Preparation method of multi-template molecularly imprinted polymer selectively adsorbing multiple pigments and application thereof in solid-phase extraction filler

Abstract

This invention discloses a method for preparing a multi-template molecularly imprinted polymer that selectively adsorbs multiple pigments and its application in solid-phase extraction packing. The method includes the following steps: mixing 50-80 mg of carboxyl-modified polymeric microspheres, 80-120 mL of template molecule-functional monomer complex, 80-110 μL of EGDMA, 50-80 μL of tetraethyl orthosilicate, 30-50 mg of AIBN, and 0.1-0.3 mL of ammonia water; degassing and deoxygenating the mixture; sealing the reaction chamber; Soxhlet extraction and elution to remove unreacted templates, crosslinking agents, and functional monomers; rinsing with an eluent; and purification and elution with a purification solution. After purification, centrifugation is performed to collect the precipitate, and the precipitate is dried to obtain the multi-template molecularly imprinted polymer that selectively adsorbs multiple pigments. This polymer is then mixed with PSA packing at a mass ratio of 2-6:5 to obtain the solid-phase extraction packing. The advantage of this method is that it effectively removes pigment matrix interference while ensuring analyte recovery.

Description

Technical Field

[0001] This invention relates to a molecularly imprinted solid-phase extraction packing material, and more particularly to a method for preparing a multi-template molecularly imprinted polymer that selectively adsorbs multiple pigments and its application in solid-phase extraction packing materials. Background Technology

[0002] Solid-phase extraction (SPE) is a technique for separating and enriching target compounds in analytical samples. Solid materials possess specific affinities, selectively adsorbing target compounds while excluding interfering substances. The target compound is eluted from the solid material by altering solvent polarity or pH, yielding a pure solution of the target compound. SPE is widely used in chemistry, biochemistry, and environmental analysis due to its high selectivity, ease of operation, and excellent enrichment effects, making it widely applicable in analytical chemistry. However, in practice, some biological or sediment samples are rich in various natural pigments. These pigments can significantly impact analytical performance in SPE because their presence triggers multiple interactions, affecting adsorption, elution, interference, and detection. Furthermore, the presence of pigments significantly affects various detectors. Excessive pigments may compete with the selected solid material for adsorption, reducing the adsorption efficiency of the target analyte. This competitive adsorption effect can severely impact the selectivity of SPE, especially when the pigment and target analyte have similar chemical properties. Therefore, solid-phase materials must be carefully selected and optimized to ensure that the selectivity and adsorption efficiency of the target analyte are not affected by pigments.

[0003] QuEChERS, developed from solid-phase extraction, offers advantages such as simple operation, short processing time, and low solvent consumption, significantly improving analytical efficiency and sample preparation convenience. A common QuEChERS method combines dispersive solid-phase extraction (d-SPE) with purification and has been applied to analyze typical contaminants in fish tissue samples. However, due to the significant differences in properties between different configurations of contaminants, traditional QuEChERS struggles to simultaneously extract and purify multiple multi-configuration contaminants from biological samples. Furthermore, aquatic biological samples often contain complex matrices, especially complex pigment species, which can interfere with the extraction and purification process, making it difficult to achieve satisfactory matrix removal and target analyte recovery. Therefore, more effective extraction methods are needed for biological sample pretreatment to reduce the matrix effect caused by complex pigments and improve the recovery and purity of the target analyte. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing a multi-template molecularly imprinted polymer that selectively adsorbs multiple pigments while ensuring the recovery rate of analytes and effectively removing the interference of pigment matrix, and its application in solid phase extraction packing.

[0005] The technical solution adopted by this invention to solve the above-mentioned technical problems is as follows: a method for preparing a multi-template molecularly imprinted polymer that selectively adsorbs multiple pigments, comprising the following steps:

[0006] (1) Mix 3-5 mL of concentrated pigment extracted from aquatic products, 80-100 mL of mixed solvent composed of toluene and acetonitrile in equal volume ratio, 30-50 mg of β-carotene, 3-5 mg of fucoxanthin and 90-120 μL of mixed functional monomers, sonicate at room temperature for 5-10 min, and shake in the dark to form a stable template molecule-functional monomer complex.

[0007] (2) Mix 50 mL of acetonitrile, 2 mL of crosslinking agent (ethylene glycol dimethacrylate) EGDMA, 13.6-22.8 μL of functional monomer MAA and 150-200 mg of initiator (azobisisobutyronitrile) AIBN, degas in an ultrasonic bath for 5-8 min, deoxygenate by purging with nitrogen for 5-8 min, seal the reaction system, stir in a constant temperature water bath at 50-70℃ for 20-28 h, cool to room temperature after heating, centrifuge and vacuum dry at 40-50℃ to obtain carboxyl-modified polymer microspheres;

[0008] (3) Mix 50-80 mg of carboxyl-modified polymeric microspheres, 80-120 mL of template molecule-functional monomer complex, 80-110 μL of LEGDMA, 50-80 μL of tetraethyl orthosilicate, 30-50 mg of AIBN, and 0.1-0.3 mL of ammonia water, degas the mixture by sonication for 5-8 min, deoxygenate it by nitrogen purging for 5-8 min, seal the reaction system, stir in a constant temperature water bath at 50-70℃, after the reaction is completed, extract by Soxhlet extraction for 8-12 h, wash away unreacted template, crosslinking agent and functional monomer, rinse with elution solution, and finally purify with purification solution. After the elution is completed, collect the precipitate by centrifugation and dry it in a vacuum drying oven at 50-70℃ for 4-6 h to obtain a multi-template molecularly imprinted polymer that selectively adsorbs multiple pigments.

[0009] Further, the extraction method of the concentrated pigment described in step (1) is as follows: Aquatic products are added to an acetonitrile solution containing 0.5 wt% formic acid at a mass-to-volume ratio of 20 g: 20 mL, sonicated for 8-12 min, and microwaved for 5-8 min at 20-30℃ and 300-500W power. Then, the pigment is extracted by shaking at 8-120 rpm for 20-40 min, and the supernatant is collected by centrifugation. The precipitate is collected and the extraction and centrifugation are repeated 2-4 times. The supernatant containing the pigment is combined to obtain the supernatant containing the pigment. The supernatant containing the pigment is concentrated by rotary evaporation to 1 / 10-1 / 15 of its volume to obtain the concentrated pigment.

[0010] Furthermore, the aquatic product mentioned is at least one of mussels, clams, oysters, razor clams, large yellow croaker, small yellow croaker, and mackerel.

[0011] Further, the mixed functional monomer mentioned in step (1) is composed of functional monomer MAA and 3-aminopropyltriethoxysilane in a volume ratio of 6:4.

[0012] Further, the eluent in step (3) is composed of methanol and acetic acid in a volume ratio of 9:1; the rinsing solution is composed of ethanol and acetonitrile in a volume ratio of 8:2; and the purification solution is methanol.

[0013] Furthermore, the various pigments mentioned include β-carotene, fucoxanthin, and polydinocyanin.

[0014] The present invention also provides the application of the above-mentioned multi-template molecularly imprinted polymer in the preparation of solid-phase extraction fillers for selective adsorption of various pigments.

[0015] Furthermore, the solid-phase extraction packing is a mixture of multi-template molecularly imprinted polymer and PSA packing in a mass ratio of 2-6:5.

[0016] This invention also provides a method for pretreatment of QuEChERS samples using the above-mentioned solid-phase extraction packing material, comprising the following steps: taking 2-10g of aquatic sample, adding 15-30mL of solvent and 3-5mL of ultrapure water, vortexing for 10-15min, adding 1-3g of NaCl and 2-4g of MgSO4, continuing vortexing for 2-5min, centrifuging, taking the upper organic phase, adding 70-100mg of solid-phase extraction packing material and 80-150mg of MgSO4, vortexing for 0.5-2min, centrifuging, taking the supernatant, filtering through a 0.22μm microporous membrane, concentrating to 0.5-2mL under N2 gas flow, and then analyzing by GC-MS / MS.

[0017] Furthermore, the solvent is at least one selected from acetonitrile, methanol, n-hexane, ethyl acetate, and acetone.

[0018] Compared with existing technologies, the advantages of this invention are as follows: This invention provides a method for preparing a multi-template molecularly imprinted polymer (D-MIP) that selectively adsorbs multiple pigments, and its application in solid-phase extraction packing materials. The D-MIP provides excellent pigment adsorption capacity through multiple target sites, while the mixed-color pigment template molecules extracted from aquatic products provide a complete pigment configuration for the diversity of pigments in organisms. The surface grafting precipitation method provides the imprinted material with excellent specific surface area, porosity, and multi-template specific sites. Compared with traditional solid-phase extraction and QuEChERS methods, solid-phase extraction and QuEChERS methods incorporating D-MIPs exhibit lower matrix effects, effectively removing pigment matrix interference while ensuring analyte recovery. Attached Figure Description

[0019] Figure 1 Photograph of the concentrated pigment extracted in Specific Embodiment 1;

[0020] Figure 2 The image shows a scanning electron microscope image of the multi-template molecularly imprinted polymer in Specific Example 1, where A represents D-MIPs and B represents D-NIPs.

[0021] Figure 3 The specific surface area and average pore size of D-MIPs and D-NIPs in Specific Embodiment 1;

[0022] Figure 4 The adsorption kinetics curves of D-MIPs and D-NIPs in Specific Example 2 are shown.

[0023] Figure 5 The results show the selective adsorption performance analysis of D-MIPs and D-NIPs in Specific Example 2;

[0024] Figure 6 This is a comparison of the adsorption effects of different solid-phase extraction packings on β-carotene and fucoxanthin in Specific Example 3, where A represents different combinations of adsorbents and B represents D-MIPs + PSA packing combinations with different amounts of D-MIPs.

[0025] Figure 7 For the comparative analysis of the effects of biological sample purification before and after in specific embodiment three, A represents the color change of the extract before and after purification for different adsorbents, and B represents the change of absorbance of the extract before and after purification.

[0026] Figure 8 The results of the effect of adding D-MIPs on the recovery rate of eight polychlorinated biphenyls are shown in Specific Example 4. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0028] Specific Example 1: Preparation of a multi-template molecularly imprinted polymer that selectively adsorbs multiple pigments, comprising the following steps:

[0029] Step 1: Add 20 mL of acetonitrile solution containing 0.5 wt% formic acid to 20 g of shellfish meat (4 g each of mussels, clams, oysters, and razor clams) or common aquatic organism samples (large yellow croaker, small yellow croaker, and mackerel). Sonicate for 10 min, then microwave extract for 5-8 min at 25℃ and 400W power. Then, shake at 100 rpm for 30 min to extract the pigment from the sample. Next, centrifuge at 9000 rpm for 15 min, collect the supernatant, and repeat the extraction and centrifugation operation 3 times. Combine the results to obtain the supernatant containing the pigment.

[0030] 60 mL of the supernatant containing the pigment was added to a 100 mL round-bottom flask and evaporated at 30 °C and 150 rpm for 40 min to obtain 5 mL of concentrated pigment. 20 μL of the concentrated pigment was placed on a thin-layer chromatography plate and immersed in 1 mL of a separation solvent composed of ethyl acetate and methanol in a 20:1 volume ratio. The main pigment species in the concentrated pigment were determined by thin-layer chromatography. The results are as follows: Figure 1 As shown, the concentrated pigments are brownish-brown and are mainly composed of carotenoids, including three types of pigments: β-carotene, fucoxanthin, and polydinolin.

[0031] Step 2: Prepare multi-template molecularly imprinted polymers (D-MIPs) using a surface grafting combined with precipitation polymerization method.

[0032] Since β-carotene, fucoxanthin, and polydinoxin all belong to the carotenoid family and have similar conjugated long chains, and β-carotene and fucoxanthin are present in higher amounts, adding β-carotene and fucoxanthin increases the template molecule content and improves the selective adsorption of β-carotene and fucoxanthin. The specific process is as follows:

[0033] (1) Mix 3-5 mL of the concentrated pigment obtained in step 1, 80-100 mL of a mixed solvent consisting of toluene and acetonitrile in equal volume ratio, 30-50 mg of β-carotene, 3-5 mg of fucoxanthin, and 90-120 μL of a mixed functional monomer consisting of functional monomers methacrylic acid (MAA) and 3-aminopropyltriethoxysilane (APTES) in a volume ratio of 6:4. Sonicate the mixture at room temperature for 5-10 min and shake it in the dark for 1 h to form a stable template molecule-functional monomer complex.

[0034] (2) Mix 50 mL of acetonitrile, 2 mL of crosslinking agent ethylene glycol dimethacrylate (EGDMA), 13.6-22.8 μL of functional monomer MAA, and 150-200 mg of initiator azobisisobutyronitrile (AIBN), degas in an ultrasonic bath for 5-8 min, deoxygenate by purging with nitrogen for 5-8 min, seal the reaction system, and heat in a 60℃ constant temperature water bath at 400 rpm for 24 h. After heating, cool to room temperature and then heat at 8 × 10⁻⁶ rpm. 3 Centrifuge for 10 min, then vacuum dry at 50 °C to obtain carboxyl-modified polymeric microspheres;

[0035] (3) Mix 50-80 mg of carboxyl-modified polymeric microspheres, 100 mL of template molecule-functional monomer complex, 80-110 μL of LEGDMA, 50-80 μL of tetraethyl orthosilicate (TEOS), 40 mg of AIBN, and 0.1-0.3 mL of ammonia water. Degas the mixture using ultrasound for 5-8 min, then deoxygenate by purging with nitrogen for 5-8 min. Seal the reaction system and incubate in a 60℃ constant temperature water bath at 300 rpm for 24 h. After the reaction, perform Soxhlet extraction for 8-12 h. Elute with 100 mL of a 9:1 volume ratio of methanol and acetic acid to remove unreacted template, crosslinking agent, and functional monomer. Rinse twice with 40 mL of an 8:2 volume ratio of ethanol and acetonitrile. Finally, use 20 mL of methanol as the purification solution for 8 h of purification elution. After elution, use 8 × 10⁻⁶... 3 Centrifuge for 20 min, collect the precipitate and dry it in a vacuum drying oven at 60℃ for 4-6 h to obtain multi-template molecularly imprinted polymers, abbreviated as D-MIPs.

[0036] The control group consisted of non-molecularly imprinted polymers (D-NIPs), synthesized using the same steps as described above, except that the pigment template molecules (concentrated pigments, β-carotene, and fucoxanthin) were replaced with an equal amount of acetonitrile. The results are as follows: Figure 2 As shown, Figure 2 A-scans and D-scans show that the MIPs are rough-surfaced, dispersed spherical structures with a diameter of approximately 0.8-1 μm. Figure 2 B shows that D-NIPs have no significant difference in surface morphology from D-MIPs, indicating that this method can prepare complete spherical polymers. Figure 3 As shown, the surface area and average pore size of D-MIPs are 447.7 μm. 2 / g, 8.47nm, D-NIPs 336.1m 2 / g, 11.21nm. Due to the imprinting effect of multiple templates, D-MIPs have a larger specific surface area and a denser and more uniform pore size than D-NIPs, which is beneficial to improving mass transfer rate and adsorption capacity.

[0037] Analysis of the adsorption performance of D-MIPs prepared by the method in Specific Example 2 and Specific Example 1 on mixed pigments.

[0038] 1. Adsorption kinetics analysis

[0039] A first mixed pigment solution was prepared by adding β-carotene and fucoxanthin to acetonitrile, wherein the initial concentration of β-carotene was 50 mg / L and the initial concentration of fucoxanthin was 20 mg / L. The absorbance of the solution was measured at wavelengths of 450 nm and 525 nm. 0-450 and A 0-525 Subsequently, 20 mg of D-MIPs and 20 mg of D-NIPs were weighed into centrifuge tubes, and 4 mL of the first mixed pigment solution was added. The mixture was then vortexed at 200 rpm for 30 min at room temperature. After adsorption, 8 × 10⁻⁶ μL of the solution was collected. 3 Centrifuge for 10 min, take 0.2 mL of the supernatant and measure its absorbance at wavelengths of 450 nm and 525 nm. 450 and A 525 The adsorption capacities Q of β-carotene by D-MIPs and D-NIPs, and the adsorption capacities Q of fucoxanthin by D-MIPs and D-NIPs, were calculated using the following method:

[0040] C β-胡萝卜素 =A 450 / A 0-450 ×120, the adsorption capacity of β-carotene Q=[C 0β-胡萝卜素 -C β-胡萝卜素 )×V] / m;

[0041] Among them, C 0β-胡萝卜素 The initial concentration of β-carotene in the first mixed pigment solution was 50 mg / L; C β-胡萝卜素 The concentration of β-carotene in the first mixed pigment solution after adsorption equilibrium is given; V is the volume of the first mixed pigment solution, with a value of 0.004 L; and m is the mass of D-MIPs or D-NIPs added, with a value of 0.02 g.

[0042] C 岩藻黄素 =A 525 / A 0-525 ×120, the adsorption capacity of fucoxanthin Q=[C 0岩藻黄素 -C 岩藻黄素 )×V] / m, where C 0岩藻黄素 The initial concentration of fucoxanthin in the first mixed pigment solution was 20 mg / L, C 岩藻黄素 The concentration of fucoxanthin in the first mixed pigment solution after adsorption equilibrium is given by V and m as described above. Each experiment was performed in triplicate, and the average value was taken. The results are as follows: Figure 4As shown, the adsorption capacity of D-MIPs for β-carotene solution and fucoxanthin increased rapidly within the first 30 minutes, reaching equilibrium around 50 minutes. Under the same adsorption conditions, the adsorption capacity of D-MIPs for both pigments was significantly better than that of D-NIPs, with adsorption capacities Q for β-carotene solution and fucoxanthin being 1.81 mg / g and 0.84 mg / g, respectively. This indicates that the surface of D-MIPs possesses a large number of selective binding sites, enabling it to adsorb different types of pigments.

[0043] 2. Adsorption Selectivity Analysis

[0044] A second mixed pigment solution with a concentration of 50 mg / L was prepared by adding β-carotene, fucoxanthin, polydinocyanine, chlorophyll A, and cholesterol to acetonitrile. The absorbance A of the solution was then measured at 450, 525, 565, 420, 663, and 416 nm (the corresponding absorption wavelengths of each pigment). 0-波长 Weigh 20.0 mg of D-MIPs and D-NIPs, add 4 mL of the second mixed pigment solution, and vortex for adsorption for 30 min. Take the supernatant and measure its absorbance at wavelengths of 450, 525, 565, 420, 663, and 416 nm. 波长 The adsorption amounts of D-MIPs and D-NIPs for different pigments were calculated using methods from adsorption kinetic analysis.

[0045] The results are as follows Figure 5 As shown, D-MIPs exhibit good adsorption capacity for carotenoids (β-carotene, fucoxanthin, and polydinocyanine), with the highest adsorption capacity of 1.88 mg / g for β-carotene. This is attributed to the specific binding sites of D-MIPs generated by the imprinting effect. D-MIPs show greater adsorption capacity for β-carotene and fucoxanthin than D-NIPs, due to the selectivity of D-MIPs for the β-terminal groups of carotenoids, making them suitable for the purification of various carotenoids in biological samples. Furthermore, D-MIPs also show a high adsorption capacity for polydinocyanine, indicating the presence of polydinocyanine pigment recognition sites in D-MIPs. Meanwhile, compared to other structural analogs, D-MIPs exhibit poor adsorption capacity for chlorophyll A and cholesterol, but higher affinity for β-carotene, fucoxanthin, and polydinocyanine. This high selectivity is due to the imprinted cavities of the template molecules on D-MIPs.

[0046] Specific Example 3: A method for pretreatment of QuEChERS samples using D-MIPs prepared by the method in the specific example, the steps of which are as follows:

[0047] Step 1: Preparation and formulation optimization of solid-phase extraction packing material for pigment adsorption.

[0048] A third mixed pigment solution was prepared by adding β-carotene and fucoxanthin to acetonitrile, with an initial concentration of 50 mg / L for both β-carotene and fucoxanthin. Eight experimental groups were set up: a blank group, a group receiving 50 mg D-MIPs, a group receiving 50 mg graphitized carbon black (GCB), and a group receiving 50 mg C. 18 50mg ethylenediamine-N-propyl silica gel (PSA), 50mg D-MIPs + 50mg PSA filler, 50mg C 18 Add 20 mL of the third mixed pigment solution to each of the two groups: +50 mg PSA filler and 50 mg GCB + 50 mg PSA filler. After vortex adsorption for 50 min, measure the absorbance values ​​A at 450 and 525 nm. 波长 The result is as follows Figure 6 As shown in Figure A, the D-MIPs+PSA combined packing material has a better purification effect than other combined packing materials.

[0049] Six experimental groups were further set up: blank group, 20 mg D-MIPs + 50 mg PSA packing material, 30 mg D-MIPs + 50 mg PSA packing material, 40 mg D-MIPs + 50 mg PSA packing material, 50 mg D-MIPs + 50 mg PSA packing material, and 60 mg D-MIPs + 50 mg PSA packing material. 20 mL of the third mixed pigment solution was added to each group. After vortex adsorption for 50 min, the absorbance values ​​(A) at 450 and 525 nm were measured. 波长 The result is as follows Figure 6 As shown in Figure B, the purification effect increases with increasing D-MIP dosage. However, there is no significant difference in pigment removal between 50 mg and 60 mg D-MIP dosages. Meanwhile, GCB and GCB+PSA also showed good pigment adsorption effects, but significantly affected the recovery rate of the target substance. Therefore, considering economic benefits and practical application, the optimal dosage of 50 mg D-MIPs + 50 mg PSA packing material combination was selected for further sample pretreatment.

[0050] Step 2: QuEChERS Sample Pretreatment

[0051] Taking the determination of pesticides (any one of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and organochlorine pesticides) in biological samples as an example, take 2-10g of the homogenized biological sample specified in step 1 of specific embodiment one into a 50mL polypropylene centrifuge tube, add 15-30mL of solvent (one or more of acetonitrile, methanol, n-hexane, ethyl acetate, and acetone) and 3-5mL of ultrapure water, vortex extract for 10-15min, add 1-3g NaCl and 2-4g MgSO4 and continue vortexing for 2-5min, then centrifuge at 6×10⁻⁶. 3Centrifuge for 10 min under g centrifugal force, take the upper organic phase, take pictures and measure its absorbance values ​​at 450 nm, 525 nm and 565 nm.

[0052] The upper organic phase was transferred to an Erlenmeyer flask, and 50 mg of D-MIPs + 50 mg of PSA packing material and 80-150 mg of anhydrous MgSO4 were added. The mixture was vortexed for 1 min, and then purified at 6 × 10⁻⁶. 3 After centrifugation at g centrifugal force for 2–5 min, the supernatant was photographed and its absorbance values ​​at 450 nm, 525 nm, and 565 nm were measured. The results are as follows: Figure 7 As shown. By Figure 7 The color change of the samples before and after the addition of D-MIPs shows that the extract with added D-MIPs is transparent and colorless. Figure 7 B shows that after the addition of D-MIPs, the absorbance values ​​of β-carotene (A450), fucoxanthin (A525), chlorophyll A (A663), and polydinocyanin (A565) all decreased, indicating that the mixed pigments were successfully adsorbed and removed by D-MIPs.

[0053] Specific Example 4: The effect of D-MIPs prepared using the method of Specific Example 1 on the recovery rate of the target analyte.

[0054] A mixed standard solution of eight polychlorinated biphenyls (PCBs) with a concentration of 0.2 mg / L was prepared using a mixed solvent of n-hexane and acetone in a volume ratio of 9:1. The eight PCBs were 1,3,5-trichlorobenzene, hexachlorobenzene, 2,2',3,5'-tetrachlorobiphenyl, 2',3,4-trichlorobenzene, 3,4,4',5-tetrachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, 2,2',3,4',5',6-hexachlorobiphenyl, and 2,2',3,4',5,5',6-hexachlorobiphenyl. After adding 1 g of NaCl and 2 g of MgSO4 to the mixed standard solution and vortexing for 2 min, the solution was then subjected to a 6 × 10⁻⁶ ppm temperature. 3 Centrifuge at 6 × 10 g for 10 min, transfer the organic phase to an Erlenmeyer flask, add 50 mg D-MIPs + 50 mg PSA packing material and 80 mg anhydrous MgSO4, vortex for 1 min, and then centrifuge at 6 × 10 g for 10 min. 3 After centrifugation at a centrifugal force of g for 2 min, the supernatant was filtered through a 0.22 μm microporous membrane and concentrated to 1 mL under a weak N2 gas flow for GC-MS / MS analysis. The results are as follows: Figure 8As shown, the recoveries of eight polychlorinated biphenyl (PCB) pesticides (1-8 corresponding to 1,3,5-trichlorobenzene, 2',3,4-trichlorobenzene, 2,2',3,5'-tetrachlorobiphenyl, 3,4,4',5-tetrachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, hexachlorobenzene, 2,2',3,4',5',6-hexachlorobiphenyl, and 2,2',3,4',5,5',6-hexachlorobiphenyl, respectively) ranged from 98.37% to 118.12%, and were not affected by the non-specific adsorption of D-MIPs. This indicates that D-MIPs can selectively adsorb pigments and will not affect the target substances. The GC-MS / MS analysis method is as follows:

[0055] GC conditions: Instrument model: Agilent 7890B-7000D; Inlet temperature set: 280℃; Injection mode: splitless injection; Injection volume: 2.0 μL; Carrier gas: high-purity helium at a flow rate of 1 mL / min. Column temperature program: 80℃ held for 1 min, then increased to 150℃ at 20℃ / min, then to 300℃ at 5℃ / min, held for 5 min; total run time: 39.5 min; Transfer line temperature: 280℃.

[0056] Mass spectrometry conditions: The ion source was an electron impact ionization (EI) source at 280℃, the quadrupole temperature was 150℃, and the solvent delay time was 4 min. The mass spectrometry method used was DMRM mode, and the selection of ion pairs for qualitative and quantitative analysis of each substance conformed to the EU four-point qualitative method.

[0057] Verification of the practical application of the QuEChERS sample pretreatment method in Specific Embodiment 5 and Specific Embodiment 3.

[0058] Eight low-concentration (1 μg / L) standards were added to the blank matrix extracts of two types of shellfish samples: 1,3,5-trichlorobenzene, hexachlorobenzene, 2,2',3,5'-tetrachlorobiphenyl, 2',3,4-trichlorobenzene, 3,4,4',5-tetrachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, 2,2',3,4',5',6-hexachlorobiphenyl, and 2,2',3,4',5,5',6-hexachlorobiphenyl. A mixed extract was constructed, and the supernatant was extracted according to step 2 of Example 3. The supernatant was filtered through a 0.22 μm microporous membrane and concentrated to 1 mL under a weak N2 gas flow for GC-MS / MS analysis. The analysis was repeated six times, and the standard deviation was calculated. Three times the standard deviation was the limit of detection, and ten times the standard deviation was the limit of quantitation. Simultaneously, eight concentration points ranging from 0.1 to 50 μg / kg were prepared in the blank matrix, and their fitted curves were used to verify the linear correlation of the calibration curve. The results are shown in Table 1, showing the linear correlation R of the eight polychlorinated biphenyls. 2All values ​​were greater than 0.9980, the recoveries ranged from 75.8% to 114.2%, the RSDs ranged from 0.3% to 6.8%, the limits of detection ranged from 0.004 to 0.116 μg / kg, and the limits of quantitation ranged from 0.014 to 0.332 μg / kg. These results indicate that the method can be well applied to the extraction of complex pigments from real biological samples without significantly affecting the target substance.

[0059] Table 18 linear ranges and linear correlation coefficients (R) for the quantitative analysis of polychlorinated biphenyls (PCBs). 2 ), limit of detection, limit of quantitation, and actual sample recovery rate (n=6)

[0060]

[0061] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A method for preparing a multi-template molecularly imprinted polymer that selectively adsorbs multiple pigments, characterized in that... Includes the following steps: (1) Mix 3-5 mL of concentrated pigment extracted from aquatic products, 80-100 mL of mixed solvent composed of toluene and acetonitrile in equal volume ratio, 30-50 mg of β-carotene, 3-5 mg of fucoxanthin and 90-120 μL of mixed functional monomers, and sonicate at room temperature for 5-10 min. Shake in the dark to form a stable template molecule-functional monomer complex. The mixed functional monomer is composed of functional monomer MAA and 3-aminopropyltriethoxysilane in a volume ratio of 6:

4. (2) Mix 50 mL acetonitrile, 2 mL crosslinking agent EGDMA, 13.6-22.8 μL functional monomer MAA and 150-200 mg initiator AIBN, degas in an ultrasonic bath for 5-8 min, deoxygenate by purging with nitrogen for 5-8 min, seal the reaction system, stir in a constant temperature water bath at 50-70 ℃ for 20-28 h, cool to room temperature after heating, centrifuge and vacuum dry at 40-50 ℃ to obtain carboxyl-modified polymer microspheres; (3) Mix 50-80 mg of carboxyl-modified polymeric microspheres, 80-120 mL of template molecule-functional monomer complex, 80-110 μL of LEGDMA, 50-80 μL of tetraethyl orthosilicate, 30-50 mg of AIBN and 0.1-0.3 mL of ammonia water, degas the mixture by sonication for 5-8 min, deoxygenate by nitrogen purging for 5-8 min, seal the reaction system, stir in a constant temperature water bath at 50-70℃, after the reaction is completed, extract by Soxhlet extraction for 8-12 h, wash away unreacted template, crosslinking agent and functional monomer with elution buffer, rinse with rinsing buffer, and finally purify with purification buffer. After elution, collect the precipitate by centrifugation and dry it in a vacuum drying oven at 50-70℃ for 4-6 h to obtain a multi-template molecularly imprinted polymer that selectively adsorbs multiple pigments.

2. The method for preparing a multi-template molecularly imprinted polymer that selectively adsorbs multiple pigments according to claim 1, characterized in that... The extraction method for the concentrated pigment described in step (1) is as follows: Aquatic products are added to an acetonitrile solution containing 0.5 wt% formic acid at a mass-to-volume ratio of 20 g: 20 mL. The mixture is sonicated for 8-12 min, microwaved for 5-8 min at 20-30℃ and 300-500W power, and then the pigment is extracted by shaking at 8-120 rpm for 20-40 min. The supernatant is then collected by centrifugation. The precipitate is collected and the extraction and centrifugation are repeated 2-4 times. The supernatant containing the pigment is then combined. The supernatant containing the pigment is concentrated by rotary evaporation to 1 / 10-1 / 15 of its volume to obtain the concentrated pigment.

3. The method for preparing a multi-template molecularly imprinted polymer that selectively adsorbs multiple pigments according to claim 2, characterized in that... The aquatic products mentioned are at least one of mussels, clams, oysters, razor clams, large yellow croaker, small yellow croaker, and mackerel.

4. The method for preparing a multi-template molecularly imprinted polymer that selectively adsorbs multiple pigments according to claim 1, characterized in that... The eluent in step (3) is composed of methanol and acetic acid in a volume ratio of 9:1; the rinsing solution is composed of ethanol and acetonitrile in a volume ratio of 8:2; and the purification solution is methanol.

5. A method for preparing a multi-template molecularly imprinted polymer that selectively adsorbs multiple pigments according to any one of claims 1-4, characterized in that: The various pigments mentioned include β-carotene, fucoxanthin, and polydinoxin.

6. The application of a multi-template molecularly imprinted polymer prepared by the method of claim 1 in the preparation of a solid-phase extraction filler for selective adsorption of multiple pigments.

7. The application according to claim 6, characterized in that: The solid-phase extraction packing material is a mixture of multi-template molecularly imprinted polymer and PSA packing material in a mass ratio of 2-6:

5.

8. A method for sample pretreatment of QuEChERS using solid-phase extraction packing material, characterized in that... The procedure includes the following steps: Take 2-10 g of aquatic sample, add 15-30 mL of solvent and 3-5 mL of ultrapure water, vortex extract for 10-15 min, add 1-3 g of NaCl and 2-4 g of MgSO4, continue vortexing for 2-5 min, centrifuge, take the upper organic phase, add 70-100 mg of the solid-phase extraction packing material described in claim 7 and 80-150 mg of MgSO4, vortex purify for 0.5-2 min, centrifuge, take the supernatant, filter through a 0.22 μm microporous membrane, concentrate to 0.5-2 mL under N2 gas flow, and then analyze by GC-MS / MS.

9. The method for pretreatment of QuEChERS samples using solid-phase extraction packing material according to claim 8, characterized in that... The solvent is at least one selected from acetonitrile, methanol, n-hexane, ethyl acetate, and acetone.

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