elution program:

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

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

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

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

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

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

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

[0097] The total analysis time was 14.5 minutes. This gradient elution procedure can effectively separate the seven neonicotinoid pesticides while ensuring a short analysis time and improving detection efficiency.

[0098] Mass spectrometry conditions:

[0099] Electrospray ionization (ESI) ion source was used for detection in positive ion scanning mode. 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 are as follows:

[0100] Ion source temperature: 550°C;

[0101] Electrospray voltage: 5000 V;

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

[0103] Gas 1 (Ion Source Gas 1) and Gas 2 (Ion Source Gas 2): both 50 psi;

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

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

[0106]

[0107]

[0108] Note: The product ions with “*” are quantification ions.

[0109] S3.2: Establish a standard curve by preparing standard working solutions of the substances to be tested.

[0110] Preparation of standard solution: Take 1mL of 7 kinds of neonicotinoid pesticide mixed standard solution (100μg / mL, purchased from Tianjin Alta Technology Co., Ltd.), dilute to 100.0mL with acetonitrile, and obtain 1000μg / L mixed standard stock solution. Take appropriate amount of mixed standard stock solution, dilute step by step with acetonitrile, and 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 200.0μg / L. These standard solutions are used to establish standard curves and realize quantitative analysis by external standard method.

[0111] S3.3: The analytes are qualitatively analyzed based on ion pairs and retention times, and quantitatively analyzed by external standard method using peak areas and standard curves.

[0112] Use the prepared mixed standard sequence working solution, analyze according to the above chromatographic and mass spectrometric conditions, draw a standard curve with the target concentration as the horizontal axis and the mass spectrometry response area as the vertical axis. According to the linear regression equation of the standard curve, calculate the concentration of each target in the sample to be tested. Use the external standard method for quantification, and compare the peak area of ​​the target in the sample with the standard curve to obtain the content of each pesticide in the sample.

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

[0114] 1. Overview of the Embodiments

[0115] This example aims to verify a method for detecting neonicotinoid pesticides in honey. The linear range, detection limit, quantitative limit, recovery rate and precision of the method were verified experimentally, proving its feasibility and reliability in actual detection.

[0116] 2. Experimental Materials and Equipment

[0117] Reagents:

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

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

[0120] Mixed standard solutions of seven neonicotinoid pesticides (100 μg / mL, purchased from Tianjin Alta Technology Co., Ltd.).

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

[0122] equipment:

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

[0124] Vortex mixer.

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

[0126] Refrigeration equipment (-35℃).

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

[0128] 3. Experimental Procedure

[0129] Sample preparation:

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

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

[0132] Extraction and purification:

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

[0134] Centrifuge at 10,000 r / min for 5 minutes at 4°C.

[0135] The mixed solution after centrifugation was frozen at -35°C for 15 minutes.

[0136] The upper extract was taken and filtered through a 0.22 μm PTFE filter membrane to obtain the sample solution to be tested.

[0137] Testing conditions:

[0138] Liquid chromatography conditions:

[0139] Chromatographic column: 2.1mm×100mm×1.7μm ACQUITY BEH C18 column.

[0140] Column temperature: 35°C.

[0141] Injection volume: 2 μL.

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

[0143] Mobile phase B: acetonitrile.

[0144] Flow rate: 0.4mL / min.

[0145] Gradient elution program:

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

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

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

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

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

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

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

[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: 5000V.

[0158] Collision dissociation energy (CAD): medium.

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

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

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

[0162] Data Analysis:

[0163] A mixed standard sequence working solution was prepared with concentrations of 0.5, 1.0, 2.0, 5.0, 10.0, 20.0, 50.0, 100.0 and 200.0 μg / L.

[0164] The standard solution and the sample solution to be tested were analyzed using a liquid chromatography-tandem mass spectrometer, and a standard curve was drawn with the target concentration as the horizontal axis and the mass spectrometry response area as the vertical axis.

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

[0166] 4. Results and Verification

[0167] 1) Linear range and detection limit:

[0168] The standard curve was drawn with the concentration of the working solution of each target standard sequence as the horizontal axis (X) and the mass spectrometry peak area as the vertical axis (Y) (Table 2). The results showed that nitenpyram and thiamethoxam were in the range of 1-200 μg / L, and clothianidin, imidacloprid, chlorthiazolin, acetamiprid and thiamethoxam were in the range of 0.5-200 μg / L. The correlation coefficients R 2 >0.9996.

[0169] The concentration of 3 times the signal-to-noise ratio (S / N) was defined as the limit of detection (LOD), and the lowest spike level with acceptable recovery (70%-120%) and precision (≤20%) was defined as the limit of quantification (LOQ). The results showed that the detection limit of this method was 0.03-0.3μg / kg, and the method quantification limit was 0.3-0.6μg / 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 without nitenpyram, thiamethoxam, clothianidin, imidacloprid, chlorpyrifos, acetamiprid and thiacloprid were selected, and spike recovery experiments were carried out at three addition levels: one times the quantitative limit concentration, 1.5μg / kg and 6μg / kg, and the average recovery rate and relative standard deviation (RSD) were calculated. The results are shown in Table 3. The results showed that the average recovery rate of the seven neonicotinoid pesticides ranged from 80.3% to 116.2%, and the RSD ranged from 4.4% to 18.5%, indicating that the accuracy and precision of this method are good and meet the requirements of pesticide residue analysis.

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

[0175]

[0176] 3) Matrix effect assessment:

[0177] The blank matrix solution was extracted by the method proposed in the present invention, and the standard curve was prepared. The matrix effect was evaluated by the ratio of the slope of the matrix curve to the slope of the solvent curve. The calculation formula was: matrix effect (%) = (matrix curve slope / solvent curve slope-1) × 100%. The absolute value of the matrix effect was less than 20%, indicating that the matrix effect was weak or negligible. If the matrix effect value was greater than 20% or less than -20%, it indicated that the matrix effect was enhanced or inhibited significantly. The matrix effects of 7 neonicotinoid pesticides in 4 kinds of honey, including multiflora, sophora japonica, osmanthus and linden, were investigated. The results showed (Table 4) that the matrix effect values ​​were between -3.8% and 12.6%, indicating that the matrix effect was not obvious or negligible.

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

[0179]

[0180] 5. Conclusion

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

[0182] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting neonicotinoid pesticides in honey, characterized in that: The following steps are involved: S1. Sample dissolution: dissolve the honey sample in EDTA-Mcilvaine buffer solution; S1.1: Accurately weigh 1.0 g of honey sample into 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: adding an extraction solvent to the sample solution, performing vortex extraction and centrifugal separation, and then freezing the sample solution to obtain the upper extract; S2.1: Add 0.6 mL of formic acid acetonitrile (v / v) with a concentration ranging from 0.5% to 1.5% as an extraction solvent to the sample solution obtained in step S1; S2.2: performing vortex extraction, the vortex time being 1.0 min to 2.0 min; S2.3: centrifuge at a speed of 8000 r / min to 12000 r / min for 3 minutes to 7 minutes, and the centrifugation temperature range is 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 extract and filter it through a filter membrane with a pore size range of 0.1 μm to 0.3 μm to obtain a sample to be tested; S3. Detection and quantification: Liquid chromatography-tandem mass spectrometry was used to detect and quantify neonicotinoid pesticides in the extract; S3.1: Use liquid chromatography-tandem mass spectrometry to detect the sample to be tested; S3.2: Establish a standard curve by preparing the standard working solution of the substance to be tested; S3.3: The analytes are qualitatively analyzed based on ion pairs and retention times, and quantitatively analyzed by external standard method using peak areas and standard curves.

2. A method for detecting neonicotinoid pesticides in honey according to claim 1, characterized in that: There are seven types of neonicotinoid pesticides, namely, nitenpyram, thiamethoxam, clothianidin, imidacloprid, chlorthiazolin, acetamiprid and thiacloprid.

3. A method for detecting neonicotinoid pesticides in honey according to claim 1, characterized in that: In the step S1.1, the honey sample is melted and mixed in a 40° C. water bath before weighing.

4. A method for detecting neonicotinoid pesticides in honey according to claim 1, characterized in that: In the step S2.1, the concentration of the extraction solvent is 1% formic acid acetonitrile (v / v); In S2.2, the vortex extraction time is 1.5 minutes; the vortex speed range is 1500r / min to 3000r / min.

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

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

7. A method for detecting neonicotinoid pesticides in honey according to claim 1, characterized in that: In step S3.1, the liquid chromatography conditions are: Using the 2.1 mm × 100 mm × 1.7 μm ACQUITY BEH C18 column; Column temperature: 35°C; Injection volume 2 μL; Mobile phase A was 0.05% (v / v) formic acid in water, and mobile phase B was acetonitrile; Mobile phase flow rate: 0.4 mL / min; The gradient elution program was: 0-4.0 min, 5% phase B; 4.0-6.0 min, 5% to 20% phase B; 6.0-8.0 min, 20% to 40% phase B; 8.0-9.0 min, 40% to 95% phase B; 9.0-11.0 min, 95% phase B; 11.0-13.5min, 95% to 5% phase B; 13.5-14.5 min, 5% phase B.

8. A method for detecting neonicotinoid pesticides in honey according to claim 1, characterized in that: In step S3.1, the mass spectrometry conditions are: The ESI ion source was used in positive ion scanning mode; Multiple ion reaction monitoring (MRM) mode; Ion source temperature 550°C; Electrospray voltage: 5000 V; The collision dissociation energy CAD is set to medium; Gas 1 and Gas 2 are both 50 psi; The curtain air (CUR) was 35 psi.

9. A method for detecting neonicotinoid pesticides in honey according to claim 1, characterized in that: In step S3.2, the concentration range of the standard working solution is 0.5-200 μg / L, and the preparation solvent is acetonitrile.

10. An application of a method for detecting neonicotinoid pesticides in honey, applied to a method for detecting neonicotinoid pesticides in honey according to any one of claims 1 to 9, characterized in that: The method was used to simultaneously detect seven neonicotinoid pesticides in honey, including nitenpyram, thiamethoxam, clothianidin, imidacloprid, chlorthiazolin, acetamiprid and thiacloprid.

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