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

Abstract

The invention discloses a preparation method of a multi-template molecularly imprinted polymer capable of selectively adsorbing various pigments and application of the multi-template molecularly imprinted polymer in solid-phase extraction filler. The preparation method is characterized by comprising the following steps: mixing 50-80mg of carboxyl modified polymeric microspheres, 80-120mL of template molecule-functional monomer compound, 80-110mu L of EGDMA, 50-80mu L of tetraethoxysilane, 30-50mg of AIBN and 0.1-0.3 mL of ammonia water; after degassing, deoxidizing and sealing reaction, performing Soxhlet extraction and elution to remove unreacted templates, cross-linking agents and functional monomers, leaching with leacheate, performing purification and elution with purification liquid, centrifuging, collecting precipitates, and drying to obtain the multi-template molecularly imprinted polymer capable of selectively adsorbing various pigments. The solid-phase extraction filler has the advantages that the pigment matrix interference is effectively removed while the analyte recovery rate is ensured.

Description

Technical Field

[0001] The invention relates to a molecular imprinting solid phase extraction filler, in particular to a preparation method of a multi-template molecular imprinting polymer for selectively absorbing multiple pigments and application of the polymer in a solid phase extraction filler. Background Art

[0002] Solid Phase Extraction (SPE) is a technique for separating and enriching target compounds in analytical samples. Solid phase materials have specific affinity and can selectively adsorb target compounds while excluding interfering substances. The target compound is eluted from the solid phase material by changing the polarity or pH value of the solvent to obtain a pure target compound solution. Solid phase extraction is widely used in chemistry, biochemistry, environmental analysis and other fields. This technology has the characteristics of high selectivity, simple operation and good enrichment effect, so it has been widely used in analytical chemistry. However, in actual operation, some biological or sediment samples are rich in a variety of natural pigments. Pigments may have a significant impact on analytical performance in SPE because the presence of pigments can trigger multiple interactions, such as effects on adsorption, elution, interference and detection. At the same time, the presence of pigments has a significant impact on various detectors. In addition, excessive pigments may compete with the selected solid phase material for adsorption, resulting in a decrease in the adsorption efficiency of the target analyte. This competitive adsorption effect may seriously affect the selectivity of SPE, especially when the pigment has similar chemical properties to the target analyte. Therefore, the solid phase material must be carefully selected and optimized to ensure selectivity and adsorption efficiency for the target analytes without interference from pigments.

[0003] The QuEChERS method was developed on the basis of solid phase extraction. It has the advantages of simple operation, short time, and low solvent consumption, which greatly improves the analysis efficiency and convenience of sample processing. The common QuEChERS method combines the extraction and purification of dispersed solid phase extraction (d-SPE) and has been applied to the analysis of typical pollutants in fish tissue samples. However, due to the large differences in the properties of different configurations of pollutants, it is difficult for traditional QuEChERS to extract and purify multiple multi-configuration pollutants from biological samples at the same time. In addition, aquatic biological samples usually contain complex matrices, especially complex pigment types, which interfere with the extraction and purification process, making it difficult to obtain satisfactory matrix removal effects and target analyte recovery rates. Therefore, when pre-treating biological samples, a more effective extraction method is needed to reduce the matrix effect produced by complex pigments and improve the recovery rate and purity of the target analytes. Summary of the invention

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

[0005] The technical solution adopted by the present invention to solve the above technical problems is: a method for preparing a multi-template molecular imprinting polymer that selectively adsorbs multiple pigments, comprising the following steps:

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

[0007] (2) 50 mL of acetonitrile, 2 mL of crosslinker (ethylene glycol dimethacrylate) EGDMA, 13.6-22.8 μL of functional monomer MAA and 150-200 mg of initiator (azobisisobutyronitrile) AIBN were mixed, degassed in an ultrasonic bath for 5-8 min, deoxygenated with nitrogen for 5-8 min, sealed the reaction system, stirred in a constant temperature water bath at 50-70° C. for 20-28 h, cooled to room temperature after heating, centrifuged and dried in a vacuum at 40-50° C. to obtain carboxyl-modified polymer microspheres;

[0008] (3) 50-80 mg of carboxyl modified polymer microspheres, 80-120 mL of template molecule-functional monomer complex, 80-110 μL of EGDMA and 50-80 μL of tetraethyl orthosilicate, 30-50 mg of AIBN and 0.1-0.3 mL of ammonia water were mixed, ultrasonically degassed for 5-8 min, nitrogen deoxygenated for 5-8 min, sealed the reaction system, and stirred in a constant temperature water bath at 50-70°C. After the reaction was completed, Soxhlet extraction was performed for 8-12 h, and the unreacted template, cross-linking agent and functional monomer were eluted to remove the unreacted template, cross-linking agent and functional monomer, and then washed with eluent, and finally purified with purification solution. After the elution was completed, the precipitate was collected by centrifugation and dried in a vacuum drying oven at 50-70°C for 4-6 h to obtain a multi-template molecular imprinting polymer that selectively adsorbs multiple pigments.

[0009] Furthermore, the extraction method of the concentrated pigment described in step (1) is as follows: adding the aquatic product in a mass volume ratio of 20g:20mL to an acetonitrile solution containing 0.5vt% formic acid, ultrasonicating for 8-12min, microwave extraction for 5-8min at 20-30°C and 300-500W, then extracting the pigment at a speed of 8-120rpm for 20-40min, centrifuging to obtain the supernatant, taking the precipitate and repeating the extraction and centrifugation operation 2-4 times, and combining to obtain the supernatant containing the pigment; rotary evaporating the supernatant containing the pigment to concentrate it to 1 / 10-1 / 15 of its volume to obtain the concentrated pigment.

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

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

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

[0013] Furthermore, the plurality of pigments include β-carotene, fucoxanthin and peridinin.

[0014] The present invention also provides the use of the multi-template molecular imprinting polymer in preparing a solid phase extraction filler for selectively adsorbing multiple pigments.

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

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

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

[0018] Compared with the prior art, the advantages of the present invention are: a preparation method of a multi-template molecular imprinted polymer for selectively adsorbing multiple pigments and its application in solid phase extraction fillers; multiple target sites of the multi-template molecular imprinted polymer provide excellent pigment adsorption capacity; at the same time, the mixed color pigment template molecules extracted from aquatic products provide a perfect pigment configuration for the pigment diversity in the organism; the surface grafting precipitation method provides excellent specific surface area, pores and multi-template specific sites for the imprinted material. Compared with traditional solid phase extraction and QuEChERS methods, the solid phase extraction and QuEChERS methods introducing D-MIPs show lower matrix effects, achieving effective removal of pigment matrix interference while ensuring the recovery rate of the analyte. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a photo of concentrated pigment extracted in specific embodiment 1;

[0020] Figure 2 is a scanning electron micrograph of the multi-template molecularly imprinted polymer in the specific embodiment 1, wherein A is D-MIPs and B is D-NIPs;

[0021] Figure 3 is the specific surface area and average pore size of D-MIPs and D-NIPs in specific embodiment 1;

[0022] Figure 4 is the adsorption kinetic curve of D-MIPs and D-NIPs in specific embodiment 2;

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

[0024] Figure 6 The comparison of the effects of different solid phase extraction fillers on the adsorption of β-carotene and fucoxanthin in the specific embodiment 3, wherein A is a combination of different adsorbents, and B is a combination of D-MIPs+PSA fillers with different D-MIPs dosages;

[0025] Figure 7 This is a comparative analysis of the effects of biological sample purification before and after purification in the specific embodiment 3, wherein A is the color change of the extract before and after purification, and B is the absorbance change of the extract before and after purification;

[0026] Figure 8 The results of the effect of adding D-MIPs on the recovery rates of eight PCBs in Example 4 are shown. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below with reference to the accompanying drawings.

[0028] Specific embodiment 1: Preparation of multi-template molecular imprinting polymers for selectively adsorbing multiple pigments, comprising the following steps:

[0029] Step 1. Add 20 mL of acetonitrile solution containing 0.5 vt% formic acid to 20 g of shellfish meat (4 g each of mussels, clams, oysters, and razor clams) or common aquatic biological samples (large yellow croaker, small yellow croaker, and bluefin croaker), ultrasonicate for 10 min, microwave extract for 5-8 min at 25 ° C and 400 W power, and then oscillate at 100 rpm for 30 min to extract the pigment in the sample; then, centrifuge at 9000 rpm for 15 min, take the supernatant, repeat the extraction and centrifugation operations 3 times, and combine 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 after rotary evaporation at 30°C and 150 rpm for 40 min, 5 mL of concentrated pigment was obtained; 20 μL of the concentrated pigment was placed on a thin layer chromatography plate and immersed in 1 mL of a separation solvent consisting of ethyl acetate and methanol in a volume ratio of 20:1, and the main pigment species in the concentrated pigment were determined by thin layer chromatography. The results are shown in Figure 1 As shown, the concentrated pigment is brown in color and is mainly composed of carotenoids, including three types of pigments: β-carotene, fucoxanthin and peridinin.

[0031] Step 2: Prepare multi-template molecular imprinted polymers (D-MIPs) by surface grafting combined with precipitation polymerization.

[0032] Since β-carotene, fucoxanthin and peridinin all belong to the carotenoid family, have similar conjugated long chains, and the content of β-carotene and fucoxanthin is high, β-carotene and fucoxanthin are added to increase the content of template molecules and improve the selective adsorption of β-carotene and fucoxanthin. The specific process is as follows:

[0033] (1) 3-5 mL of the concentrated pigment obtained in step 1, 80-100 mL of a mixed solvent consisting of toluene and acetonitrile in an 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 methacrylic acid (MAA) and 3-aminopropyltriethoxysilane (APTES) in a volume ratio of 6:4 were mixed, dissolved by ultrasonication at room temperature for 5-10 min, and shaken in the dark for 1 h to form a stable template molecule-functional monomer complex;

[0034] (2) 50 mL of acetonitrile, 2 mL of crosslinker ethylene glycol dimethacrylate (EGDMA), 13.6-22.8 μL of functional monomer MAA and 150-200 mg of initiator azobisisobutyronitrile (AIBN) were mixed, degassed in an ultrasonic bath for 5-8 min, deoxygenated with nitrogen for 5-8 min, and the reaction system was sealed and placed in a constant temperature water bath at 60°C and stirred at 400 rpm for 24 h. After heating, the mixture was cooled to room temperature and heated to 8 × 10 3 g for 10 min and vacuum dried at 50 °C to obtain carboxyl-modified polymer microspheres;

[0035] (3) 50-80 mg of carboxyl-modified polymer microspheres, 100 mL of template molecule-functional monomer complex, 80-110 μL of EGDMA, 50-80 μL of tetraethyl orthosilicate (TEOS), 40 mg of AIBN and 0.1-0.3 mL of ammonia water were mixed, and then ultrasonically degassed for 5-8 min, and nitrogen was passed through for deoxygenation for 5-8 min. The reaction system was sealed and placed in a 60°C constant temperature water bath with sealed stirring at 300 rpm for 24 h. After the reaction was completed, Soxhlet extraction was performed for 8-12 h. After the unreacted template, cross-linking agent and functional monomer were removed by elution with 100 mL of an eluent composed of methanol and acetic acid in a volume ratio of 9:1, the microspheres were washed twice with 40 mL of an eluent composed of ethanol and acetonitrile in a volume ratio of 8:2. Finally, 20 mL of methanol was used as a purification solution for 8 h of purification and elution. After the elution was completed, 8×10 3 The mixture was centrifuged at 400 °C for 20 min, and the precipitate was collected and dried in a vacuum oven at 60 °C for 4-6 h to obtain multi-template molecular imprinting polymers, abbreviated as D-MIPs.

[0036] The synthesis method of the control group, D-Non-imprinted polymers (D-NIPs), was the same as the above steps, except that the pigment template molecules (concentrated pigment, β-carotene and fucoxanthin) were replaced by an equal amount of acetonitrile. Figure 2 As shown, Figure 2 A shows that MIPs are dispersed spherical with rough surface and a diameter of about 0.8-1 μm. Figure 2 B shows that the surface morphology of D-NIPs is not significantly different from that of D-MIPs, indicating that this method can prepare complete spherical polymers. Figure 3 As shown in Figure 2, the surface area and average pore size of D-MIPs are 447.7 m 2 / g, 8.47nm, D-NIPs is 336.1m 2 / g, 11.21nm. Due to the imprinting effect of multiple templates, D-MIPs have a larger specific surface area and more dense and uniform pore size than D-NIPs, which is beneficial to improving the mass transfer rate and adsorption capacity.

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

[0038] 1. Adsorption kinetics analysis

[0039] β-carotene and fucoxanthin were added to acetonitrile to prepare a first mixed pigment solution, wherein the initial concentration of β-carotene was 50 mg / L, the initial concentration of fucoxanthin was 20 mg / L, and the absorbance thereof was measured at wavelengths of 450 nm and 525 nm as A 0-450 and A 0-525 Then, 20 mg of D-MIPs and 20 mg of D-NIPs were weighed into centrifuge tubes, 4 mL of the first mixed pigment solution was added, and vortex adsorption was performed at 200 rpm for 30 min at room temperature. 3 The supernatant was centrifuged at 4000 g for 10 min, and 0.2 mL of the supernatant was measured at 450 nm and 525 nm for absorbance. 450 and A 525 , calculate the adsorption amount Q of D-MIPs and D-NIPs on β-carotene, and the adsorption amount Q of D-MIPs and D-NIPs on fucoxanthin. The calculation method is as follows:

[0040] C β-胡萝卜素 =A 450 / A 0-450 ×120, the adsorption amount 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 is the initial concentration of β-carotene in the first mixed pigment solution, which is 50 mg / L; C β-胡萝卜素 is the concentration of β-carotene in the first mixed pigment solution after adsorption equilibrium; V is the volume of the first mixed pigment solution, which is 0.004 L; m is the mass of the added D-MIPs or D-NIPs, which is 0.02 g.

[0042] C 岩藻黄素 =A 525 / A 0-525 ×120, the adsorption amount of fucoxanthin Q = [C 0岩藻黄素 -C 岩藻黄素 )×V] / m, where C 0岩藻黄素 is the initial concentration of fucoxanthin in the first mixed pigment solution, which is 20 mg / L, C 岩藻黄素 is the concentration of fucoxanthin in the first mixed pigment solution after adsorption equilibrium, V and m are the same as above. Each group of experiments was repeated three times and the average value was taken. Figure 4As shown in the figure, the adsorption of β-carotene solution and fucoxanthin by D-MIPs increased rapidly in the first 30 minutes and reached equilibrium at about 50 minutes. Under the same adsorption conditions, the adsorption capacity of D-MIPs for the two pigments was much better than that of D-NIPs, and the adsorption amounts Q for β-carotene solution and fucoxanthin were 1.81 mg / g and 0.84 mg / g, respectively. This shows that the surface of D-MIPs has a large number of selective binding sites and can adsorb different types of pigments.

[0043] 2. Adsorption selectivity analysis

[0044] β-carotene, fucoxanthin, peridinin, metacholine, chlorophyll A and cholesterol were added to acetonitrile to prepare a second mixed pigment solution with a pigment concentration of 50 mg / L. The absorbance A at 450, 525, 565, 420, 663 and 416 nm (corresponding absorption wavelengths of each pigment) was measured respectively. 0-波长 Weigh 20.0 mg of D-MIPs and D-NIPs, add 4 mL of the second mixed pigment solution, vortex adsorb for 30 min, take the supernatant and measure its absorbance value A at 450, 525, 565, 420, 663, and 416 nm wavelengths respectively. 波长 The adsorption amount of D-MIPs and D-NIPs on different pigments was calculated according to the method in adsorption kinetic analysis.

[0045] The results are as follows Figure 5 As shown in the figure, D-MIPs have good adsorption capacity for carotenoids (β-carotene, fucoxanthin and peridinin), with the maximum adsorption capacity of β-carotene being 1.88 mg / g, which is attributed to the specific binding sites of D-MIPs produced by the imprinting effect. D-MIPs have greater adsorption capacity for β-carotene and fucoxanthin than D-NIPs, which is due to the selectivity of D-MIPs for the β-terminal group of carotenoids and can be used to purify a variety of carotenoids in biological samples; in addition, D-MIPs also have a higher adsorption capacity for peridinin, which indicates that D-MIPs have a peridinin pigment recognition site. At the same time, compared with other structural analogs, D-MIPs show poor adsorption capacity for chlorophyll A and cholesterol, and higher affinity for β-carotene, fucoxanthin and peridinin. This high selectivity is due to the imprinting holes of the template molecules on D-MIPs.

[0046] Specific embodiment 3: A method for pre-treating a QuEChERS sample using D-MIPs prepared by the method of the specific embodiment, the steps are as follows:

[0047] Step 1: Preparation and formulation optimization of solid phase extraction filler for adsorption of pigments

[0048] β-carotene and fucoxanthin were added to acetonitrile to prepare a third mixed pigment solution, wherein the initial concentration of β-carotene was 50 mg / L, and the initial concentration of fucoxanthin was 50 mg / L. Eight experimental groups were set up, namely, a blank group, 50 mg D-MIPs, 50 mg graphitized carbon black (GCB), 50 mg C 18 、50mg ethylenediamine-N-propyl silica gel (PSA), 50mg D-MIPs+50mg PSA filler, 50mg C 18 +50mg PSA filler and 50mg GCB+50mg PSA filler, add 20mL of the third mixed pigment solution to each group, vortex adsorb for 50min and measure the absorbance value A at 450 and 525nm 波长 The results are as follows Figure 6 As shown in A, the D-MIPs+PSA combined filler has a better purification effect than other combined fillers.

[0049] Six experimental groups were further set up, namely, blank group, 20 mg D-MIPs + 50 mg PSA filler, 30 mg D-MIPs + 50 mg PSA filler, 40 mg D-MIPs + 50 mg PSA filler, 50 mg D-MIPs + 50 mg PSA filler and 60 mg D-MIPs + 50 mg PSA filler. 20 mL of the third mixed pigment solution was added to each group, and the absorbance values ​​A at 450 and 525 nm were measured after vortex adsorption for 50 min. 波长 The results are as follows Figure 6 As shown in Figure B, the purification effect increases with the increase in the amount of D-MIPs. However, the difference in pigment removal between 50 mg and 60 mg D-MIPs is not significant. At the same time, the use of GCB and GCB+PSA also has a good effect on pigment adsorption, but it will significantly affect the recovery rate of the target substance. Therefore, considering the economic benefits and actual use effects, the optimal dosage of 50 mg D-MIPs + 50 mg PSA filler combination is selected for further sample pretreatment.

[0050] Step 2: QuEChERS sample preparation

[0051] Taking the determination of pesticides (any one of polycyclic aromatic hydrocarbons, polychlorinated biphenyls, and organochlorine pesticides) in biological samples as an example, 2-10 g of the homogenized biological sample specified in step 1 of the specific embodiment 1 was placed in a 50 mL polypropylene centrifuge tube, 15-30 mL of solvent (the solvent is one or more of acetonitrile, methanol, n-hexane, ethyl acetate, and acetone) and 3-5 mL of ultrapure water were added, and vortexed for 10-15 min. 1-3 g of NaCl and 2-4 g of MgSO4 were added and vortexed for 2-5 min. 3The mixture was centrifuged at 0.5 g for 10 min, and the upper organic phase was taken for photographing and its absorbance at 450 nm, 525 nm, and 565 nm was measured;

[0052] The upper organic phase was transferred to a conical glass bottle, and 50 mg of D-MIPs + 50 mg of PSA filler combination and 80-150 mg of anhydrous MgSO4 were added. The mixture was vortexed for 1 min and heated at 6 × 10 3 After centrifugation at 0.5 g for 2 to 5 minutes, take the supernatant, take a picture and measure its absorbance at 450 nm, 525 nm and 565 nm. Figure 7 As shown. Figure 7 A The color change of the sample before and after adding D-MIPs shows that the extract with D-MIPs is transparent and colorless. Figure 7 B shows that after adding D-MIPs, the absorbance values ​​of β-carotene (A450), fucoxanthin (A525), chlorophyll A (A663) and peridinin (A565) all decreased, indicating that the mixed pigments were successfully adsorbed and removed by D-MIPs.

[0053] Specific Example 4: Effect of D-MIPs prepared by the method of Specific Example 1 on the recovery rate of the target substance

[0054] A mixed solvent consisting of n-hexane and acetone in a volume ratio of 9:1 was used to prepare a mixed standard solution of 8 PCBs with a concentration of 0.2 mg / L. The 8 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. 1 g NaCl and 2 g MgSO4 were added to the mixed standard solution and vortexed for 2 min. The mixed standard solution was stirred at 6 × 10 3 The mixture was centrifuged at 6 × 10 °C for 10 min, and the organic phase was transferred to a conical glass bottle. 50 mg of D-MIPs + 50 mg of PSA filler combination and 80 mg of anhydrous MgSO4 were added. The mixture was vortexed for 1 min and then heated at 6 × 10 °C for 2 h. 3 After centrifugation at 1.5 g for 2 min, the supernatant was filtered through a 0.22 μm microporous membrane and concentrated to 1 mL under a weak N2 flow for GC-MS / MS analysis. Figure 8As shown, the recoveries of eight polychlorinated biphenyl pesticides (1-8 correspond 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, 2,2',3,4',5,5',6-hexachlorobiphenyl) were all between 98.37% and 118.12%, and were not affected by the nonspecific adsorption of D-MIPs, indicating 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: The instrument model is Agilent 7890B-7000D, the injection port temperature is set at 280℃, the injection mode is splitless injection, the injection volume is 2.0μL, and the carrier gas is high-purity helium at a flow rate of 1mL / min. The column temperature program is 80℃ for 1min, then increased to 150℃ at a rate of 20℃ / min, and increased to 300℃ at 5℃ / min, and maintained for 5min. The total running time is 39.5min; the transfer line temperature is 280℃;

[0056] Mass spectrometry conditions: the ion source is an electron impact ion source (EI), the temperature is 280°C, the quadrupole temperature is 150°C, and the solvent delay time is 4 minutes. The mass spectrometry method uses the DMRM mode, and the selection of qualitative and quantitative ion pairs for each substance complies with the EU four-point qualitative method.

[0057] Specific embodiment 5: Practical application verification of the QuEChERS sample pretreatment method in specific embodiment 3.

[0058] Eight low concentration (1 μg / L) standards were added to the blank matrix extracts of the two shellfish biological samples, namely 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, 2,2',3,4',5,5',6-hexachlorobiphenyl, to construct a mixed extract, and the supernatant was extracted according to the method of step 2 in specific embodiment 3, and the supernatant was filtered through a 0.22 μm microporous filter membrane, concentrated to 1 mL under a weak N2 gas flow, and then analyzed by GC-MS / MS. Repeat the analysis 6 times, calculate the standard deviation, three times the standard deviation is the method detection limit, and ten times the standard deviation is the quantification limit. At the same time, 8 concentration points of 0.1-50 μg / kg were prepared in the blank matrix, and the fitting curve was used to verify the linear correlation of the calibration curve. The results are shown in Table 1. The linear correlation R 2The values ​​were all greater than 0.9980, the recoveries were between 75.8% and 114.2%, the RSDs were between 0.3% and 6.8%, the limits of detection were between 0.004 and 0.116 μg / kg, and the limits of quantification were between 0.014 and 0.332 μg / kg. These results indicate that this method can be well applied to the extraction of complex pigments in actual biological samples and has no significant effect on the target substances.

[0059] Table 1 Quantitative linear range and linear correlation coefficient (R 2 ), detection limit, quantification limit and actual sample recovery rate (n=6)

[0060]

[0061] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A method for preparing a multi-template molecularly imprinted polymer for selectively adsorbing multiple pigments, characterized in that The following steps are involved: (1) 3-5 mL of concentrated pigment extracted from aquatic products, 80-100 mL of a mixed solvent composed of toluene and acetonitrile in equal volume ratios, 30-50 mg of β-carotene, 3-5 mg of fucoxanthin, and 90-120 μL of a mixed functional monomer were mixed, dissolved by ultrasonication at room temperature for 5-10 min, and shaken in the dark to form a stable template molecule-functional monomer complex; (2) 50 mL of acetonitrile, 2 mL of cross-linking agent EGDMA, 13.6-22.8 μL of functional monomer MAA and 150-200 mg of initiator AIBN were mixed, degassed in an ultrasonic bath for 5-8 min, deoxygenated with nitrogen for 5-8 min, sealed the reaction system, stirred in a constant temperature water bath at 50-70° C. for 20-28 h, cooled to room temperature after heating, centrifuged and dried in a vacuum at 40-50° C. to obtain carboxyl-modified polymer microspheres; (3) 50-80 mg of carboxyl modified polymer microspheres, 80-120 mL of template molecule-functional monomer complex, 80-110 μL of EGDMA and 50-80 μL of tetraethyl orthosilicate, 30-50 mg of AIBN and 0.1-0.3 mL of ammonia water were mixed, ultrasonically degassed for 5-8 min, nitrogen deoxygenated for 5-8 min, sealed the reaction system, and stirred in a constant temperature water bath at 50-70°C. After the reaction was completed, Soxhlet extraction was performed for 8-12 h, and the unreacted template, cross-linking agent and functional monomer were eluted to remove the unreacted template, cross-linking agent and functional monomer, and then washed with eluent, and finally purified with purification solution. After the elution was completed, the precipitate was collected by centrifugation and dried in a vacuum drying oven at 50-70°C for 4-6 h to obtain a multi-template molecular imprinting polymer that selectively adsorbs multiple pigments.

2. The method for preparing a multi-template molecularly imprinted polymer for selectively adsorbing multiple pigments according to claim 1, characterized in that The extraction method of the concentrated pigment described in step (1) is as follows: adding the aquatic product in a mass volume ratio of 20g:20mL to an acetonitrile solution containing 0.5vt% formic acid, ultrasonicating for 8-12min, microwave extraction for 5-8min at 20-30°C and 300-500W, then extracting the pigment at a speed of 8-120rpm for 20-40min, centrifuging to obtain the supernatant, taking the precipitate and repeating the extraction and centrifugation operation 2-4 times, and combining to obtain the supernatant containing the pigment; rotary evaporating the supernatant containing the pigment to concentrate it to 1 / 10-1 / 15 of its volume to obtain the concentrated pigment.

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

4. The method for preparing a multi-template molecularly imprinted polymer for selectively adsorbing multiple pigments according to claim 1, characterized in that The mixed functional monomer described in step (1) is composed of functional monomer MAA and 3-aminopropyltriethoxysilane in a volume ratio of 6:

4.

5. The method for preparing a multi-template molecularly imprinted polymer for selectively adsorbing 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 rinse solution is composed of ethanol and acetonitrile in a volume ratio of 8:2; and the purification solution is methanol.

6. A method for preparing a multi-template molecularly imprinted polymer for selectively adsorbing multiple pigments according to any one of claims 1 to 5, characterized in that: The plurality of pigments include beta-carotene, fucoxanthin and peridinin.

7. Use of the multi-template molecularly imprinted polymer according to claim 1 in preparing a solid phase extraction filler for selectively adsorbing multiple pigments.

8. Use of the multi-template molecularly imprinted polymer according to claim 7 in preparing a solid phase extraction filler for selectively adsorbing multiple pigments, characterized in that: The solid phase extraction filler is a mixture of multi-template molecular imprinting polymer and PSA filler in a mass ratio of 2-6:

5.

9. A method for QuEChERS sample pretreatment using the solid phase extraction filler according to claim 8, characterized in that The method comprises the following steps: taking 2-10 g of aquatic samples, adding 15-30 mL of solvent and 3-5 mL of ultrapure water, vortexing and extracting for 10-15 minutes, adding 1-3 g of NaCl and 2-4 g of MgSO4, and continuing to vortex for 2-5 minutes, centrifuging and taking the upper organic phase, adding 70-100 mg of solid phase extraction filler and 80-150 mg of MgSO4, vortexing and purifying for 0.5-2 minutes, centrifuging and taking the supernatant, filtering through a 0.22 μm microporous filter membrane, concentrating to 0.5-2 mL under a N2 gas flow, and waiting for GC-MS / MS analysis.

10. The method for QuEChERS sample pretreatment using solid phase extraction filler according to claim 9, characterized in that The solvent is at least one of acetonitrile, methanol, n-hexane, ethyl acetate and acetone.

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