Method for purifying honey and detecting pesticide residue of honey by using amino derivatization mesoporous material

Abstract

The invention belongs to the field of pesticide residue detection, and particularly relates to a method for detecting pesticide residue of honey purified by an amino derivatization mesoporous material, which comprises the following steps: sample extraction, sample purification, sample detection, quantification by using an external standard method, and liquid chromatography gradient elution, mass spectrometry conditions are that an electrospray ion source is used, and simultaneous scanning is performed in a positive ion mode and a negative ion mode, multi-reaction monitoring is carried out, and different retention times, parent ions, daughter ions, declustering voltages and collision energy are determined according to different pesticides. The method has the advantages of simple pretreatment, good effect of removing interfering substances such as sugar and impurities, reduction of matrix interference, no adsorption of pesticide residues, improvement of the detection accuracy and sensitivity, no need of adding expensive isotope internal standard in the detection process, low cost, short detection time, and easy popularization and application. The method has a wide application prospect in detection of pesticide residues in high-sugar-content food such as honey.

Description

Technical Field

[0001] The present invention belongs to the field of pesticide residue detection, and particularly relates to a method for purifying honey with an amino-derivatized mesoporous material to detect its pesticide residue content. Background Art

[0002] SBA-15 is a mesoporous molecular sieve, a mesoporous material synthesized by Academician Zhao Dongyuan of Fudan University. It has a unique two-dimensional hexagonal through-hole structure, with a relatively large pore diameter (6 - 11 nm), a relatively high specific surface area (550 - 600 m² / g), and good hydrothermal stability. QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) is a rapid sample pretreatment and purification technology for agricultural product detection that has been newly developed internationally in recent years.

[0003] Honey is a natural sweet substance that bees collect the nectar, secretions or honeydew of plants, mix with their own secretions, and fully brew. Its color varies from water white (nearly colorless) to dark (dark brown) depending on the honey source variety. It has a unique taste and smell, no abnormal smell, and is in a viscous fluid state at room temperature, or partially or completely crystallized. Honey has medicinal, edible, and tonic health care values, so it is deeply loved by consumers. Since it is inevitable for bees to come into contact with plant flowers contaminated with pesticides during the honey collection process, resulting in the detection of pesticide residues in honey. According to investigations, more than 75% of the honey globally contains residues of at least one pesticide. Therefore, it is necessary to pay attention to the pesticide residues in domestically produced honey. There have been a large number of literature reports on the detection of pesticide residues in honey, and relevant national standards have also been formulated. Among them, the commonly used detection methods are gas chromatography, liquid chromatography, mass spectrometry, immunoassay, etc. There are two key steps in the process of detecting pesticide residues in honey, sample pretreatment and detection method.

[0004] Sample pretreatment is the most time-consuming step and is crucial for reducing errors caused by the low concentration or uneven distribution of analytes in the honey matrix. The pretreatment methods for honey samples include liquid-liquid extraction, dispersive liquid-liquid microextraction, solid-phase extraction, QuEChERS method and other sample pretreatment methods. Both the extraction method and the solid-phase extraction method use a large amount of reagents, with cumbersome operation steps and high costs. The QuEChERS method has the characteristics of being fast, simple, cheap, effective, reliable, and safe compared to other methods, and is the preferred choice for the pretreatment method of pesticide residue detection at present. Honey is mainly composed of fructose and glucose. Generally, the content of fructose and glucose in honey samples is more than 60%, followed by proteins, enzymes, amino acids, organic acids, minerals, vitamins, flavor compounds, phenolic compounds, flavonoids, etc. These substances not only have a matrix effect on the accuracy of pesticide residue detection, but also have a certain adsorption on pesticides, affecting the recovery rate. Moreover, if these substances cannot be removed well, they will also contaminate the pesticide residue detection equipment, increasing the cost of equipment maintenance. The purification agents [such as PSA (N-propylethylenediamine), NH 2 (aminopropyl) packing] used in the traditional QuEChERS method have limited adsorption capacity for sugars and impurities, resulting in a significant matrix effect on the detection accuracy. In addition, existing adsorption materials (such as silica gel, unfunctionalized SBA-15) are difficult to efficiently remove impurities such as sugars and retain target pesticides simultaneously due to the lack of selective adsorption groups.

[0005] There have been a large number of literature reports on the detection of pesticide residues in honey, and relevant national detection standards have also been formulated. The commonly used detection methods include gas chromatography, liquid chromatography, gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, immunoassay, etc. Immunoassay is suitable for the screening of single pesticides. Due to the cross-reaction between antibodies and false positive or false negative reactions, it cannot be used for the simultaneous detection of multiple pesticides and the final confirmation. Gas chromatography and gas chromatography-mass spectrometry are widely used in the detection of pesticide residues, but they can only detect low-boiling pesticides, and the application range is relatively narrow. For example, "GB 23200.97—2016 National Food Safety Standard Determination of Residues of 5 Organophosphorus Pesticides in Honey Gas Chromatography" is only applicable to the determination of the residues of trichlorfon, fenchlorphos, chlorpyrifos, malathion, and coumaphos in honey; "GB 23200.7—2016 National Food Safety Standard Determination of Residues of 497 Pesticides and Related Chemicals in Honey, Fruit Juice and Fruit Wine Gas Chromatography-Mass Spectrometry" does not include dinotefuran, carbendazim, thiamethoxam, clothianidin, imidacloprid, acetamiprid, fosthiazate, forchlorfenuron, flumorph, azoxystrobin, dimethomorph, cyproconazole, cyazofamid, diflubenzuron, flusilazole, phoxim, pyraclostrobin, indoxacarb, emamectin benzoate, tolfenpyrad, flonicamid, sulfone of fipronil, sulfoxide of fipronil. Liquid chromatography is also used in the detection of pesticide residues, but due to the long analysis time, the need to consume a large amount of organic solvents during operation, and the difficulty in separating some of the miscellaneous peaks generated during detection, the detection and qualitative ability is weak, resulting in false positives. UPLC MS / MS (ultra-high performance liquid chromatography-tandem mass spectrometry) has the advantages of high separation efficiency, fast separation speed, and simple quantitative analysis of chromatography, and also has the characteristics of high sensitivity and strong qualitative ability of mass spectrometry, and can simultaneously achieve the detection purposes of qualitative and quantitative analysis. At present, this method has developed rapidly in the simultaneous detection of multiple pesticide residues. For example, "GB / T 20771—2008 Determination of Residues of 486 Pesticides and Related Chemicals in Honey Liquid Chromatography-Tandem Mass Spectrometry" uses dichloromethane for extraction, purifies through a solid-phase extraction column, and elutes with acetonitrile + toluene (3+1). This method requires 15 mL of acetone, 80 mL of dichloromethane, and 35 mL of acetonitrile + toluene (3+1) mixed solution for each sample detection - the consumption of organic solvents is relatively large, posing risks to operators and the environment. Summary of the Invention

[0006] Objective of the Invention: Aiming at some problems existing in the prior art, the present invention provides a method for purifying honey with an amino-derivatized mesoporous material to detect its pesticide residue content. This method has a simple pretreatment, good effects in removing sugars, impurities and other interfering substances, reduces matrix interference, does not adsorb pesticide residues, and improves the detection accuracy and sensitivity. The detection process does not require the addition of expensive isotope internal standards, has low costs and short detection time, makes up for the deficiencies in the detection technology of pesticide residues in high-sugar-content foods such as honey, and also has the application prospect of being extended to the detection of fruit products, meat products, etc. with relatively high sugar content.

[0007] Technical Solution: To achieve the above objective, the method for purifying honey with an amino-derivatized mesoporous material to detect its pesticide residue content according to the present invention includes the following steps: (1) Sample extraction Weigh a honey sample, dissolve the sample with water, add an acetonitrile solution containing 0.5% (by volume) acetic acid as the extraction solution, vortex and mix evenly, add a mixed inorganic salt, vortex and mix evenly, and centrifuge to obtain the first sample supernatant.

[0008] (2) Sample purification Pipette the first supernatant, add a purifying agent containing an amino-derivatized SBA-15 material thereto, mix evenly and centrifuge to obtain the second supernatant.

[0009] (3) UPLC-MS / MS detection Filter the second supernatant through a microporous filter membrane and then conduct the detection. At the same time, prepare a mixed pesticide standard solution for quantitative analysis.

[0010] Among them, the pesticides detected in the sample treatment in step (1) include any one or more of propamocarb, dinotefuran, carbendazim, thiamethoxam, clothianidin, imidacloprid, acetamiprid, fosthiazate, forchlorfenuron, flumorph, azoxystrobin, dimethomorph, cyproconazole, cyazofamid, diflubenzuron, flusilazole, propiconazole, phoxim, pyraclostrobin, diniconazole, indoxacarb, emamectin benzoate, tolfenpyrad, chlorpyrifos, propargite, fipronil, flonicamid, fipronil sulfone, fipronil sulfoxide.

[0011] Among them, the mass of the honey sample required in step (1) is 1 - 5 g, and the volume of the acetonitrile solution containing 0.5% (by volume) acetic acid is 5 - 25 mL.

[0012] Preferably, in step (1), the mass of the honey sample is 2 g, the volume of water is 5 mL, and the volume of the acetonitrile solution containing 0.5% (by volume) acetic acid is 10 mL.

[0013] Among them, the mixed inorganic salt in step (1) is anhydrous magnesium sulfate (the addition amount is 2 - 10 g) and anhydrous sodium acetate (the addition amount is 0.5 - 2.5 g).

[0014] Preferably, in the step (1), the composition and proportion of the mixed inorganic salts are: 4 g of anhydrous magnesium sulfate + 1 g of anhydrous sodium acetate.

[0015] Among them, in the step (2), the process of amine-derivatized SBA-15 is as follows: using a material synthesized from SBA-15 and any one of the silane coupling agents 3-aminopropyltriethoxysilane, N-(2-aminoethyl)-3-aminopropyltriethoxysilane, and diethylenetriaminepropyltrimethoxysilane. Among them, the material synthesized with 3-aminopropyltriethoxysilane is 1N-SBA-15, the material synthesized with N-(2-aminoethyl)-3-aminopropyltriethoxysilane is 2N-SBA-15, and the material synthesized with diethylenetriaminepropyltrimethoxysilane is 3N-SBA-15. Among them, the amine-derivatized SBA-15 that plays a major purification role in the QuEChERS purifying agent is one of 1N-SBA-15, 2N-SBA-15, or 3N-SBA-15.

[0016] Among them, in the step (2), the composition of the purifying agent is amine-derivatized SBA-15 and anhydrous magnesium sulfate, and the addition amounts are 50 - 250 mg and 150 - 750 mg respectively.

[0017] Preferably, in the step (2), the composition and proportion of the purifying agent are: 100 mg of amine-derivatized SBA-15, 300 mg of anhydrous magnesium sulfate.

[0018] Among them, in the step (2), the ratio of the purifying agent to the supernatant No. 1 of the absorbed sample is that 100 - 200 mg of the purifying agent needs to be added per milliliter of the supernatant No. 1.

[0019] Among them, in the step (3), the sample adopts the UPLC-MS / MS detection method and quantitative analysis is carried out by the external standard method. The liquid chromatography conditions are as follows: mobile phase A is a 0.1% (volume ratio) formic acid aqueous solution containing 2 mmol / L ammonium formate, and B is methanol; flow rate: 0.2 - 0.5 mL / min; column temperature: 30 - 40 °C; injection volume: 1.0 - 5.0 μL; the elution gradient program is shown in Table 1-1: Table 1-1 Elution Gradient Program

[0020] Preferably, in the step (3), the sample adopts the UPLC-MS / MS detection method and quantitative analysis is carried out by the external standard method. The liquid chromatography conditions are as follows: (1) Mobile phase: A is a 0.1% (volume ratio) formic acid aqueous solution containing 2 mmol / L ammonium formate, and B is methanol; flow rate: 0.40 mL / min; column temperature: 40 °C; injection volume: 1.0 μL.

[0021] The elution gradient program is shown in Table 1-2: Table 1-2 Elution Gradient Program

[0022] In step (3), the sample is detected by UPLC-MS / MS, and quantified by the external standard method. The mass spectrometry conditions are as follows: Ion source type: electrospray ionization source (ESI), scanning mode: simultaneous scanning in positive and negative ion modes, multiple reaction monitoring (MRM), electrospray voltage (IS): 5000 - 5500 V in positive ion mode, -4500 - 4000 V in negative ion mode, collision gas (CAD): Medium, ion source temperature (TEM): 400 - 550 °C.

[0023] Preferably, in step (3), the sample is detected by UPLC-MS / MS, and quantified by the external standard method. The mass spectrometry conditions are as follows: Ion source type: electrospray ionization source (ESI), scanning mode: simultaneous scanning in positive and negative ion modes, multiple reaction monitoring (MRM), electrospray voltage (IS): 5500 V in positive ion mode, -4500 V in negative ion mode, collision gas (CAD): Medium, ion source temperature (TEM): 450 °C.

[0024] The multiple reaction monitoring ion pairs and mass spectrometry related parameters of the pesticides in step (3) are shown in Table 1-3: Table 1-3 Mass Spectrometry Parameter Table

[0025] Advantages of the present invention: In the honey sample pretreatment method provided by the present invention, the amino-derivatized SBA-15 used as a purifying agent, compared with the PSA solid phase extraction filler and NH2 solid phase extraction filler methods used in traditional QuEChERS, the three amino-derivatized SBA-15 used in this method as purifying agents for pretreatment have a fructose removal rate higher than 80% and a glucose removal rate higher than 95%. The effect is significantly better than the average fructose removal rate of 50% and the average glucose removal rate of 76% of the traditional QuEChERS method. Moreover, the impurity removal rate after improvement (48% - 52%) is also higher than the impurity removal rate of the traditional treatment method (17% - 27%), significantly reducing the interference caused by the matrix effect; it is beneficial for the detection equipment to more accurately detect the pesticide residues in honey samples.

[0026] Applying the present invention for the pretreatment of honey samples can effectively remove glucose, fructose and other impurities, reduce the contamination of samples to detection equipment, and lower the equipment maintenance cost. In the present invention, the sample pretreatment operation based on amino-functionalized SBA-15 is simpler, superior to the cumbersome solid-phase extraction method in the existing pesticide residue standards for honey, saves a large amount of organic reagents, and makes the detection process more environmentally friendly. Description of the Drawings

[0027] To more clearly illustrate the technical solutions and embodiments of the present invention, the drawings to be used will be briefly introduced below.

[0028] Figure 1 It is a schematic diagram of the structures of three amino-functionalized SBA-15 purifying agents; Figure 2 It is the total ion current chromatogram of 29 pesticides; Figure 3 It is a comparison chart of the recovery rates of 29 pesticides extracted with different solvents; Figure 4 It is a comparison chart of the fructose removal rates of 5 purifying agents; Figure 5 It is a comparison chart of the glucose removal rates of 5 purifying agents; Figure 6 It is a chart of the impurity removal rates of different purifying agents during the pretreatment of honey detection; Figure 7 It is the total ion current mass spectrum without pretreatment of the present invention (maximum signal value = 1.7e9 cps); Figure 8 It is the total ion current mass spectrum after pretreatment of the present invention (maximum signal value = 3.2e8 cps). Examples

[0029] The embodiments of the technical solutions of the present invention will be described in detail below with reference to the drawings. The following examples are only used to more clearly illustrate the technical solutions of the present invention, so they are only examples and cannot be used to limit the protection scope of the present invention. It should be noted that unless otherwise specified, the technical terms or scientific terms used in this application should have the ordinary meanings understood by those skilled in the technical field to which the present invention belongs. The instruments and reagents used in this embodiment are as follows.

[0030] Instruments: Liquid chromatography-tandem mass spectrometer, ultra-high performance liquid chromatography, triple quadrupole / hybrid linear ion trap mass spectrometer, high performance liquid chromatography-evaporative light scattering detector, ultrapure water machine, bench-top high-speed refrigerated centrifuge, vortex oscillator, electronic balance, heating magnetic stirrer with constant temperature, nitrogen blowing concentration device, suction filtration device.

[0031] Reagents: Methanol, acetonitrile, ethyl acetate, and acetone were all of chromatographic grade; formic acid (purity 85.0%); ammonium acetate (purity 98.0%); toluene, anhydrous magnesium sulfate, sodium chloride, sodium citrate, and disodium hydrogen citrate were all analytical reagent grade; SBA-15 (pore size 6 - 11 nm); 3-aminopropyltriethoxysilane (CAS: 919-30-2); N-(2-aminoethyl)-3-aminopropyltriethoxysilane (CAS: 5089-72-5), diethylenetriaminepropyltrimethoxysilane (CAS: 35141-30-1); PSA solid-phase extraction packing, NH2 solid-phase extraction packing; 29 pesticide residue standard solutions with a concentration of 1000 μg / mL (propamocarb, dinotefuran, carbendazim, thiamethoxam, clothianidin, imidacloprid, acetamiprid, fosthiazate, forchlorfenuron, flumorph, azoxystrobin, dimethomorph, cyproconazole, cyazofamid, diflubenzuron, flusilazole, propiconazole, phoxim, pyraclostrobin, diniconazole, indoxacarb, emamectin benzoate, tolfenpyrad, chlorpyrifos, propargite, fipronil, flonicamid, fipronil sulfone, fipronil sulfoxide).

[0032] Example 1 Synthesis of three amino-derivatized SBA-15s The process of amino-derivatized SBA-15 is as follows: materials synthesized by using SBA-15 and any one of the silane coupling agents 3-aminopropyltriethoxysilane, N-(2-aminoethyl)-3-aminopropyltriethoxysilane, and diethylenetriaminepropyltrimethoxysilane. Among them, the one synthesized with 3-aminopropyltriethoxysilane is 1N-SBA-15, the one synthesized with N-(2-aminoethyl)-3-aminopropyltriethoxysilane is 2N-SBA-15, and the one synthesized with diethylenetriaminepropyltrimethoxysilane is 3N-SBA-15. Figure 1 Figure shows the structural schematic diagrams of three amino-derivatized SBA-15 purifying agents. The synthesis process is as follows: Place 3 g of SBA-15 in an oven at 105 °C to remove moisture and activate the material. After 12 h, cool it to room temperature, add 150 mL of toluene, and add 3 g of silane coupling agent (3-aminopropyltriethoxysilane, N-(2-aminoethyl)-3-aminopropyltriethoxysilane, and diethylenetriaminepropyltrimethoxysilane respectively) under stirring. React under nitrogen protection at 110 °C by reflux for 24 h. After cooling, filter with a 0.45 μm organic microporous membrane. Add the filter cake to 100 mL of methanol, stir vigorously for 30 min, filter, wash the filter cake with methanol, and dry it in vacuum at 70 °C for 12 h. The obtained powder is amino-derivatized SBA-15, namely 1N-SBA-15, 2N-SBA-15, and 3N-SBA-15 respectively.

[0033] Example 2 Selection of extraction solvent: Considering that honey has a high sugar content and is viscous in state, it is impossible to dissolve the sample directly by adding organic solvents for extraction. However, honey is easily soluble in water. It is chosen to first dissolve 2 g of the sample in 5 mL of water, and then use a reagent that can be separated from water for liquid-liquid extraction to effectively retain a large amount of sugar in the aqueous phase and extract the target pesticide residues into the organic phase. 4 g of anhydrous magnesium sulfate and 1 g of anhydrous sodium acetate are added to separate the organic phase from the aqueous phase. When selecting the extraction solvent, it is screened from acetone, acetonitrile, methanol, and ethyl acetate. Acetone and methanol are miscible with water and cannot be layered, and they will bring a large amount of sugar into the solvent. After adding salt and vortexing, acetonitrile and ethyl acetate can be layered from the aqueous phase. The blank sample spiked recovery rates of acetonitrile and ethyl acetate are tested. Ethyl acetate is not easy to dissolve sugars with high polarity. However, it is found that the recovery rates of pesticides with high polarity such as carbendazim, propamocarb, acetamiprid, thiamethoxam, and dinotefuran in ethyl acetate are relatively low (8.7% - 68%). Only substances with relatively low polarity such as fipronil and its three metabolites have better recovery rates, such as Figure 3 The comparison chart of the recovery rates of 29 pesticides extracted by different solvents shows that the experimental results show that the extraction efficiency of acetonitrile is significantly better than that of ethyl acetate. Also, because the effect of removing sugars is the best under acidic conditions in the subsequent process, it is chosen to add acetic acid to acetonitrile to prepare acetonitrile containing 0.5% acetic acid (volume ratio) as the extraction solution.

[0034] Example 3 Investigation of the effect of removing sugars (fructose and glucose): Weigh multiple samples, each sample is 2 g of honey. Add 5 mL of water, vortex to dissolve the sample, then add 10 mL of acetonitrile solution containing 0.5% acetic acid (volume ratio), vortex and extract again for 1 min. Add 4 g of anhydrous magnesium sulfate and 1 g of sodium acetate, shake vigorously for 1 min, and then centrifuge at 5000 r / min for 5 min. After collecting all the supernatant acetonitrile layers and mixing them evenly, take 3.0 mL and add it to the purification powder containing 300 mg of anhydrous magnesium sulfate and containing 10 mg, 25 mg, 50 mg, 100 mg, and 200 mg respectively (they are PSA solid-phase extraction packing, NH2 solid-phase extraction packing, N-SBA-15, 2N-SBA-15, 3N-SBA-15), vortex and mix evenly for 1 min, centrifuge at 5000 r / min for 5 min, and then pass through a 0.22 μm organic microporous filter membrane into the injection vial. The liquid chromatography evaporative light scattering detector uses a power function equation to draw a standard curve. The chromatographic and detector conditions refer to the national standard "National Food Safety Standard - Determination of Fructose, Glucose, Sucrose, Maltose, and Lactose in Foods" (GB 5009.8 - 2023) to determine the fructose and glucose contents in 3 mL of the organic extraction solution, and compare it with the untreated extraction solution to evaluate the effects of different purifying agents on removing fructose and glucose.

[0035] Compared with the untreated extraction solution, the five substances with -NH2 groups, namely traditional PSA solid-phase extraction packing, NH2 solid-phase extraction packing, and 1N-SBA-15, 2N-SBA-15, and 3N-SBA-15, all have polar interactions and anion exchange effects. In the medium-polarity solvent ethyl acetate - acetonitrile solution, -NH2 can form hydrogen bonds with -OH in fructose and glucose, thereby adsorbing saccharide substances. Under acidic conditions, -NH2 can be protonated, enhancing the hydrogen bond force and facilitating the adsorption and removal of saccharide substances. The aminated SBA-15, due to its large surface area and porous hexagonal mesoporous structure of its own material, has more functional sites than PSA packing and NH2 packing, so the adsorption effect on fructose and glucose is obvious. From Figure 4 and Figure 5 The comparison chart shows that the effect of removing fructose and glucose with the same mass in the three derivatized SBA-15 materials is significantly greater than that of PSA packing and NH2 packing. When using 100 mg and 200 mg of the three different amine-derivatized SBA-15 purifying agents, the difference in the effect of removing fructose and glucose is not significant. When using 100 mg, the removal rate of fructose is higher than 75%, and the removal rate of glucose is higher than 95%. While the average removal rates of fructose by traditional PSA solid-phase extraction packing and NH2 solid-phase extraction packing are 53% and 63% respectively, and the average removal rates of glucose are 76% and 75% respectively. Therefore, 100 mg of the amine-derivatized SBA-15 purifying agent is finally selected as the optimal treatment amount.

[0036] Example 4 Investigation of impurity removal: Weigh multiple samples, each sample being 2 g of honey. Collect and combine the extraction solution in the same sugar removal experiment. Add 10 mL of each part to a solution containing 500 mg of anhydrous magnesium sulfate and 300 mg of purification powder (which are PSA packing, NH2 packing, molecular sieve SBA-15, 1N-SBA-15, 2N-SBA-15, and 3N-SBA-15 respectively), vortex and mix for 1 min, centrifuge at 5000 r / min for 5 min, then filter through a 0.22 μm organic microporous filter membrane into a test tube. Precisely measure 7.0 mL and place it in a weighed centrifuge tube, blow to dry with nitrogen, weigh again, and subtract the weight of the centrifuge tube to obtain the dry weight of the purified matrix. For another part of the extraction solution, treat the sample without adding a purifying agent to obtain the dry weight of the unpurified matrix. The impurity removal rate is calculated as follows:

[0037] The impurity removal rate determination through the dry weight of the matrix is used to evaluate the impurity removal effect of the purifying agent. From Figure 6From the impurity removal rates of different purifying agents during the pretreatment of honey detection, it can be seen that the impurity removal effect of SBA-15 after amino derivatization is significantly better than the other three. The average impurity removal rates of the three amino-derivatized SBA-15s (48%-52%) are more than 25% higher than those of the PSA solid-phase extraction packing material, NH2 solid-phase extraction packing material method used in traditional QuEChERS, and the impurity removal rates of the undervivatized SBA-15 (17%-27%). Combining with the sugar removal experiment, it is understood that the derivatized SBA-15 material shows high adsorption for saccharide substances and other impurities under the combined action of functional groups and porous structures, which is beneficial to purifying the sample and reducing matrix interference.

[0038] Example 5 Comparing the total ion current chromatograms to investigate the impurity removal effect: The negative honey sample was scanned comprehensively using a liquid chromatography-tandem mass spectrometer, and the total ion current mass spectrum of the sample was obtained within the mass range of 100-1000 daltons. By comparing the total ion current signal intensities of the sample treated with amino-derivatized SBA-15 and the sample not treated with a purifying agent, it can be seen that there are obvious differences between the sample treated with amino-derivatized SBA-15 and the untreated sample. Figure 7 is the total ion current mass spectrum of the honey sample without pretreatment (maximum signal value = 1.7e9 cps), and there are multiple interference peaks within 0-5 min. Figure 8 is the total ion current mass spectrum of the honey sample pretreated with amino-derivatized SBA-15 agent (maximum signal value = 3.2e8 cps), and the interference peaks are not obvious within 0-5 min. And compared with Figure 7 the maximum signal intensity value is reduced by about 5 times, significantly reducing the interference of the sample matrix on the detection, which is beneficial to the detection of the target substance and improving the detection sensitivity.

[0039] Example 6 Matrix effect investigation: The matrix effects of the standard curves prepared with negative sample matrices treated with PSA packing material, NH2 packing material, 1N-SBA-15, 2N-SBA-15, and 3N-SBA-15 in the concentration range of 2.0-200.0 ng / mL and the standard curve prepared with acetonitrile were evaluated according to the following formula:

[0040] The matrix effect is caused by other impurity components co-eluted with the analyte, which affects the ionization during the electrospray ionization process, and is manifested as matrix enhancement or suppression. Endogenous substances such as saccharides and phenols contained in honey are all sources of matrix effects during the detection of pesticide residues. When the matrix effect enhancement or suppression exceeds 20%, it is considered that the matrix effect has a significant impact on quantitative detection and affects the experimental results.

[0041] After detecting 29 pesticides and through calculation, the matrix effects of the samples treated with three kinds of amino-functionalized SBA-15 as shown in the table were all within 10%. For the samples treated with traditional PSA filler and NH2 filler, the matrix effects of some pesticides exceeded 20%, which proved that the purification with amino-functionalized SBA-15 reduced matrix interference and had a better effect than PSA filler and NH2 filler. The results are shown in Table 1-4.

[0042] Table 1-4 Matrix Effects of 29 Pesticides

[0043] Example 7 Investigation of the limits of quantitation, linear regression, recovery rate, and precision: Accurately pipette 0.20 mL of 29 kinds of pesticide residue standard solutions with a concentration of 1000 μg / mL (propamocarb, dinotefuran, carbendazim, thiamethoxam, clothianidin, imidacloprid, acetamiprid, fosthiazate, forchlorfenuron, flumorph, azoxystrobin, dimethomorph, cyproconazole, cyazofamid, diflubenzuron, flusilazole, propiconazole, phoxim, pyraclostrobin, diniconazole, indoxacarb, emamectin benzoate, tolfenpyrad, chlorpyrifos, propargite, fipronil, flonicamid, fipronil sulfone, fipronil sulfoxide) into 20-mL volumetric flasks respectively, and make up the volume to the mark with acetonitrile to prepare a pesticide residue mixed standard stock solution with a concentration of 10 μg / mL. Then accurately pipette 0.50 mL of the above stock solution into a 10-mL volumetric flask and make up the volume to the mark with acetonitrile to prepare a pesticide residue mixed standard working solution with a concentration of 500 ng / mL. Prepare 7 series of mixed standard solutions with concentrations of 2.0 - 200.0 ng / mL using the mixed standard working solution, and carry out the determination according to the UPLC-MS / MS conditions. Figure 2 It is the total ion current chromatogram of 29 pesticides. With the mass concentration (X, ng / mL) as the abscissa and the peak area (Y) as the ordinate, the standard curves of each compound are plotted. Prepare a blank honey extract solution with a spiked level of 0.025 mg / kg, and the corresponding concentration is 5.0 ng / mL. Calculate the method limit of quantitation (limits of quantitation, LOQ) with a 10-fold signal-to-noise ratio. The linear equations, correlation coefficients r, and limits of quantitation of 29 pesticides are shown in Table 1-5. The linear equations of 29 pesticides are good, and the correlation coefficients r all reach above 0.995, meeting the quantitative requirements. Their limits of quantitation are 0.005 - 0.025 mg / kg.

[0044] Table 1-5 Calibration Curves, Correlation Coefficients, and Limits of Quantitation of 29 Pesticides

[0045] Add a mixed standard solution of 29 pesticides to the honey blank sample at a spiking level of 0.025 mg / kg, and repeat 6 parallel experiments on 1N-SBA-15, 2N-SBA-15, and 3N-SBA-15. Calculate the average recovery rate and relative standard deviation (RSD). The results are shown in Table 1-6. It can be seen that the recovery rates of the 29 pesticides determined in the samples treated with the three amino-functionalized SBA-15s are between 82.9% and 108.5%, and the relative standard deviation is less than 10%. The recovery rates and RSDs of the 6 parallel experiments in the experimental results meet the requirements of GB / T 27404-2008, indicating that the accuracy and repeatability of this method are good.

[0046] Table 1-6 Comparison of Recovery Rates of 29 Pesticides

Claims

1. A method for purifying honey using amine-derivatized mesoporous materials to detect pesticide residues, characterized in that: The method uses amine-derivatized SBA-15 to purify the sample, extract the pesticide residues in the honey sample, and then analyze it by UPLC-MS / MS, comprising the following steps: Step A, sample extraction: weigh a honey sample, add 2 to 5 times the weight of water to dissolve the sample, add an acetonitrile solution containing 0.5% (volume ratio) acetic acid as an extract, vortex mix, add mixed inorganic salts, vortex mix, and centrifuge to obtain sample supernatant No. 1; Step B, sample purification: aspirate supernatant No. 1, add the purifying agent containing amine-derivatized SBA-15 thereto, mix well, and centrifuge to obtain supernatant No. 2; Step C, UPLC-MS / MS detection: The supernatant is passed through a microporous filter membrane for detection, and a mixed standard solution of pesticide residues is prepared for quantitative analysis.

2. The method for detecting pesticide residues in honey based on amine-derivatized SBA-15 according to claim 1, characterized in that: The pesticides include any one or more of propamocarb, dinotefuran, carbendazim, thiamethoxam, clothianidin, imidacloprid, acetamiprid, thiathiazolin, chlorfenapyr, flumorph, azoxystrobin, dimethomorph, cyproconazole, cyazofamid, diflubenzuron, flusilazole, propiconazole, phoxim, pyraclostrobin, diniconazole, indoxacarb, emamectin benzoate, tolfenpyrad, chlorpyrifos, fenpropimorph, fipronil, flunitrile, fipronil sulfone, and fipronil sulfoxide.

3. The method for purifying honey and detecting pesticide residues using amine-derivatized mesoporous materials according to claim 1, characterized in that: The mass of the honey sample required in step A is 1-5 g, and the volume of the acetonitrile solution of 0.5% (volume ratio) acetic acid is 5-25 mL.

4. The method for purifying honey and detecting pesticide residues using amine-derivatized mesoporous materials according to claim 1, characterized in that: The mixed inorganic salt in step A is anhydrous magnesium sulfate, added in an amount of 2 to 10 g; anhydrous sodium acetate, added in an amount of 0.5 to 2.5 g.

5. The method for purifying honey and detecting pesticide residues using amine-derivatized mesoporous materials according to claim 1, characterized in that: In the step B, the amino-derivatized SBA-15 is a material synthesized by using SBA-15 and any one of the silane coupling agents 3-aminopropyltriethoxysilane, N-aminoethyl-3-aminopropyltriethoxysilane and diethylenetriaminopropyltrimethoxysilane, wherein 1N-SBA-15 is synthesized with 3-aminopropyltriethoxysilane, 2N-SBA-15 is synthesized with N-aminoethyl-3-aminopropyltriethoxysilane, and 3N-SBA-15 is synthesized with diethylenetriaminopropyltrimethoxysilane.

6. The method for purifying honey and detecting pesticide residues using amine-derivatized mesoporous materials according to claim 1, characterized in that: The purifier in step B is composed of amino-derivatized SBA-15 and anhydrous magnesium sulfate, and the added amounts are 50-250 mg and 150-750 mg respectively.

7. The method for purifying honey and detecting pesticide residues using amine-derivatized mesoporous materials according to claim 1, characterized in that: In the step B, the ratio of the purifier to the supernatant No. 1 of the sample is 100-200 mg of the purifier per milliliter of the supernatant No.

1.

8. The method for purifying honey and detecting pesticide residues using amine-derivatized mesoporous materials according to claim 1, characterized in that: In the step C, the sample is detected by UPLC-MS / MS method and quantified by external standard method, and the chromatographic conditions are as follows: mobile phase A is 0.1% (volume ratio) formic acid aqueous solution containing 2 mmol / L ammonium formate, B is methanol, flow rate is 0.2-0.5 mL / min, gradient elution; mass spectrometry conditions are: simultaneous scanning of positive ion and negative ion modes.

9. The method for purifying honey and detecting pesticide residues using amine-derivatized mesoporous materials according to claim 1, characterized in that: In the step C, different pesticides have their own characteristic multiple reaction monitoring ion pairs, mass spectrometry declustering voltages and collision energies.

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