A microporous organic network hollow fiber membrane for efficient extraction of okadaic acid and application thereof

Abstract

This invention discloses a microporous organic network hollow fiber membrane for efficient extraction of okadaic acid and its applications, belonging to the field of marine food safety detection technology. The fiber membrane is prepared by a combination of electrospinning, in-situ growth, and sacrificial template methods. This fiber membrane possesses a hollow fiber structure, high stability, and high extraction efficiency, and can be applied to the efficient extraction of trace amounts of okadaic acid from shellfish samples. Furthermore, by coupling it with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), rapid and accurate detection of okadaic acid in shellfish samples can be achieved, demonstrating broad application prospects in the field of aquatic food safety screening.

Description

Technical Field

[0001] This invention belongs to the field of marine food safety testing technology, specifically relating to a hollow fiber membrane based on microporous organic network (MONs) materials, and its application in the efficient extraction and detection of trace amounts of okada (soft sponge) acid in shellfish soft tissue samples. Background Technology

[0002] Okada acid (OA) is a lipid-soluble toxin produced by toxic marine algae and accumulated in shellfish such as scallops, oysters, mussels, and clams. It is characterized by high toxicity, low content, and complex matrix interference. Currently, sample pretreatment for OA detection mainly relies on solid phase extraction (SPE) column technology, but there are obvious defects in actual shellfish sample detection: (1) The shellfish matrix contains a large number of interfering substances such as proteins, polysaccharides, lipids, and pigments. Conventional extraction materials are severely non-specifically adsorbed, which can easily cause SPE column blockage and decreased extraction performance, resulting in false positives and low recovery rates; (2) Commercial extraction materials have insufficient recognition sites for OA, making it difficult to achieve efficient extraction of ultra-trace OA, and thus failing to meet the food safety limit detection requirements.

[0003] Membrane solid-phase extraction (F-SPE) is an important derivative of SPE technology. This technology uses functionalized membrane materials as adsorption carriers, replacing the granular packing material of traditional column-based SPE. The adsorption, enrichment, and matrix separation of the target analyte are completed through membrane permeation and filtration, combining the enrichment and purification advantages of solid-phase extraction with the high throughput and low resistance characteristics of membrane materials. In the F-SPE technology system, the selection of membrane materials is a core and critical step, as their comprehensive performance directly determines the extraction efficiency of the target analyte, thus decisively influencing the recovery rate of sample pretreatment and even the sensitivity of subsequent analytical detection methods.

[0004] Microporous organic networks (MONs) possess advantages such as high specific surface area, ordered pore structure, functionalizability, strong physicochemical stability, ease of forming π-π stacking with target analytes, hydrophobic interactions, and hydrogen bonding, demonstrating excellent enrichment effects and application prospects in sample pretreatment. However, currently available MONs membranes are mostly concentrated in gas separation, water treatment, and heavy metal adsorption, and no MONs-based extraction membranes specifically designed for treating the complex matrices of shellfish have yet emerged. Therefore, developing a MONs-based OA-specific extraction membrane to address the problems of strong matrix interference and low extraction efficiency in shellfish samples is of significant practical importance for improving the level of marine aquatic safety testing. Summary of the Invention

[0005] To address the problems of severe matrix interference and low extraction efficiency in current okadaic acid pretreatment, this invention provides a hollow fiber membrane based on microporous organic network materials (MONs). This fiber membrane has the characteristics of high specific surface area, ordered pores, strong toxin affinity, and resistance to matrix interference, enabling efficient extraction and detection of ultra-trace amounts of okadaic acid in shellfish samples.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: One objective of this invention is to protect a microporous organic network hollow fiber membrane for efficient extraction of okadaic acid. This membrane is prepared using a combination of electrospinning, in-situ growth, and sacrificial template methods. Specifically, 1,4-diiodobenzene is first inserted into a polyacrylonitrile fiber membrane via electrospinning. Then, 1,3,5-tris(4'-ethynylphenyl)benzene is bonded to the polyacrylonitrile fiber membrane via in-situ growth. Finally, the polyacrylonitrile substrate is removed using a sacrificial template method, thereby obtaining the microporous organic network hollow fiber extraction membrane.

[0007] Furthermore, its preparation method specifically includes the following steps: (1) Preparation of 1,4-diiodobenzene / polyacrylonitrile fiber membrane by electrospinning: Polyacrylonitrile and 1,4-diiodobenzene were mixed and dissolved in N,N-dimethylformamide and stirred at room temperature for 3 h to obtain a uniform electrospinning solution. Then, 1,4-diiodobenzene / polyacrylonitrile fiber membrane was prepared by electrospinning and dried at room temperature. (2) Preparation of microporous organic network / polyacrylonitrile fiber membrane by in-situ growth method: 1,3,5-tris(4'-ethynylphenyl)benzene, bis(triphenylphosphine)palladium dichloride and cuprous iodide were mixed and dissolved in a mixed solution of triethylamine and toluene, and the 1,4-diiodobenzene / polyacrylonitrile fiber membrane obtained in step (1) was added to immerse it in the solution. Then the reaction was carried out under nitrogen protection. After the reaction, the membrane was taken out, washed with dichloromethane, acetone and methanol in sequence and then dried to obtain microporous organic network / polyacrylonitrile fiber membrane; (3) Preparation of microporous organic network hollow fiber membrane by sacrificial template method: The microporous organic network / polyacrylonitrile fiber membrane obtained in step (2) is added to N,N-dimethylformamide at 70℃ and soaked for 8 h and then vacuum dried to obtain microporous organic network hollow fiber membrane.

[0008] Furthermore, the average molecular weight of the polyacrylonitrile used in step (1) is 150,000 to 250,000.

[0009] Furthermore, the mass ratio of polyacrylonitrile and 1,4-diiodobenzene used in step (1) is 1:(1.0~1.5).

[0010] Furthermore, the mass concentration of polyacrylonitrile in the electrospinning solution obtained in step (1) is 10%~15%.

[0011] Furthermore, the electrospinning process parameters in step (1) are as follows: working voltage is 15~20 kV, distance from the tip of the spinning needle to the collector is 13~18 cm, and solution propulsion flow rate is 0.5~1.0 mL / h.

[0012] Furthermore, the room temperature drying time in step (1) is 12 h.

[0013] Furthermore, the mass ratio of 1,3,5-tris(4'-ethynylphenyl)benzene, bis(triphenylphosphine)palladium dichloride and cuprous iodide used in step (2) is 1:(0.4~0.6):(0.1~0.175).

[0014] Furthermore, in step (2), the volume ratio of triethylamine to toluene in the mixed solution is 1:1.

[0015] Furthermore, the reaction in step (2) is carried out at a temperature of 85-95°C for 24 hours.

[0016] Furthermore, the drying temperature in step (2) is 70°C and the time is 12 h.

[0017] Furthermore, the vacuum drying temperature in step (3) is 70°C and the time is 12 h.

[0018] Another object of the present invention is to protect the application of the hollow fiber membrane in the microporous organic network.

[0019] Furthermore, the fiber membrane is suitable for both organic and aqueous phase systems and can be directly used for the efficient extraction of crude extracts from shellfish such as scallops, oysters, mussels, and clams. Therefore, it can be used for the extraction and detection of trace amounts of okadaic acid in shellfish samples.

[0020] Furthermore, the application method involves taking soft tissue from shellfish samples, using methanol as the extractant, and homogenizing, sonicating, and centrifuging to obtain a crude extract. Then, the crude extract is subjected to membrane solid-phase extraction using the microporous organic network hollow fiber membrane. After extraction, the fiber membrane is rinsed with ultrapure water, the rinsing solution is discarded, and then eluted with methanol. The methanol eluent is collected, diluted to a fixed volume, and then detected by high performance liquid chromatography-tandem mass spectrometry.

[0021] The fiber membrane obtained in this invention is a porous homogeneous membrane structure composed of functionalized microporous organic network materials. It can achieve size exclusion and specific adsorption of okadaic acid, and its functional groups are alkynyl groups and benzene rings, which can form π-π interactions and hydrophobic interactions with okadaic acid molecules, achieving highly efficient adsorption of okadaic acid. Simultaneously, the fiber membrane has a hollow fiber structure with abundant binding sites, enabling efficient adsorption of okadaic acid in shellfish samples while repelling interfering substances such as proteins, lipids, and polysaccharides in the matrix, significantly reducing the matrix effect and improving extraction selectivity. Therefore, it is suitable for the extraction of trace amounts of okadaic acid from shellfish soft tissue samples. Furthermore, the membrane structure formed by the interwoven hollow fibers possesses both good mechanical strength and pore connectivity, ensuring high flux of the fiber membrane.

[0022] Compared with existing technologies, this invention has the following significant technical advantages and practical application value: (1) Highly efficient adsorption of target substances, significantly improving extraction efficiency: By designing monomer molecules of microporous organic network materials, multiple binding functional sites are introduced to form multiple interactions with okadaic acid, thus the extraction capacity of okadaic acid is much higher than that of traditional hydrophobic extraction materials. (2) Resistant to interference from complex shellfish matrix and high detection accuracy: The porous channels of functionalized microporous organic network materials can achieve size exclusion of macromolecular interferences such as proteins, lipids, and pigments, and greatly reduce matrix effect; (3) Strong trace enrichment ability, meeting the limit detection requirements: Microporous organic network materials have high specific surface area and well-developed pore structure, which can enrich ultra-trace Okada acid in the sample to the response range of the detection instrument, with a detection limit as low as 0.0005 μg / kg, which is far below the national food safety limit standard. (4) High extraction efficiency, suitable for batch sample detection: The fiber membrane has good pore connectivity and high throughput. The extraction time is only 3~5 min and the flow rate can reach 2.5 mL / min, which is much faster than the traditional solid phase extraction column. Moreover, the batch reproducibility RSD≤5.2% can realize high throughput and rapid processing of shellfish samples. (5) The preparation process is simple and can be mass-produced: The combination of electrospinning technology, in-situ growth method and sacrificial template method is adopted. The reaction conditions are mild and no complicated equipment is required. The pore structure can be precisely controlled. The prepared fiber membrane has high mechanical strength and good repeatability, which is suitable for industrial-scale production. (6) High versatility and compatibility with mainstream detection technologies: It can be used in conjunction with high performance liquid chromatography-tandem mass spectrometry without the need to modify the detection equipment, and is easy to promote and apply in aquatic product quality inspection institutions. Attached Figure Description

[0023] Figure 1 Scanning electron microscope image of the microporous organic network hollow fiber membrane prepared for the example.

[0024] Figure 2 Transmission electron microscopy (TEM) image of the microporous organic network hollow fiber membrane prepared for the example.

[0025] Figure 3 Scanning electron microscope (SEM) image of the microporous organic network / polyacrylonitrile hybrid membrane prepared for comparison.

[0026] Figure 4 Solid-state nuclear magnetic resonance spectrum of the microporous organic network hollow fiber membrane prepared for the example.

[0027] Figure 5 The nitrogen isothermal adsorption / desorption curves of the microporous organic network hollow fiber membrane prepared for the example are shown.

[0028] Figure 6 The pore size distribution diagram of the microporous organic network hollow fiber membrane prepared for the example.

[0029] Figure 7 The adsorption kinetic curve of the microporous organic network hollow fiber membrane prepared for the example is shown.

[0030] Figure 8 The figure shows the matrix interference resistance of the microporous organic network hollow fiber membrane prepared for the example.

[0031] Figure 9 The stability of the microporous organic network hollow fiber membranes prepared for different batches of examples in terms of Okada acid extraction effect is shown in the figure.

[0032] Figure 10 This is a photograph of a membrane solid-phase extraction device prepared using the microporous organic network hollow fiber membrane prepared in the examples.

[0033] Figure 11 This is a standard working curve of Okayama acid extraction using a microporous organic network hollow fiber membrane established in the application examples.

[0034] Figure 12 The image shows a chromatogram obtained from the extraction and detection of Okada acid in scallop samples using a microporous organic network hollow fiber membrane in an application example. Detailed Implementation

[0035] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0036] Example: Preparation of hollow fiber membranes in microporous organic networks (1) Preparation of 1,4-diiodobenzene / polyacrylonitrile fiber membrane by electrospinning: 1.5 g of polyacrylonitrile (average molecular weight Mw=150000) and 1.5 g of 1,4-diiodobenzene were dissolved together in 10 mL of N,N-dimethylformamide and stirred continuously at room temperature for 3 h until a uniformly dispersed electrospinning solution without obvious particles was formed; the parameters of the electrospinning machine were adjusted, namely, the working voltage was set to 18 kV, the distance from the tip of the spinning needle to the current collector was 15 cm, and the solution feed rate was 0.8 mL / h. Then, the obtained electrospinning solution was used for electrospinning to prepare a 1,4-diiodobenzene / polyacrylonitrile fiber membrane with a high 1,4-diiodobenzene loading rate; the prepared 1,4-diiodobenzene / polyacrylonitrile fiber membrane was dried at room temperature for 12 h to remove residual solvent.

[0037] (2) Preparation of microporous organic network / polyacrylonitrile fiber membrane by in-situ growth method: 0.3 g of 1,3,5-tris(4'-ethynylphenyl)benzene, 0.14 g of bis(triphenylphosphine)palladium dichloride and 0.038 g of cuprous iodide were added to a mixed solution of 20 ml toluene and 20 ml triethylamine. The mixture was ultrasonically dispersed for 5 min to ensure uniform mixing of all components. Then the prepared 1,4-diiodobenzene / polyacrylonitrile fiber membrane was added. The system was evacuated to remove air, and then nitrogen was introduced for protection. The entire system was then placed in an oven at 90 °C for 24 h. After the reaction, the microporous organic network / polyacrylonitrile fiber membrane was obtained. The obtained microporous organic network / polyacrylonitrile fiber membrane was washed twice each with dichloromethane, acetone and methanol to remove unreacted raw materials. After washing, it was placed in a vacuum drying oven at 70 °C for 12 h.

[0038] (3) Preparation of microporous organic network hollow fiber membrane by sacrificial template method: The dried microporous organic network / polyacrylonitrile fiber membrane was added to N,N-dimethyldiamide at 70℃ and soaked for 8 h to fully dissolve and remove the polyacrylonitrile template in the fiber membrane; after soaking, the system was placed in a vacuum drying oven at 70℃ and dried for 12 h to remove residual solvent and obtain microporous organic network hollow fiber membrane.

[0039] Preparation of comparative microporous organic network / polyacrylonitrile hybrid membranes (1) Preparation of polyacrylonitrile fiber membrane by electrospinning: 1.5 g of polyacrylonitrile (average molecular weight Mw=150000) was dissolved in 10 mL of N,N-dimethylformamide and stirred continuously at room temperature for 3 h until a uniformly dispersed electrospinning solution without obvious particles was formed; the parameters of the electrospinning machine were adjusted, namely, the working voltage was set to 18 kV, the distance from the tip of the spinning needle to the current collector was 15 cm, and the solution feed rate was 0.8 mL / h. Then, the obtained electrospinning solution was used to perform electrospinning operation to prepare polyacrylonitrile fiber membrane; the prepared polyacrylonitrile fiber membrane was dried at room temperature for 12 h to remove residual solvent.

[0040] (2) Preparation of microporous organic network / polyacrylonitrile hybrid membrane by in-situ growth method: 0.3 g of 1,3,5-tris(4'-ethynylphenyl)benzene, 1.5 g of 1,4-diiodobenzene, 0.14 g of bis(triphenylphosphine)palladium dichloride and 0.038 g of cuprous iodide were added to a mixed solution of 20 ml to to ensure uniform mixing of the components. Then the prepared polyacrylonitrile fiber membrane was added, and the system was evacuated to remove air. Nitrogen gas was then introduced for protection. The entire system was then placed in an oven at 90 °C for 24 h to react. After the reaction, a microporous organic network / polyacrylonitrile hybrid membrane was obtained. The obtained microporous organic network / polyacrylonitrile hybrid membrane was washed twice each with dichloromethane, acetone and methanol to remove unreacted raw materials. After washing, it was placed in a vacuum drying oven at 70 °C for 12 h to dry.

[0041] 1. Membrane structure characterization Figure 1 , 2 The figures show transmission electron microscopy (TEM) and ultra-high resolution field emission scanning electron microscopy (FESEM) images of the microporous organic network hollow fiber membrane prepared in the examples. As shown in the figures, the fiber membrane prepared in the examples is formed by interwoven fibers, and these fibers are interconnected hollow cavities. This structural feature effectively accelerates the interfacial diffusion mass transfer rate, thereby improving the extraction efficiency of the extraction membrane for okadaic acid.

[0042] Figure 3 The image shows a scanning electron microscope (SEM) image of the microporous organic network / polyacrylonitrile hybrid membrane prepared for comparison. As shown in the figure, the hybrid membrane prepared for comparison is formed by the growth of microporous organic network material on the surface of polyacrylonitrile fiber membrane. This causes the microporous organic network material to agglomerate in the gaps between the polyacrylonitrile fibers. This agglomeration directly leads to the blockage of the pores in the hybrid membrane, which is detrimental to improving the extraction efficiency of the membrane.

[0043] Figure 4The solid-state nuclear magnetic resonance (NMR) spectrum of the microporous organic network hollow fiber membrane prepared for this example is shown in the figure. As illustrated, the resonance peaks at 122 ppm and 130 ppm in the solid-state NMR spectrum correspond to the carbon atoms (C=C) of the benzene ring double bond in the structures of 1,3,5-tris(4'-ethynylphenyl)benzene and 1,4-diiodobenzene, respectively. The characteristic peak at 90 ppm originates from the bridging carbon site (C≡C) formed after the alkynyl coupling reaction between 1,3,5-tris(4'-ethynylphenyl)benzene and 1,4-diiodobenzene. Furthermore, the characteristic signal of the nitrile carbon (C≡N) in polyacrylonitrile (PAN) is not present at 118 ppm in this spectrum, indicating that the PAN component in the obtained fiber membrane has been completely removed. In summary, this invention successfully achieved the preparation of a microporous organic network hollow fiber membrane.

[0044] Figure 5 The nitrogen isotherm adsorption / desorption curves of the microporous organic network hollow fiber membrane prepared for this example are shown in the figure. As can be seen, the prepared fiber membrane exhibits typical characteristics of a type II isotherm. Calculations using the Langmuir model yielded a specific surface area of ​​500.24 m². 2 / g. This excellent specific surface area can effectively improve the adsorption capacity of the fiber membrane and significantly enhance its extraction performance for okadaic acid.

[0045] Figure 6 The figure shows the pore size distribution of the microporous organic network hollow fiber membrane prepared for this example. Pore size analysis results indicate that the prepared fiber membrane has a porous structure, and its pore size is mainly composed of three parts: the ordered pores of the microporous organic network material, the hollow cavities of the fibers themselves, and the skeletal structure formed by the interwoven fibers. The ordered pore size of the microporous organic network material is 2.2 nm. This size of ordered pores can effectively exclude large molecular interferences in the system, ensuring the extraction performance of the fiber membrane.

[0046] 2. Characterization of membrane extraction performance a) To investigate the maximum adsorption capacity of the microporous organic network hollow fiber membrane prepared in the examples for Okada sponge acid, the following experimental verification method was used: The fiber membrane was cut into circular pieces (13 mm in diameter) and placed in a filter. 18 mL of a 1 μg / mL Okada sponge acid standard solution was taken and passed through the fiber membrane in the filter at a flow rate of 0.3 mL / min for adsorption treatment (adsorption time controlled from 1 to 60 min). The supernatant obtained at different adsorption time points was collected, and each supernatant was analyzed by high performance liquid chromatography-tandem mass spectrometry to calculate the adsorption capacity (Q). t The experimental data were fitted using a pseudo-first-order adsorption kinetic formula.

[0047] Adsorption capacity (Q) t The formula for Q is:t =(C0-C t )V / m, where: Q t (μg / g) represents the adsorption amount at adsorption time t; C0 (μg / mL) represents the initial concentration of the Okada sponge acid standard solution; C t (μg / mL) is the concentration of Okada sponge acid at adsorption time t; V (mL) is the total volume of the sample solution used in the adsorption process; m (g) is the mass of the fiber membrane.

[0048] The pseudo-first-order adsorption kinetics formula is: ln(Q) e -Q t )=lnQ e -k1t, where: Q e (μg / g) represents the equilibrium adsorption capacity; Q t (μg / g) represents the adsorption amount at adsorption time t; t (min) represents the adsorption time; k1 (min) represents the adsorption amount at adsorption time t. -1 ) is the pseudo-first-order adsorption rate constant.

[0049] Figure 7 The fitting curve obtained by fitting the experimental data using the above adsorption kinetic model shows that the maximum adsorption capacity (Qmax) of the prepared fiber membrane for okadaic acid is 1851.6 μg / g. This data fully demonstrates that the obtained fiber membrane has a high adsorption capacity for okadaic acid, providing reliable data support for its practical application.

[0050] (b) To verify the resistance of the fiber membrane prepared in the examples to interference from complex matrices, several common and abundant biological matrices were selected for interference testing. The biological matrices were glucose, vitamin C, oleic acid, and bovine serum albumin (BSA). The experimental method was as follows: Different biological matrices with a concentration of 500.0 pg / mL were added to a 250.0 pg / mL Okada sponge acid standard solution for extraction and recovery. The treated solutions were then analyzed using high-performance liquid chromatography-tandem mass spectrometry. The extraction conditions are as follows: First, the fiber membrane is rinsed and activated sequentially with 1 mL of ultrapure water and 1 mL of methanol. Then, 10 mL of a standard solution of Okada sponge acid with added biological matrix is ​​passed through the fiber membrane at a flow rate of 2.5 mL / min to ensure that the Okada sponge acid in the solution is fully adsorbed by the fiber membrane. Next, the fiber membrane is rinsed with 1 mL of ultrapure water to remove matrix impurities adsorbed on the surface of the fiber membrane. Finally, the fiber membrane is eluted with 1 mL of methanol at a flow rate of 1.0 mL / min, and all eluent is collected. The eluent is then purged to near dryness with nitrogen and diluted to 0.2 mL with methanol to obtain the test solution, which is used for subsequent detection and analysis by high performance liquid chromatography-tandem mass spectrometry.

[0051] Figure 8 The results of the matrix interference resistance experiment show that the fiber membrane prepared in the examples still achieved an extraction recovery rate of over 80% for okadaic acid in systems containing glucose, vitamin C, oleic acid, and bovine serum albumin (BSA), respectively. These experimental results demonstrate that the prepared fiber membrane possesses excellent anti-interference capabilities, effectively resisting interference from impurities in complex matrices, further ensuring its extraction reliability and stability in practical complex systems.

[0052] c) To evaluate the reproducibility of the fiber membranes prepared in the examples, six different batches of the fiber membranes were selected, and extraction and recovery experiments of Okada sponge acid were conducted under the extraction conditions described above to verify the consistency of performance among different batches of fiber membranes. The results are shown in […]. Figure 9 .

[0053] like Figure 9 As shown, there was no significant difference in the extraction effect of okadaic acid among different batches of fiber membranes, with a relative standard deviation (RSD) ≤ 5.3%. These experimental results confirm that the prepared fiber membrane possesses good reproducibility, ensuring consistent performance across different batches and providing strong support for its large-scale production and practical application.

[0054] Application Examples: Microporous Organic Network Hollow Fiber Membranes for the Extraction and Detection of Okada Acid in Scallops

[0055] The microporous organic network hollow fiber membrane prepared in the examples was used for membrane solid-phase extraction, combined with high performance liquid chromatography-tandem mass spectrometry, to detect the content of okadaic acid in scallops. The specific steps are as follows: (1) Preparation of standard solutions: Prepare Okada spongic acid standard solutions with concentrations of 0.2, 0.5, 1.0, 2.0, 5.0, 10.0, 50.0, 100.0, 250.0, 500.0 and 1000.0 pg / mL respectively, and make up to 25 mL.

[0056] (2) Preparation of crude extract of shellfish sample: Weigh 2 g of homogenized scallop soft tissue into a 50 mL centrifuge tube, add 9 mL of methanol, vortex mix for 1 min, ultrasonically extract for 10 min, centrifuge at 8000 r / min for 5 min, then take the supernatant into a 20 mL graduated glass tube, add 9 mL of methanol to the residue and repeat the extraction once, combine the supernatants, and make up to 20 mL with methanol to obtain the crude extract of the sample.

[0057] (3) Construction of membrane solid phase extraction device: Cut the microporous organic network hollow fiber membrane prepared in the example into a circular piece (13 mm in diameter) and fill it into the inside of the stainless steel filter head to ensure that the extraction membrane is tightly attached to the inner wall of the stainless steel filter head without loosening; then install the stainless steel filter head filled with fiber membrane at the front outlet of the syringe. After the assembly is firm, fix the syringe to the injection pump to ensure that the whole device is tightly connected and there is no leakage during operation.

[0058] (4) Membrane solid phase extraction: First, the activated fiber membrane is rinsed with 1 mL of ultrapure water and 1 mL of methanol in sequence; then, 10 mL of standard solution or crude sample extract is passed through the fiber membrane at a flow rate of 2.5 mL / min to allow Okada acid to be adsorbed by the fiber membrane; then, the fiber membrane is rinsed with 1 mL of ultrapure water to remove matrix impurities adsorbed on the surface; finally, 1 mL of methanol is used to elute at a flow rate of 1.0 mL / min, all eluent is collected, the eluent is blown to near dryness with nitrogen, and the volume is adjusted to 0.2 mL with methanol to obtain the standard test solution or sample test solution.

[0059] (5) High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS): The obtained test solution was detected by HPLC-MS / MS, and quantification was performed using the external standard method. Instruments and analytical conditions: HPLC-MS / MS analysis was performed on an Accela liquid chromatography system (Thermo Fisher, USA) and a TSQ Quantum triple quadrupole mass spectrometer (Thermo Fisher, USA). Chromatographic conditions: The chromatographic column was a Thermo Fisher Hypersil GOLD aQ (150 × 2.1 mm, 5 μm); mobile phase A was an aqueous solution containing 0.1% formic acid, and mobile phase B was an acetonitrile solution containing 0.1% formic acid, with isocratic elution using 20% ​​B phase; the total run time was 5 min. The column temperature was room temperature, the injection volume was 10 μL, and the flow rate was 200 μL / min. Mass spectrometry conditions: Ion source: electrospray ionization source, positive ion mode, selected reaction monitoring mode; spray voltage: +3000 V, capillary temperature: 350℃, evaporation temperature: 300℃; sheath gas: nitrogen, purity 99.999%, pressure 35 bar; auxiliary gas: nitrogen, purity 99.999%, pressure 10 bar; collision gas: helium, purity 99.999%, pressure 1.5 bar. The quantitative ion pair for DA was 312.33 > 266.42, and the collision energy was 16 eV.

[0060] (6) Plotting the standard working curve: Plot the standard working curve with the concentration of the Okada sponge acid standard solution as the abscissa and its peak area as the ordinate.

[0061] (7) Actual sample analysis: The content of Okada sponge acid in the sample was calculated using the obtained linear equation.

[0062] Figure 11 This study aims to extract a series of okadaic acid standard solutions using a microporous organic network hollow fiber membrane and plot the resulting standard working curves. As shown in the figure, the linear equation is y = 127.6182x + 1112.2249, with a linear range of 2.0–1000.0 pg / mL, a linear correlation coefficient (R) of 0.9995, and a detection limit of 0.5 pg / mL. This indicates that the method exhibits excellent linearity and a low detection limit, enabling highly sensitive detection of okadaic acid.

[0063] Figure 12 The chromatograms obtained after extraction and detection of okadaic acid in scallops using a microporous organic network hollow fiber membrane were analyzed. As shown in the figure, the peak area corresponding to okadaic acid is 8678.88. Substituting this peak area into the obtained linear equation, the content of okadaic acid in the scallop sample was calculated to be 74.13 μg / kg.

[0064] In addition, spiked recovery experiments and precision tests were conducted on scallop samples at three levels (10 pg / mL, 50 pg / mL, and 250 pg / mL). The spiked recoveries were found to be within the range of 84.2% to 108.0%, and the precision (RSD) was ≤9.4% (n=6, where n is the number of parallel experiments). These experimental results demonstrate that the prepared microporous organic network hollow fiber membrane can be effectively applied to the extraction and quantitative detection of okadaic acid in actual shellfish samples, exhibiting excellent quantitative detection accuracy and precision.

[0065] To further verify the improved effect of the okadaic acid detection method established by the present invention using a microporous organic network hollow fiber membrane, the method of the present invention (i.e., the method of using the above-mentioned microporous organic network hollow fiber membrane for membrane solid-phase extraction to extract and detect okadaic acid in shellfish samples) and the solid-phase extraction column determination method specified in the "National Food Safety Standard for Determination of Diarrheal Shellfish Toxins" were used to extract and detect okadaic acid in scallops, and the detection data were compared and analyzed. The results are shown in Table 1.

[0066] Table 1 Comparison of results from different methods

[0067] As shown in Table 1, when using the microporous organic network hollow fiber membrane of this invention for membrane solid-phase extraction, thanks to its excellent and efficient extraction performance, the extraction time is shortened by nearly 60% compared to traditional solid-phase extraction columns. Furthermore, the detection limit of the method is far lower than the detection limit of the solid-phase extraction column method specified in the national food safety standards. The above comparative experimental results fully demonstrate that applying the microporous organic network hollow fiber membrane of this invention to membrane solid-phase extraction can achieve efficient extraction and accurate detection of okadaic acid in shellfish, possessing good technical feasibility and significant advantages over existing conventional detection methods.

[0068] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A microporous organic network hollow fiber membrane for efficient extraction of okada acid, characterized in that, Its preparation method includes the following steps: (1) Electrospinning: Polyacrylonitrile and 1,4-diiodobenzene were mixed and dissolved in N,N-dimethylformamide and stirred at room temperature for 3 hours to obtain a uniform electrospinning solution. Then, 1,4-diiodobenzene / polyacrylonitrile fiber membranes were prepared by electrospinning and dried at room temperature. (2) In-situ growth: 1,3,5-tris(4'-ethynylphenyl)benzene, bis(triphenylphosphine)palladium dichloride and cuprous iodide were mixed and dissolved in a mixed solution of triethylamine and toluene, and the 1,4-diiodobenzene / polyacrylonitrile fiber membrane obtained in step (1) was added to immerse it in the solution. Then the reaction was carried out under nitrogen protection. After the reaction, the membrane was taken out, washed with dichloromethane, acetone and methanol in sequence and then dried to obtain a microporous organic network / polyacrylonitrile fiber membrane. (3) Template sacrifice: The microporous organic network / polyacrylonitrile fiber membrane obtained in step (2) is added to N,N-dimethylformamide at 70°C and soaked for 8 h, and then vacuum dried to obtain a microporous organic network hollow fiber membrane.

2. The microporous organic network hollow fiber membrane according to claim 1, characterized in that, The mass ratio of polyacrylonitrile and 1,4-diiodobenzene used in step (1) is 1:(1.0~1.5).

3. The microporous organic network hollow fiber membrane according to claim 1, characterized in that, The mass concentration of polyacrylonitrile in the electrospinning solution obtained in step (1) is 10%~15%.

4. The microporous organic network hollow fiber membrane according to claim 1, characterized in that, The electrospinning process parameters in step (1) are: working voltage of 15~20 kV, distance from the tip of the spinning needle to the collector of 13~18 cm, and solution propulsion flow rate of 0.5~1.0 mL / h.

5. The microporous organic network hollow fiber membrane according to claim 1, characterized in that, The mass ratio of 1,3,5-tris(4'-ethynylphenyl)benzene, bis(triphenylphosphine)palladium dichloride and cuprous iodide used in step (2) is 1:(0.4~0.6):(0.1~0.175).

6. The microporous organic network hollow fiber membrane according to claim 1, characterized in that, In step (2), the volume ratio of triethylamine to toluene in the mixed solution is 1:

1.

7. The microporous organic network hollow fiber membrane according to claim 1, characterized in that, The reaction in step (2) is carried out at a temperature of 85~95℃ for 24 hours.

8. The application of a microporous organic network hollow fiber membrane as described in any one of claims 1-7 in the efficient extraction and detection of trace amounts of okadaic acid in shellfish samples.

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