Polymer microspheres and application thereof in extraction and determination of nitrofuran metabolites in aquatic products

Abstract

The invention discloses polymer microspheres and application thereof in extraction and determination of nitrofuran metabolites in aquatic products, and belongs to the field of analysis. According to the invention, the mesoporous GMA-EGDMA polymer microspheres rich in cis-diol are synthesized and are used for boric acid affinity extraction; meanwhile, 4-fluoro-3-formylphenylboronic acid is used for derivatization of NFMs so as to carry out pretreatment on an aquatic product sample. The GMA-EGDMA polymer microsphere disclosed by the invention shows excellent reusability, and still keeps good performance after six times of adsorption-desorption cycles; the boric acid affinity extraction method does not need to use an extraction solvent, so that the pretreatment process is simplified, and a mild extraction environment is kept. The determination method provided by the invention provides an economic, simple, rapid and sensitive alternative scheme for monitoring NFMs residues in aquatic products.

Description

Technical Field

[0001] The present invention relates to the field of analysis, and in particular to a polymer microsphere and an application thereof in extracting and determining nitrofuran metabolites in aquatic products. Background Art

[0002] Aquatic products are among the most widely consumed foods worldwide, but they can be contaminated with substances used in aquaculture for disease control, such as nitrofuran drugs. These drugs are used to enhance the antimicrobial properties of aquatic products. Nitrofuran compounds are a class of synthetic broad-spectrum antimicrobial drugs that have been widely used in the past, primarily including furaltadone, nitrofurotoin, furazolidone, and nitrofuroxol. Nitrofuran drugs are rapidly metabolized in aquaculture, with their major metabolites including 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 1-aminohydantoin (AHD), 3-amino-2-oxazolidinone (AOZ), and semicarbazide (SEM). These metabolites may have significant carcinogenic, teratogenic, and mutagenic effects. Consequently, the use of nitrofuran compounds in aquaculture has been strictly prohibited in regions such as Europe, the United States, Japan, and China. Monitoring their presence in aquatic products is crucial to ensuring food safety. Due to their rapid metabolism, nitrofuran metabolites (NFMs) are typically analyzed as tissue-bound residues. These metabolites are typically isolated and extracted by acid hydrolysis, followed by derivatization using reagents such as 2-nitrobenzaldehyde (2-NBA) [National Food Safety Standard GB31656.13-2021] and 2,4-dinitrophenylhydrazine, enabling their separation and detection by liquid chromatography-tandem mass spectrometry (HPLC-MS / MS).

[0003] To reduce matrix interference and ensure reliable analysis, an appropriate sample preparation procedure is crucial. Among advanced sample pretreatment methods such as liquid-liquid extraction (LLE), solid phase extraction (SPE), and dispersive solid phase extraction (DSPE), LLE is the most commonly used technique for NFMs analysis. However, LLE requires the transfer of residues from the acid hydrolysis solution to an organic solvent phase (such as ethyl acetate used in the national food safety standard GB 31656.13-2021). This process usually requires a large amount of organic solvent, which increases costs and environmental issues. In comparison, SPE is more environmentally friendly, but usually requires dedicated equipment. DSPE is simpler and does not require such equipment, but still requires centrifugation for phase separation, which can be cumbersome for high-throughput analysis.

[0004] Adsorbent selection is another key factor in achieving excellent DSPE results. Recent advances have introduced novel adsorbents such as metal-organic frameworks (MOFs), multi-walled carbon nanotubes (MWCNTs), molecularly imprinted polymers, and porous polymers. Developing suitable adsorbents for sample preparation using DSPE is crucial for the determination of nitrofuran metabolites in aquatic products. Summary of the Invention

[0005] The present invention provides polymer microspheres and their use in the extraction and determination of nitrofuran metabolites in aquatic products. Cis-diol-rich mesoporous GMA-EGDMA polymer microspheres were synthesized for boric acid affinity extraction. Furthermore, NFMs were derivatized with 4-fluoro-3-formylphenylboronic acid (FFBA, CAS No. 374538-01-9) to facilitate the immobilization of the boronic acid ligand on their molecular structure. Boric acid affinity extraction eliminates the need for extraction solvents and centrifugation, simplifying pretreatment and maintaining a mild extraction environment.

[0006] The present invention first provides a method for preparing cis-diol-modified polymer microspheres, comprising the following steps:

[0007] (1) mixing glycidyl methacrylate, ethylene glycol dimethacrylate, ethyl 2-bromopropionate, CuBr2, n-octanol, dichloromethane, and N,N,N',N',N"-pentamethyldiethylenetriamine to obtain a mixture A;

[0008] (2) mixing an aqueous solution containing polyvinyl alcohol, sodium lauryl sulfate and ascorbic acid with the mixture A, and reacting the mixture to obtain polymer microspheres;

[0009] (3) mixing the polymer microspheres and a sulfuric acid solution and hydrolyzing them to obtain the cis-diol-modified polymer microspheres.

[0010] In the above-mentioned preparation method, the mass percentage concentration of the glycidyl methacrylate in the mixture A is 8% to 15%, specifically 11.9%;

[0011] The mass percentage concentration of the ethylene glycol dimethacrylate is 8% to 15%, specifically 11.4%;

[0012] The mass percentage concentration of the ethyl 2-bromopropionate is 0.05% to 0.2%, specifically 0.1%;

[0013] The mass percentage concentration of the CuBr2 is 8% to 15%, specifically 11.9%;

[0014] The mass percentage concentration of the N,N,N',N',N"-pentamethyldiethylenetriamine is 0.3% to 0.8%, specifically 0.6%;

[0015] The volume ratio of n-octanol to dichloromethane is 1:1 to 1:4, specifically 1:1;

[0016] In the aqueous solution containing polyvinyl alcohol, sodium lauryl sulfate and ascorbic acid, based on the total volume of the aqueous solution, the concentration of the polyvinyl alcohol is 20-40 mg / mL, specifically 30 mg / mL; the concentration of the sodium lauryl sulfate is 0.5-3 mg / mL, specifically 1 mg / mL; the concentration of the ascorbic acid is 8-12 mg / mL, specifically 9.4 mg / mL;

[0017] The volume ratio of the aqueous solution containing polyvinyl alcohol, sodium lauryl sulfate and ascorbic acid to the mixture A is 16:1 to 16:5, specifically 16:3.

[0018] In the above preparation method, in step (2), the reaction temperature is 50-70°C, specifically 60°C; the reaction time is 6-10h, specifically 8h;

[0019] In step (3), the concentration of the sulfuric acid solution is 0.2 to 0.8 mol / L, specifically 0.5 mol / L;

[0020] The hydrolysis temperature is 50-70°C, specifically 60°C;

[0021] The hydrolysis time is 3 to 6 hours, specifically 5 hours.

[0022] The present invention further provides cis-diol-modified polymer microspheres prepared by the above preparation method.

[0023] Finally, the present invention provides a method for determining nitrofuran metabolites in aquatic products, comprising sample pretreatment, wherein the sample pretreatment comprises the following steps:

[0024] (1) mixing a sample of aquatic product to be tested, a hydrochloric acid solution, and a 4-fluoro-3-formylphenylboronic acid solution for hydrolysis and derivatization, adjusting the pH value of the mixture, and centrifuging to obtain a supernatant;

[0025] (2) The cis-diol-modified polymer microspheres are used as fillers to prepare a push rod type shaking type purification column, and the supernatant is loaded into the push rod type shaking type purification column for extraction and elution to obtain an eluate, which is the test solution.

[0026] In the above method, the volume ratio of the mass of the aquatic product sample to be tested to the hydrochloric acid solution is 1g:2-4mL, specifically 1g:2.5mL;

[0027] The volume ratio of the aquatic product sample to be tested to the 4-fluoro-3-formylphenylboronic acid solution is 1 g:80-120 μL, specifically 1 g:100 μL;

[0028] The concentration of the hydrochloric acid solution is 0.1-0.3 M, specifically 0.2 M;

[0029] The concentration of the 4-fluoro-3-formylphenylboronic acid solution is 30-60 mM, specifically 50 mM;

[0030] The solvent of the 4-fluoro-3-formylphenylboronic acid solution is methanol;

[0031] In step (1), the hydrolysis temperature is 30-40°C, specifically 37°C, and the time is 10-20h, specifically 16h;

[0032] Use K2HPO4 solution to adjust the pH value of the mixed solution to 7-9, specifically 8;

[0033] The centrifugal speed is 5000-10000 rpm, specifically 8000 rpm;

[0034] The centrifugation time is 5 to 15 minutes, specifically 10 minutes.

[0035] In the above method, the mass ratio of the cis-diol-modified polymer microspheres to the aquatic product sample in step (2) is 1:10 to 1:30, specifically 1:20;

[0036] In step (2), the extraction time is 8 to 12 minutes, specifically 10 minutes;

[0037] The elution solvent used for the elution is acetonitrile with a pH value of 2 to 5, specifically, a pH value of 2;

[0038] The elution time is 1 to 6 minutes, specifically 5 minutes;

[0039] The push rod type oscillating purification column is recorded in ZL202020843439.1.

[0040] The above method further comprises a step of washing with a methanol solution having a volume percentage concentration of 2% to 6% (specifically 5%) before elution;

[0041] After the elution, the eluate is evaporated and then redissolved in a methanol solution with a volume percentage concentration of 2% to 20% (specifically 5%) to obtain the test solution.

[0042] In the above method, the method for determining nitrofuran metabolites in aquatic products adopts the internal standard method for determination;

[0043] The method further comprises a sample detection step, which is to detect the liquid to be tested by using an ultra-high performance liquid chromatography-triple quadrupole / composite linear ion trap mass spectrometer.

[0044] In the above method, the detection conditions are as follows:

[0045] High performance liquid chromatography conditions: Accuity Chromatographic separation was performed using a BEH C18 column; mobile phases: phase A was 2 mM ammonium acetate solution, phase B was methanol; gradient elution; column temperature was 40°C; flow rate was 0.3 mL / min; injection volume was 5.0 μL;

[0046] The gradient elution conditions are as follows: 0-1 min, 25% phase B; 1-1.5 min, 25%-70% phase B; 1.5-4.5 min, 70% phase B; 4.5-5 min, 70%-25% phase B; 5-6 min, 25% phase B;

[0047] The mass spectrometry conditions were as follows: ionization was performed in positive ion mode with a spray voltage of 5.5 kV; the ion source temperature was 500°C; the curtain gas, ion source gas 1, and ion source gas 2 were set to 35, 55, and 35 psi, respectively; and MS data were acquired in multiple reaction monitoring mode.

[0048] The present invention has the following advantages:

[0049] (1) Compared with China's national food safety standard GB 31656.13-2021, the boric acid affinity extraction method does not require the use of extraction solvents and centrifugation, simplifies the pretreatment process, and maintains a mild extraction environment.

[0050] (2) GMA-EGDMA polymer microspheres exhibited excellent reusability and maintained good performance after six adsorption-desorption cycles.

[0051] (3) The method of the present invention NFMs is 0.5-50 μg kg -1 Good linearity (R 2 >0.999), and the limit of quantification was 0.5 μg kg -1 The recoveries of spiked tilapia samples ranged from 94.2% to 110.7%, with intra-day and inter-day relative standard deviations (RSDs) lower than 5.9%.

[0052] (4) The method of the present invention allows direct processing of hydrochloric acid hydrolysis solutions without the need for centrifugation and ethyl acetate extraction; the performance of the method was successfully verified by analyzing NFMs in fish samples, demonstrating its potential as a rapid, efficient, and environmentally friendly method for monitoring nitrofuran residues in aquatic products.

[0053] (5) The determination method of the present invention provides an economical, simple, rapid and sensitive alternative for monitoring NFMs residues in aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1This is the preparation process of cis-diol-modified GMA-EGDMA polymer microspheres.

[0055] Figure 2 Scanning electron microscopy and FTIR characterization of cis-diol modified GMA-EGDMA polymer microspheres.

[0056] Figure 3 is the pore size distribution of cis-diol-modified GMA-EGDMA polymer microspheres determined by the BET method.

[0057] Figure 4 This is a confocal laser scanning microscopy image of cis-diol-modified GMA-EGDMA polymer microspheres incubated with FITC-BSA solution (0.5 mg / mL) for 20 minutes; Figure 4 (a) is the surface and (b) is the interior.

[0058] Figure 5 Schematic diagram of the sample purification process.

[0059] Figure 6 For AOZ-FFBA, AHD-FFBA, AMOZ-FFBA and SEM-FFBA (1.0μg L -1 ) is a typical chromatogram.

[0060] Figure 7 The effects of different parameters on the DSPE extraction recovery rate are shown in Figure 2. Figure 7 (a) is the type of eluent, including I: acetonitrile; II: 10% water-acetonitrile; III: 20% water-acetonitrile; IV: methanol; (b) is the volume of the eluent; (c) is the pH value of the eluent; and (d) is the amount of adsorbent used.

[0061] Figure 8 Six adsorption-desorption cycles of cis-diol-modified GMA-EGDMA polymer microspheres.

[0062] Figure 9 It is the matrix effect of nitrofuran metabolites. DETAILED DESCRIPTION

[0063] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention.

[0064] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0065] The quantitative tests in the following examples were performed in triplicate unless otherwise specified, and the results were averaged.

[0066] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0067] Example 1. Preparation of cis-diol-modified GMA-EGDMA polymer microspheres

[0068] Glycidyl methacrylate (GMA, CAS No. 106-91-2) and ethylene glycol dimethacrylate (EGDMA, CAS No. 97-90-5) used below were purchased from Sigma-Aldrich; ethyl 2-bromopropionate (EBP, CAS No. 535-11-5) was purchased from Alfa Aesar; N,N,N',N',N"-pentamethyldiethylenetriamine (PMDETA, CAS No. 3030-47-5) was provided by Beijing Bailingwei Chemical Co., Ltd.; sodium lauryl sulfate (SLS, CAS No. 151-21-3) and polyvinyl alcohol (PVA, CAS No. 9002-89-5) were purchased from Beijing Chemical Plant; FITC-bovine serum albumin (FITC-BSA) was purchased from Beijing Philip Morris Biotechnology Co., Ltd.

[0069] The preparation process of cis-diol modified GMA-EGDMA polymer microspheres is as follows Figure 1 shown.

[0070] A mixture of 38.5 mmol GMA, 26.5 mmol EGDMA, 0.017 mL EBP, 0.29 g CuBr2, 10 mL n-octanol and 20 mL dichloromethane was added to an ampoule. The ampoule was sealed with a rubber septum and degassed with nitrogen (N2). Subsequently, 1.3 mmol PMDETA was quickly added, and the mixture was shaken for 30 s to obtain mixture A. In another container, 4.8 g PVA, 0.16 g SLS and 1.5 g ascorbic acid were dissolved in 160 mL water and then added to mixture A. After incubation at 60 ° C for 8 h, the microspheres were collected by filtration and subsequently hydrolyzed at 60 ° C for 5 h in 0.5 M sulfuric acid (mass volume concentration of 10% (w / v)). The hydrolyzed microspheres were washed with water to a pH of about 7.0 and then dried to obtain cis-diol-modified GMA-EGDMA polymer microspheres.

[0071] Scanning electron microscope (SEM) (JSM-IT700HR, Japan JEOL company) is used to check the morphology of the GMA-EGDMA polymer microspheres modified by cis-diol. Micromeritics ASAP 2460 analyzer (U.S. Micromeritics company) is used to measure specific surface area and pore size distribution by nitrogen adsorption-desorption isotherm. Confocal laser scanning microscope (CLSM) imaging is used to characterize the protein restriction entry characteristics of the GMA-EGDMA polymer microspheres modified by cis-diol. GMA-EGDMA polymer microspheres modified by cis-diol are hatched 20min with 5mg / mL FITC-BSA. Unabsorbed FITC-BSA is removed by centrifugation at 8000rpm. Inverted fluorescence microscope (ECHO, RVL-100-M, U.S.) is used to characterize microspheres. In order to check the internal structure, microspheres are crushed, and internal fluorescence is observed by CLSM under the same conditions.

[0072] The morphology of the microspheres was characterized by SEM. Figure 2 As shown in (a) and (b), the cis-diol-modified GMA-EGDMA polymer microspheres not only have regular spherical shapes but also have relatively smooth surfaces. The nitrogen adsorption-desorption isotherm shows a type IV curve with an H2 hysteresis loop ( Figure 2 c), indicating the presence of mesoporous structures in the microspheres. The average pore size determined using the Brunauer-Emmett-Teller (BET) method was 19.49 nm ( Figure 3 The total pore volume and mesopore volume are 0.581547 cm 3 / g and 0.58054cm 3 / g, and a BET surface area of 119.36 cm 2 / g. The cis-diol groups on the surface of GMA-EGDMA polymer microspheres are the key factor for the selective adsorption of FFBA-derivatized NFMs. The presence of cis-diol groups on the surface of GMA-EGDMA polymer microspheres was confirmed by FI-TR spectroscopy. Figure 2 As shown in (d), the -OH stretching vibration is at 3500 cm -1 The peak at 906 cm-1 confirmed the presence of cis-diol groups on the surface of GMA-EGDMA polymer microspheres. -1 The peak at 100 nm disappeared. Based on these results, it is shown that this embodiment successfully prepared GMA-EGDMA polymer microspheres with a surface rich in cis-diol groups. In addition, the cis-diol-modified GMA-EGDMA polymer microspheres have an average pore size of 19.49 nm, and the outer layer is coated with a hydrophilic polymer brush composed of cis-diol groups, which also exhibits size exclusion ability, effectively preventing the entry of macromolecules such as proteins. Figure 4 As shown in the figure, confocal laser scanning microscopy (CLSM) clearly shows the capture of FITC-BSA on the surface of cis-diol-modified GMA-EGDMA polymer microspheres. After 20 minutes of incubation, the outer shell of the cis-diol-modified GMA-EGDMA polymer microspheres was completely covered with aggregated FITC-BSA, while no BSA adsorption was observed inside the microspheres.

[0073] The following four NFMs (AOZ, AMOZ, SEM, AHD) were used as mixed external standard solutions (all at a concentration of 100 mg / L) and mixed internal standard solutions (AOZ-D4, AMOZ-D5, SEM- 13 C 15 N2, AHD- 13 C3) (all concentrations were 100 mg / L) were purchased from Tianjin Alta Technology Co., Ltd.

[0074] Example 2

[0075] The sample purification process is shown in the following figure: Figure 5 As shown, the details are as follows:

[0076] 1. Sample Collection and Derivatization

[0077] Fresh tilapia samples were purchased from supermarkets in Beijing, China. Homogenized fish muscle tissue (2.0 g) was transferred to a 15 mL polypropylene centrifuge tube. Hydrolysis and derivatization were then performed by adding 5 mL of 0.2 M hydrochloric acid and 200 μL of 50 mM FFBA (4-fluoro-3-formylphenylboronic acid) methanol solution. The mixture was shaken for 2 minutes and incubated at 37°C for approximately 16 hours. After cooling to room temperature, the pH was adjusted to approximately 8 using 8.0 mL of 1 M K2HPO4 solution. The mixture was centrifuged at 8000 rpm for 10 minutes, and the supernatant was transferred to a push-rod shaker for vortex-based DSPE.

[0078] 2. Vortex-based DSPE program

[0079] The following purification is performed using the push rod oscillating purification column in patent ZL202020843439.1, wherein the filler is 100 mg of cis-diol-modified GMA-EGDMA polymer microspheres prepared in Example 1.

[0080] (i) Sample loading: Pull the piston of a push-rod shaking purification column (20 mL) upward to the sample loading port. Use a pipette to add the supernatant to be purified into the column tube through the sample loading port and place it on a tube rack.

[0081] (ii) Extraction: The push rod shaking type cleanup column was vortexed at 700 rpm for 10 min.

[0082] (iii) Washing: Push the piston to expel and discard the supernatant. Pull the piston of the push-rod shaking cleanup column upward to the sample loading port. Use a pipette to add 2 mL of 5% methanol (v / v) aqueous solution to the column tube through the loading port. Vortex at 1200 rpm for 5 min. Expel the wash solution by pushing the column piston.

[0083] (iv) Elution: Pull the plunger of the push-rod shaking cleanup column upward to the sample loading port. Use a pipette to add 3 mL of acetonitrile (pH 2.0, adjusted with 0.2 M HCl) through the sample loading port to the column tube for elution. After vortexing at 1500 rpm for 5 min (elution time), the eluate was collected by pushing the plunger and transferred to a centrifuge tube. The eluate was concentrated to near dryness with liquid nitrogen and redissolved in 1 mL of 5% methanol (v / v) in water. The final solution was filtered through a 0.22 μm membrane and analyzed by HPLC-QTRAP-MS / MS.

[0084] 3. Testing conditions

[0085] Analytes were detected using an AB SCIEX HPLC system coupled to a QTRAP 6500 triple quadrupole / linear ion trap mass spectrometer.

[0086] The HPLC conditions were as follows: Accuity Chromatographic separation was performed on a BEH C18 column (50 mm × 2.1 mm, 1.7 μm). The mobile phase consisted of eluent A (2 mM ammonium acetate solution) and eluent B (methanol). The gradient elution method is shown in Table 1. The LC column was set at 40°C with a flow rate of 0.3 mL / min. The LC injection volume was 5.0 μL.

[0087] Table 1 Liquid chromatography gradient elution conditions for target compounds

[0088] Time / min Flow / (ml / min) A / ％ B / ％ 0 0.3 75.0 25.0 1.00 0.3 75.0 25.0 1.50 0.3 30.0 70.0 4.50 0.3 30.0 70.0 5.00 0.3 75.0 25.0 6.00 0.3 75.0 25.0

[0089] Mass spectrometry conditions were as follows: ionization was performed in positive ion mode with a spray voltage of 5.5 kV. The ion source temperature was set to 500°C. Curtain gas, ion source gas 1, and ion source gas 2 were set to 35, 55, and 35 psi, respectively. MS data were acquired in multiple reaction monitoring (MRM) mode. The optimized MS parameters for the NFMs derivatives are shown in Table 2.

[0090] Table 2 Chemical information and mass spectrometry parameters of FFBA-derived NFMs

[0091]

[0092] NFMs were isolated and identified using HPLC-QTRAP-MS / MS, a technique widely recognized as an effective method for detecting drug residues in fish samples. After derivatization with FFBA reagents, NFMs exhibited amine groups as shown in Table 2. Based on their chemical properties, MS identification was performed using positive ion mode. The molecular weights of the four NFM derivatives were verified using a QTRAP 6500MS detector. Typical product ion spectra and molecular ion peaks (m / z, [M]) of these derivatives are shown in Table 2. + H] + )like Figure 1 Furthermore, comparison of the measured molecular weights with the exact molecular weights listed in Table 2 confirms the accuracy of our measurements, thereby validating FFBA as an effective derivatization agent for NFMs. MS parameters were further evaluated, and a precursor ion and two product ions were selected for each NFM derivative, as shown in Table 2.

[0093] In HPLC-QTRAP-MS / MS analysis, the signal intensity is affected not only by the MS parameters but also by the HPLC separation conditions. Methanol is preferred as the eluent over acetonitrile and the Accuity HPLC separation was performed on a BEH C18 column (50 mm × 2.1 mm, 1.7 μm). The mobile phase consisted of methanol (phase B) and 2 mM ammonium acetate (phase A). Optimal chromatographic separation was achieved under specific gradient elution conditions.

[0094] Pipette 50 μL of the standard solution (20 μg / L) into a centrifuge tube. Except for not adding blank fish meat, the above methods 1-3 were used to extract and detect the FFBA derivatives of the four nitrofuran metabolites in the sample. The chromatograms are shown in Figure 2. Figure 6 As shown, complete separation was demonstrated within 3 minutes;

[0095] Example 3

[0096] 1. Optimization of Dispersive Solid Phase Extraction (DSPE) Procedure

[0097] To optimize the dispersive solid-phase extraction procedure and evaluate the extraction efficiency of cis-diol-modified GMA-EGDMA polymer microspheres, several key parameters affecting the extraction efficiency of cis-diol-modified GMA-EGDMA polymer microspheres were systematically investigated. These parameters included the type of eluent used for the sample solution, the volume of the eluent, the pH of the eluent, and the amount of adsorbent (cis-diol-modified GMA-EGDMA polymer microspheres).

[0098] The specific experimental methods are as follows:

[0099] Homogenized fish muscle tissue (2.0 g) was transferred to a 15 mL polypropylene centrifuge tube. 50 μL of a standard solution (100 μg / L, prepared by diluting a mixed external standard solution with methanol) was pipetted into the centrifuge tube to prepare a spiked concentration of 2.5 μg kg -1 The muscle samples of tilapia were subjected to the same extraction and purification treatment and detection conditions as in Example 2. The effects of different parameters on the DSPE extraction recovery are shown in FIG. Figure 7 Based on these results, the optimal conditions were as follows: acetonitrile as the elution solvent, an elution solvent volume of 3 mL, an eluent pH of 2.0, an adsorbent dosage of 100.0 mg, and an elution time of 5 min. Furthermore, prior to elution, the adsorbent was washed with a 5% (v / v) methanol solution in water to remove physically adsorbed substances, thereby improving the selectivity of the extraction process.

[0100] 2. Reusability

[0101] In addition to exhibiting excellent DSPE performance, the cis-diol-modified GMA-EGDMA polymer microspheres also showed remarkable reusability. To evaluate their regeneration ability, six consecutive adsorption-desorption cycles were performed. -1 Tilapia muscle samples with the analytes of interest were used to evaluate the reusability of the microspheres.

[0102] The specific experimental methods are as follows:

[0103] Homogenized fish muscle tissue (2.0 g) was transferred to a 15 mL polypropylene centrifuge tube. 50 μL of a standard solution (100 μg / L, prepared by diluting a mixed external standard solution with methanol) was pipetted into the centrifuge tube to prepare a spiked concentration of 2.5 μg kg -1 The tilapia muscle samples were subjected to the same extraction, purification and detection conditions as in Example 2, and the reusability of the microspheres was evaluated by the changes in the peak areas of the four nitrofuran metabolites.

[0104] The results are as follows Figure 8As shown, the adsorption efficiencies of AHD-FFBA and AOZ-FFBA on GMA-EGDMA polymer microspheres remained high, with only a slight downward trend, reaching 95.9% and 94.5% of their initial adsorption capacities, respectively. Notably, the adsorption efficiencies of SEM-FFBA and AMOZ-FFBA remained virtually unchanged, at 103.8% and 98.4% of their initial values, respectively. The excellent reusability of the cis-diol-modified GMA-EGDMA polymer microspheres is attributed to their high structural stability, derived from a three-dimensional cross-linked mesoporous structure and the presence of abundant cis-diol groups. These groups facilitate the formation of new boronate bonds with FFBA-derived NFMs. These properties contribute to the excellent regeneration performance of the microspheres. Based on these results, cis-diol-modified GMA-EGDMA polymer microspheres demonstrate significant potential for NFM sample pretreatment, providing a robust and sustainable solution for analytical workflows.

[0105] Example 4

[0106] The analytical parameters of the developed method were validated, including matrix effect (ME), linearity, limit of quantification (LOQ), recovery, and precision.

[0107] 1. Matrix Effect

[0108] To evaluate matrix effects, tilapia muscle samples were spiked with 10 μg kg -1 The specific method is as follows:

[0109] 50 μL of standard solution (400 μg / L, obtained by diluting a mixed external standard solution with methanol) was transferred to a centrifuge tube. The same extraction, purification, and detection conditions as in Example 2 were used, except that no blank fish was added. The peak areas of the four nitrofuran metabolites in the solvent were obtained.

[0110] Homogenized fish muscle tissue (2.0 g) was transferred to a 15 mL polypropylene centrifuge tube. 50 μL of a standard solution (400 μg / L, prepared by diluting a mixed external standard solution with methanol) was pipetted into the centrifuge tube to prepare a spiked concentration of 10 μg kg -1 Tilapia muscle samples were subjected to the same extraction, purification, and detection conditions as in Example 2 to obtain the peak areas of four nitrofuran metabolites in the fish sample matrix. To evaluate the ability of the adsorbent to remove matrix interference, the matrix effect (ME) was calculated using the following formula:

[0111] ME(%)=(Area m / Area s -1)×100

[0112] Area mand Area s The mean peak areas of each NFM in fish sample matrix and solvent are shown respectively. The ME of each NFM was calculated by spiking fish sample matrix with 10 μg / kg of analyte (n=6).

[0113] like Figure 9 As shown in Figure 3, significant ion suppression was observed, with matrix effect values ranging from -30% to -50%, which was attributed to the presence of matrix interference. To improve quantitative accuracy, isotope internal standard calibration was used as the preferred calibration strategy for quantitative analysis.

[0114] 3. Method Validation

[0115] (1) Preparation of isotope calibration standard curve

[0116] In a 50 mL centrifuge tube, accurately add 100.0 μL of mixed standard working solution (including SEM, AOZ, AHD, AMOZ, all at a concentration of 10.0 μg / L, obtained by diluting the mixed external standard solution with methanol; taking the concentration point of the standard curve with a concentration of 1.0 μg / L as an example), accurately add 50 μL of mixed internal standard working solution (including SEM- 13 C- 15 N2, AOZ-D4, AHD- 13 C3 and AMOZ-D5, both at a concentration of 200.0 μg / L, were obtained by diluting a mixed internal standard solution with methanol. The same extraction, purification, and detection conditions as in Example 2 were used, except that no blank fish was added.

[0117] The same method as above was used to prepare mixed standard working solutions with concentrations of 0.5 μg / L, 1.0 μg / L, 2.0 μg / L, 5.0 μg / L, 10.0 μg / L, 20.0 μg / L and 50.0 μg / L, and they were detected by liquid chromatography tandem mass spectrometry. The concentration value was used as the horizontal axis, and the area ratio of the target compound quantitative ion chromatographic peak and the corresponding isotope internal standard peak measured on the mass spectrometer was used as the vertical axis. A linear equation was prepared and the correlation coefficient was obtained, as shown in Table 3.

[0118] Table 3 Standard curves and correlation coefficients of nitrofuran metabolites

[0119]

[0120] (2) Sample testing

[0121] The tilapia muscle tissue used for development and verification of the present invention was ground and crushed with a homogenizer and stored frozen at -20°C. A blank tilapia sample was spiked (2.0 μg / kg) and tested under the same extraction, purification, and detection conditions as in Example 2.

[0122] (3) The accuracy test of the method of the present invention uses the sample spike recovery and relative standard deviation as indicators. The experimental method is the same as the extraction, purification and detection conditions in Example 2. The spike recovery of the blank tilapia sample is calculated based on the percentage of the concentration measured by the instrument to the target concentration; the relative standard deviation is calculated by the percentage of the relative deviation of the concentration measured in 6 parallel samples of the same compound to the average value, see Table 4. According to the European Commission Regulation SANTE / 11312 / 2021, the limit of quantification (LOQ) is defined as the lowest spike concentration that produces acceptable accuracy and precision, which is determined to be 0.5 μg kg -1 . These LOQs are consistent with the requirements of the newly released Chinese national standard GB 31656.13-2021, making this method suitable for monitoring the illegal use of nitrofuran compounds in Chinese aquatic products. The recoveries of the four target compounds ranged from 103.1% to 112.6%, with intra-day and inter-day RSDs below 11.1%. This meets the requirements of GB / T 27404-2008 "Laboratory Quality Control Specification for Physical and Chemical Analysis of Foods" for a recovery range of 60% to 120% when the content of the measured component is less than 0.1 mg / kg, and a relative standard deviation range of 30% to 21% when the content of the measured component is 1 to 10 μg / kg. This shows that this method has high correctness and precision, and good accuracy.

[0123] Spike recovery = C / C0 × 100%

[0124] Where C is the concentration measured by the instrument and C0 is the spiked concentration.

[0125] Relative standard deviation = S / C 平 ×100%

[0126] Where S is the standard deviation of the measured concentration, C 平 is the average value of the measured concentrations.

[0127] Table 4 Recovery and precision test results of four nitrofuran metabolite derivatives (n=3)

[0128]

[0129] The liquid chromatography and mass spectrometry conditions for detection are the same as those in Example 2.

[0130] Comparison with GB 31656.13-2021: The GB 31656.13-2021 method involves a five-step sample pretreatment process before HPLC-MS / MS detection, including hydrolysis and derivatization, pH adjustment, extraction with 8 mL of ethyl acetate, nitrogen drying and reconstitution, and purification by high-speed centrifugation (14,000 rpm). In contrast, the method of the present invention simplifies the process to four steps: hydrolysis and derivatization, pH adjustment, boric acid affinity extraction, and nitrogen drying and reconstitution. The main advantages of the method of the present invention are as follows:

[0131] (i) Streamlined workflow: The sample pretreatment protocol was reduced from five steps to four, eliminating the need for high-speed centrifugation before HPLC-MS / MS analysis, thereby improving efficiency.

[0132] (ii) Implementation of green chemistry: By directly extracting the hydrochloric acid hydrolysis solution using boronic acid affinity interaction, our method avoids the use of 8 mL of ethyl acetate, which is a flammable and hazardous solvent that may pose risks to human health and the environment.

[0133] (iii) Reduced equipment dependency: The developed method eliminates the need for high-speed centrifugation, further simplifying the process and reducing equipment requirements.

[0134] Overall, the method of the present invention shows significant potential in monitoring NFMs in aquatic products, providing a more efficient, environmentally friendly and user-friendly alternative to existing technologies.

[0135] This study presents a novel method based on cis-diol-modified GMA-EGDMA polymer microspheres for the direct enrichment of FFBA-derived NFMs from hydrochloric acid hydrolyzed fish samples. This specific enrichment strategy is based on boronic acid affinity interactions and is achieved by introducing boronic acid ligands into the NFM molecular structure through FFBA derivatization. A key innovation of this purification strategy is the replacement of traditional dispersive solid-phase extraction (DSPE) with vortex-based DSPE, eliminating the need for centrifugation and enabling high-throughput sample pretreatment.

[0136] This is the first reported vortex-based DSPE method, allowing for manual, parallel purification of multiple samples without centrifugation. The microspheres exhibit superior selectivity for FFBA-derived NFMs compared to control derivatives such as 2-NBA, which lack the boronic acid ligand. The developed method simplifies the sample pretreatment of NFMs into three key steps: hydrolysis, derivatization, and vortex-based DSPE purification. This simplified method is simpler and faster than traditional SPE.

Claims

1. A method for preparing cis-diol-modified polymer microspheres, comprising the following steps: (1) mixing glycidyl methacrylate, ethylene glycol dimethacrylate, ethyl 2-bromopropionate, CuBr2, n-octanol, dichloromethane, and N,N,N',N',N"-pentamethyldiethylenetriamine to obtain a mixture A; (2) mixing an aqueous solution containing polyvinyl alcohol, sodium lauryl sulfate and ascorbic acid with the mixture A, and reacting the mixture to obtain polymer microspheres; (3) mixing the polymer microspheres and a sulfuric acid solution and hydrolyzing them to obtain the cis-diol-modified polymer microspheres.

2. The preparation method according to claim 1, wherein: In the mixture A, the mass percentage concentration of the glycidyl methacrylate is 8% to 15%; The mass percentage concentration of the ethylene glycol dimethacrylate is 8% to 15%; The mass percentage concentration of the ethyl 2-bromopropionate is 0.05% to 0.2%; The mass percentage concentration of the CuBr2 is 8% to 15%; The mass percentage concentration of the N,N,N',N',N"-pentamethyldiethylenetriamine is 0.3% to 0.8%; The volume ratio of n-octanol to dichloromethane is 1:1 to 1:4; In the aqueous solution containing polyvinyl alcohol, sodium lauryl sulfate and ascorbic acid, based on the total volume of the aqueous solution, the concentration of the polyvinyl alcohol is 20-40 mg / mL; the concentration of the sodium lauryl sulfate is 0.5-3 mg / mL; and the concentration of the ascorbic acid is 8-12 mg / mL; The volume ratio of the aqueous solution containing polyvinyl alcohol, sodium lauryl sulfate and ascorbic acid to the mixture A is 16:1 to 16:

5.

3. The preparation method according to claim 1 or 2, characterized in that: In step (2), the reaction temperature is 50-70° C. and the reaction time is 6-10 h; In step (3), the concentration of the sulfuric acid solution is 0.2 to 0.8 mol / L; The hydrolysis temperature is 50-70°C; The hydrolysis time is 3 to 6 hours.

4. Cis-diol-modified polymer microspheres prepared by the preparation method according to any one of claims 1 to 3.

5. A method for determining nitrofuran metabolites in aquatic products, comprising sample pretreatment, characterized in that: The sample pretreatment comprises the following steps: (1) mixing a sample of aquatic product to be tested, a hydrochloric acid solution, and a 4-fluoro-3-formylphenylboronic acid solution for hydrolysis and derivatization, adjusting the pH value of the mixture, and centrifuging to obtain a supernatant; (2) The cis-diol-modified polymer microspheres of claim 4 are used as fillers to prepare a push rod type shaking type purification column, and the supernatant is loaded into the push rod type shaking type purification column for extraction and elution to obtain an eluate, which is the test solution.

6. The method according to claim 5, characterized in that: The mass ratio of the aquatic product sample to be tested to the volume ratio of the hydrochloric acid solution is 1g:2-4mL; The volume ratio of the aquatic product sample to be tested to the 4-fluoro-3-formylphenylboronic acid solution is 1 g: 80-120 μL; The concentration of the hydrochloric acid solution is 0.1 to 0.3 M; The concentration of the 4-fluoro-3-formylphenylboronic acid solution is 30-60 mM; The solvent of the 4-fluoro-3-formylphenylboronic acid solution is methanol; In step (1), the hydrolysis temperature is 30-40° C. and the time is 10-20 h; Use K2HPO4 solution to adjust the pH value of the mixture to 7-9; The centrifugal speed is 5000-10000 rpm; The centrifugation time is 5 to 15 minutes.

7. The method according to claim 5 or 6, characterized in that: The mass ratio of the cis-diol-modified polymer microspheres to the aquatic product sample in step (2) is 1:10 to 1:30; In step (2), the extraction time is 8 to 12 minutes; The elution solvent used for the elution is acetonitrile with a pH value of 2 to 5; The elution time is 1 to 6 minutes; The push rod type oscillating purification column is recorded in ZL202020843439.

1.

8. The method according to any one of claims 5 to 7, characterized in that: Before the elution, a washing step is performed using a methanol solution with a volume percentage concentration of 2% to 6%; After the elution, the eluate is evaporated and then redissolved in a methanol solution with a volume percentage concentration of 2% to 20% to obtain the solution to be tested.

9. The method according to any one of claims 5 to 8, characterized in that: The method for determining nitrofuran metabolites in aquatic products adopts the internal standard method for determination; The method further comprises a sample detection step, which is to detect the liquid to be tested by using an ultra-high performance liquid chromatography-triple quadrupole / composite linear ion trap mass spectrometer.

10. The method according to claim 9, characterized in that: The conditions of the test are as follows: High performance liquid chromatography conditions: Accuity Chromatographic separation was performed using a BEH C18 column; mobile phases: phase A was 2 mM ammonium acetate solution, phase B was methanol; gradient elution; column temperature was 40°C; flow rate was 0.3 mL / min; injection volume was 5.0 μL; The gradient elution conditions are as follows: 0-1 min, 25% phase B; 1-1.5 min, 25%-70% phase B; 1.5-4.5 min, 70% phase B; 4.5-5 min, 70%-25% phase B; 5-6 min, 25% phase B; Mass spectrometry conditions: ionization was performed in positive ion mode with a spray voltage of 5.5 kV and an ion source temperature of 500 °C. The curtain gas, ion source gas 1, and ion source gas 2 were set to 35, 55, and 35 psi, respectively; MS data were acquired in multiple reaction monitoring mode.

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