Preparation method and application of sponge for analysis pretreatment of pesticide in feed

Abstract

The invention discloses a preparation method of sponge for pesticide analysis pretreatment in feed, which comprises the following steps: dissolving dopamine hydrochloride, dodecanethiol and ethylenediamine-N-propyl silane in a mixed solution of Tris-HCl and ethanol to obtain a pretreatment solution; immersing a melamine sponge into the pretreatment solution, repeatedly mixing and extruding, and then carrying out ultrasonic treatment together to obtain a pretreated melamine sponge; the pretreated melamine sponge is placed in a glass bottle to be sealed, then placed in a drying oven to be subjected to heat preservation, washed with ethyl alcohol and dried, and the melamine sponge adsorbent PSA / pDA / DM (at) MeS is obtained. The melamine sponge adsorbent is also applied to pesticide analysis pretreatment and detection. The prepared melamine sponge adsorbent can efficiently and selectively adsorb grease, protein and other complex matrixes in feed, the problems that pesticide residue analyte is large in loss, low in recovery rate, tedious in purification process, difficult to efficiently remove various complex matrixes and the like are solved, and the application prospect is wide.

Description

Technical Field

[0001] The present invention relates to the technical field of analysis and detection, in particular to a preparation method of a melamine sponge adsorbent and application of the melamine sponge adsorbent in pre-analysis treatment and detection of organophosphorus pesticides in feed. Background Art

[0002] With the growing global demand for animal-derived foods, the livestock industry frequently uses organophosphorus pesticides to increase production. The misuse of these pesticides can lead to drug residues in food, compromising food safety. Long-term consumption of foods with excessive residues can also be harmful to human health. Therefore, the development of highly sensitive technologies for the detection of trace organophosphorus pesticides is crucial. Currently, gas chromatography-tandem mass spectrometry (GC-MS) is a commonly used method for detecting organophosphorus pesticide residues due to its high sensitivity, selectivity, and accuracy. However, its analytical accuracy is highly dependent on sample pretreatment.

[0003] QuEChERS is a widely used rapid sample pretreatment method. Due to its advantages of rapidity, simplicity, efficiency, accuracy, and good reproducibility, it has been widely applied to the detection of various residues in food samples. However, this method was initially applied mainly to agricultural products with simple matrices, such as fruits and vegetables, where the traditional QuEChERS method could produce good results. With the continuous development of the QuEChERS method, existing traditional standard sample pretreatment methods can no longer meet the detection requirements of complex matrices (such as organic fertilizers and feed). As the source of animal food production, the quality and safety of feed directly affect the nutritional value and safety of animal products. When detecting multiple pesticide residues in complex feed matrices, if the matrix is not completely cleaned up, single-polarity capillary columns will have problems such as poor resolution, affecting the quantitative and accurate detection of pesticide residues. In particular, the error is greater for trace pesticide residue analysis. Therefore, efficient cleanup pretreatment of complex matrices in feed is a core component of establishing highly sensitive feed pesticide residue detection methods.

[0004] In recent years, researchers in related fields have developed various adsorbents for matrix purification processes, mainly including octadecylsilane (C18), ethylenediamine-N-propylsilane (PSA), graphitized carbon black (GCB), multi-walled carbon nanotubes (WMCNT) and graphene. Although these adsorbents have obvious advantages in sample purification, matrix separation requires an additional high-speed centrifugation process or the assistance of an external strong magnetic field, which greatly increases the difficulty and time of pre-treatment. In addition, traditional adsorbents mostly rely on physical adsorption or a single force (such as hydrophobic effect), which makes it difficult to deal with multiple interferences in feed at the same time. Based on this, different magnetic nanomaterials have been developed in succession for matrix purification, which can be quickly recovered by a magnetic field within seconds. However, magnetic nanomaterials still have problems of particle agglomeration and incomplete recovery in some cases.

[0005] Therefore, solving the problems of existing methods for detecting organophosphorus pesticide residues in feed, such as large analyte loss, low recovery rate, cumbersome purification process, difficult to recycle purification materials, difficulty in efficiently removing multiple complex matrices, and lack of evaluation of matrix effects, is a technical problem that urgently needs to be solved. Summary of the Invention

[0006] The purpose of the present invention is to provide a sponge preparation method and application for pretreatment of pesticide analysis in feed, so as to solve the problems of existing methods for detecting pesticide residues in feed proposed in the above background technology, such as large analyte loss, low recovery rate, cumbersome purification process, difficult recovery of purification materials, difficulty in efficiently removing multiple complex matrices, and lack of evaluation of matrix effects, thereby realizing the construction of a highly sensitive method for detecting trace organophosphorus pesticides in feed.

[0007] In a first aspect, the present invention provides a method for preparing a sponge for pre-treatment of pesticide analysis in feed, comprising the following steps:

[0008] S1. Weigh 1.6-1.7 g of dopamine hydrochloride (pDA), 380-400 μL of dodecanethiol (DM), and 80-100 mg of ethylenediamine-N-propylsilane (PSA) and dissolve them in 200 mL of a mixture of tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) and ethanol to obtain a pretreatment solution.

[0009] S2. Immerse 7 to 8 pieces of melamine sponges (MeS) in the pretreatment solution, repeatedly mix and squeeze them 6 to 8 times, and then place them together in an ultrasonic device for ultrasonic treatment to obtain pretreated melamine sponges.

[0010] S3. The pretreated melamine sponge was placed in a sealed glass bottle, kept warm in an oven at 35-38°C for 12 hours, and then naturally cooled. After washing twice with 99% ethanol, it was dried in an oven at 55-60°C for 24-25 hours to obtain the melamine sponge adsorbent PSA / pDA / DM@MeS modified with ethylenediamine-N-propylsilane / polydopamine / dodecanethiol.

[0011] As a preferred technical solution of the present invention, in step S1: the concentration of Tris-HCl is 10 mM and the pH is 8.5; the concentration of ethanol is 99%; and the volume ratio of Tris-HCl to ethanol is 3:1.

[0012] As a preferred technical solution of the present invention, the length, width and height of the melamine sponge are 1.0-1.5 cm, 1.0-1.5 cm and 1.0-1.5 cm respectively.

[0013] As a preferred technical solution of the present invention, the frequency of the ultrasonic equipment is 40-42 kHz, the temperature is 30-35° C., and the ultrasonic treatment time is 30-32 minutes.

[0014] In a second aspect, the present invention further provides an application of a sponge for pre-analysis treatment of pesticides in feed, wherein the method steps for applying the sponge in pre-analysis treatment and detection of pesticides are as follows:

[0015] Step 1. Sample extraction: Weigh 2.0-2.2 g of feed sample, place it in a 50 mL centrifuge tube, add 8-10 mL of ultrapure water, vortex at 2000 rpm for 3 min, let it stand for 5 min, then add 10 mL of acetonitrile, vortex at 2000 rpm for 3 min to obtain the sample extraction mixture.

[0016] Step 2: Dehydration: Add 4.0-4.2 g of anhydrous magnesium sulfate and 1.0-1.2 g of sodium chloride to the sample extraction mixture, vortex at 2000 rpm for 3 min, centrifuge at 4000 rpm for 10-12 min, and take the supernatant for purification.

[0017] Step 3: Purification: Weigh 3 mL of the supernatant prepared in step 2 and place it into a disposable syringe containing 2 to 3 pieces of PSA / pDA / DM@MeS adsorbent, and squeeze and purify to obtain the purified solution.

[0018] Step 4: Detection: The purified solution is nitrogen-purged and the sample solution obtained after redissolution is filtered and then detected by gas chromatography-tandem mass spectrometry (GC-MS).

[0019] As a preferred technical solution of the present invention, the temperature of the nitrogen blowing is 30° C., and the nitrogen blowing state is to blow until 0.10 to 0.15 mL of the purified liquid remains.

[0020] As a preferred technical solution of the present invention, the re-dissolving reagent is n-hexane, and the fixed volume is 1 mL.

[0021] As a preferred technical solution of the present invention, the pore size of the filter membrane used for filtration is 0.22 μm.

[0022] As a preferred technical solution of the present invention, the gas chromatography parameters for the gas chromatography-tandem mass spectrometry (GC-MS) detection are: using an HP-5X capillary column, an injection port temperature of 250°C, helium as the carrier gas, and an injection volume of 1 μL; in the temperature rising program, the initial temperature is 100°C, maintained for 2 minutes, and then increased to 270°C at 10°C / min and maintained for 5 minutes; the mass spectrometry conditions are: the chromatographic and mass spectrometry interface temperature is 280°C, the solvent delay is 2.0 minutes, and the ion source temperature is 230°C.

[0023] As a preferred technical solution of the present invention, the purity of the helium is ≥99.999% and the flow rate is 1.2 mL / min.

[0024] As a preferred technical solution of the present invention, the gas chromatography-tandem mass spectrometry detection method is as follows: after creating a sample sequence in a gas chromatography-tandem mass spectrometry workstation, the injection bottle is placed in the automatic injector and the injection volume is set to 1 μL; the sequence is started, and the system injects and collects data.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. The melamine sponge adsorbent PSA / pDA / DM@MeS of the present invention is a multifunctional material based on polydopamine (PDA) modification and dodecanethiol (DM) and ethylenediamine-N-propylsilane (PSA) functionalization, and has the unique triple characteristics of "hydrophobic-lipophilic-specific recognition". Among them, the long-chain alkyl group of dodecanethiol gives the material excellent hydrophobic properties, which can efficiently adsorb lipid interfering substances in feed; the phenolic hydroxyl group, amino group and other functional groups provided by the polydopamine layer can form hydrogen bonds and π-π stacking effects with proteins; ethylenediamine-N-propylsilane has amino functional groups, which can adsorb polar interfering substances such as organic acids, sugars, and phenols through hydrogen bonds and electrostatic effects. This multi-mechanism synergistic effect enables the melamine sponge adsorbent to simultaneously achieve efficient adsorption of multiple polar and non-polar organophosphorus pesticides in feed, solving the problem that existing commercial adsorbents mostly rely on physical adsorption or a single force (such as hydrophobic effect) to deal with multiple interfering substances in feed at the same time, as well as the problem of large analyte loss. The melamine sponge adsorbent PSA / pDA / DM@MeS, developed in this paper, requires only simple squeezing or soaking for adsorption and desorption, eliminating the need for complex steps like centrifugation or column chromatography. This simplifies the pretreatment process for organophosphorus pesticides in feed, improves detection efficiency, and overcomes the technical challenges of existing detection methods, which are high cost, time-consuming, and require large amounts of organic solvents. Furthermore, melamine sponges are inexpensive and readily available, significantly reducing detection costs.

[0027] 2. The present invention provides a pretreatment method for organophosphorus pesticide residues in feed, employing a melamine sponge adsorbent to construct an improved QuECHERS pretreatment method. This method purifies samples through simple extrusion, eliminating the need for complex procedures such as centrifugation and column chromatography. This significantly shortens pretreatment time, enables efficient pretreatment of feed samples, and improves detection efficiency. Furthermore, the melamine sponge adsorbent used in the purification process is easily recyclable, reducing detection costs while minimizing environmental pollution.

[0028] 3. The present invention also provides a method for detecting organophosphorus pesticide residues in feed, which adopts the improved QuECHERS method combined with gas chromatography-tandem mass spectrometry (GC-MS) for detection. This method solves the problems of incomplete purification of high-fat feed matrix, insufficient detection sensitivity, and low recovery rate of the traditional QuEChERS method, and provides a reliable technical means for feed quality and safety supervision. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a flow chart for preparing the melamine sponge adsorbent PSA / pDA / DM@MeS of the present invention;

[0030] Figure 2 This is the pretreatment method and detection steps for organophosphorus pesticide residues in feed in the present invention;

[0031] Figure 3 This is the gas chromatography-mass spectrometry of organophosphorus pesticide standards;

[0032] Figure 4 This is a graph showing the effects of different extraction reagents on the recovery of organophosphorus pesticides;

[0033] Figure 5 This is a diagram showing the influence of different extraction reagents on the matrix effect of organophosphorus pesticides;

[0034] Figure 6 This is a graph showing the effect of different reconstitution reagents on the recovery rate of organophosphorus pesticides;

[0035] Figure 7 This is a diagram showing the influence of different reconstitution reagents on the matrix effect of organophosphorus pesticides;

[0036] Figure 8 Effect of different amounts of PDA, DM and PSA on the recovery of organophosphorus pesticides on PSA / PDA / DM@MeS;

[0037] Figure 9 This is a diagram showing the effects of PDA / DM@MeS, PSA@MeS, and PSA / PDA / DM@MeS melamine sponges on the recovery rates of organophosphorus pesticides. DETAILED DESCRIPTION

[0038] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.

[0039] The present invention combines the adhesiveness and secondary reactivity of dopamine hydrochloride with melamine sponge to produce a composite material of ethylenediamine-N-propylsilane / polydopamine / dodecanethiol-modified melamine sponge (PSA / pDA / DM@MeS). Dopamine can be oxidized under weakly alkaline conditions to form a quinone structure, which then undergoes polymerization to form a polydopamine layer. This polydopamine layer contains highly reactive quinone structures and hydroxyl groups, exhibiting secondary reactivity and serving as a secondary reaction platform for covalently immobilizing amino and thiol molecules. The present invention selects organic molecules dodecanethiol (DM) with long-chain alkane groups and ethylenediamine-N-propylsilane (PSA) containing a large number of amino groups. The thiol (-SH) and primary amine (-NH2) groups undergo Michael addition reaction and Schiff base reaction with pDA nanoparticles, respectively. The obtained polydopamine-ethylenediamine-N-propylsilane (pDA-PSA) and polydopamine-dodecanethiol (pDA-DM) are deposited on the surface of melamine sponge (MeS) to prepare the melamine sponge adsorbent PSA / pDA / DM@MeS.

[0040] The present invention utilizes the synergistic effect of the adhesiveness of dopamine and the low surface energy of dodecanethiol, combined with the specific functional groups of ethylenediamine-N-propylsilane, to enable the melamine sponge adsorbent PSA / pDA / DM@MeS to efficiently and selectively adsorb complex matrices such as oil and protein in feed, solving the problems of "polar and non-polar purification cannot be achieved at the same time" and low detection signal values caused by matrix contamination in traditional methods. Moreover, the material can be quickly purified with just simple extrusion, effectively improving pretreatment efficiency and solving the problems of existing methods such as cumbersome operation, long time consumption, large amount of purification agents and a wide variety of types.

[0041] like Figure 1 As shown, a method for preparing a sponge for pretreatment of pesticide analysis in feed includes the following steps: S1. dissolving dopamine hydrochloride (pDA), dodecanethiol (DM), and ethylenediamine-N-propylsilane (PSA) in 200 mL of a mixture of Tris-HCl and ethanol to obtain a pretreatment solution; S2. immersing a melamine sponge in the pretreatment solution, repeatedly mixing and squeezing the mixture, and then placing the mixture together in an ultrasonic device for ultrasonic treatment to obtain a pretreated melamine sponge; S3. placing the pretreated melamine sponge (MeS) in a glass bottle, sealing it, and then placing it in an oven at a temperature of 35-38°C for 12 hours, then naturally cooling it, and washing it twice with 99% ethanol, and then drying it in an oven at 55-60°C for 24-25 hours to obtain a melamine sponge adsorbent PSA / pDA / DM@MeS.

[0042] Figure 2This is the pretreatment method and detection steps for organophosphorus pesticide residues in feed of Example 1; take 2.0g of feed sample, place it in a 50mL centrifuge tube, add 8mL of ultrapure water, vortex at 2000rpm for 3min, and let it stand for 5min to fully infiltrate and disperse the sample; add 10mL of acetonitrile, vortex at 2000rpm for 3min, and extract; add 4.0g of anhydrous magnesium sulfate and 1.0g of sodium chloride, vortex at 2000rpm for 3min, centrifuge at 4000rpm for 10min, and take 3mL of supernatant for purification; add 3mL of supernatant to a disposable syringe containing melamine sponge and squeeze it for purification; take the purified supernatant and blow it with nitrogen at 30°C until 0.10mL of the purified liquid remains, use the reconstitution reagent n-hexane to reconstitute the volume to 1mL, pass it through a filter membrane with a pore size of 0.22um, and determine it by machine (GC-MS).

[0043] The raw materials used in the present invention are all commercially available raw materials.

[0044] Example 1:

[0045] A method for preparing a sponge for pre-treatment of pesticide analysis in feed comprises the following steps:

[0046] S1. Weigh 1.6 g of dopamine hydrochloride (pDA), 400 μL of dodecanethiol (DM), and 100 mg of ethylenediamine-N-propylsilane (PSA) and dissolve them in 200 mL of a mixture of Tris-HCl (10 mM, pH 8.5) and ethanol (99%) (the volume ratio of Tris-HCl to ethanol is 3:1) to obtain a pretreatment solution.

[0047] S2. Eight pieces of melamine sponges (the length, width, and height of the melamine sponges were 1.0 cm, 1.0 cm, and 1.0 cm, respectively) were immersed in the pretreatment solution, mixed and squeezed repeatedly for six times, and then placed together in an ultrasonic device at a frequency of 40 kHz and a temperature of 30°C for ultrasonic treatment for 32 minutes to obtain pretreated melamine sponges.

[0048] S3. The pretreated melamine sponge was placed in a sealed glass bottle, kept warm in an oven at 35°C for 12 h, and then naturally cooled. After washing twice with 99% ethanol, it was dried in an oven at 60°C for 24 h to obtain the melamine sponge adsorbent PSA / pDA / DM@MeS modified with ethylenediamine-N-propylsilane / polydopamine / dodecanethiol.

[0049] The method steps for using melamine sponge in pesticide pre-analysis treatment and detection are as follows:

[0050] Step 1. Sample extraction: Weigh 2.0 g of feed sample, place it in a 50 mL centrifuge tube, add 8 mL of ultrapure water, vortex at 2000 rpm for 3 min, let it stand for 5 min to fully infiltrate and disperse the sample, then add 10 mL of acetonitrile, vortex at 2000 rpm for 3 min to obtain the sample extraction mixture.

[0051] Step 2: Dehydration: Add 4.0 g of anhydrous magnesium sulfate and 1.0 g of sodium chloride to the sample extraction mixture, vortex at 2000 rpm for 3 min, centrifuge at 4000 rpm for 10 min, and take the supernatant for purification.

[0052] Step 3: Purification: Weigh 3 mL of the supernatant prepared in step 2 and place it into a disposable syringe containing two pieces of PSA / pDA / DM@MeS adsorbent. Squeeze and purify to obtain the purified solution.

[0053] Step 4: Detection: Blow nitrogen at 30°C until 0.10 mL of the purified liquid remains. Use the reconstitution reagent n-hexane to reconstitute the volume to 1 mL, pass it through a filter membrane with a pore size of 0.22 μm, create a sample sequence in the gas chromatography-tandem mass spectrometry workstation, place the injection bottle into the automatic injector, and set the injection volume to 1 μL; start the sequence run, and the system injects and collects data.

[0054] The gas chromatography parameters for gas chromatography-tandem mass spectrometry (GC-MS) detection were as follows: an HP-5X capillary column, an injection port temperature of 250°C, helium as the carrier gas (helium purity ≥99.999%, flow rate of 1.2 mL / min), and an injection volume of 1 μL; in the temperature program, the initial temperature was maintained at 100°C for 2 min, then increased to 270°C at a rate of 10°C / min and maintained for 5 min; the mass spectrometry conditions were: chromatographic and mass spectrometry interface temperature of 280°C, solvent delay of 2.0 min, and ion source temperature of 230°C.

[0055] Example 2:

[0056] A method for preparing a sponge for pre-treatment of pesticide analysis in feed comprises the following steps:

[0057] S1. Weigh 1.7 g of dopamine hydrochloride (pDA), 380 μL of dodecanethiol (DM), and 80 mg of ethylenediamine-N-propylsilane (PSA) and dissolve them in 200 mL of a mixture of Tris-HCl (10 mM, pH 8.5) and ethanol (99%) (the volume ratio of Tris-HCl to ethanol is 3:1) to obtain a pretreatment solution.

[0058] S2. Eight pieces of melamine sponges (the length, width, and height of the melamine sponges were 1.5 cm, 1.5 cm, and 1.5 cm, respectively) were immersed in the pretreatment solution, mixed and squeezed repeatedly eight times, and then placed together in an ultrasonic device at a frequency of 42 kHz and a temperature of 35°C for ultrasonic treatment for 30 minutes to obtain pretreated melamine sponges.

[0059] S3. The pretreated melamine sponge was placed in a sealed glass bottle, kept warm in an oven at 38°C for 12 h, and then naturally cooled. After washing twice with 99% ethanol, it was dried in an oven at 55°C for 25 h to obtain the melamine sponge adsorbent PSA / pDA / DM@MeS modified with ethylenediamine-N-propylsilane / polydopamine / dodecanethiol.

[0060] The method steps for using melamine sponge in pesticide pre-analysis treatment and detection are as follows:

[0061] Step 1. Sample extraction: Weigh 2.2 g of feed sample, place it in a 50 mL centrifuge tube, add 10 mL of ultrapure water, vortex at 2000 rpm for 3 min, let it stand for 5 min to fully infiltrate and disperse the sample, then add 10 mL of acetonitrile, vortex at 2000 rpm for 3 min to obtain the sample extraction mixture.

[0062] Step 2: Dehydration: Add 4.2 g of anhydrous magnesium sulfate and 1.2 g of sodium chloride to the sample extraction mixture, vortex at 2000 rpm for 3 min, centrifuge at 4000 rpm for 12 min, and take the supernatant for purification.

[0063] Step 3: Purification: Weigh 3 mL of the supernatant prepared in step 2 and place it into a disposable syringe containing two pieces of PSA / pDA / DM@MeS adsorbent. Squeeze and purify to obtain the purified solution.

[0064] Step 4: Detection: Blow nitrogen at 30°C until 0.15 mL of the purified liquid remains. Use the reconstitution reagent n-hexane to reconstitute the volume to 1 mL, pass it through a filter membrane with a pore size of 0.22 μm, create a sample sequence in the gas chromatography-tandem mass spectrometry workstation, place the injection bottle into the automatic injector, and set the injection volume to 1 μL; start the sequence run, and the system injects and collects data.

[0065] The gas chromatography parameters for gas chromatography-tandem mass spectrometry (GC-MS) detection were as follows: an HP-5X capillary column, an injection port temperature of 250°C, helium as the carrier gas (helium purity ≥99.999%, flow rate of 1.2 mL / min), and an injection volume of 1 μL; in the temperature program, the initial temperature was maintained at 100°C for 2 min, then increased to 270°C at a rate of 10°C / min and maintained for 5 min; the mass spectrometry conditions were: chromatographic and mass spectrometry interface temperature of 280°C, solvent delay of 2.0 min, and ion source temperature of 230°C.

[0066] Example 3:

[0067] A method for preparing a sponge for pre-treatment of pesticide analysis in feed comprises the following steps:

[0068] S1. Weigh 1.6 g of dopamine hydrochloride (pDA), 390 μL of dodecanethiol (DM), and 90 mg of ethylenediamine-N-propylsilane (PSA) and dissolve them in 200 mL of a mixture of Tris-HCl (10 mM, pH 8.5) and ethanol (99%) (the volume ratio of Tris-HCl to ethanol is 3:1) to obtain a pretreatment solution.

[0069] S2. Eight pieces of melamine sponges (the length, width, and height of the melamine sponges were 1.0 cm, 1.0 cm, and 1.0 cm, respectively) were immersed in the pretreatment solution, mixed and squeezed repeatedly for six times, and then placed together in an ultrasonic device at a frequency of 40 kHz and a temperature of 32°C for ultrasonic treatment for 30 minutes to obtain pretreated melamine sponges.

[0070] S3. The pretreated melamine sponge was placed in a sealed glass bottle, kept warm in an oven at 36°C for 12 h, and then naturally cooled. After washing twice with 99% ethanol, it was dried in an oven at 58°C for 24 h to obtain the melamine sponge adsorbent PSA / pDA / DM@MeS modified with ethylenediamine-N-propylsilane / polydopamine / dodecanethiol.

[0071] The method steps for using melamine sponge in pesticide pre-analysis treatment and detection are as follows:

[0072] Step 1. Sample extraction: Weigh 2.0 g of feed sample, place it in a 50 mL centrifuge tube, add 9 mL of ultrapure water, vortex at 2000 rpm for 3 min, let it stand for 5 min to fully infiltrate and disperse the sample, then add 10 mL of acetonitrile, vortex at 2000 rpm for 3 min to obtain the sample extraction mixture.

[0073] Step 2: Dehydration: Add 4.1 g of anhydrous magnesium sulfate and 1.1 g of sodium chloride to the sample extraction mixture, vortex at 2000 rpm for 3 min, centrifuge at 4000 rpm for 11 min, and take the supernatant for purification.

[0074] Step 3: Purification: Weigh 3 mL of the supernatant prepared in step 2 and place it into a disposable syringe containing 3 pieces of PSA / pDA / DM@MeS adsorbent, and squeeze and purify to obtain the purified solution.

[0075] Step 4: Detection: Blow nitrogen at 30°C until 0.10 mL of the purified liquid remains. Use the reconstitution reagent n-hexane to reconstitute the volume to 1 mL, pass it through a filter membrane with a pore size of 0.22 μm, create a sample sequence in the gas chromatography-tandem mass spectrometry workstation, place the injection bottle into the automatic injector, and set the injection volume to 1 μL; start the sequence run, and the system injects and collects data.

[0076] The gas chromatography parameters for gas chromatography-tandem mass spectrometry (GC-MS) detection were as follows: an HP-5X capillary column, an injection port temperature of 250°C, helium as the carrier gas (helium purity ≥99.999%, flow rate of 1.2 mL / min), and an injection volume of 1 μL; in the temperature program, the initial temperature was maintained at 100°C for 2 min, then increased to 270°C at a rate of 10°C / min and maintained for 5 min; the mass spectrometry conditions were: chromatographic and mass spectrometry interface temperature of 280°C, solvent delay of 2.0 min, and ion source temperature of 230°C.

[0077] Comparative Example 1:

[0078] The difference from Example 1 is that in step S1 , dopamine hydrochloride (pDA) is 1.4 g, dodecanethiol (DM) is 350 μL, and ethylenediamine-N-propylsilane (PSA) is 80 mg.

[0079] Comparative Example 2:

[0080] The difference from Example 1 is that in step S1 , dopamine hydrochloride (pDA) is 1.8 g, dodecanethiol (DM) is 450 μL, and ethylenediamine-N-propylsilane (PSA) is 120 mg.

[0081] Comparative Example 3:

[0082] A method for preparing a sponge for pre-treatment of pesticide analysis in feed comprises the following steps:

[0083] S1. Weigh 1.6 g of dopamine hydrochloride (pDA) and 400 μL of dodecanethiol (DM) and dissolve them in 200 mL of a mixture of Tris-HCl (10 mM, pH 8.5) and ethanol (99%) (the volume ratio of Tris-HCl to ethanol is 3:1) to obtain a pretreatment solution.

[0084] S2. Eight pieces of melamine sponges (the length, width, and height of the melamine sponges were 1.0 cm, 1.0 cm, and 1.0 cm, respectively) were immersed in the pretreatment solution, mixed and squeezed repeatedly for six times, and then placed together in an ultrasonic device at a frequency of 40 kHz and a temperature of 30°C for ultrasonic treatment for 32 minutes to obtain pretreated melamine sponges.

[0085] S3. The pretreated melamine sponge (MeS) was placed in a sealed glass bottle, then placed in an oven at 35°C for 12 hours and allowed to cool naturally. After washing twice with 99% ethanol, it was dried in an oven at 60°C for 24 hours to obtain a polydopamine / dodecanethiol-modified melamine sponge (pDA / DM@MeS).

[0086] Other methods and steps for using melamine sponge in pesticide pre-analysis treatment and detection are the same as those in Example 1.

[0087] Comparative Example 4:

[0088] A method for preparing a sponge for pre-treatment of pesticide analysis in feed comprises the following steps:

[0089] S1. Weigh 100 mg of ethylenediamine-N-propylsilane (PSA) and dissolve it in 200 mL of a mixture of Tris-HCl (10 mM, pH 8.5) and ethanol (99%) (the volume ratio of Tris-HCl to ethanol is 3:1) to obtain a pretreatment solution.

[0090] S2. Eight pieces of melamine sponges (the length, width, and height of the melamine sponges were 1.0 cm, 1.0 cm, and 1.0 cm, respectively) were immersed in the pretreatment solution, mixed and squeezed repeatedly for six times, and then placed together in an ultrasonic device at a frequency of 40 kHz and a temperature of 30°C for ultrasonic treatment for 32 minutes to obtain pretreated melamine sponges.

[0091] S3. The pretreated melamine sponge (MeS) was placed in a sealed glass bottle, then placed in an oven at 35°C for 12 hours and allowed to cool naturally. After washing twice with 99% ethanol, it was dried in an oven at 60°C for 24 hours to obtain ethylenediamine-N-propylsilane-modified melamine sponge (PSA@MeS).

[0092] Other methods and steps for using melamine sponge in pesticide pre-analysis treatment and detection are the same as those in Example 1.

[0093] Figure 3 This is the gas chromatography-mass spectrometry of the organophosphorus pesticide standard. The gas chromatography parameters are as follows: an HP-5X capillary column (30m×0.25mm, 0.25μm) is used; the injection port temperature is 250℃; the carrier gas is helium with a purity of ≥99.999% and a flow rate of 1.2mL / min; the injection method is splitless injection, and the injection volume is 1μL; in the temperature ramp program, the initial temperature is 100℃, maintained for 2min; then increased to 270℃ at 10℃ / min and maintained for 5min; the mass spectrometry conditions are: the chromatographic and mass spectrometry interface temperature is 280℃; the solvent delay is 2.0min; and the ion source temperature is 230℃. Figure 3The organophosphorus pesticide peaks displayed are complete, sharp, and have a stable baseline, indicating excellent separation. All 24 organophosphorus pesticides elute within 5 to 21 minutes. Retention times and characteristic ions allow for qualitative analysis of each pesticide peak. Furthermore, no tailing or unidentified peaks are observed, demonstrating excellent column efficiency.

[0094] Figure 4 The figure shows the effect of different extraction reagents, acetonitrile, n-hexane, ethyl acetate and acetone, on the recovery rate of organophosphorus pesticides. Figure 4 It can be seen that when the extraction reagent is acetonitrile, the recovery rate of 75% of the target substance is within the acceptable range of 80%-120%. When the extraction reagent is n-hexane, ethyl acetate and acetone, the recovery rates of 16.7%, 29.2% and 16.7% of the target substance are respectively within the acceptable range of 80%-120%. It can be clearly seen that better recovery effect can be achieved when using acetonitrile extraction.

[0095] Figure 5 The effects of different extraction reagents, acetonitrile, n-hexane, ethyl acetate, and acetone, on the matrix effect of organophosphorus pesticides were studied. Figure 5 It can be seen that the influence of the four different extraction reagents on the matrix effect is not much different, and the distribution of matrix effect is concentrated between 60-80%, which is a strong matrix effect. The comprehensive recovery results consider the selection of acetonitrile as the extraction reagent.

[0096] Figure 6 The effects of different reconstitution reagents, acetone, n-hexane and ethyl acetate, on the recovery of organophosphorus pesticides are shown in Figure 2. Figure 6 It can be seen that when the reconstitution reagent is acetone or n-hexane, 75% of the target recovery rate is within the acceptable range of 80%-120%, while when ethyl acetate is used for reconstitution, only 8.3% of the target recovery rate is within the acceptable range of 80%-120%.

[0097] Figure 7 The effects of different reconstitution reagents, acetone, n-hexane, and ethyl acetate, on the matrix effect of organophosphorus pesticides are shown in the figure. Figure 7 It can be seen that when reconstituted with acetone, only 4.2% of the target substances had a matrix effect lower than 50%, indicating that only 4.2% of the target substances were within the range of medium or low matrix effects; when reconstituted with n-hexane, 16.7% of the target substances had a matrix effect lower than 50%; when reconstituted with ethyl acetate, 20.8% of the target substances had a matrix effect lower than 50%. Although using ethyl acetate as a reconstitution reagent can achieve a lower matrix effect, the recovery rate of the target substances reconstituted with ethyl acetate is poorer than that of acetone and n-hexane. Therefore, n-hexane was selected as the reconstitution reagent after comprehensive consideration.

[0098] Figure 8The effects of PSA / PDA / DM@MeS prepared with different amounts of PDA, DM and PSA on the recovery of organophosphorus pesticides are shown in the figure. Figure 8 As shown, the ethylenediamine-N-propylsilane / polydopamine / dodecanethiol modified melamine sponge composite materials (PSA / pDA / DM@MeS) prepared in Examples 1, 2 and 3 have better overall performance in the recovery rate of organophosphorus pesticides in feed than the melamine sponge materials prepared in Comparative Examples 1 and 2. In Example 1, 75% of the target substances have a recovery rate within the acceptable range of 80-120%, in Example 2, 71% of the target substances have a recovery rate within the acceptable range of 80-120%, and in Example 3, 66.7% of the target substances have a recovery rate within the acceptable range of 80-120%. In Comparative Examples 1 and 2, respectively, only 16.7% and 8.3% of the target substances have a recovery rate within the acceptable range of 80-120%, which is lower than the results of the embodiments. The melamine sponge composite materials prepared in Examples 1, 2, and 3 can achieve better recovery rates when treating feed samples. The modification of PSA and DM is relatively more sufficient, and the functional groups on the surface of the material are more completely covered. Therefore, the matrix is removed more completely, showing an overall higher recovery rate. The polydopa layer contains highly reactive quinone structures and hydroxyl groups, which have secondary reactivity and can be used as a secondary reaction platform to achieve covalent immobilization of amino and thiol groups. Under alkaline conditions, the quinone structure in pDA can undergo Michael addition reaction with DM containing thiol (-SH) to form a CS bond; and undergo Schiff base reaction with PSA containing primary amine (-NH2) to generate an imine bond (C=N), but there may be a competitive binding mechanism between the two. When the amount of pDA is insufficient, PSA and DM are not fully modified, and the functional groups on the material surface are not fully covered, resulting in incomplete matrix removal and an overall lower recovery rate; while excessive pDA will cause DM to form an excessively thick hydrophobic layer. The excessive hydrophobicity causes the amino group of PSA to be sterically shielded by the long-chain alkyl group of dodecanethiol, resulting in a decrease in the adsorption efficiency on polar matrices.

[0099] The effects of feed pretreatment on the recovery rate of organophosphorus pesticides using the PSA / PDA / DM@MeS melamine sponge prepared in Example 1, the PDA / DM@MeS melamine sponge prepared in Comparative Example 3, and the PSA@MeS melamine sponge prepared in Comparative Example 4 were compared. Figure 9 As shown. Figure 9It can be seen that the ethylenediamine-N-propylsilane / polydopamine / dodecanethiol-modified melamine sponge composite material (PSA / pDA / DM@MeS) prepared in Example 1 has a better recovery rate for organophosphorus pesticides in feed than the sponge materials prepared in Comparative Examples 3 and 4. In Example 1, 75% of the target substance recovery rate is within the acceptable range of 80-120%, while in Comparative Example 3, only 4.2% of the target substance recovery rate is within the acceptable range of 80-120%, and in Comparative Example 4, 58.3% of the target substance recovery rate is within the acceptable range of 80-120%. Using the PSA / PDA / DM@MeS melamine sponge to treat feed samples can achieve a better recovery rate, indicating that the matrix purification effect of the PSA / PDA / DM@MeS melamine sponge is better than that of melamine sponges modified with only PSA or a combination of PDA and DM. As can be seen, PSA, which possesses primary and secondary amino groups, can form strong hydrogen bonds with certain matrix components. PSA has the ability to remove organic acids, some sugars, and fatty acids. However, melamine sponges modified solely with PSA have insufficient adsorption capacity for nonpolar interferents, and directly grafted PSA tends to aggregate and fall off after repeated extrusion. While the abundant quinone groups on the surface of polydopamine (pDA)-modified melamine sponge materials can adsorb some polar interferents (such as proteins) through hydrogen bonding and π-π stacking, the lack of long-chain alkyl groups limits their adsorption efficiency for nonpolar interferents (such as lipids). In contrast, pDA / DM-modified materials, which covalently bond low-surface-energy small molecules via the Michael addition reaction to form a superhydrophobic layer, significantly enhance their adsorption capacity for nonpolar substances. However, the steric hindrance of the long-chain alkyl groups of dodecanethiol (DM) reduces the number of amino-binding sites on the material surface, resulting in a decrease in adsorption efficiency for polar matrices. Therefore, achieving co-cleanup of multicomponent matrices can improve the recovery rate of target compounds.

[0100] In summary, the melamine sponge adsorbent PSA / pDA / DM@MeS modified with ethylenediamine-N-propylsilane / polydopamine / dodecanethiol prepared by the present invention can efficiently and selectively adsorb complex matrices such as oil, protein, etc. in feed, solving the problem of "polar and non-polar purification cannot be achieved at the same time" in traditional methods and the low signal value of the detection results caused by matrix contamination; and the material only requires simple extrusion to achieve rapid purification, effectively improving the pretreatment efficiency, and solving the problems of the existing methods such as cumbersome operation, long time consumption, large amount of purification agents, and many types, and has broad application prospects.

[0101] The above are only specific embodiments of the present invention, but the technical features of the present invention are not limited thereto. Any simple changes, equivalent substitutions, or modifications based on the present invention to solve substantially the same technical problems and achieve substantially the same technical effects are all included in the scope of protection of the present invention.

Claims

1. A method for preparing a sponge for pre-treatment of pesticide analysis in feed, characterized in that: The following steps are involved: S1. Weigh 1.6-1.7 g of dopamine hydrochloride, 380-400 μL of dodecanethiol, and 80-100 mg of ethylenediamine-N-propylsilane and dissolve them in 200 mL of a mixture of Tris-HCl and ethanol to obtain a pretreatment solution. S2. Immerse 7 to 8 pieces of melamine sponge in the pretreatment solution and repeatedly mix and squeeze 6 to 8 times and then place them together in an ultrasonic device for ultrasonic treatment to obtain a pre-treated melamine sponge; S3. The pretreated melamine sponge was placed in a sealed glass bottle, kept warm in an oven at 35-38°C for 12 hours, and then naturally cooled. After washing twice with 99% ethanol, it was dried in an oven at 55-60°C for 24-25 hours to obtain the melamine sponge adsorbent PSA / pDA / DM@MeS modified with ethylenediamine-N-propylsilane / polydopamine / dodecanethiol.

2. The method for preparing a sponge for pre-treatment of pesticide analysis in feed according to claim 1, characterized in that: In step S1, the concentration of Tris-HCl is 10 mM and the pH is 8.5; the concentration of ethanol is 99%; and the volume ratio of Tris-HCl to ethanol is 3:

1.

3. The method for preparing a sponge for pre-treatment of pesticide analysis in feed according to claim 1, characterized in that: The length, width and height of the melamine sponge are 1.0-1.5 cm, 1.0-1.5 cm and 1.0-1.5 cm respectively.

4. The method for preparing a sponge for pre-treatment of pesticide analysis in feed according to claim 1, characterized in that: The frequency of the ultrasonic equipment is 40-42 KHz, the temperature is 30-35° C., and the ultrasonic treatment time is 30-32 minutes.

5. Use of the sponge for pre-analysis treatment of pesticides in feed according to any one of claims 1 to 4 in pre-analysis treatment and detection of pesticides.

6. Use of the sponge for pre-analysis treatment of pesticides in feed according to claim 5 in pre-analysis treatment and detection of pesticides, characterized in that: The method steps for using the sponge in pre-analysis treatment and detection of pesticides are as follows: Step 1, sample extraction: Weigh 2.0-2.2 g of feed sample, place in a 50 mL centrifuge tube, add 8-10 mL of ultrapure water, vortex at 2000 rpm for 3 min, let stand for 5 min, then add 10 mL of acetonitrile, vortex at 2000 rpm for 3 min to obtain the sample extraction mixture; Step 2: Dehydration: Add 4.0-4.2 g of anhydrous magnesium sulfate and 1.0-1.2 g of sodium chloride to the sample extraction mixture, vortex at 2000 rpm for 3 min, centrifuge at 4000 rpm for 10-12 min, and collect the supernatant for purification; Step 3: Purification: Weigh 3 mL of the supernatant prepared in step 2 and place it into a disposable syringe containing 2 to 3 pieces of PSA / pDA / DM@MeS adsorbent, and squeeze and purify to obtain the purified solution; Step 4: Detection: The purified solution is nitrogen-purged and re-dissolved to obtain a sample solution to be tested, which is then filtered and detected by gas chromatography-tandem mass spectrometry.

7. Use of the sponge for pre-analysis treatment of pesticides in feed according to claim 6 in pre-analysis treatment and detection of pesticides, characterized in that: The nitrogen blowing temperature is 30°C, and the nitrogen blowing state is blown until 0.10-0.15 mL of the purified liquid remains; The reconstitution reagent is n-hexane, and the fixed volume is 1 mL; The pore size of the filter membrane used in the filtration is 0.22 μm.

8. Use of the sponge for pre-analysis treatment of pesticides in feed according to claim 6 in pre-analysis treatment and detection of pesticides, characterized in that: The gas chromatography parameters for the gas chromatography-tandem mass spectrometry detection are: an HP-5X capillary column, an injection port temperature of 250°C, helium as the carrier gas, and an injection volume of 1 μL; in the temperature ramp, the initial temperature is 100°C, maintained for 2 minutes, then increased to 270°C at a rate of 10°C / min and maintained for 5 minutes; the mass spectrometry conditions are: a chromatographic and mass spectrometry interface temperature of 280°C, a solvent delay of 2.0 minutes, and an ion source temperature of 230°C.

9. Use of the sponge for pre-analysis treatment of pesticides in feed according to claim 8 in pre-analysis treatment and detection of pesticides, characterized in that: The purity of the helium gas is ≥99.999%, and the flow rate is 1.2 mL / min.

10. Use of the sponge for pre-analysis treatment of pesticides in feed according to claim 6 or 8 in pre-analysis treatment and detection of pesticides, characterized in that: The gas chromatography-tandem mass spectrometry detection method is as follows: after creating a sample sequence in a gas chromatography-tandem mass spectrometry workstation, the injection bottle is placed in the automatic injector and the injection volume is set to 1 μL; the sequence is started, and the system injects samples and collects data.

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