CN109839452B - Molecular imprinting stirring rod and preparation method and application thereof - Google Patents
Molecular imprinting stirring rod and preparation method and application thereof Download PDFInfo
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- CN109839452B CN109839452B CN201711230617.2A CN201711230617A CN109839452B CN 109839452 B CN109839452 B CN 109839452B CN 201711230617 A CN201711230617 A CN 201711230617A CN 109839452 B CN109839452 B CN 109839452B
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Abstract
The invention discloses a molecularly imprinted stirring rod and a preparation method and application thereof, wherein the molecularly imprinted stirring rod comprises a glass stirring rod with a silanized iron core on the surface and a molecularly imprinted coating covering the glass stirring rod. The molecular imprinting stirring rod can be used for detecting beta-receptor agonist drugs in animal-derived food, is beneficial to simplifying detection steps, reducing matrix interference, improving detection efficiency, detection accuracy and sensitivity, is environment-friendly, and has good scientific research value, practical application value and development prospect.
Description
Technical Field
The invention belongs to the fields of analytical chemistry and detection, relates to a molecular imprinting solid-phase microextraction technology and application, and particularly relates to a molecular imprinting stirring rod and a preparation method and application thereof.
Background
The beta-receptor stimulant is a kind of chemically synthesized phenylethanolamines, and can significantly increase the lean meat percentage and weight gain of animals and improve feed conversion, thereby reducing the cost. After people eat animal tissues with high content of beta-stimulant, poisoning symptoms such as muscle tremor, myalgia, nausea, light headedness and the like can occur, and serious people endanger life. The Ministry of agriculture, publication No. 235, animal food maximum veterinary drug residue Limit, stipulates that such drugs are not detectable in all animal derived foods. Abroad, the european union has also issued a series of regulations that prescribe the MRL of clenbuterol in animal tissues. However, the beta-receptor agonist drugs are various in types, and more than 20 types are clinically applied and available. In order to obtain greater economic benefit and avoid government regulation and legal penalty, part of illegal vendors illegally use other beta-receptor agonist drugs in the animal breeding process, which brings great challenges to the conventional detection and daily regulation of the beta-receptor agonist drugs in the animal breeding process and animal-derived food. Therefore, the research establishes a screening technology and a confirmation method with high flux and high sensitivity of the beta-receptor stimulant drugs, provides technical support for improving detection efficiency and monitoring means, and has important significance for monitoring the use of the beta-receptor stimulant drugs in feed, breeding links and animal products.
At present, the beta-receptor stimulant drugs in feed, animal food and biological materials are mainly analyzed by an immune screening method and an instrument confirmation method. The immune screening method comprises an enzyme-linked immunosorbent assay (ELISA), a colloidal gold test strip and the like. The method has the advantages of simple and convenient operation, high screening speed, convenient on-line real-time analysis and mobile analysis, and the defect of inaccurate quantification or the qualitative analysis result of false positive and false negative. The instrumental analysis methods include Liquid Chromatography (LC), Capillary Electrophoresis (CE), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the like. The LC-MS/MS is used as a detection platform with high detection sensitivity, wide application range and strong reliability and is widely applied to the determination of beta-receptor agonists and metabolites thereof in feed, animal tissues, hair, blood and animal urine. The main advantages of the instrument detection are high accuracy of analysis results and high requirements on operation technology, and complex pretreatment is required for biological sample matrixes. The impact of sample matrix effects on the accuracy and precision of confirmatory assay results remains a significant challenge in mass spectrometry. Therefore, the research of the sample pretreatment technology is very important in the detection of the beta-receptor agonist.
Sample pretreatment process research has become one of the leading issues in today's analytical chemistry. Samples faced by modern analytical science tend to be complicated, and a serious challenge is provided for sample pretreatment technology, and a pretreatment method is required to have high selectivity, rapidness, simplicity, no solvent or less solvent and the like. Traditional sample pretreatment methods such as liquid-liquid extraction, soxhlet extraction, column chromatography and the like all require the use of more organic solvents and are complex to operate, and Solid Phase Extraction (SPE) can well concentrate and purify a sample, but the selectivity is not high when trace components in a complex biological sample are treated.
The Solid phase micro-extraction (SPME) is a sample pretreatment method which can efficiently extract and enrich analytes in micro-volume polymers or organic solvents, integrates sampling, extraction, concentration and sample injection into a whole, has no (little) solvent and is easy to be used with other technologies on line. Molecularly Imprinted Polymers (MIPs) refer to polymers obtained by pre-assembling a target molecule to be separated with a functional monomer by covalent or non-covalent interactions, and then copolymerizing the target molecule with a crosslinking agent. After the target molecules are removed, a 'hole' which is spatially complementary to the target molecules and has a predetermined action site is formed in the polymer, so that the polymer has a 'memory' effect on the spatial structure of the target molecules and can be used for recognizing the imprinted molecules in a complex sample with high selectivity. The molecular imprinting solid phase microextraction technology (MIP-SPME) integrates the characteristics of good stability and high selectivity of a molecular imprinting polymer with the solid phase microextraction technology, and realizes the selective separation and enrichment of a compound with a similar structure in a complex sample. The micro-extraction technology is developed rapidly once being provided, and different forms such as fiber solid phase micro-extraction, in-tube solid phase micro-extraction, stirring rod adsorption extraction and the like exist at present.
Stir bar adsorption extraction (SBSE) is a novel solid phase microextraction sample pretreatment technique, proposed by Baltussen et al 1999, which was commercialized by Gerstel GmbH 2000. The technology has the advantages of large stationary phase volume, high extraction capacity, no need of an additional stirrer, avoidance of competitive adsorption, realization of extraction and enrichment while stirring, and the like, and is widely applied to pretreatment of food, environment and biological sample analysis. The molecular imprinting stirring rod adsorption extraction technology (MIP-SBSE) combines the advantages of MIP and SBSE technologies, a MIP coating with a molecular recognition function is fixedly supported on the surface of the stirring rod, selective extraction of trace analytes in a complex matrix is realized while stirring, and compared with fiber MI-SPME, the MI-SBSE coating has the advantages that the volume of a stationary phase is more than 50 times larger, and higher extraction capacity and extraction efficiency are realized.
However, solid phase microextraction techniques are rarely used in current methods for the detection of β -agonists in foods of animal origin. Aiming at the problems of complicated operation, poor selectivity and low sensitivity in the pretreatment process of the beta-stimulant detection sample in the current animal-derived food, the search for an effective detection technology becomes urgent.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides the molecular imprinting stirring rod which can effectively simplify the detection steps, reduce the matrix interference, improve the detection efficiency, the detection accuracy and the detection sensitivity and is environment-friendly, and the preparation method and the application thereof.
In order to solve the technical problems, the invention adopts the following technical scheme:
the molecularly imprinted stirring rod comprises a stirring rod and a molecularly imprinted coating covering the stirring rod, wherein the molecularly imprinted coating is mainly prepared by using clenbuterol as a template molecule, methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross-linking agent, methanol as a pore-forming agent and azodiisobutyronitrile as an initiator to prepare a molecularly imprinted polymer, and then eluting the template molecule in the molecularly imprinted polymer to prepare the molecularly imprinted polymer, wherein the stirring rod is a surface silanized iron-core glass stirring rod.
As a general technical concept, the present invention also provides a method for preparing a molecular imprinting stirring rod, comprising the steps of:
dissolving template molecule clenbuterol in methanol, adding functional monomer methacrylic acid, polymerizing, adding crosslinking agent ethylene glycol dimethacrylate and initiator azobisisobutyronitrile, mixing and carrying out ultrasonic treatment to obtain a mixed solution; and (3) placing the iron core glass stirring rod with the silanized surface into the obtained mixed solution, performing secondary ultrasonic treatment, reacting under the conditions of nitrogen atmosphere and water bath heating, and forming a molecular imprinting coating on the iron core glass stirring rod with the silanized surface to obtain the molecular imprinting stirring rod.
In the preparation method of the molecular imprinting stirring rod, preferably, the addition ratio of the clenbuterol, the methacrylic acid, the ethylene glycol dimethacrylate, the methanol and the azobisisobutyronitrile is 0.25 mmol-1 mmol: 1 mmol-4 mmol: 4 mmol-16 mmol: 5 mL: 15 mg-30 mg;
and/or the water bath heating temperature is 50-75 ℃, and the reaction time is 4-36 h.
In the preparation method of the molecular imprinting stirring rod, preferably, the adding ratio of the clenbuterol, the methacrylic acid, the ethylene glycol dimethacrylate, the methanol and the azobisisobutyronitrile is 0.5mmol, 2mmol, 8mmol, 5mL and 15 mg;
and/or the water solution is heated at 70 ℃ for 24-36 h.
In the preparation method of the molecular imprinting stirring rod, preferably, the polymerization time is 12-24 h, the ultrasonic treatment time is 10-15 min, and the secondary ultrasonic treatment time is 5-10 min.
In the above method for preparing a molecularly imprinted stir bar, preferably, the preparation process of the surface silanized iron-core-containing glass stir bar comprises: soaking an iron core-containing glass stirring rod in NaOH solution to expose silanol groups on the surface of the iron core-containing glass stirring rod to the maximum extent, then washing the iron core-containing glass stirring rod with distilled water, then soaking the iron core-containing glass stirring rod in hydrochloric acid solution to remove residual alkali liquor on the surface of the iron core-containing glass stirring rod, then washing the iron core-containing glass stirring rod with distilled water and drying the iron core-containing glass stirring rod, soaking the dried iron core-containing glass stirring rod in acetone solution of 3- (methacryloyloxy) propyl trimethoxy silane to silanize the surface of the iron core-containing glass stirring rod, and obtaining the iron core-containing glass stirring rod with silanized surface after methanol washing and nitrogen flow drying;
and/or the clenbuterol is clenbuterol after pretreatment, and the pretreatment process comprises the following steps: adding clenbuterol hydrochloride into water, then adding hydrochloric acid, fully dissolving, neutralizing with NaOH solution, controlling the pH value to be 9-12, extracting twice with trichloromethane, and evaporating to dryness to obtain clenbuterol crystalline powder, namely the pretreated clenbuterol.
As a general technical concept, the invention also provides an application of the molecular imprinting stirring rod or the molecular imprinting stirring rod prepared by the preparation method in detection of beta-receptor agonist drugs in animal-derived food.
In the above application, preferably, the application comprises the following steps: extracting a target object to be detected in an animal-derived food sample by trichloroacetic acid, adjusting the pH value of an extraction solution, extracting and enriching the target object to be detected by using an isopropanol-ethyl acetate mixed solution, blowing nitrogen for concentration, dissolving residues by using an extraction solvent, then placing a molecular imprinting stirring rod into the obtained sample solution, performing adsorption extraction on the target object to be detected under stirring, and finally resolving the target object to be detected by using a resolving solvent for high performance liquid chromatography tandem mass spectrometry.
In the above application, preferably, the extraction solvent includes water, methanol, acetonitrile, toluene, acetone or ethanol, the desorption solvent includes methanol, water, a methanol-acetic acid mixed solution or an acetic acid solution, the time of adsorption and extraction is 15min to 120min, the time of desorption is 5min to 30min, the stirring speed is 100r/min to 800r/min, and the temperature of adsorption and extraction is room temperature to 60 ℃.
In the above application, preferably, the target to be detected includes one or more of clenbuterol, ractopamine, salbutamol, brombuterol, terbutaline and mabuterol,
when the beta-receptor agonist drug is clenbuterol, the linear equation is that y is 10124+2.65 x 106x, linear range of 0.5 to 35 μ g/L,
when the beta-receptor agonist drug is salbutamol, the linear equation is that y is 20563+1.05 x 106x, linear range of 1 to 35 μ g/L,
when the beta-receptor agonist drug is ractopamine, the linear equation is that y is 489612+2.96 multiplied by 106x, linear range of 1 to 35 μ g/L,
when the beta-receptor agonist drug is bromobuterol, the linear equation is that y is 52461+ 2.35X 106x, linear range of 1 to 35 μ g/L,
when the beta-receptor agonist drug is terbutaline, the linear equation is that y is 89764+1.89 x 106x, linear range of 1 to 35 μ g/L,
when the beta-receptor agonist drug is the mabuterol, the linear equation is that y is 78456+ 1.03X 106x, linear range is 1-35 mu g/L.
In the above application, more preferably, the extraction solvent is toluene, the desorption solvent is an acetic acid solution, the time of adsorption and extraction is 60min, the time of desorption is 10min, the stirring speed is 300r/min, and the temperature of adsorption and extraction is room temperature.
Compared with the prior art, the invention has the advantages that:
the invention develops the detection research of beta-receptor stimulant drugs in animal derived food based on the molecularly imprinted stir bar solid-phase microextraction technology and the mass spectrometry analysis technology. Firstly, clenbuterol is used as a template molecule, methacrylic acid is used as a functional monomer, ethylene glycol dimethacrylate is used as a cross-linking agent, methanol is used as a pore-forming agent, azobisisobutyronitrile is used as an initiator, a molecularly imprinted stirring rod with clenbuterol as the template molecule is successfully prepared on a surface silanized iron-core glass stirring rod, and preparation conditions and adsorption and extraction conditions are optimized. According to the invention, a scanning electron microscope and a Fourier infrared spectrum are used for carrying out structural morphology characterization on the polymer coating, and the properties such as selective adsorption and adsorption capacity of the molecular imprinting stirring rod are evaluated through static adsorption and dynamic selection experiments, so that the molecular imprinting stirring rod with excellent morphology and performance is obtained.
The invention applies a molecular imprinting stirring rod to the detection of 6 beta-receptor agonist drugs such as clenbuterol and analogues thereof in animal-derived food, extracts, purifies and enriches animal-derived food samples, and researches and establishes a proof analysis method for 6 beta-receptor agonist drugs such as clenbuterol, ractopamine, salbutamol, brombuterol, terbutaline and mabuterol in the animal-derived food by combining high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Experimental results show that the linear range of the clenbuterol is 0.5-35 mu g/L, and the ractopamine, the salbutamol, the brombuterol, the terbutaline and the mabuterol have good linear relation in the concentration range of 1-35 mu g/L. The detection limit is calculated by 3 times of signal-to-noise ratio, and the detection limit of the above 6 compounds is 0.20-0.35 mu g/kg. The relative standard deviation (n ═ 3) is between 2.6% and 4.8%. The method is applied to the determination of 6 beta-stimulants in pork and feed products, the recovery rates of the method are respectively 70.6-86.5% and 70.2-88.9%, and the relative standard deviation is 3.9-5.5%. Experiments verify the accuracy and precision of the method of the molecular imprinting stirring rod in the detection application, and a good detection effect is obtained.
In conclusion, the scheme and the achievement of the invention have good scientific research value, practical application value and development prospect, not only reduce the experiment cost and reagent consumption, reduce the emission and the pollution to the environment, but also simplify the experiment steps, reduce the matrix interference, realize the high-efficiency selective separation and enrichment of the template molecules and the structural analogs, and improve the detection efficiency, the accuracy and the sensitivity.
Drawings
FIG. 1 is a schematic illustration of a surface silanized iron core-containing glass stir bar submerged in a glass sleeve in example 1 of the present invention.
FIG. 2 is a comparison of a molecular imprinting stir bar and a blank glass bar in example 2 of the present invention.
FIG. 3 is an infrared spectrum of clenbuterol (a), NIP (b), MIP (c) of non-eluted template molecule, MIP (d) of eluted template molecule in example 2 of the present invention.
FIG. 4 is a scanning electron microscope image of MIP at 200 times (a) and 5000 times (b) magnification, NIP at 200 times (c) and 5000 times (d) magnification in example 2 of the present invention.
FIG. 5 is a schematic diagram of an experimental setup for the SBSE procedure of the molecular imprinting stir bar in example 3 of the present invention.
Fig. 6 shows the extraction ratio of clenbuterol in different extraction solvents according to example 3 of the present invention (n-3).
Fig. 7 shows the recovery rate of clenbuterol in different resolving solvents in example 3 of the present invention.
FIG. 8 shows the adsorption kinetics of the stirring bar for molecular imprinting in the solution of clenbuterol in example 3 of the present invention.
FIG. 9 shows the analytic kinetics of the molecularly imprinted stir bar of example 3 of the present invention in a clenbuterol solution.
FIG. 10 shows the extraction capacity of clenbuterol molecular imprinted stir bar and non-molecular imprinted stir bar at different concentrations in example 3 of the present invention.
FIG. 11 shows the molecular imprinting and non-molecular imprinting extractions of different compounds of example 3 of the present invention at a concentration level of 20. mu.g/L.
Illustration of the drawings:
1. a glass sleeve; 2. an iron core; 3. a surface silanized iron-core-containing glass stirring rod; 4. a round bottom flask; 5. a sample solution; 6. a MIP stir bar; 7. a magnetic stirrer.
Detailed Description
The invention is further described below with reference to the drawings and specific preferred embodiments of the description, without thereby limiting the scope of protection of the invention.
The materials and equipment used in the following examples are commercially available.
Example 1: molecular imprinting stirring rod and preparation method thereof
The molecularly imprinted stirring rod comprises a stirring rod and a molecularly imprinted coating covering the stirring rod, wherein the molecularly imprinted coating is prepared by mainly using clenbuterol as a template molecule, methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross-linking agent, methanol as a pore-forming agent and azobisisobutyronitrile as an initiator to prepare a molecularly imprinted polymer, and then eluting the template molecule in the molecularly imprinted polymer to prepare the molecularly imprinted polymer, wherein the stirring rod is a surface silanized iron-core glass stirring rod.
The preparation method of the molecular imprinting stirring rod comprises the following steps:
(1) pretreatment of glass rods
A glass capillary tube with an outer diameter of 3mm is taken, one end of the glass capillary tube is sintered on an alcohol blast lamp and then cut into a length of 2cm, an iron core 2 (formed by cutting a clip) is placed in the glass capillary tube, and one end of the glass capillary tube is sintered to be used as a coated carrier. The glass rods were pretreated before coating as follows: firstly, soaking the glass rod in 1.0mol/L NaOH solution for 12 hours to expose silanol groups on the surface of the glass rod to the maximum extent. After washing with distilled water, the glass rod was immersed in a 0.10mol/L hydrochloric acid solution for 1 hour to remove residual alkali on the surface of the glass rod. And washing the glass rod for 3-5 times by using distilled water, and then drying the glass rod in an oven at 150 ℃ for 1 h. And then soaking the glass rod in 30 percent acetone solution of 3- (methacryloyloxy) propyl trimethoxy silane (silane coupling agent KH-570) for 3h to silanize the surface of the glass rod so as to form a double bond structure to enable the glass rod and the molecularly imprinted polymer to be combined more firmly. After soaking, the glass rod is cleaned by methanol and dried by nitrogen flow, and the pretreated glass rod, namely the iron core-containing glass stirring rod 3 with silanized surface is obtained for standby.
(2) Clenbuterol hydrochloride pretreatment
Adding a certain amount of clenbuterol hydrochloride into 20mL of water, adding 2mL of 0.1mol/L hydrochloric acid to fully dissolve the clenbuterol hydrochloride, neutralizing with NaOH aqueous solution, controlling the pH value to be about 12, extracting twice with chloroform with the same volume, and evaporating to dryness to obtain white or almost white clenbuterol crystalline powder. The above-mentioned amount means that the amount of processing can be selected according to actual requirements.
(3) Preparation of molecularly imprinted stir bar
Weighing 0.1385g (0.5mmol) of template molecule clenbuterol CL in a 50mL polytetrafluoroethylene centrifuge tube with a stopper, adding 5mL of methanol, dissolving, adding 0.18mL (2mmol) of functional monomer MAA, uniformly mixing, and polymerizing for 12 h. Then adding 1.6mL (8mmol) of crosslinking agent ethylene glycol dimethacrylate EGDMA and 15mg of initiator azobisisobutyronitrile AIBN, uniformly mixing, and performing ultrasonic treatment for 10min to obtain a mixed solution. And (2) taking a proper amount of the mixed solution, adding the mixed solution into a glass capillary (namely a glass sleeve 1) with the inner diameter of 5mm and the length of 10cm, sintering and sealing one end of the glass capillary, and immersing the iron core-containing glass stirring rod 3 with silanized surface obtained in the step (1) as shown in a schematic diagram of fig. 1. And performing ultrasonic treatment for 5min again, introducing nitrogen to remove residual air in the tube, and immediately sintering and sealing the other end of the glass tube. And then placing the mixture in a water bath at 60 ℃ for reaction for 24 hours to form a molecular imprinting coating on the stirring rod to obtain the molecular imprinting stirring rod, namely the MIP stirring rod. The glass tube was carefully removed, and the stirring rod was removed and immersed in a 9: 1 by volume mixture of methanol and acetic acid until no CL was detected. Synthesis of a blank molecularly imprinted polymer (NIP) was performed as described above except that the template molecule CL was not added.
Example 2: optimization of preparation conditions of molecular imprinting stirring rod
The preparation conditions of the molecular imprinting stirring rod are optimized, the preparation process is the same as that of the embodiment 1, and the optimization contents are as follows:
1. selection of polymerization reagents
In the preparation process of the molecular imprinting stirring rod, the selection of a functional monomer, a cross-linking agent, a pore-forming agent and an initiator is very important, and the quality of a coating is directly related. After a large number of experimental analyses, the following comparison of materials was focused on: the methacrylic acid MAA and the 4-vinylpyridine (4-VP) are used as functional monomers for comparison, experiments show that the MAA is more favorable for the polymerization of the functional monomers and template molecules, and the prepared coating is more compact and has higher imprinting ratio. Experiments compared the imprinting effect of two crosslinks Divinylbenzene (DVB) and ethylene glycol dimethacrylate EGDMA. Better crosslinking effect and more uniform coating can be obtained by adopting EGDMA compared with DVB. In addition, according to the solubility of clenbuterol, four solvents of methanol, chloroform, toluene and acetonitrile are selected as pore-forming agents in experiments, and experiments show that the solubility of clenbuterol in methanol and chloroform is better than that in toluene and acetonitrile, the coating prepared in the methanol solvent is more uniform and compact than other solvents, and the prepared coating has better adsorption performance. The initiator is azodiisobutyronitrile AIBN which is commonly used. Therefore, experiments confirm that MAA is finally selected as a functional monomer, EGDMA is selected as a cross-linking agent, methanol is selected as a pore-forming agent, and AIBN is selected as an initiator.
2. Optimization of polymer dosage and proportion
The amount and the ratio of the polymerization reagent were adjusted according to Table 1, and the optimum amount and ratio were selected according to the appearance of the coating and the adsorption performance thereof. As can be seen from the table, the amount of the cross-linking agent directly affects the quality of the coating. When the cross-linking agent is less, the coating is thinner, the cross-linking is insufficient, the firmness is poor, and the coating is easy to peel off; when the amount of the crosslinking agent is large, although the coating is firmly bonded, the local crosslinking is excessive, the coating is not uniform, and the active sites of the coating are covered by the crosslinking agent and reduced, thereby reducing the adsorption performance. When the molar ratio of the template molecules to the functional monomers to the cross-linking agent is 1: 4: 16, the prepared coating is uniform and compact, and has good adsorption performance and optimal coating quality.
When the dosage of the polymerization reagent is reduced in equal proportion, polymerization sites are correspondingly reduced, and the adsorption performance is reduced; when the amount of the polymerization reagent is increased in an equal proportion, the difficulty in eluting the template molecules is increased due to the increase of the amount of the crosslinking agent. Therefore, the amounts of template molecule CL, functional monomer MAA and cross-linking agent EGDMA are finally determined to be 0.5, 2.0 and 8.0mmol respectively.
TABLE 1 optimization of the amount and ratio of reagents for the polymerization reaction of the molecularly imprinted stir bars
3. Optimization of water bath heating time and temperature
In the preparation process of the molecular imprinting coating, the heating time and temperature of the water bath (namely the polymerization time and temperature of reactants in the heating process of the water bath) also have certain influence on the quality of the coating. Experiments were performed according to the protocol of table 2 to determine the optimal polymerization time and temperature for heating in the water bath. As can be seen from Table 2, when the polymerization temperature is too low or the polymerization time is too short, the resulting coating is thin and easily peeled off; when the polymerization temperature is too high or the polymerization time is too long, the polymerization reaction is too violent, the local crosslinking is excessive, the coating is not uniform, and the coating is easy to peel off. Therefore, the final determined polymerization temperature was chosen to be 70 ℃ and the polymerization time 24 h. Figure 2 shows MIP stir bars prepared under optimal coating reagents and optimal coating conditions, as well as blank glass rods. As can be seen from FIG. 2, the stir bar coating is uniform, dense, and has a certain thickness.
TABLE 2 optimization of polymerization time and temperature for molecularly imprinted coatings
| Serial number | Polymerization temperature (. degree. C.) | Polymerization time (h) | Test results |
| 1 | 50 | ＞24 | Thin coating, incomplete polymerization and easy peeling |
| 2 | 60 | 4 | The coating is thin and easy to peel off |
| 3 | 60 | 8 | Uniform and less |
| 4 | 60 | 24 | Uniform and less |
| 5 | 70 | 24 | Uniform and |
| 6 | 70 | 36 | Uniform and |
| 7 | 75 | 12 | Uneven coating and easy peeling |
The following are characterizations of the molecularly imprinted stir bar:
the chemical structure and the composition of the molecular imprinting stirring rod coating prepared under the optimal condition are characterized and determined by adopting a Fourier transform infrared instrument: the stir bar coated powder was dried under an infrared lamp. And then taking Clenbuterol (CL), an NIP stirring rod coating, an eluted template molecule MIP and MIP stirring rod coating powder without the eluted template molecule, and preparing samples by adopting a potassium bromide tabletting method. In the range of 400-4000 cm-1The infrared absorption spectrum of the sample is scanned in the wave number range, and the difference of the chemical composition of the sample is researched. The infrared spectrum is shown in FIG. 3. In FIG. 3, the spectra a, b, c, d represent clenbuterol, NIP, MIP without eluting template molecule, MIP with eluting template molecule, respectively. As can be seen from FIGS. 3(b) and (d), the eluted template MIP and NIP coating have the same infrared absorption spectrum at 1729cm-1All have strong adsorption peaks, which are mainly from C ═ O groups in the crosslinking agent EGDMA. Furthermore, at 3448cm-1All had absorption peaks from O-H stretching vibration at 2970cm-1Has an absorption peak from O-H stretching vibration at 1636cm-1Has absorption peaks from C ═ C stretching vibration at 1456 and 1385cm-1All have absorption peaks from C-H bending vibration at 1261cm-1There is absorption of vibration from C-O expansion and contractionPeak at 1156cm-1There is an absorption peak from C-O-C stretching vibration. As is clear from FIGS. 3(c) and (d), the IR absorption spectrum of the template molecule MIP not eluted was compared with that of the template molecule MIP eluted except at 1520cm-1There is no obvious difference in other places. At 1520cm-1The absorption peak is mainly from the stretching vibration of C ═ C on the benzene ring of the template molecule clenbuterol.
In conclusion, the infrared absorption spectrum result shows that the MIP and the NIP coating have very similar chemical structures, and the template molecules do not participate in chemical polymerization and are combined with the functional monomer only through the action of hydrogen bonds.
And characterizing the surface morphologies of the MIP stirring rod coating and the NIP stirring rod coating by adopting a scanning electron microscope. The scanning electron micrograph is shown in FIG. 4. FIGS. 4(a) and (c) are scanning electron micrographs of MIP and NIP at 200 Xmagnification, respectively. As shown in the figure, the coating particles on the surfaces of the MIP and the NIP stirring rod are fine and uniform. FIGS. 4(b) and (d) are SEM images of MIP and NIP magnified 5000 times, respectively. As shown in the figure, compared with NIP, the surface of the MIP coating has more mutually-crosslinked and loose structures with gaps, and the structure with high porosity is more favorable for absorbing mass transfer of an object to be detected in the stationary phase during extraction, so that the extraction rate is improved.
Example 3 SBSE Process and Condition optimization of molecularly imprinted stir bars
An application method of the molecularly imprinted stir bar, namely the adsorption extraction and desorption process of the molecularly imprinted stir bar, adopts the conditions of the embodiment 1 to prepare the molecularly imprinted stir bar, and comprises the following steps:
preparing a toluene mixture solution of clenbuterol, ractopamine, salbutamol, terbutaline and mabuterol (sample solution 5), placing the solution in a round-bottomed flask 4, placing a MIP stirring rod 6 and a NIP stirring rod (not shown in figure 5) in the solution, and stirring and adsorbing the solution on a magnetic stirrer 7 at room temperature at a rotating speed of 500r/min for 1h, wherein the schematic diagram is shown in figure 5. Then taking out, adsorbing the surface solution with filter paper, placing in a lining tube, adding 200 μ L of 10% volume fraction acetic acid solution, and performing ultrasonic treatment for 10min to obtain an analytic solution. The stir bar was removed and immersed in a 9: 1 mixture of methanol and acetic acid for the next use. The analysis solution was passed through a 0.22 μm filter and measured by LC-MS/MS method.
In order to obtain the optimal adsorption and extraction effect, the adsorption and extraction conditions of the molecular imprinting stirring rod are optimized as follows:
1. selection of extraction solvent and resolution solvent
Respectively preparing clenbuterol standard solution with the concentration of 10ng/mL by using water, methanol, acetonitrile, toluene, acetone and ethanol, then placing a molecular imprinting stirring rod (MIP stirring rod) in the solution, stirring and extracting for 1h, respectively comparing the clenbuterol content in the standard solution before and after extraction, calculating the extraction rate, and inspecting the extraction performance of the MIP stirring rod in different solvents. As can be seen from fig. 6, the extraction rate of clenbuterol in toluene solvent was higher with MIP stir bar. Thus, toluene was chosen as the extraction solvent.
Methanol, water, methanol-acetic acid (9: 1, v/v) and water-acetic acid (9: 1, v/v) are selected as desorption solvents, the recovery rates of different desorption solvents after desorption are compared, and the desorption effects of different desorption solvents are examined. As can be seen from FIG. 7, the extraction recovery of clenbuterol MIP stir bars is significantly higher in water-acetic acid (9: 1, v/v) solvent than other solvents, and therefore water-acetic acid (9: 1, v/v) solvent was chosen as the desorption solvent.
2. Optimization of adsorption and desorption times
And (3) placing an MIP stirring rod in 5mL of clenbuterol standard solution with the concentration of 10ng/mL, stirring and adsorbing for 15min, 30min, 45 min, 60min, 90 min and 120min, respectively measuring the content of clenbuterol in the extraction solution at different time points, and calculating the adsorption rate. The results of the adsorption time study are shown in FIG. 8. The amount of adsorption increased with time before the extraction equilibrium was reached. When the time reaches 60min, the increase of the adsorption amount is not obvious, and the adsorption balance is reached. In order to reduce the influence of adsorption time fluctuation on the adsorption quantity and ensure the basic balance of adsorption and extraction, 60min is selected as the optimal adsorption time.
Water-acetic acid (9: 1, v/v) is selected as a desorption solvent, ultrasonic desorption is carried out, 5min, 10min, 15min, 20min and 30min are respectively selected as desorption time, the recovery rates of clenbuterol at different desorption time points are compared, and the optimal desorption time is selected. As shown in fig. 9, desorption equilibrium can be reached by ultrasonic desorption for 10min, and therefore, 10min is selected as the desorption time.
3. Selection of stirring speed and extraction temperature
The recovery rate of clenbuterol at different stirring speeds of 100, 200, 300, 400, 500, 600, 800r/min and the like is investigated in an experiment, and the experimental result shows that when the rotating speed of a stirring rod is below 300r/min, the stirring speed is increased, and the extraction amount is increased; when the rotating speed of the stirring rod is between 300 and 800r/min, the change of the rotating speed has no obvious influence on the adsorption performance of the stirring rod. Considering that the wear of the stirring rod is severe at high stirring speed, which affects the service life of the stirring rod, the rotating speed of the stirring rod is determined to be 300r/min in the test.
Other conditions are fixed, and the extraction effects of clenbuterol at different extraction temperatures of 20, 25, 40, 50, 60 ℃ and the like are compared. The experimental result shows that when the adsorption temperature is 20-60 ℃, the temperature change has no obvious influence on the adsorption performance. This can be interpreted as: the diffusion coefficient of the analyte becomes larger when the temperature rises, and the extraction efficiency is improved; however, the efficiency of extraction is reduced by the reduced partition coefficient of the analyte between the two phases. In consideration of the convenience of the adsorption operation, the adsorption test is performed at room temperature.
The following are the performance evaluations of the molecularly imprinted stir bar:
1. adsorption capacity
Toluene is used as a solvent to prepare a clenbuterol standard solution with the concentration of 1-60 mu g/mL. And (3) inspecting the adsorption capacity of the clenbuterol molecularly imprinted stir bar MIP on the target molecule clenbuterol by adopting a static adsorption test, and taking NIP as reference. Respectively placing the MIP and the NIP stirring rod in clenbuterol toluene solutions with different concentrations, wherein the extraction volume is 5mL, the extraction time is 60min, and respectively measuring the extraction amount. The extraction results are shown in FIG. 10. The test result shows that the extraction amount of the MIP stirring rod and the NIP stirring rod is gradually increased along with the increase of the CL concentration of the extraction solution, and when the CL standard solution concentration is increased to 35 mu g/L, the extraction amount of the MIP stirring rod and the NIP stirring rod is saturated. The amount of MIP stirrer extracted at this time was 67. mu.g, which was 3.76 times the amount of NIP stirrer extracted at 17.8. mu.g. This difference in extraction yields derives from the different extraction mechanisms of MIP stir bar coatings and NIP stir bar coatings. When the MIP stirring rod is prepared, hydrogen bond action is generated between the template molecules and the functional monomers, the ordered arrangement of the hydrogen bond structure is fixed in the polymerization process, and as the template molecules are eluted from the polymer, a left cavity is formed in the space occupied by the template molecules originally, and the cavity structure can be used for selectively identifying the target object again. The coating of the NIP stirring rod does not have the specific hole structure, the extraction mechanism is nonspecific adsorption, and the extraction capacity of the coating on the target is obviously lower than that of the MIP stirring rod.
2. Clenbuterol molecular imprinting stirring rod selective adsorption test
In order to further examine the selective adsorption performance of the clenbuterol molecular imprinting stirring rod (MIP), clenbuterol molecular structural analogs and other interfering substances are selected as extraction media, specifically clenbuterol, ractopamine, brombuterol, mabuterol, terbutaline, salbutamol, benzyl alcohol and acrylamide, and meanwhile, in order to avoid adsorption saturation and competitive adsorption, extraction solutions of the different substances are respectively prepared, wherein the concentrations are all 20 mu g/L. The adsorption extractions of MIP and NIP in different extraction media are shown in figure 11. The extraction amount of clenbuterol and structural analogues thereof is obviously higher than that of other interferents, and the MIP extraction amount of clenbuterol, ractopamine, salbutamol, brombuterol, terbutaline and mabuterol is respectively 3.8, 2.9, 3.1, 3.5, 3.2 and 3.3 times of the NIP extraction amount. The amount of extracted interfering substances, which differ structurally and chemically from clenbuterol, was barely detectable. Therefore, the high selective adsorption performance of the template molecules and the structural analogs thereof is caused by the specific recognition sites formed by the template molecules and the polymers in the preparation process of the molecular imprinting coating. The difference between the molecular structure and the molecular interface directly affects the adsorption performance. In a competitive adsorption system, the higher the degree of matching between the target and the hole structure, the smaller the molecular volume, the more easily the target enters the hole structure, and the higher the probability of being recognized by forming a hydrogen bond with the recognition site of the hole, and therefore, the higher the extraction rate.
3. Solvent resistance test of Kroteron molecular imprinting stirring rod
Respectively soaking MIP and NIP in water, methanol, acetonitrile, acetone, dichloromethane, benzene, toluene, 10% acetic acid solution, dimethyl sulfoxide, etc. for 30 min. Test results show that the MIP and NIP coatings are well preserved in the above solvents, no evidence of peeling or flaking occurs, and no residue is found in the solvents. This indicates that the stir bar molecularly imprinted coating has good solvent resistance and is suitable for high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) analysis.
4. Service life of Krotiro molecular imprinting stirring rod
The template molecules of the MIP are eluted by water-acetic acid (9: 1, v/v), and the MIP is placed in dry air and stored for 3 months, and still maintains good adsorption performance. After 40 times of repeated use, there was no significant damage to the MIP and NIP coatings.
EXAMPLE 4 practical application of the molecularly imprinted stir bar
The method for detecting 6 beta-receptor agonist drugs such as clenbuterol and structural analogues thereof in animal derived food by using the molecularly imprinted stir bar comprises the following steps:
lean and feed samples were purchased on the market for testing. Firstly, crushing a sample, then respectively weighing 2g of the sample into a 50mL polytetrafluoroethylene centrifugal tube, adding a mixed standard solution of clenbuterol, ractopamine, salbutamol, brombuterol, terbutaline and mabuterol, setting two labeling levels, wherein the labeling levels are 5 and 10 mu g/kg respectively, and simultaneously, paralleling each labeling level for 5 times, and standing for 30min after labeling to ensure that the standard sample is fully adsorbed. The sample was then pre-treated and extracted twice with trichloroacetic acid (15mL, 2% volume) by sonication for 15min each time. Combining the extracting solutions, and adjusting the pH value to 9-10 by using 1mol/L NaOH solution. Then 2g of sodium chloride is added, the mixture is evenly mixed by vortex, the mixture is centrifuged for 5min at 4000r/min, the supernatant is transferred into another centrifuge tube and extracted twice by 15mL of isopropanol-ethyl acetate (60: 40, v/v), the organic phases are combined and blown to be nearly dry in a water bath at 50 ℃ by nitrogen. 5mL of toluene was added to dissolve the residue, and the solution was analyzed by extraction with a molecular imprinting stirrer. And (3) placing a molecular imprinting stirring rod in the toluene solution of the sample, stirring at the rotating speed of 300r/min, adsorbing and extracting for 1h, taking out the stirring rod, placing in a lining tube, adding 200 mu L of 10% acetic acid solution by volume fraction, and carrying out ultrasonic treatment for 10min to obtain an analytic solution. The stir bar was removed and immersed in a 9: 1 mixture of methanol and acetic acid for the next use. The analysis solution passes through a filter membrane with the diameter of 0.22 mu m and is determined by LC-MS/MS.
1. HPLC-MS/MS conditions
(1) HPLC conditions
A chromatographic column: hypersil GOLDTMaQ column (100 mm. times.2.1 mm, 1.9 μm). Mobile phase: the solvent A is formic acid aqueous solution with volume fraction of 0.1%, and the solvent B is acetonitrile; gradient elution procedure: 0-1 min, 90-40% A, 1-4 min, 40-5% A, 4-5 min, 5% A, 5.0-5.1 min, 5% A-90% A, 5.1-7.0 min, 90% A. Flow rate: 0.3 mL/min; column temperature: 35 ℃; sample introduction amount: 10 μ L.
(2) MS/MS conditions
An ion source: electrospray electric ion source (ESI); the scanning mode is as follows: scanning positive ions; auxiliary gas pressure: 10 Arb; sheath gas pressure: 40 Arb; ion spray voltage (IS): 3500V; temperature of the atomizer: 300 ℃; ion source temperature: 350 ℃; collision gas (argon) pressure: 1.5 mtorr; the detection mode is as follows: multiple Reaction Monitoring (MRM) scan mode. The mass spectrometry parameters of the beta-receptor agonist drug obtained by optimization under the positive ion mode are shown in table 3.
TABLE 36 Mass Spectrometry parameters of beta receptor agonists in multiple reaction monitoring mode
2. Methodology validation
Respectively sucking 5mL of clenbuterol, ractopamine, salbutamol, brombuterol, terbutaline and mabuterol mixed standard solutions with different concentrations into a round-bottomed flask, measuring the adsorption capacity of the MIP in the mixed standard solutions with different concentrations according to the operation steps of the molecular imprinting stirring rod detection method, and inspecting the linear range of the analysis method. The test results are shown in table 4. Experimental results show that the linear range of the clenbuterol is 0.5-35 mu g/L, and the ractopamine, the salbutamol, the brombuterol, the terbutaline and the mabuterol have good linear relation in the concentration range of 1-35 mu g/L. The detection limit is calculated by 3 times of signal-to-noise ratio, and the detection limit of the above 6 compounds is between 0.20 mu g/kg and 0.35 mu g/kg. The relative standard deviation (n is 3) is between 2.6% and 4.8%.
TABLE 4 measurement of Linear Range and detection of beta-receptor agonists by molecular imprinting stir bar adsorption extraction-HPLC tandem mass spectrometry
Limit and relative standard deviation
3. Sample analysis
Pork and feed products supplemented with mixed beta agonists at different concentration levels were tested. The measurement results are shown in Table 5. As can be seen from Table 5, the method can be successfully applied to the determination of the beta-stimulant in pork and feed products, the recovery rates are respectively 70.6-86.5% and 70.2-88.9%, and the relative standard deviation is between 3.9-5.5%.
TABLE 5 recovery of beta-receptor agonist addition in pork and feed samples
The method and the national standard detection method GB/T22286-2008 'determination of residual amount of multiple beta-receptor agonists in animal-derived food liquid chromatography tandem mass spectrometry' are respectively applied to the same clenbuterol quality control sample to detect the Clenbuterol (CL) content, and the detection results are shown in Table 6. As can be seen from Table 6, the test results of the method of the present invention are slightly lower than those measured by the national standards, but within the satisfactory value range, the test requirements are satisfied.
TABLE 6 comparison of the test results of quality control samples by the method of the present invention and the national standard method
| Quality control sample CL assigned value (μ g/kg) | 3.43±1.00 |
| CL test results (μ g/kg) of the method of the invention | 2.56 |
| GB/T22286-2008CL test results (ug/kg) | 2.78 |
By combining the embodiments, the invention successfully prepares the molecularly imprinted stir bar taking the clenbuterol as the template molecule and applies the molecularly imprinted stir bar to the detection of 6 beta-receptor agonist drugs such as clenbuterol and structural analogues thereof in animal-derived food. Meanwhile, the accuracy and precision of the method for detecting the beta-stimulant in the application of the molecular imprinting stirring rod are verified through experiments, the recovery rates of the method for detecting the beta-stimulant in the pork and feed products are respectively 70.6% -86.5% and 70.2% -88.9%, the relative standard deviation is 3.9% -5.5%, and a good detection effect is obtained. The scheme and the achievement of the invention have good scientific research value, practical application value and development prospect, not only reduce the experiment cost and reagent consumption, reduce the emission and the pollution to the environment, but also simplify the experiment steps, reduce the matrix interference, realize the selective separation and enrichment of template molecules and structural analogues, and improve the detection efficiency, the accuracy and the sensitivity.
The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. Although the present invention has been described with reference to the preferred embodiments, it is not intended to be limited thereto. Those skilled in the art can make many possible variations and modifications to the disclosed embodiments, or equivalent modifications, without departing from the spirit and scope of the invention, using the methods and techniques disclosed above. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments according to the technical essence of the present invention are still within the scope of the protection of the technical solution of the present invention.
Claims (4)
1. The application of the molecular imprinting stirring rod in detecting beta-receptor agonist drugs in animal-derived food is characterized by comprising the following steps of: extracting a target object to be detected in an animal-derived food sample by trichloroacetic acid, adjusting the pH value of an extraction solution to 9-10, adding sodium chloride, mixing uniformly, extracting and enriching the target object to be detected by using an isopropanol-ethyl acetate mixed solution with the volume ratio of 6: 4, dissolving residues by using an extraction solvent after nitrogen blowing concentration, then placing a molecular imprinting stirring rod in the obtained sample solution, performing adsorption extraction on the target object to be detected under stirring, and finally resolving the target object to be detected by using an resolving solvent to perform high performance liquid chromatography tandem mass spectrometry;
the parameters of the HPLC tandem mass spectrometry are as follows:
a chromatographic column: aQ column, mobile phase: the solvent A is formic acid aqueous solution with volume fraction of 0.1%, and the solvent B is acetonitrile; gradient elution procedure: 0-1 min, 90% A-40% A, 1-4 min, 40% A-5% A, 4-5 min, 5% A, 5.0-5.1 min, 5% A-90% A, 5.1-7.0 min, 90% A, flow rate: 0.3 mL/min; column temperature: 35 ℃; sample introduction amount: 10 muL;
an ion source: an electrospray ionization source; the scanning mode is as follows: scanning positive ions; auxiliary gas pressure: 10 Arb; sheath gas pressure: 40 Arb; ion spray voltage: 3500V; temperature of the atomizer: 300 ℃; ion source temperature: 350 ℃; collision gas argon pressure: 1.5 mtorr; the detection mode is as follows: multiple reaction monitoring scan mode, other mass spectrometry parameters were as follows:
the extraction solvent is toluene, the desorption solvent is methanol-acetic acid mixed solution or acetic acid solution with volume fraction of 10% in the volume ratio of 9: 1, the adsorption and extraction time is 15min to 120min, the desorption time is 5min to 30min, the stirring speed is 100r/min to 800r/min, and the adsorption and extraction temperature is room temperature to 60 ℃;
the target to be detected comprises clenbuterol, ractopamine, salbutamol, brombuterol, terbutaline and mabuterol,
when the beta-receptor agonist drug is clenbuterol, the linear equation is y =10124+2.65 × 106 x, linear range of 0.5 to 35 μ g/L,
when the beta-receptor agonist drug is salbutamol, the linear equation is y =20563+1.05 × 106 x, linear range of 1 to 35 μ g/L,
when the beta-receptor agonist drug is ractopamine, the linear equation is y =489612+2.96 × 106 x, linear range of 1 to 35 μ g/L,
when the beta-receptor agonist drug is bromobuterol, the linear equation is y =52461+2.35 × 106 x, linear range of 1 to 35 μ g/L,
when the beta-receptor agonist drug is terbutaline, the linear equation is y =89764+1.89 × 106 x, linear range of 1 to 35 μ g/L,
when the beta-receptor agonist drug is mabuterol, the linear equation is y =78456+1.03 × 106 x, linear range1-35 mug/L;
the molecular imprinting stirring rod comprises a stirring rod and a molecular imprinting coating covering the stirring rod, wherein the molecular imprinting coating is mainly prepared by using clenbuterol as a template molecule, methacrylic acid as a functional monomer, ethylene glycol dimethacrylate as a cross-linking agent, methanol as a pore-forming agent and azobisisobutyronitrile as an initiator to prepare a molecular imprinting polymer, and then eluting the template molecule in the molecular imprinting polymer to prepare the molecular imprinting polymer, wherein the stirring rod is a surface silanized iron-core-containing glass stirring rod;
the preparation method of the molecular imprinting stirring rod comprises the following steps:
dissolving template molecule clenbuterol in methanol, adding functional monomer methacrylic acid, polymerizing, adding crosslinking agent ethylene glycol dimethacrylate and initiator azobisisobutyronitrile, mixing and carrying out ultrasonic treatment to obtain a mixed solution; placing the iron core glass stirring rod with the silanized surface into the obtained mixed solution, performing secondary ultrasonic treatment, reacting under the conditions of nitrogen atmosphere and water bath heating, and forming a molecular imprinting coating on the iron core glass stirring rod with the silanized surface to obtain a molecular imprinting stirring rod;
the addition ratio of the clenbuterol, the methacrylic acid, the ethylene glycol dimethacrylate, the methanol and the azobisisobutyronitrile is 0.5mmol, 2mmol, 8mmol, 5mL and 15 mg;
the temperature of the water bath heating is 70 ℃, and the reaction time is 24-36 h.
2. The use according to claim 1, wherein the time of the adsorption extraction is 60min, the time of the desorption is 10min, the stirring speed is 300r/min, and the temperature of the adsorption extraction is room temperature.
3. The use according to claim 1, wherein the polymerization time is 12-24 h, the sonication time is 10-15 min, and the re-sonication time is 5-10 min.
4. The use according to claim 1, wherein the surface silanized iron core-containing glass stirring rod is prepared by: soaking an iron core-containing glass stirring rod in NaOH solution to expose silanol groups on the surface of the iron core-containing glass stirring rod to the maximum extent, then washing the iron core-containing glass stirring rod with distilled water, soaking the iron core-containing glass stirring rod in hydrochloric acid solution to remove residual alkali liquor on the surface of the iron core-containing glass stirring rod, washing the iron core-containing glass stirring rod with distilled water and drying the iron core-containing glass stirring rod, soaking the dried iron core-containing glass stirring rod in acetone solution of 3- (methacryloyloxy) propyl trimethoxy silane to silanize the surface of the iron core-containing glass stirring rod, and cleaning the iron core-containing glass stirring rod with methanol and drying the stirring rod with nitrogen flow to obtain the iron core-containing glass stirring rod with silanized surface.
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