A method for analyzing lipid-soluble shellfish toxins based on solid-phase extraction using polystyrene nanofibers

Abstract

A method for analyzing lipophilic shellfish toxins based on solid-phase extraction using polystyrene nanofibers is disclosed. This method first extracts lipophilic shellfish toxins from the sample using methanol, then uses polystyrene nanofibers as a solid-phase extraction adsorbent to purify the matrix and enrich the lipophilic shellfish toxins in aquatic products. Finally, high-performance liquid chromatography-triple quadrupole tandem mass spectrometry (HPLC-MS / MS) is employed for detection, enabling the detection of 12 lipophilic shellfish toxins in aquatic products. This invention is the first to introduce nanofiber solid-phase extraction into the detection of lipophilic shellfish toxins. Through hydrophobic interactions and π-π interactions between benzene-ring-containing, hydrophobic polystyrene nanofibers and target analytes containing polyethers or macrolides, efficient enrichment and purification are achieved, removing matrix interference and concentrating the target analytes. The method's sensitivity is nearly 100 times higher than that of the national standard method. Furthermore, this method uses less organic solvent, effectively saving sample pretreatment time and improving detection efficiency.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and specifically to a method for analyzing lipid-soluble shellfish toxins based on solid-phase extraction using polystyrene nanofibers. Background Technology

[0002] Fat-soluble shellfish toxins are mainly compounds containing polyethers or macrolides. According to the toxicity mechanism, most of them are diarrhetic shellfish toxins, such as okata spongiformis toxin and its derivatives, fin algae toxin and protodinium alginate toxin. They have high specific toxicity to the nervous or cardiovascular systems of organisms. Diarrhea, dizziness, vomiting, and abdominal distension and pain will occur in humans 30 minutes to several hours after ingestion. Long-term consumption can cause cancer of the digestive system.

[0003] Currently, detection technologies for fat-soluble shellfish toxins in aquatic products, both domestically and internationally, are mainly divided into three categories: bioanalytical testing, enzyme-linked immunosorbent assay (ELISA), and high-performance liquid chromatography (HPLC). Bioanalytical testing includes mouse bioassays and cytotoxicity tests, while HPLC includes HPLC-fluorescence detection and HPLC-tandem mass spectrometry (HPLC-MS / MS). With the rapid development of mass spectrometry technology, HPLC-MS / MS has been introduced into the field of shellfish toxin detection due to its advantages such as fast analysis speed, high sensitivity, and good selectivity. Because shellfish products have complex matrices, interfering substances such as proteins, amino acids, and minerals in the matrix can easily affect the ionization of target analytes, producing matrix effects that alter the sensitivity and accuracy of the instrument. Therefore, developing pretreatment techniques suitable for the extraction, purification, and enrichment of shellfish toxins in aquatic product matrices is of great significance for the accuracy and reliability of detection results.

[0004] Currently, shellfish toxins in aquatic products are generally extracted with solvents first, followed by purification using solid-phase extraction columns. For example, the national standard GB5009.212-2016, "National Food Safety Standard - Determination of Diarrhea-causing Shellfish Toxins in Shellfish," uses a general-purpose solid-phase extraction column for the detection of diarrhea-causing shellfish toxins, but the method has low selectivity and sensitivity. Chinese patent CN 106706829 B discloses a method for determining diarrhea-causing shellfish toxins in shellfish using immunoaffinity purification-liquid chromatography-tandem mass spectrometry. This method employs 80% methanol-water extraction, followed by dilution with phosphate buffer, and purification and enrichment using an immunoaffinity column. Although this method has low matrix interference and good purification effect, its selectivity is too high, detecting only three target compounds: okata fuscinic acid and its derivative, fin algae toxin. Chinese patent CN108872412 B discloses a UPLC-MS / MS detection method for fat-soluble shellfish toxins based on the QuEChERS method using graphene. This method, based on methanol / ethanol / isopropanol extraction combined with cryogenic degreasing and graphene oxide purification, enables the simultaneous detection of nine fat-soluble shellfish toxins. However, this pretreatment method primarily serves a purification function and does not concentrate or enrich the target analytes, thus limiting the method's sensitivity. The detection limit is 0.10–1.47 μg / kg, and the quantitation limit is 0.32–4.92 μg / kg.

[0005] Electrospun nanofiber solid-phase extraction (SPE) technology uses the possible forms, structures, and physicochemical properties of target analytes as clues. It utilizes electrospinning technology to prepare material systems that can interact with target analytes in multiple ways, achieving synergistic effects including coordination bonds, hydrogen bonds, van der Waals forces, and hydrophilic / hydrophobic forces across both short and long ranges. This results in materials with highly efficient dual-mode adsorption and desorption of target analytes. Compared to micron-sized solid particle adsorption media, fiber adsorption media have advantages such as high porosity, strong permeability, and rapid diffusion of target analytes. In nanofiber SPE devices with equivalent adsorption efficiency, the amount of nanofiber media used is only one-tenth that of conventional adsorption media, and the amount of reagents used is also reduced by more than an order of magnitude. According to research, there are currently no publicly reported applications of nanofibers in the detection of shellfish toxins, either domestically or internationally. This invention, based on the structural characteristics of lipid-soluble shellfish toxins, designs and prepares polystyrene nanofibers that can interact with target analytes containing polyethers or macrolides in multiple ways. Combined with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS / MS), it achieves accurate and rapid detection of 12 lipid-soluble shellfish toxins in aquatic products. Summary of the Invention

[0006] Technical problems solved: Addressing the issues of complex matrices and numerous interfering substances in existing shellfish products, as well as the large amounts of organic solvents, cumbersome operations, and low sensitivity of current pretreatment methods, this invention provides a method for analyzing lipophilic shellfish toxins based on solid-phase extraction using polystyrene nanofibers. This method combines nanofiber solid-phase extraction with ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS / MS) for the detection of shellfish toxins in aquatic products. By designing and preparing hydrophobic polystyrene nanofibers containing benzene rings, hydrophobic interactions and π-π interactions occur between these nanofibers and lipophilic shellfish toxins containing polyethers or macrolides, improving the selectivity and sensitivity of the method and demonstrating promising application prospects.

[0007] Technical solution: A method for analyzing lipid-soluble shellfish toxins based on solid-phase extraction using polystyrene nanofibers, comprising the following steps:

[0008] Step 1, Sample preparation: Weigh the thoroughly homogenized soft tissue of aquatic products after washing and draining, add methanol for extraction, take the supernatant, and repeat the extraction of the residue with methanol at least once. Combine the supernatants, blow dry with nitrogen, and then redissolve with 5% methanol solution.

[0009] Step 2, Solid-phase extraction: Polystyrene nanofibers are packed into a solid-phase extraction column, which is activated with methanol and water in sequence. Then, the reconstituted extract is added to the activated solid-phase extraction column. After the liquid flows out, it is washed and eluted to obtain the test solution. The washing solution is a 5% methanol solution and the elution solution is a 1% ammonia-methanol solution.

[0010] Step 3: Quantitative analysis: The test solution was detected by ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, and the external standard method was used for quantification. The content of the target analyte was obtained according to the standard curve.

[0011] The fat-soluble shellfish toxins mentioned are 12 types, specifically: okadaic acid (OA), cycloiminotoxin (GYM), fin algae toxin 1 (DTX1), protodinium alginate shellfish toxin 1 (AZA1), fin algae toxin 2 (DTX2), homopolymerized scallop toxin (h-YTX), scallop toxin (YTX), protodinium alginate shellfish toxin 3 (AZA3), scallop toxin 2 (PTX2), protodinium alginate shellfish toxin 2 (AZA2), scallop toxin (PnTX), and 13-norspirolactone (SPX1).

[0012] Preferably, the aquatic product in step one is shellfish. After cleaning and draining, it is thoroughly homogenized as follows: Rinse the shell with clean water to remove impurities and take out all soft tissues. Then rinse with ultrapure water to remove mud and sand. After draining the water, cut it into pieces and thoroughly homogenize it.

[0013] Preferably, in step one, the mass ratio of the soft tissue of the aquatic product to the volume of methanol used for extraction is 1 g: 4.5 mL, and the extraction conditions are: vortexing for 1 min, ultrasonic extraction at 25~30℃ for 10 min, and centrifugation at 4℃ and 7500 r / min for 10 min.

[0014] Preferably, in step two, the mass ratio of the polystyrene nanofibers packed into the solid-phase extraction column to the volume ratio of the reconstitution solution, the rinsing solution, and the elution solution is 10 mg: 1 mL: 1 mL: 0.2 mL.

[0015] Preferably, in step three, the liquid chromatography conditions are as follows: Column: C 18 Column dimensions: 2.1 × 100 mm, 1.7 µm; Mobile phase: A is an aqueous solution containing 0.01% ammonia and 2 mmol / L ammonium formate; B is an aqueous solution of 95% acetonitrile containing 0.01% ammonia and 2 mmol / L ammonium formate; Gradient elution program: 0–2 min, 30% B; 2–10 min, 30%–90% B; 10–12 min, 90% B; 12–14 min, 90%–30% B; 14–16 min, 30% B; Flow rate: 0.3 mL / min; Column temperature: 40℃; Injection volume: 2 μL.

[0016] Preferably, in step three, the mass spectrometry conditions are as follows: ionization mode: electrospray ionization mode (ESI); mass spectrometry scanning mode: multiple reaction monitoring (MRM); gas temperature: 300 °C; gas flow rate: 5 L / min; nebulizer gas pressure: 45 psi; sheath gas (N2) temperature: 250 °C; sheath gas (N2) flow rate: 11 L / min; Dell: 15 ms; capillary voltage: 3500 V.

[0017] As a preferred method, the mass spectrometry scanning mode for the 12 lipid-soluble shellfish toxins was MRM mode, and the quantitative ion pairs were: OA (okadaic acid) 827.4 / 723.4, GYM (cycloiminotoxin) 508.3 / 490.3, DTX1 (fin algae toxin 1) 841.5 / 737.5, AZA1 (protodinium alginate shellfish toxin 1) 842.5 / 824.5, DTX2 (fin algae toxin 2) 827.4 / 723.4, homopolymerized scallop toxin h-YTX 1177.5 / 971.4, YTX (scallop toxin) 1163.5 / 957.2, AZA3 (protodinium alginate shellfish toxin 3) 828.4 / 810.4, PTX2 (scallop toxin 2) 876.4 / 823.4, and AZA2 (protodinium alginate shellfish toxin 2). 856.4 / 838.4, Jiangyaoqing toxin PnTX 694.4 / 164.0, 13-norspirolactone SPX1 692.3 / 674.3.

[0018] Preferably, in step three, the external standard method is used for quantification, and the content of the target substance is obtained according to the standard curve as follows:

[0019] S1. Dilute 200 μg / L of the mixed standard intermediate solution with methanol to prepare a series of standard solutions with concentrations of 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, and 0.1 μg / L. Perform liquid chromatography-tandem mass spectrometry (LC-MS / MS) to determine the concentrations of the target analytes. Plot a standard curve with the target analyte concentration on the x-axis and the corresponding peak area on the y-axis to obtain the corresponding linear regression equation. The mass concentration of each target analyte in the mixed standard intermediate solution is 200 μg / L.

[0020] S2. Substitute the detection results from step three into the corresponding linear regression equation in S1 for calculation to obtain the content of 12 fat-soluble shellfish toxins.

[0021] Beneficial effects: (1) This invention introduces nanofiber solid phase extraction into the detection of fat-soluble shellfish toxins for the first time. Through the hydrophobic interaction and π-π interaction between the benzene ring-containing hydrophobic polystyrene nanofibers and the target substances containing polyether or macrolides, efficient enrichment and purification are achieved, thereby removing matrix interference and concentrating the target substances. A qualitative and quantitative analysis method for 12 fat-soluble shellfish toxins has been established.

[0022] (2) The sensitivity of this method is nearly 100 times higher than that of the method in the national standard.

[0023] (3) In addition, this method uses less organic solvent, effectively saves sample pretreatment time, and improves detection efficiency. Attached Figure Description

[0024] Figure 1 The figures show scanning electron microscope (SEM) and transmission electron microscope (TEM) images of PS fibers and PS-PPy fibers. In the figures, A is a scanning electron microscope image of PS fibers; B is a TEM image of PS fibers; C is a scanning electron microscope image of PS-PPy fibers; and D is a TEM image of PS-PPy fibers.

[0025] Figure 2 The figure shows the extraction ion chromatograms of 12 fat-soluble shellfish toxins, where AL represents AZA1, h-YTX, AZA2, OA, AZA3, SPX1, DTX1, PnTX, DTX2, PTX2, GYM and YTX toxins, respectively.

[0026] Figure 3 The effect of different nanofiber solid-phase extraction columns on the adsorption efficiency of lipid-soluble shellfish toxins.

[0027] Figure 4 The effect of different eluents on the extraction efficiency of nanofibers.

[0028] Figure 5 Matrix effect of spiked samples with different matrix concentrations. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are within the scope of the present invention. Experimental methods and reagents not specifically described in the embodiments are performed according to conventional conditions in the art.

[0030] Unless otherwise specified, all instruments and materials used in the specific embodiments of this instruction manual are from commercially available products.

[0031] 1. Instruments

[0032] Agilent 6470 Triple Quad LC-MS / MS, with electrospray ionization (ESI) source and MassHunter software data processing system, Agilent Technologies, USA; Centrifuge 5430R cryogenic high-speed centrifuge, Eppendorf, Germany; ML304T analytical balance (Mettler-Toledo Instruments (Shanghai) Co., Ltd.); Centrifuge 5430R cryogenic high-speed centrifuge (Eppendorf, Germany); N-EVAP 112 nitrogen evaporator (Organomation, USA); Milli-Q ultrapure water system (Millipore, USA); ultrasonic instrument, Shanghai Qiqian Electronic Technology Co., Ltd.; 0.22 µm organic phase filter membrane, Shanghai Anpu Experimental Technology Co., Ltd.; Vortex-Genie 2 vortex oscillator (Scientific Industries, USA).

[0033] 2. Raw materials and reagents

[0034] Standards: okadaic acid (10.08 mg / L), cycloiminotoxin (2.50 mg / L), fin algae toxin 1 (10.01 mg / L), protodinium alginate shellfish toxin 1 (1.22 mg / L), fin algae toxin 2 (3.83 mg / L), homopolymerized scallop toxin (5.05 mg / L), scallop toxin (5.01 mg / L), protodinium alginate shellfish toxin 3 (1.18 mg / L), scallop toxin 2 (4.40 mg / L), protodinium alginate shellfish toxin 2 (1.19 mg / L), scallop toxin (1.92 mg / L), and 13-norspirolactone (5.01 mg / L) were purchased from Qingdao Puribang Biotechnology Co., Ltd. Specific information is shown in Table 1.

[0035] Table 1. Basic Information on 12 Fat-Soluble Shellfish Toxins

[0036]

[0037] Reagents: Methanol and acetonitrile were mass spectrometry grade and purchased from Shanghai Anpu Experimental Technology Co., Ltd.; ammonia and ammonium formate were analytical grade and purchased from Sinopharm Group; formic acid (analytical grade) was from Aladdin; laboratory water was ultrapure water, using a Milli-Q ultrapure water system from Merck AG, Germany.

[0038] Polystyrene nanofibers (PS) were purchased from Suzhou Dongqi Biotechnology Co., Ltd. The preparation process of polystyrene-polypyrrole nanofibers (PS-PPy) was as follows: PS nanofibers were immersed in a 0.04 mol / L pyrrole solution and vigorously shaken at room temperature to disperse the fibers. The mixture was then sonicated for 30 min. An equal volume of 0.08 mol / L ferric chloride aqueous solution was added, and the mixture was shaken and sonicated for another 30 min to polymerize the pyrrole monomers onto the fiber surface, forming a black coating. The black fibers were washed with excess ultrapure water, and finally washed with an appropriate amount of ethanol and dried to obtain PS-PPy nanofibers. The morphology of the polystyrene nanofibers and polystyrene-polypyrrole nanofibers was scanned and characterized by high-resolution transmission electron microscopy and field emission scanning electron microscopy. The scanning electron microscopy and transmission electron microscopy results of the fibers are shown in [Figure number missing]. Figure 1 The PS nanofibers have a diameter of approximately 840 nm, are uniformly distributed, and have a smooth surface. The PS-PPy nanofibers have a diameter of approximately 860 nm, but the surface of the fibers is polymerized with polypyrrole particles, resulting in an uneven surface and more fiber breakage.

[0039] Packing and activation of the nanofiber solid-phase extraction column: Weigh 10 mg of polystyrene nanofibers, evenly place them at the bottom of the column, and compact them. Add 200 μL of methanol to the bottom of the column, and use an airtight syringe to force the methanol out of the purification column.

[0040] Example 1: Determination of the content of 12 fat-soluble shellfish toxins in shellfish products

[0041] Step 1: Sample Preparation: Rinse fresh clam shells with clean water to remove impurities. Remove all soft tissue and rinse with ultrapure water to remove sand. Drain the water, chop finely, and homogenize thoroughly. Accurately weigh 1 g (accurate to 0.1 mg) into a centrifuge tube, add 4.5 mL of methanol, vortex for 1 min, sonicate for 10 min, centrifuge at 7500 r / min at 4℃ for 10 min, and transfer the supernatant to a 15 mL centrifuge tube. Add 4.5 mL of methanol to the residue and repeat the extraction once. Combine the extracts, bring the volume to 10 mL with methanol, dry under nitrogen, and reconstitute with 1.0 mL of 5% methanol.

[0042] Step 2, Solid-Phase Extraction: Weigh 10 mg of fiber and feed it to the lower end of the solid-phase extraction column using a solid metal cylinder, compressing it into a small solid-phase extraction column. Activate the solid-phase extraction column by passing 200 μL of methanol and 400 μL of water through the fiber in sequence. Transfer the extract into the activated PS solid-phase extraction column. After the liquid elutes, rinse with 1.0 mL of 5% methanol solution, and finally elute with 0.2 mL of 1% ammonia-methanol solution for analysis by liquid chromatography-tandem mass spectrometry.

[0043] Step 3, Quantitative Analysis: High-performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS / MS) was used for detection, and quantification was performed using the external standard method based on the standard curve. The HPLC-MS / MS detection conditions are as follows:

[0044] Column: Waters, ACQUITY UPLC BEH C 18 (2.1 × 100 mm, 1.7 µm); Mobile phase: A (containing 0.01% ammonia, 2 mmol / L ammonium formate aqueous solution) and B (containing 0.01% ammonia, 2 mmol / L ammonium formate in 95% acetonitrile solution); Gradient elution program: 0–2 min, 30% B; 2–10 min, 30%–90% B; 10–12 min, 90% B; 12–14 min, 90–30% B; 14–16 min, 30% B; Flow rate: 0.3 mL / min; Column temperature: 40℃; Injection volume: 2 μL.

[0045] Ionization mode: Electrospray ionization (ESI); Mass spectrometry mode: Multiple reaction monitoring (MRM); Gas temperature: 300 °C; Gas flow rate: 5 L / min; Nebulizer gas pressure: 45 psi; Sheath gas (N2) temperature: 250 °C; Sheath gas (N2) flow rate: 11 L / min; Dell: 15 ms; Capillary voltage: 3500 V. Specific mass spectrometry parameters are shown in Table 2.

[0046] Table 2. Mass spectrometry parameters of 12 fat-soluble shellfish toxins

[0047]

[0048] Note: * indicates quantitative ions.

[0049] The quantitative analysis using the external standard method based on the standard curve is as follows:

[0050] S1. Dilute 200 μg / L of the mixed standard intermediate solution with methanol to prepare a series of standard solutions with concentrations of 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, and 0.1 μg / L. Perform liquid chromatography-tandem mass spectrometry (LC-MS / MS) on these solutions. Plot a standard curve with the target analyte concentration (X, μg / L) on the x-axis and the corresponding peak area (Y) on the y-axis to obtain the corresponding linear regression equation.

[0051] S2. Substitute the detection results from step three into the corresponding linear regression equation in S1 for calculation to obtain the content of 12 fat-soluble shellfish toxins.

[0052] In this embodiment, the mixed standard intermediate solution is prepared as follows: transfer appropriate volumes of standard solutions of different concentrations into volumetric flasks to prepare a mixed standard intermediate solution with a mass concentration of 200 μg / L for each target analyte, and store it in a -20 ℃ refrigerator for later use.

[0053] The method for quantifying shellfish toxins in this embodiment is as follows: Under optimized chromatographic and mass spectrometric conditions, a series of standard solutions of lipid-soluble shellfish toxins diluted with methanol were mixed and analyzed by liquid chromatography-tandem mass spectrometry. The extraction ion chromatograms of the 12 lipid-soluble shellfish toxins are shown below. Figure 2 A standard curve was plotted with the target analyte concentration on the x-axis (X, μg / L) and the corresponding peak area on the y-axis. The linear equation, correlation coefficient (r), and linear range of the target analyte are shown in Table 3.

[0054] Table 3. Linear range, linear equation, and correlation coefficient of 12 fat-soluble shellfish toxins.

[0055]

[0056] Example 2: Optimization of Extraction Conditions

[0057] Lipid-soluble shellfish toxicants, mainly compounds containing polyethers or macrolides, are soluble in nonpolar solvents, hydrophobic, and mostly nonpolar or weakly polar molecules. Based on the hydrophobic characteristics of lipid-soluble shellfish toxicants, this invention selected PS and PS-PPy nanofibers and tested the two materials using a contact angle meter. The results showed that the contact angle of PS nanofibers was 134.2°, and that of PS-PPy nanofibers was 120.4°. A contact angle greater than 90° indicates that the solid surface is hydrophobic, meaning that liquids do not easily wet the solid and easily move on the surface. A larger contact angle indicates better hydrophobic properties of the material.

[0058] One 1.0 mL of a 10 μg / L mixed standard solution diluted with 5% methanol was passed through PS and PS-PPy nanofiber solid-phase extraction columns, respectively. The residue at the bottom of the column was collected, and the content of the target analyte was determined and compared with that of the 10 μg / L standard solution. The adsorption efficiency was calculated according to the formula "(1 - mass spectrometric response of the target analyte remaining in the post-column solution / mass spectrometric response of the standard solution) * 100%". The results are shown in the figure. Figure 3 The adsorption efficiency of 12 fat-soluble shellfish toxins by PS nanofiber solid-phase extraction column was greater than 98%, while the adsorption efficiency of PS-PPy nanofiber for GYM was relatively low. This may be because the target analyte is a cyclic imine structure with more hydrophobic groups. Therefore, PS nanofiber has better selectivity for the target analyte.

[0059] According to literature reports, elution with alkaline solutions is beneficial for improving the extraction efficiency of fat-soluble shellfish toxins. This invention patent investigated the elution effects of 0.2 mL methanol, 0.3% ammonia-methanol, and 1% ammonia-methanol on 12 fat-soluble shellfish toxins. The extraction efficiency was calculated by the mass spectrometry response ratio of the eluent to a 50 μg / L mixed standard solution diluted with the corresponding solvent. The results are shown in […]. Figure 4 1% ammonia-methanol solution showed good extraction efficiency for all 12 target compounds, ranging from 80% to 120%, so 1% ammonia-methanol solution was chosen for elution.

[0060] Example 3: Investigating the matrix effect of methods

[0061] To investigate the matrix effect in the detection of fat-soluble shellfish toxins in aquatic products, blank clam samples were processed according to steps one and two of Example 1 to obtain a blank sample matrix. Mixed standard solutions of 10 μg / L, 50 μg / L, and 100 μg / L were prepared using the blank sample matrix and 1% ammonia-methanol, respectively. Detection was performed under the chromatographic-mass spectrometric conditions described in step three. The matrix effect was calculated using the response ratio of the matrix-spikened sample to the standard solution. The matrix effect test results for 12 fat-soluble shellfish toxins ranged from 75.4% to 117.5%. Although the matrix had a slight inhibitory effect on DTX1, DTX2, OA, and YTX, and a slight enhancing effect on PTX2, overall, the matrix effect was within an acceptable range (see [link to relevant documentation]). Figure 5 ).

[0062] Example 4: Examining the method's limit of detection and limit of quantitation

[0063] The standard spiking method was used, adding standard solutions to blank clam samples with progressively decreasing spiking concentrations. Detection was performed according to steps one through three of Example 1. The limits of detection (LOD, S / N ≥ 3) and limits of quantitation (LOQ, S / N ≥ 10) were calculated based on the signal-to-noise ratio. Results showed that the LODs for the 12 fat-soluble shellfish toxins ranged from 0.02 to 0.50 µg / kg, and the LOQs ranged from 0.06 to 1.65 µg / kg (see Table 4). The limits of detection and quantitation for OA, DTX-1, and DTX-2 in the national standard GB 5009.212-2016, "National Food Safety Standard - Determination of Diarrheal Shellfish Toxins in Shellfish," are 10 µg / kg and 30 µg / kg, respectively. The sensitivity of the present invention far exceeds the detection requirements of the national standard.

[0064] Table 4. Limits of detection and limits of quantitation for 12 fat-soluble shellfish toxins.

[0065]

[0066] Example 5: Investigating the recovery and stability of the method

[0067] Three concentrations of mixed standard solutions (4.0 µg / kg, 20.0 µg / kg, and 40.0 µg / kg) were added to blank clam samples. Six samples were prepared in parallel for each addition level, and the tests were performed according to steps one to three of Example 1. The recoveries and relative standard deviations (RSDs) were calculated. The results showed that the recoveries of the 12 fat-soluble shellfish toxins ranged from 65.7% to 114.2%, with RSDs ranging from 2.7% to 14.2% (see Table 5).

[0068] Table 5. Recovery rates and relative standard deviations of 12 fat-soluble shellfish toxins (n ​​= 6)

[0069]

[0070] Example 6: Actual Sample Testing

[0071] This method was applied to detect the content of 12 fat-soluble shellfish toxins in 10 batches of commercially available aquatic products, and none were detected. The reasons for this may be twofold: firstly, the local area is not coastal; secondly, the products were purchased outside of a red tide season. To further validate and apply this method, standard quality control samples were tested, and the results are shown in Table 6.

[0072] Table 6 Recovery rates and relative standard deviations of soluble shellfish toxins (n ​​= 6)

[0073]

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for analyzing lipid-soluble shellfish toxins based on solid-phase extraction using polystyrene nanofibers, characterized in that, The steps are as follows: Step 1, Sample preparation: Weigh the thoroughly homogenized soft tissue of aquatic products after washing and draining, add methanol for extraction, take the supernatant, and repeat the extraction of the residue with methanol at least once. Combine the supernatants, blow dry with nitrogen, and then redissolve with 5% methanol solution. Step 2, Solid-phase extraction: Polystyrene nanofibers are packed into a solid-phase extraction column, which is activated with methanol and water in sequence. Then, the reconstituted extract is added to the activated solid-phase extraction column. After the liquid flows out, it is washed and eluted to obtain the test solution. The washing solution is a 5% methanol solution and the elution solution is a 1% ammonia-methanol solution. Step 3, Quantitative Analysis: The test solution was analyzed using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS). The content of the target analyte was quantified using the external standard method based on the standard curve. The HPLC conditions included: column: C 18 , 2.1×100 mm, 1.7 µm; Mobile phase: A is an aqueous solution containing 0.01% ammonia and 2 mmol / L ammonium formate; B is an aqueous solution of 95% acetonitrile containing 0.01% ammonia and 2 mmol / L ammonium formate; Gradient elution program: 0~2 min, 30% B; 2~10 min, 30%~90% B; 10~12 min, 90% B; 12~14 min, 90~30% B; 14~16 min, 30% B; The fat-soluble shellfish toxins mentioned therein are 12 types, specifically okadaic acid, cycloiminotoxin, fin algae toxin 1, protodinium alginate shellfish toxin 1, fin algae toxin 2, homopolymerized scallop toxin, scallop toxin, protodinium alginate shellfish toxin 3, scallop toxin 2, protodinium alginate shellfish toxin 2, scallop toxin, and 13-norspirolactone.

2. The method for analyzing lipid-soluble shellfish toxins based on solid-phase extraction using polystyrene nanofibers according to claim 1, characterized in that, The aquatic products in step one are shellfish. After cleaning and draining, they are thoroughly homogenized as follows: Rinse the shells with clean water to remove impurities and take out all soft tissues. Then rinse with ultrapure water to remove mud and sand, drain the water, cut into pieces and thoroughly homogenize.

3. The method for analyzing lipid-soluble shellfish toxins based on solid-phase extraction using polystyrene nanofibers according to claim 1, characterized in that, In step one, the mass ratio of the soft tissue of the aquatic product to the volume of methanol used for extraction is 1 g: 4.5 mL. The extraction conditions are: vortexing for 1 min, ultrasonic extraction at 25~30℃ for 10 min, and centrifugation at 4℃ and 7500 r / min for 10 min.

4. The method for analyzing lipid-soluble shellfish toxins based on solid-phase extraction using polystyrene nanofibers according to claim 1, characterized in that, In step two, the mass ratio of the polystyrene nanofibers packed into the solid-phase extraction column to the volume ratio of the reconstitution solution, the rinsing solution, and the elution solution is 10 mg: 1 mL: 1 mL: 0.2 mL.

5. The method for analyzing lipid-soluble shellfish toxins based on solid-phase extraction of polystyrene nanofibers according to claim 1, characterized in that, In step three, the liquid chromatography conditions also include: flow rate: 0.3 mL / min; column temperature: 40℃; injection volume: 2 μL.

6. The method for analyzing lipid-soluble shellfish toxins based on solid-phase extraction using polystyrene nanofibers according to claim 1, characterized in that, In step three, the mass spectrometry conditions are as follows: ionization mode: electrospray ionization mode (ESI); mass spectrometry scanning mode: multiple reaction monitoring (MRM); gas temperature: 300 °C; gas flow rate: 5 L / min; nebulizer gas pressure: 45 psi; sheath gas N2 temperature: 250 °C; sheath gas N2 flow rate: 11 L / min; Dell: 15 ms; capillary voltage: 3500 V.

7. The method for analyzing lipid-soluble shellfish toxins based on solid-phase extraction of polystyrene nanofibers according to claim 6, characterized in that, Mass spectrometry (MRM) was used to scan 12 lipid-soluble shellfish toxins. The quantitative ion pairs were: OA (okadaic acid) 827.4 / 723.4, GYM (cycloiminotoxin) 508.3 / 490.3, DTX1 (fin algae toxin 1) 841.5 / 737.5, AZA1 (protodinium alginate shellfish toxin 1) 842.5 / 824.5, DTX2 (fin algae toxin 2) 827.4 / 723.4, homopolymerized scallop toxin h-YTX1 1177.5 / 971.4, YTX (scallop toxin) 1163.5 / 957.2, AZA3 (protodinium alginate shellfish toxin 3) 828.4 / 810.4, PTX2 (scallop toxin 2) 876.4 / 823.4, and AZA2 (protodinium alginate shellfish toxin 2). 856.4 / 838.4, Jiangyaoqing toxin PnTX 694.4 / 164.0, 13-norspirolactone SPX1 692.3 / 674.

3.

8. The method for analyzing lipid-soluble shellfish toxins based on solid-phase extraction of polystyrene nanofibers according to claim 1, characterized in that, In step three, the external standard method is used for quantification, and the content of the target substance is obtained according to the standard curve as follows: S1. Dilute 200 μg / L of the mixed standard intermediate solution with methanol to prepare a series of standard solutions with concentrations of 100, 50, 20, 10, 5, 2, 1, 0.5, 0.2, and 0.1 μg / L. Perform liquid chromatography-tandem mass spectrometry (LC-MS / MS) to determine the concentrations of the target analytes. Plot a standard curve with the target analyte concentration on the x-axis and the corresponding peak area on the y-axis to obtain the corresponding linear regression equation. The mass concentration of each target analyte in the mixed standard intermediate solution is 200 μg / L. S2. Substitute the detection results from step three into the corresponding linear regression equation in S1 for calculation to obtain the content of 12 fat-soluble shellfish toxins.

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