Fluoroquinolone compound separation-ionization integration device and application thereof

Abstract

The application discloses a fluoroquinolone compound separation-ionization integrated device and a preparation method and application thereof, wherein the method for preparing the compound separation-ionization integrated device comprises the following steps: performing a silicon hydroxylation treatment on a substrate to obtain a silicon-hydroxylated substrate; performing an amino modification treatment on at least part of the surface of the silicon-hydroxylated substrate to obtain an amino-modified substrate; performing a grafting treatment on the amino-modified substrate to obtain a grafted substrate; contacting a fluoroquinolone template molecule with a first monomer compound, a second monomer compound and a pore-forming agent to perform a prepolymerization reaction, so as to obtain a prepolymer embedded with the fluoroquinolone compound; performing a polymerization reaction on the prepolymer and the grafted substrate to obtain a polymer-coated substrate; contacting the polymer-coated substrate with a crosslinking agent to perform a self-polymerization treatment, so as to obtain a device primary product; and performing an elution treatment on the device primary product to obtain the fluoroquinolone compound separation-ionization integrated device.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, and more specifically, to an integrated device for the separation and ionization of fluoroquinolone compounds, its preparation method, and its application. Background Technology

[0002] Fluoroquinolone antibiotics are an important class of antibiotics with broad-spectrum activity against both Gram-positive and Gram-negative bacteria. They are widely used in livestock and poultry farming, playing a vital role in animal disease prevention and control, improving feed utilization, and promoting livestock and poultry growth. However, with the excessive use of antibiotics, especially their unscientific abuse and failure to adhere to withdrawal periods, problems such as bacterial resistance and antibiotic residues in food substrates have become increasingly prominent, attracting significant public attention.

[0003] Currently, the main methods used for antibiotic residue detection include microbial inhibition assays, immunoassays, high-performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC-MS). Among these methods, microbial inhibition assays are simple to operate, but have large detection errors and are time-consuming, making them more suitable for screening experiments; immunoassays have low detection limits and high specificity, but target a single individual, making them more suitable for qualitative or semi-quantitative detection; chromatographic and mass spectrometric equipment are precise, with high detection sensitivity and accuracy, and can simultaneously perform qualitative and quantitative detection of antibiotic residues, but they have high requirements for the analytical samples, requiring extensive pretreatment operations such as extraction, purification, and enrichment, consuming large amounts of organic solvents and time, making them difficult to meet the requirements for rapid on-site detection.

[0004] Therefore, methods for the specific extraction of fluoroquinolone compounds need further investigation. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. Therefore, one objective of the present invention is to provide a method for preparing an integrated device for the separation and ionization of fluoroquinolone compounds. The device prepared by this method has good selectivity, enabling the specific extraction of fluoroquinolone compounds and their direct ionization as a solid-phase substrate for mass spectrometry detection.

[0006] Therefore, according to one aspect of the present invention, the present invention provides a method for preparing an integrated device for the separation and ionization of fluoroquinolone compounds. According to an embodiment of the present invention, the method includes: silanizing a substrate to modify at least a portion of the surface of the acidified substrate with silanol groups to obtain a silanized substrate; modifying at least a portion of the surface of the silanized substrate with amino groups to obtain an amino-modified substrate; grafting the amino-modified substrate to obtain a grafted substrate; contacting a fluoroquinolone template molecule with a first monomer compound, a second monomer compound, and a porogen to perform a prepolymerization reaction to obtain a prepolymer incorporating the fluoroquinolone compound; polymerizing the prepolymer with the grafted substrate to obtain a polymer-coated substrate; contacting the polymer-coated substrate with a crosslinking agent to perform a self-polymerization treatment to form an imprint cavity to obtain a preliminary device; and eluting the preliminary device to obtain the integrated device for the separation and ionization of fluoroquinolone compounds.

[0007] The separation and ionization integrated device prepared by the method of the present invention has a uniform coating and exhibits specific adsorption and enrichment of fluoroquinolone compounds and their structural analogs. It has strong extraction and enrichment specificity and is suitable for detection by analytical methods such as open solid substrate electrospray ionization mass spectrometry. It is particularly suitable for the enrichment and detection of fluoroquinolone chemicals in complex matrix materials and has broad application prospects in the fields of rapid detection and trace detection.

[0008] In addition, the method for preparing the integrated separation and ionization device according to the above embodiments of the present invention may also have the following additional technical features:

[0009] According to an embodiment of the present invention, the first monomer compound is dimethylaminoethyl methacrylate (DMA), and the second monomer compound is tetravinylphenylboronic acid (VPBA).

[0010] According to an embodiment of the present invention, the crosslinking agent is dopamine hydrochloride and bovine serum albumin.

[0011] According to an embodiment of the present invention, the fluoroquinolone template molecule is enrofloxacin.

[0012] According to an embodiment of the present invention, the crosslinking reaction further includes: a porogen, wherein the porogen is a phosphate buffer containing acetonitrile and methanol, preferably, the volume ratio of acetonitrile:methanol:phosphate buffer in the porogen is 1:0.8-1.2:1.

[0013] According to an embodiment of the present invention, the molar ratio of the fluoroquinolone template molecule to the first monomer compound, the second monomer compound and the crosslinking agent is 1:0.8-1.2:3-5:10-20.

[0014] According to an embodiment of the present invention, the substrate has at least one angular end, and preferably, the substrate is an isosceles triangle.

[0015] According to an embodiment of the present invention, the substrate is a stainless steel plate, a wood plate, or a bamboo plate.

[0016] According to an embodiment of the present invention, the substrate is a stainless steel plate.

[0017] According to an embodiment of the present invention, the method further includes: acidifying the substrate before the silanization treatment to obtain an acidified substrate.

[0018] According to an embodiment of the present invention, the method includes: immersing the substrate in a 1.5-2.5 mol / L sulfuric acid solution, sonicating it for 3-5 hours, rinsing the substrate surface with water until neutral, to obtain the acidified substrate; immersing the acidified substrate in a mixed solution of ethanol and ammonia, adding 1-3 mL of tetraethyl orthosilicate and shaking at room temperature for 10-15 hours to perform the silanization treatment, so that at least part of the surface of the acidified substrate is silanized, rinsing with ultrapure water and ethanol, and drying with nitrogen to obtain the silanized substrate; immersing the silanized substrate in anhydrous n-hexane solution, adding 3-aminopropyltriethoxysilane, stirring at room temperature for 1.5-2.5 hours to perform the amino modification treatment, so as to modify the silanized surface with amino groups, to obtain an amino-modified substrate; and then... The substrate is contacted with polyethyleneimine, NaBH4, and glutaraldehyde for a prepolymerization reaction of 1.5-2.5 hours to obtain a prepolymer incorporating the fluoroquinolone compound. The fluoroquinolone template molecule is placed in the porogen and mixed with the first and second monomer compounds for 1.5-2.5 hours for the prepolymerization reaction to obtain the prepolymer incorporating the fluoroquinolone compound. The prepolymer is then contacted with the grafted substrate and shaken overnight for polymerization to obtain a polymer-coated substrate. Under alkaline conditions, the polymer-coated substrate is contacted with a crosslinking agent for 7-8 hours for self-polymerization to form an imprint cavity, obtaining a preliminary device. The preliminary device is then eluted to obtain the integrated fluoroquinolone compound separation and ionization device.

[0019] According to an embodiment of the present invention, the eluent is a methanol solution containing 10% formic acid.

[0020] Furthermore, according to another aspect of the present invention, an integrated device for the separation and ionization of fluoroquinolone compounds is provided. According to an embodiment of the present invention, the device is obtained using the aforementioned preparation method.

[0021] The compound separation and ionization integrated device according to embodiments of the present invention has a uniform coating and exhibits specific adsorption and enrichment effects on fluoroquinolone compounds and their structural analogs. It has strong extraction and enrichment specificity and can be detected by analytical methods such as open solid substrate electrospray ionization mass spectrometry. It is particularly suitable for the enrichment and detection of specific chemicals in complex matrix materials and has broad application prospects.

[0022] According to another aspect of the present invention, a method for extracting fluoroquinolone compounds is provided. According to an embodiment of the present invention, the method includes: immersing at least a portion of the aforementioned integrated fluoroquinolone compound separation and ionization device into a solution of the fluoroquinolone compound to be tested for extraction, so as to enrich the fluoroquinolone compound on the integrated fluoroquinolone compound separation and ionization device.

[0023] The method for extracting fluoroquinolone compounds according to embodiments of the present invention utilizes the aforementioned apparatus for extraction and enrichment, exhibiting good specific adsorption and enrichment effects on fluoroquinolone compounds and their structural analogs, with fast extraction speed and strong specificity. The apparatus containing the compound to be extracted obtained by this extraction method achieves ionization of the target analyte and allows it to be detected by mass spectrometry, avoiding the use of liquid chromatography and simplifying the detection steps.

[0024] According to an embodiment of the present invention, the extraction process takes 30-70 minutes, preferably 50 minutes.

[0025] According to another aspect of the present invention, an integrated mass spectrometry ionization system for the separation and ionization of fluoroquinolone antibiotics is provided. According to an embodiment of the present invention, the system includes: the aforementioned integrated fluoroquinolone compound separation and ionization device; and an analytical detector. Thus, the extracted and enriched analyte is directly analyzed and detected by the analytical detector, resulting in high sensitivity and speed, particularly suitable for rapid and sensitive detection of target analytes in complex matrices.

[0026] According to an embodiment of the present invention, the analytical instrument is an open-type mass spectrometer.

[0027] According to another aspect of the present invention, a method for qualitative / quantitative detection of fluoroquinolone compounds is provided. According to an embodiment of the present invention, the method includes: enriching fluoroquinolone compounds using the aforementioned method for extracting fluoroquinolone compounds to obtain an apparatus for enriching the fluoroquinolone compounds; and analyzing the apparatus for enriching the fluoroquinolone compounds using an analytical analyzer to perform qualitative / quantitative detection of the fluoroquinolone compounds.

[0028] According to the detection method of the present invention, the extracted and enriched device is directly analyzed and detected, resulting in low background noise, high sensitivity and fast speed of detection, which is especially suitable for rapid and sensitive detection of target analytes in complex matrices.

[0029] According to an embodiment of the present invention, the analytical instrument is an open-type mass spectrometer.

[0030] According to an embodiment of the present invention, the spray solvent for the open-type mass spectrometry detection is a mixed solution of methanol and formic acid.

[0031] According to an embodiment of the present invention, the ionization voltage of the open-type mass spectrometry detection is 3.6 kV.

[0032] According to an embodiment of the present invention, the detection conditions for the open mass spectrometry detection are as follows: multiple reaction monitoring (MRM); electrospray voltage (IS): 5500V; nebulizer gas pressure (GS1): 55psi; auxiliary gas pressure (GS2): 50psi; curtain gas pressure (CUR): 20psi; ion source temperature (TEM): 550℃; residence time (DT): 100ms.

[0033] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0034] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0035] Figure 1 A schematic diagram of a fluoroquinolone compound detection device according to an embodiment of the present invention is shown;

[0036] Figure 2 A schematic diagram showing the detection results of different extraction times of the integrated separation and ionization apparatus according to an embodiment of the present invention is displayed;

[0037] Figure 3 A schematic diagram showing the detection results of different spray solvents according to an embodiment of the present invention is provided.

[0038] Figure 4 A schematic diagram showing the detection results of different spray voltages according to an embodiment of the present invention is displayed;

[0039] Figure 5 A schematic diagram showing the detection results of an integrated separation and ionization apparatus for different modified groups according to an embodiment of the present invention is displayed.

[0040] Figure 6The diagram shows the adsorption results of two elements using different crosslinking agents on FQs in aqueous solution and spiked milk samples according to an embodiment of the present invention. Detailed Implementation

[0041] Embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0042] In the description of this invention, the terms "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and do not require that this invention must be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting this invention.

[0043] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0044] According to one aspect of the present invention, a method for preparing an integrated device for the separation and ionization of fluoroquinolone compounds is provided. The device prepared by the method according to the embodiments of the present invention has a uniform coating, exhibits specific adsorption and enrichment of the compound to be extracted and its structural analogues, demonstrates strong extraction and enrichment specificity, and is suitable for detection by analytical methods such as open-substrate electrospray ionization mass spectrometry. It is particularly suitable for the enrichment and detection of specific chemicals in complex matrix substances, such as the detection of fluoroquinolone compounds in dairy products and meat, and has broad application prospects in the fields of rapid detection and trace detection. The preparation method according to the embodiments of the present invention is simple in process, low in production cost, requires no special equipment, and is easy to implement industrially.

[0045] To facilitate understanding of the aforementioned method for preparing an integrated compound separation and ionization device, the preparation method is explained and illustrated according to embodiments of the present invention:

[0046] S100 silanization treatment

[0047] According to an embodiment of the present invention, the substrate is subjected to silanolization treatment, so that at least a portion of the surface of the acidified substrate is modified with silanol groups to obtain a silanolized substrate. This facilitates subsequent modification with amino and dendritic hyperbranched structures.

[0048] According to an embodiment of the present invention, the substrate is a stainless steel plate, a wooden board, or a bamboo board. This facilitates cross-linking polymer coating and reduces deformation. The inventors have found that when using an inert material as a solid substrate, copper clips are typically required for fixation and conductivity to apply high voltage to the solid substrate. This increases the risk of substrate movement and affects experimental parallelism. Furthermore, our previous experiments showed that porous membranes as solid-phase substrates exhibit higher background interference, while stainless steel sheets show lower background noise. Moreover, due to the conductivity of stainless steel sheets, high voltage can be directly applied to them, thus eliminating the need for copper clips in this experiment and further simplifying the experimental procedure. Therefore, according to a preferred embodiment of the present invention, the substrate is a stainless steel plate. According to an embodiment of the present invention, if the substrate is a stainless steel plate, the method further includes: acidifying the substrate before the silanization treatment to obtain an acidified substrate. This eliminates interference from other ions on the substrate surface.

[0049] According to an embodiment of the present invention, the substrate has at least one angular end, that is, the substrate has a angular tip shape in its top view. Preferably, the substrate is an isosceles triangle. This facilitates fixation on the analytical instrument, placing it at the horizontal front end of the instrument's sample inlet. The sprayed solvent can elute the target analyte adsorbed on the substrate, and through the action of a high-voltage power supply, ionize it at the tip to form a Taylor cone spray, which directly enters the mass spectrometer for detection. Specifically, the stainless steel sheet needs to be cut into a suitable isosceles triangle with a leg length of 2cm, a base of 1cm, and a thickness of 0.3mm.

[0050] According to an embodiment of the present invention, alkylation treatment can be performed using an alkaline solution of tetraethyl orthosilicate (TEOS). Specifically, a dry substrate is placed in a water-ethanol-ammonia solution (4:50:5) and shaken, and 2 mL of tetraethyl orthosilicate (TEOS) is slowly added dropwise to modify the surface of the stainless steel sheet with silanol groups (SS@OH).

[0051] S200 amino modification treatment

[0052] According to an embodiment of the present invention, at least a portion of the surface of the silanized substrate is subjected to amino modification treatment to obtain an amino-modified substrate. This facilitates subsequent grafting and polymerization reactions.

[0053] According to an embodiment of the present invention, amino modification is performed using 3-aminopropyltriethoxysilane (APTES). Specifically, the silanolized substrate can be placed in anhydrous n-hexane solution, and 1% (w / w) of 3-aminopropyltriethoxysilane (APTES) can be added and stirred at room temperature for 2 hours to modify the silanol surface with amino groups (SS@OH@NH2).

[0054] S300 grafting treatment

[0055] According to an embodiment of the present invention, the amino-modified substrate is subjected to grafting treatment to obtain a grafted substrate. This facilitates the bonding of the prepolymer to the substrate.

[0056] According to an embodiment of the present invention, grafting is performed using polyethyleneimine (PEI). Specifically, an amino-modified substrate, 10 mL of a 1 mg / mL polyethyleneimine (PEI) aqueous solution, and 30 mg of NaBH4 are added to 10 mL of a 1% (V / V) glutaraldehyde aqueous solution and shaken at room temperature for 3 h to graft a dendritic hyperbranched structure (SS@OH@NH2@PEI) onto the surface of a stainless steel sheet.

[0057] S400 prepolymerization reaction

[0058] According to an embodiment of the present invention, a fluoroquinolone template molecule is contacted with a first monomer compound, a second monomer compound, and a pore-forming agent to undergo a prepolymerization reaction in order to obtain a prepolymer incorporating the fluoroquinolone compound, thereby forming a cavity that matches the spatial configuration and chemical bonds of the fluoroquinolone compound, facilitating the entry of the fluoroquinolone compound and its analogues to be extracted into the cavity during subsequent extraction processes.

[0059] According to an embodiment of the present invention, the first monomer compound is dimethylaminoethyl methacrylate (DMA), and the second monomer compound is tetravinylphenylboronic acid (VPBA). DMA exhibits pH-responsive properties; changes in solution pH cause a change in the hydrophilicity of DMA, thereby affecting the water absorption capacity of the imprinted cavity formed by DMA. However, during the prepolymerization process of DMA with the template molecule, it can only act as a hydrogen bond acceptor and cannot bind to all functional sites of the template molecule. VPBA, as a complementary functional monomer, can act as a hydrogen bond donor, binding to the remaining sites of the template molecule, thereby improving the affinity of the imprinted polymer for the target molecule. Furthermore, VPBA itself has boron affinity and pH-responsive properties. The combination of DMA and VPBA as functional monomers enhances the pH-responsive properties of the resulting imprinted polymer.

[0060] According to an embodiment of the present invention, the fluoroquinolone template molecule is enrofloxacin. Enrofloxacin is widely used and its structure contains key functional groups of fluoroquinolone compounds. Using enrofloxacin as a template molecule, enrofloxacin and its analogues, such as ofloxacin, ciprofloxacin, norfloxacin, sarafloxacin, enoxacin, lomefloxacin, and pefloxacin, can be specifically extracted.

[0061] According to an embodiment of the present invention, the molar ratio of the fluoroquinolone template molecule to the first monomer compound, the second monomer compound, and the crosslinking agent is 1:0.8-1.2:3-5:10-20. Therefore, the appropriate ratio of monomer compound to crosslinking agent effectively initiates the crosslinking polymerization reaction while avoiding excessive crosslinking. The resulting molecularly imprinted layer exhibits good imprinting effect, possessing both pH-responsiveness and the ability to restrict the entry of macromolecules such as proteins.

[0062] S500 polymerization reaction

[0063] According to an embodiment of the present invention, the prepolymer is polymerized with the grafted substrate to obtain a substrate coated with the polymer. Thus, the crosslinked material forming the pores for the extracted fluoroquinolone compounds is polymerized with the dendritic hyperbranched structure on the substrate surface through a polymerization reaction, and attached to the substrate surface.

[0064] According to an embodiment of the present invention, the grafted substrate is immersed in the prepolymer and subjected to a shaking reaction overnight.

[0065] S600 self-polymerization process

[0066] According to an embodiment of the present invention, the polymer-coated substrate and a crosslinking agent are brought into contact and subjected to self-polymerization to form imprinted cavities, thereby obtaining a preliminary device. Thus, the crosslinking agent is used to fix the pores of fluoroquinolone compounds.

[0067] According to an embodiment of the present invention, the crosslinking agent is dopamine hydrochloride and bovine serum albumin (BSA), preferably, the mass ratio of dopamine hydrochloride to BSA is 1:1. Thus, dopamine exhibits excellent biocompatibility and can self-polymerize in an alkaline environment; BSA has good hydrophilicity and an isoelectric point of approximately 4.7, similar to the isoelectric point of major proteins such as casein in dairy products, and can prevent protein precipitation on the MIP surface through electrostatic repulsion, and does not interfere with the adsorption of small molecules.

[0068] According to an embodiment of the present invention, the crosslinking reaction further includes a porogen, wherein the porogen is a phosphate buffer of acetonitrile and methanol, preferably, the volume ratio of acetonitrile:methanol:phosphate buffer of the porogen is 1:0.8-1.2:1, and more preferably, the pH value of the porogen is 8.5. This results in good dissolution of the monomer compound and the template, and facilitates the self-polymerization of dopamine in the crosslinking agent to form polydopamine.

[0069] S700 elution process

[0070] According to an embodiment of the present invention, the initial sample of the device is subjected to an elution treatment to obtain the integrated fluoroquinolone compound separation and ionization device. Thus, the elution treatment removes the fluoroquinolone compounds intercalated on the crosslinked polymer, i.e., forms a coating with specific pores, thereby obtaining the integrated fluoroquinolone compound separation and ionization device.

[0071] According to an embodiment of the present invention, elution is performed using an eluent, specifically, the eluent is a methanol solution containing 10% formic acid.

[0072] Specifically, according to embodiments of the present invention, the method for preparing the integrated device for separating and ionizing fluoroquinolone compounds may include: immersing the substrate in a 1.5-2.5 mol / L sulfuric acid solution, sonicating it for 3-5 hours, and then rinsing the substrate surface with water until neutral to obtain the acidified substrate; immersing the acidified substrate in a mixed solution of ethanol and ammonia, adding 1-3 mL of tetraethyl orthosilicate and shaking at room temperature for 10-15 hours to perform the silanization treatment, so that at least part of the surface of the acidified substrate is silanized, rinsing with ultrapure water and ethanol, and drying with nitrogen to obtain the silanized substrate; immersing the silanized substrate in anhydrous n-hexane solution, adding 3-aminopropyltriethoxysilane, and stirring at room temperature for 1.5-2.5 hours to perform the amino modification treatment, so as to modify the silanized surface with amino groups. An amino-modified substrate is obtained; the amino-modified substrate is contacted with polyethyleneimine, NaBH4, and glutaraldehyde to undergo a prepolymerization reaction for 1.5-2.5 hours to obtain a hyperbranched polymer-modified substrate; the fluoroquinolone template molecule, the first monomer compound, and the second monomer compound in the porogen are mixed for 1.5-2.5 hours to undergo the prepolymerization reaction to obtain a prepolymer incorporating the fluoroquinolone compound; the prepolymer is contacted with the grafted substrate, shaken overnight, and subjected to a polymerization reaction to obtain a polymer-coated substrate; under alkaline conditions, the polymer-coated substrate is contacted with a crosslinking agent for 7-8 hours to undergo self-polymerization treatment to form an imprint cavity, obtaining a preliminary device; the preliminary device is eluted to obtain the integrated fluoroquinolone compound separation and ionization device.

[0073] Furthermore, according to another aspect of the present invention, an integrated device for the separation and ionization of fluoroquinolone compounds is provided. According to an embodiment of the present invention, the integrated device for the separation and ionization of fluoroquinolone compounds is obtained using the aforementioned preparation method.

[0074] The fluoroquinolone compound separation and ionization integrated device according to embodiments of the present invention has a uniform coating, exhibits specific adsorption and enrichment of the compound to be extracted and its structural analogues, has strong extraction and enrichment specificity, and is suitable for detection by analytical methods such as open solid substrate electrospray ionization mass spectrometry. It is particularly suitable for the enrichment and detection of specific chemicals in complex matrix materials and has broad application prospects.

[0075] According to another aspect of the present invention, a method for extracting fluoroquinolone compounds is provided. According to an embodiment of the present invention, the method includes: immersing at least a portion of the integrated fluoroquinolone compound separation and ionization device into a solution of the fluoroquinolone compound to be tested for extraction, so as to enrich the fluoroquinolone compound on the integrated fluoroquinolone compound separation and ionization device.

[0076] The method for extracting the target compound according to embodiments of the present invention utilizes the aforementioned device for extraction and enrichment, exhibiting good specific adsorption and enrichment effects on the target compound and its structural analogs, high speed, and strong extraction and enrichment specificity. The device containing the target compound obtained by this extraction method can be detected by analytical methods such as open-type solid substrate electrospray mass spectrometry, and is particularly suitable for the enrichment and detection of specific chemicals in complex matrix materials, with broad application prospects.

[0077] According to an embodiment of the present invention, the extraction process takes 30-70 minutes, preferably 50 minutes.

[0078] According to another aspect of the present invention, a method for extracting fluoroquinolone compounds is provided. According to an embodiment of the present invention, the method includes: immersing at least a portion of the aforementioned integrated fluoroquinolone compound separation and ionization device into a solution of the fluoroquinolone compound to be tested for extraction, so as to enrich the fluoroquinolone compound on the integrated fluoroquinolone compound separation and ionization device.

[0079] The method for extracting fluoroquinolone compounds according to embodiments of the present invention utilizes the aforementioned apparatus for extraction and enrichment, exhibiting good specific adsorption and enrichment effects on fluoroquinolone compounds and their structural analogs, with high speed and strong extraction and enrichment specificity. The apparatus containing the compounds to be extracted obtained by this extraction method can be directly used for detection by analytical methods such as open solid substrate electrospray ionization mass spectrometry, and is particularly suitable for the enrichment and detection of specific chemicals in complex matrix materials, showing broad application prospects.

[0080] According to an embodiment of the present invention, the extraction process takes 30-70 minutes, preferably 50 minutes. This results in a large adsorption capacity.

[0081] According to another aspect of the present invention, an integrated mass spectrometry ionization system for the separation and ionization of fluoroquinolone compounds is provided. According to an embodiment of the present invention, the system includes: the aforementioned integrated fluoroquinolone compound separation and ionization device; and an analytical detector. Thus, the extracted and enriched compound is directly analyzed and detected by the analytical detector, resulting in high sensitivity and speed, and is particularly suitable for rapid and sensitive detection of target analytes in complex matrices.

[0082] According to an embodiment of the present invention, the analytical instrument is an open-type mass spectrometer.

[0083] According to another aspect of the present invention, a method for qualitative / quantitative detection of fluoroquinolone compounds is provided. According to an embodiment of the present invention, the method includes: enriching fluoroquinolone compounds using the aforementioned method for extracting fluoroquinolone compounds to obtain an apparatus for enriching the fluoroquinolone compounds; and analyzing the apparatus for enriching the fluoroquinolone compounds using an analytical analyzer to perform qualitative / quantitative detection of the fluoroquinolone compounds.

[0084] According to the detection method of the present invention, the extracted and enriched device is directly analyzed and detected, resulting in low background noise, high sensitivity and fast speed of detection, which is especially suitable for rapid and sensitive detection of target analytes in complex matrices.

[0085] According to an embodiment of the present invention, the analytical instrument is an open-type mass spectrometer.

[0086] According to an embodiment of the present invention, the spray solvent for the open mass spectrometry detection is a mixed solution of methanol and formic acid, and preferably a solvent with a methanol-formic acid mixture ratio of 9:1 (v:v) provides better elution effect.

[0087] According to an embodiment of the present invention, the ionization voltage for the open-type mass spectrometry detection is 3.6 kV. When the voltage is too low, the analytes eluted by the eluent cannot be ionized, resulting in no signal being detected within the detection time. However, when the voltage is too high, the spray movement speed is too fast, and the analytes cannot be fully ionized, causing the instrument to be unable to collect all effective signals. When the ionization voltage is 3.6 kV, most compounds obtain stable and high signal intensities.

[0088] According to an embodiment of the present invention, the detection conditions for the open-type mass spectrometry detection are as follows: multiple reaction monitoring (MRM); electrospray voltage (IS): 5500V; nebulizer gas pressure (GS1): 55psi; auxiliary gas pressure (GS2): 50psi; curtain gas pressure (CUR): 20psi; ion source temperature (TEM): 550℃; residence time (DT): 100ms. The present invention will now be described with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and should not be construed as limiting the present invention.

[0089] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or standards used that do not specify the manufacturer are all commercially available conventional products, such as those purchased from Sigma-Aldrich.

[0090] The reagents and standards used in this invention are shown in Table 1.

[0091] Table 1 Reagents and Standards

[0092]

[0093]

[0094] Example 1

[0095] Using the method of this invention, an integrated device for the separation and ionization of fluoroquinolone compounds is prepared using enrofloxacin as a template molecule, as detailed below:

[0096] 1. Cut the stainless steel sheet into an isosceles triangle with a base of 1cm and a side of 2cm.

[0097] 2. The stainless steel sheet is first acidified with 1.5-2.5 mol / L sulfuric acid, and after sonication for 4 hours, the sulfuric acid solution is poured out and the sheet is washed repeatedly with deionized water until it is neutral.

[0098] 3. Place the cleaned stainless steel sheet in a water-ethanol-ammonia solution (volume ratio 4:50:5) and shake. Slowly add 2 mL of tetraethyl orthosilicate (TEOS) to modify the surface of the stainless steel sheet with silanol groups (SS@OH). After reacting at room temperature for 12 h, wash the sheet three times alternately with deionized water and anhydrous ethanol, and then dry it overnight in a 60°C oven.

[0099] 4. Place the dried stainless steel sheet into anhydrous n-hexane solution, add 1% (w / w) of 3-aminopropyltriethoxysilane (APTES), stir at room temperature for 2 hours to modify the silanol surface with amino groups (SS@OH@NH2), wash twice with anhydrous n-hexane, ethanol and deionized water respectively after the reaction, and place in an oven at 120℃ for 1 hour to cure.

[0100] 5. Add SS@OH@NH2, 10 mL of 1 mg / mL polyethyleneimine (PEI) aqueous solution, and 30 mg of NaBH4 to 10 mL of 1% (V / V) glutaraldehyde aqueous solution and shake at room temperature for 3 h to graft a dendritic hyperbranched structure (SS@OH@NH2@PEI) onto the surface of a stainless steel sheet.

[0101] 6. Add 1 mmol ENR, 1 mmol DMA and 3 mmol VPBA to 200 mL of methanol and acetonitrile mixture (1:1, v:v) for prepolymerization for 2 h, then add a hyperbranched stainless steel sheet and shake to react overnight.

[0102] 7. Add 15 mmol of dopamine hydrochloride and an equal mass of bovine serum albumin mixed aqueous solution, and adjust the pH of the mixed solution to 8.5 with 200 mL of phosphate buffer. Allow dopamine to self-polymerize in alkaline solution for 7 h to fix the imprinted cavity (MIPs) composed of template molecules and functional monomers, and fix hydrophilic materials outside the MIPs.

[0103] Comparative Example 1

[0104] The separation and ionization integrated device was prepared using the method of Example 1, except that the template molecule ENR was not added in step (f), and the NIPs device was prepared.

[0105] Example 2

[0106] In this embodiment, using Figure 1 The analytical apparatus shown was used to analyze spiked milk (5 mL of milk containing 10 ppb of fluoroquinolone antibiotic standard), as detailed below:

[0107] I. General Testing Methods

[0108] The integrated separation and ionization device was directly placed into a 5 mL milk sample containing 10 ppb of fluoroquinolone antibiotics, and adsorption was performed by shaking at 240 rpm for 50 min. After adsorption, excess interfering substances on the element surface were rinsed with deionized water, and excess moisture was blotted dry with lens paper. The bottom edge of the dried device was connected to the sample clamp, and the tip was aligned with the mass spectrometer inlet, approximately 5 mm away (see schematic diagram). Figure 1(As shown). 20 μL of elution solvent was dropped onto the surface of the element. After standing for 10 seconds, the high-voltage power supply was turned on, and a voltage of 2-3.8 kV was applied, preferably +3.6 kV. This propelled the elution solvent rapidly toward the tip of the element, forming a large number of Taylor cones at the tip. Finally, the solvent entered the mass spectrometry analysis. The mass spectrometry detection conditions included: detection mode: multiple reaction monitoring (MRM); electrospray voltage (IS): 5500 V; nebulizer gas pressure (GS1): 55 psi; auxiliary gas pressure (GS2): 50 psi; curtain gas pressure (CUR): 20 psi; ion source temperature (TEM): 550 °C; residence time (DT): 100 ms.

[0109] The MRM mass spectrometry parameters of the four FQs are shown in Table 2, with the highest peak value of the quantitative ion used as the metric.

[0110] Table 2. MRM detection parameters for the four types of FQs to be tested.

[0111]

[0112] * Represents quantitative ions

[0113] II. Condition Optimization

[0114] 1. Optimized extraction time

[0115] Extraction time is an important factor in the extraction process. In order to obtain a better extraction time, the effects of different extraction times of 5, 10, 15, 20, 25, 30, 40, 50, 60 and 70 min on the extraction yield were studied. Five parallel experiments were conducted, and the extraction yield was detected according to the detection steps in Example 2.

[0116] The results are as follows Figure 2 As shown, within 5-50 min, the signal intensity of the four FQs also increased with the increase of extraction time; then, when the extraction time exceeded 50 min, the signal intensity of the other three antibiotics, except OFL, no longer increased. This is mainly because the analytes in the sample solution and the analytes adsorbed on the integrated element reached a dynamic equilibrium. Therefore, an extraction time of 50 min can obtain a larger adsorption capacity.

[0117] 2. Adsorption performance optimization

[0118] Spray ionization conditions determine the amount of target analytes eluted from the integrated separation and ionization device and the detectable signal of the target analytes, playing a crucial role in open-cell mass spectrometry analysis. This study investigated two experimental parameters: spray voltage and spray solvent, comparing the signal intensities of four different fractional ionization (FQs) to determine optimal spray ionization conditions.

[0119] (1) Optimization of spray solvent

[0120] To more thoroughly elute the analytes adsorbed on the integrated element, the effect of adding different proportions of formic acid to methanol as a spray solvent on the FQs signal intensity was investigated. The detection method described above was used in three parallel analyses. The results are as follows: Figure 3 As shown, the solvent elution effect is better when methanol and formic acid are mixed in a 9:1 (v:v) ratio.

[0121] (2) Spray voltage optimization

[0122] Spray voltage significantly affects the analyte signal in open-type mass spectrometry. This study compares different voltages. When the voltage is too low, the analyte eluted by the eluent cannot be ionized, resulting in no detectable signal within the detection time. Conversely, when the voltage is too high, the spray velocity is too fast, preventing sufficient ionization of the analyte and preventing the instrument from collecting all effective signals. Following the aforementioned mass spectrometry detection method, the signal intensity of four different fractional quanta (FQs) was measured by applying different voltages, with each voltage measured three times. The results are as follows: Figure 4 As shown, the signal intensity of the four ionization voltages (FQs) increases with increasing applied voltage. When the absolute voltage increases to 3.6 kV, CIP, ENR, and OFL achieve the highest signal intensity, while NOR achieves the highest signal intensity at 3.7 kV. Afterward, the signal intensity of the analytes decreases with increasing absolute voltage. Therefore, 3.6 kV is chosen as the ionization voltage to ensure stable and high signal intensity for most compounds.

[0123] (3) Adsorption performance

[0124] To overcome the problem of small specific surface area of ​​planar solid substrates, this embodiment modifies the surface of a stainless steel sheet with a dendritic hyperbranched PEI structure, increasing the number of active sites on the stainless steel sheet surface. To test the enhancement of the adsorption capacity of the element by the hyperbranched structure, the test signal intensity was compared between MIPs synthesized directly on the surface of amino-modified stainless steel sheet (APTES-MIP) and MIPs synthesized after attaching the hyperbranched structure to the surface of stainless steel sheet (PEI-MIP). Simultaneously, to detect the specific adsorption capacity of the element for FQs, the intensity differences of the test signals between PEI-MIP and PEI-NIP, and between APTES-MIP and APTES-NIP were compared. The test results are as follows: Figure 5 PEI-MIP exhibits the strongest adsorption capacity for the four types of free radicals (FQs), while MIPs show superior adsorption capacity compared to NIPs. Experimental results demonstrate that the analytical detection device of this invention can indeed achieve specific adsorption of FQs and possesses a large number of adsorption sites, effectively enhancing detection capabilities.

[0125] 3. BSA access restriction capability

[0126] To test the exclusion ability of bovine serum albumin (BSA) for macromolecules such as proteins in food matrices, two cross-linking devices were prepared: one using dopamine and BSA as a co-crosslinking agent (DA+BSA-MIP), and the other using only dopamine as a crosslinking agent (DA-MIP). The two devices were then used to adsorb free radicals (FQs) from aqueous solutions and spiked milk samples, respectively. The results are as follows: Figure 6 As shown, in aqueous solution, the presence or absence of BSA in the crosslinking agent has little effect on the adsorption results, but BSA does exhibit a certain promoting effect on the adsorption of FQs. In spiked milk, the detection signal intensity of the DA+BSA-MIP modified stainless steel substrate is more than 3 times higher than that of the DA-MIP modified stainless steel substrate, indicating that BSA can significantly improve the adsorption performance of the element for FQs in a protein environment.

[0127] 4. Methodological validation of mass spectrometry detection

[0128] Following the method in Example 2, four different concentrations of free quantitation (FQs) in a blank milk matrix were detected: ciprofloxacin (CIP), enrofloxacin (ENR), norfloxacin (NOR), and ofloxacin (OFL). The linear range, LOD, and LOQ of the standard curves were obtained. The results are shown in Table 3. The linear ranges of CIP, ENR, and OFL were all in the range of 2-200 μg / L, and the linear range of NOR was 5-200 μg / L. The correlation coefficients (R) of the four analytes were also measured. 2 All values ​​were greater than 0.9940, LOD was 0.5-1.0 μg / L, and LOQ was 2.0-5.0 μg / L. The experimental results show that the detection method of the present invention can be used for the direct detection of milk samples and is accurate in quantification.

[0129] Table 3 Performance parameters of this method for directly detecting four FQs in milk

[0130]

[0131] Example 3

[0132] Milk samples were purchased from a local supermarket. 5 mL of milk sample was placed in a centrifuge tube, and the prepared apparatus was placed inside the tube for agitation and adsorption. Following the above-described procedure and optimized parameters, detection was performed, and no fluoroquinolone antibiotic residues were detected. To test the applicability of the developed method, different concentrations of fluoroquinolone antibiotics were added to the milk samples, and spiked recovery experiments were conducted. The results are shown in Table 4, indicating that the developed method has good applicability and can directly detect fluoroquinolone antibiotic residues in high-protein biological matrices such as milk.

[0133] Table 4. Recovery rates and RSDs of the four free radicals (FQs) in spiked milk detected by this method.

[0134]

[0135] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0136] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A method for preparing a device for separation and ionization of fluoroquinolone compounds, characterized by, The method comprises: silicon hydroxylation treatment is performed on the substrate to modify the surface of the substrate with silicon hydroxyl groups to obtain a silicon hydroxylated substrate; amino modification treatment is performed on at least part of the surface of the silicon hydroxylated substrate to obtain an amino modified substrate; grafting treatment is performed on the amino modified substrate to obtain a grafted substrate; a fluoroquinolone template molecule is contacted with a first monomer compound, a second monomer compound and a porogen to perform a prepolymerization reaction to obtain a prepolymer in which the fluoroquinolone compound is embedded; the prepolymer is contacted with the grafted substrate to perform a polymerization reaction to obtain a polymer-coated substrate; the polymer-coated substrate is contacted with a crosslinking agent to perform a self-polymerization treatment to form an imprint cavity and obtain a device prototype; and the device prototype is subjected to elution treatment to obtain a device for separating and ionizing the fluoroquinolone compound, the first monomer compound is dimethylaminoethyl methacrylate, and the second monomer compound is tetra-vinyl benzene boronic acid, the crosslinking agent is dopamine hydrochloride and bovine serum albumin, the fluoroquinolone template molecule is enrofloxacin, the self-polymerization treatment further comprises: a porogen, wherein the porogen is a phosphate buffer solution containing acetonitrile and methanol, and the volume ratio of acetonitrile, methanol and phosphate buffer solution in the porogen is 1:0.8-1.2:

1.

2. The method of claim 1, wherein, The molar ratio of the fluoroquinolone template molecule to the first monomer compound, the second monomer compound and the crosslinking agent is 1:0.8-1.2:3-5:10-20.

3. The method of claim 1, wherein, The substrate has at least one angular end.

4. The method of claim 3, wherein, The substrate is an isosceles triangle.

5. The method of claim 1, wherein, The substrate is a stainless steel plate, a wooden plate or a bamboo plate.

6. The method of claim 1, wherein, The method further comprises: Before the silicon hydroxylation treatment, the substrate is subjected to acidification treatment to obtain an acidified substrate.

7. The method according to any one of claims 1 to 6, characterized in that, The method comprises: immersing the substrate in a 1.5-2.5 mol / L sulfuric acid solution, performing ultrasonic treatment for 3-5 hours, then rinsing the surface of the substrate with water until neutralization to obtain the acidified substrate; immersing the acidified substrate in a mixed solution of ethanol and ammonia water, adding 1-3 mL of tetraethyl orthosilicate dropwise, and oscillating at room temperature for 10-15 hours to perform the silicon hydroxylation treatment to silicon hydroxylate at least part of the surface of the acidified substrate, then rinsing with ultrapure water and ethanol, and blowing dry with nitrogen to obtain the silicon hydroxylated substrate; immersing the silicon hydroxylated substrate in anhydrous n-hexane solution, adding 3-aminopropyltriethoxysilane, and stirring at room temperature for 1.5-2.5 hours to perform the amino modification treatment to modify the surface of the silicon hydroxyl group with amino groups to obtain the amino modified substrate; contacting the amino modified substrate with polyethyleneimine, NaBH4 and glutaraldehyde to perform the prepolymerization reaction for 1.5-2.5 hours to obtain a hyperbranched polymer modified substrate; immersing the fluoroquinolone template molecule in the porogen, mixing the first monomer compound and the second monomer compound for 1.5-2.5 hours to perform the prepolymerization reaction to obtain a prepolymer in which the fluoroquinolone compound is embedded; contacting the prepolymer with the grafted substrate, shaking overnight, and polymerizing to obtain a polymer-coated substrate; contacting the polymer-coated substrate with a crosslinking agent under alkaline conditions for 7-8 hours to perform a self-polymerization process to form a printing cavity and obtain a device precursor; and performing a substrate elution process on the device precursor to obtain the fluoroquinolone compound separation and ionization integrated device.

8. The method of claim 1, wherein, The eluent of the elution process is a 10% formic acid methanol solution.

9. An integrated device for the separation and ionization of fluoroquinolone compounds, characterized in that, The device is obtained by the method of any one of claims 1-8 for preparing a fluoroquinolone compound separation and ionization integrated device.

10. A method of extracting a fluoroquinolone compound, characterized by, It comprises: immersing at least part of the fluoroquinolone compound separation and ionization integrated device of claim 9 into a solution of the fluoroquinolone compound to be tested to perform an extraction process to enrich the fluoroquinolone compound on the fluoroquinolone compound separation and ionization integrated device.

11. The method of claim 10, wherein, The extraction process is performed for 30-70 minutes.

12. The method of claim 11, wherein, The extraction process is performed for 50 minutes.

13. A fluoroquinolone antibiotic separation-ionization integrated mass spectrometry ionization system, characterized in that, It comprises: the fluoroquinolone compound separation and ionization integrated device of claim 9; and an analytical detector. The analytical detector is an open mass spectrometry detector.

14. The system of claim 13, wherein, It comprises:

15. A method for qualitatively / quantitatively detecting a fluoroquinolone compound, characterized by, enriching the fluoroquinolone compound by the method of claim 10 for extracting the fluoroquinolone compound to obtain a separation and ionization integrated device enriched with the fluoroquinolone compound; and performing an analysis process on the device enriched with the fluoroquinolone compound by the analytical detector to qualitatively / quantitatively detect the fluoroquinolone compound. The analytical detector is an open mass spectrometry detector.

16. The method of claim 15, wherein, The spray solvent of the open mass spectrometry detector is a mixed solution of methanol and formic acid.

17. The method of claim 16, wherein, The ionization voltage of the open mass spectrometry detector is 3.6 kV.

18. The method of claim 16, wherein, The detection conditions of the open mass spectrometry detector are: multiple reaction monitoring (MRM); electrospray voltage (IS): 5500 V; atomization gas pressure (GS1): 55 psi; auxiliary gas pressure (GS2): 50 psi; curtain gas pressure (CUR): 20 psi; 19. The method of claim 16, wherein, ion source temperature (TEM): 550℃; residence time (DT): 100 ms. ​

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