Method for extracting and detecting microcystic toxins in water

Abstract

The invention discloses a method for extracting and detecting microcystic toxins in water, and belongs to the technical field of water quality detection.The method comprises the steps that cyclodextrin and a compound with a quaternary ammonium salt structure are polymerized, and a porous cyclodextrin polymer with cations is obtained and serves as a cation modified solid-phase extraction column; the covalently-bound and non-covalently-bound microcystic toxins are converted into free microcystic toxins through enzymolysis and the cation modified solid-phase extraction column, the recovery rate is high, and the detection accuracy is improved.

Description

Technical Field

[0001] This invention belongs to the field of water quality testing technology, specifically relating to a method for extracting and detecting microcystin in water. Background Technology

[0002] Microcystins are specific metabolic products of algae blooming in water. When microcystin cells die, they are released and pollute water bodies. One of the most significant hazards of cyanobacterial bloom pollution is the production and release of various algal toxins, primarily microcystins, in the water. Microcystins are currently known as the most toxic and harmful freshwater cyanobacterial toxins. Current analytical methods for microcystins in aquatic environments include liquid chromatography-mass spectrometry, liquid chromatography, and enzyme-linked immunosorbent assay (ELISA). The low concentrations and multiple variants of microcystins in aquatic environments necessitate appropriate sample processing (enrichment, concentration, purification, etc.) before instrumental analysis to ensure the accuracy and precision of the analytical results.

[0003] Solid-phase extraction (SPE), as a sample pretreatment technique for microcystin, relies on the differences in interactions between functional groups on a solid adsorbent and different components. It retains the target component on the adsorbent while removing interfering components. Although most microcystins are dissolved in water in a free state, they also exist in a bound state (bound to proteins, polysaccharides, or cell debris). Bound microcystins mainly form complexes with macromolecules through covalent or non-covalent binding, with protein complexes being the most prevalent. Currently, SPE only considers the free state in microcystin sample pretreatment, neglecting the capture of the bound state. This leads to an underestimation of the total toxin content, masking the actual toxicity risk. Furthermore, bound microcystins have low mobility, easily depositing in sediment or adsorbing onto suspended particles, prolonging their environmental persistence and forming a long-term toxic reservoir. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, the present invention provides a method for extracting microcystin from water. By enzymatic hydrolysis and cation exchange column chromatography, covalently bound and non-covalently bound microcystin are converted into a free state, resulting in high recovery rate and improved detection accuracy.

[0005] The technical solution for achieving the objective of this invention is as follows: A method for extracting microcystin from water, comprising the following steps:

[0006] S1. Water sample collection: After collecting the water sample, pass it through a filter membrane to remove suspended particles and collect the filtrate; place the resulting filter residue in water, centrifuge it, extract the supernatant, and combine it with the filtrate;

[0007] S2. Enzymatic hydrolysis: Add protease and pH buffer to the filtrate obtained in S1, adjust the pH to 7.0-8.0, shake for 3-4 hours, and then inactivate at 60-70℃ to obtain the sample to be extracted;

[0008] S3. Solid-phase extraction: Before use, the cationic modified solid-phase extraction column is activated with methanol and ultrapure water in sequence. The sample to be extracted obtained in S2 is passed through the cationic modified solid-phase extraction column, then washed with ultrapure water, and finally eluted with eluent. The eluent is concentrated and then diluted with ultrapure water. After passing through the filter membrane, the extraction is completed and the test solution is obtained.

[0009] The cationic modified solid-phase extraction column is packed with a cationic solid adsorbent; the preparation method of the cationic solid adsorbent is as follows:

[0010] (1) Preparation of quaternary ammonium salt cationic compound: 1 eq of 3,5-difluoropyridine was dispersed in acetonitrile, and then at least 1.5 eq of iodomethane was added. The mixture was reacted by microwave at a temperature of 100-120°C for at least 2 h. After cooling to room temperature, the mixture was concentrated, tetrahydrofuran was added, the mixture was filtered, the solid was collected, and washed with tetrahydrofuran at least twice. After drying, the quaternary ammonium salt cationic compound was obtained.

[0011] (2) Preparation of cationic solid adsorbent: Under inert gas protection, 1 eq of cyclodextrin, 2-4 eq of the quaternary ammonium salt cationic compound obtained in step (1) and 8-12 eq of potassium carbonate were weighed into a reaction vessel. A mixed solvent of tetrahydrofuran and N,N-dimethylformamide was added. After reacting at 80-90℃ for 1-2 days, the mixture was cooled and filtered. The solid was washed successively with 1-2 mol / L hydrochloric acid solution, water and tetrahydrofuran. After drying, the cationic solid adsorbent was obtained.

[0012] Preferably, the filter membrane in step S1 is a 0.45μm microporous filter membrane; the filter membrane in step S3 is a 0.22μm microporous filter membrane.

[0013] Preferably, the content of the protease is 1-2 mg / mL.

[0014] Preferably, the protease in step S2 is proteinase K, and the pH buffer is phosphate buffer.

[0015] Preferably, the cyclodextrin is one or more of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

[0016] Preferably, in step S3, the column flow rate of the sample to be extracted is 0.5–1.0 mL / min; the volume ratio of methanol to ultrapure water is 1:1, the flow rate is 1.0–1.5 mL / min, and the activation time is 4–8 min.

[0017] Preferably, the volume of ultrapure water used for rinsing is 3-8 mL; the eluent is a methanol solution containing ammonium hydroxide, the mass fraction of the ammonium hydroxide is 25-35 wt.%, the elution flow rate is 0.2-0.4 mL / min, and the elution time is 10-15 min.

[0018] Preferably, the mass of the cationic solid adsorbent in the cationic modified solid phase extraction column is (1 / 500) to (1 / 1500) of the mass of the water sample.

[0019] This application also discloses a method for detecting microcystin in water, which uses high performance liquid chromatography to analyze and detect the test solution; the test solution is the test solution obtained by the above-mentioned extraction method for microcystin in water.

[0020] Beneficial effects

[0021] This invention offers the following advantages: It provides a method for extracting microcystin from water, converting covalently and non-covalently bound microcystin into a free state through enzymatic hydrolysis and a cationic column, resulting in high recovery rates and improved detection accuracy. A porous cyclodextrin polymer with a quaternary ammonium salt structure is obtained by polymerizing cyclodextrin with the polymer. The porous structure of this polymer effectively adsorbs free microcystin, while the positively charged quaternary ammonium salt structure adsorbs negatively charged microcystin through electrostatic interactions, simultaneously repelling positively charged proteins, thus converting non-covalently bound microcystin to a free state. Furthermore, the introduction of a pyridine structure allows for π-π interactions with the benzene ring structure in microcystin, further enhancing recovery. Finally, the enzymatic hydrolysis step dissociates the covalently bound microcystin into a free state. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the synthesis route of the cationic solid adsorbent of the present invention;

[0023] Figure 2 The NMR spectrum of the quaternary ammonium salt cationic compound prepared in Example 1 of this invention;

[0024] Figure 3 The infrared spectrum of cyclodextrin and cationic solid adsorbent of this invention is shown. Detailed Implementation

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0027] The raw materials and equipment used in the embodiments and comparative examples are described below:

[0028] Proteinase K: enzyme activity ≥30u / mg, purchased from Yuanye Biotechnology;

[0029] γ-Cyclodextrin: purchased from Yuanye Biotechnology;

[0030] 3,5-Difluoropyridine: purchased from Aladdin Shanghai;

[0031] Iodomethane: Purchased from Yuanye Biotechnology;

[0032] MC-LR: Beijing Wokai Biotechnology Co., Ltd.

[0033] Phycocyanin: Purchased from Shanghai Maclean;

[0034] Water sample testing: Water samples were collected from parts of Taihu Lake.

[0035] Preparation Example 1

[0036] Quaternary ammonium salt cationic compound: 1 eq of 3,5-difluoropyridine was dispersed in acetonitrile, and then 2.0 eq of iodomethane was added. The mixture was reacted by microwave at 120 °C for 3 h. After cooling to room temperature, the mixture was concentrated, tetrahydrofuran was added, the mixture was filtered, the solid was collected, washed 3 times with tetrahydrofuran, and dried to obtain the quaternary ammonium salt cationic compound.

[0037] Preparation Example 2

[0038] Cationic solid adsorbent: Under nitrogen protection, 1 eq of γ-cyclodextrin, 3 eq of the quaternary ammonium salt cationic compound obtained in Preparation Example 1, and 10 eq of potassium carbonate were weighed into a reaction vessel. A mixed solvent of tetrahydrofuran and N,N-dimethylformamide in a volume ratio of 9:1 was added. After reacting at 90°C for 2 days, the mixture was cooled and filtered. The solid was washed successively with 1-2 mol / L hydrochloric acid solution, water, and tetrahydrofuran. After drying, the cationic solid adsorbent was obtained.

[0039] Preparation Example 3

[0040] The difference between the preparation method in Example 2 and the method in Preparation Example 2 is that the quaternary ammonium salt cationic compound is replaced with 3,5-difluoropyridine, thus obtaining a non-cationic solid adsorbent.

[0041] The following are the test methods for performance parameters involved in this invention:

[0042] Microcystin determination method: The examples and comparative examples were determined according to the high performance liquid chromatography method in GB / T20466-2006 "Determination of Microcystin in Water";

[0043] Spike recovery: Spiked solutions were prepared using examples and comparative examples, analyzed, and the recovery rate of free state was calculated using peak area;

[0044] Determination of bound state recovery: 0.25 mL of phycocyanin (1 mg / mL) was mixed with 0.5 mL of MC-LR (1 μg / L) and added to a 500 mL volumetric flask. The volume was then calibrated to 500 mL and incubated at 37 °C for 2 h to form a protein-MC complex. The final concentration of MC-LR was 1 μg / L and the final concentration of phycocyanin was 0.5 mg / L (simulating high-density algal bloom). The bound state was extracted using the extraction methods described in the examples and comparative examples and then determined by high performance liquid chromatography. The bound state recovery rate was calculated as: (Actual content measured by high performance liquid chromatography / (1 μg / L)) × 100%.

[0045] Example 1

[0046] A method for extracting microcystin from water, comprising the following steps:

[0047] S1. Water sample collection: After collecting water samples from the same area, pass them through a 0.45μm microporous membrane to remove suspended particles and collect the filtrate; place the resulting filter residue in water, centrifuge, extract the supernatant, and combine it with the filtrate;

[0048] S2. Enzymatic hydrolysis: Add proteinase K and phosphate buffer to the filtrate obtained in S1, adjust the pH to 7.0-8.0, shake at 37℃ for 3 hours, and then inactivate at 60℃ to obtain the sample to be extracted.

[0049] S3. Solid-phase extraction: Before use, the cationic modified solid-phase extraction column containing 80 mg of the cationic solid adsorbent obtained in Preparation Example 2 was activated sequentially with equal volumes of methanol and ultrapure water. 50 mL of the sample to be extracted obtained in S2 was weighed and passed through the cationic modified solid-phase extraction column at a flow rate of 0.5 mL / min. Then, it was washed with 5 mL of ultrapure water at a flow rate of 0.2 mL / min. Finally, it was eluted with a methanol solution containing 30 wt.% ammonium hydroxide at a flow rate of 0.3 mL / min for 10 min. After the eluent was concentrated, it was brought to a final volume of 0.5 mL with ultrapure water and passed through a 0.22 μm microporous membrane to complete the extraction and obtain the test solution.

[0050] Comparative Example 1

[0051] The difference between the extraction method in Example 1 and the method in Example 2 is that the cationic solid adsorbent obtained in step S3 of Preparation Example 2 is replaced with the non-cationic solid adsorbent obtained in Preparation Example 3.

[0052] Comparative Example 2

[0053] The difference between this method and the extraction method in Example 1 is that the enzymatic hydrolysis step S2 is not performed.

[0054] Comparative Example 3

[0055] The difference between the extraction method in Example 1 and the method in Example 2 is that the cationic solid adsorbent obtained in step S3 is replaced with a C18 solid phase extraction column.

[0056] Table 1 Summary of Sample Testing

[0057]

[0058] As shown in Table 1, Example 1 exhibits good spike recovery and bound state recovery. Comparative Example 1 shows that when the cationic solid adsorbent is replaced with a non-cationic solid adsorbent, both spike recovery and bound state recovery decrease. Comparative Example 2 shows that without the enzymatic hydrolysis step, the spike recovery decreases by 10.8% compared to Example 1, while the bound state recovery decreases significantly by 20.2%. Comparative Example 3 shows that the spike recovery and bound state recovery using the C18 solid-phase extraction column are lower than those of Example 1 and Comparative Example 1, indicating that its recovery and separation efficiency for both free and bound microcystin toxins are inferior to the cationic modified solid-phase extraction column prepared in this invention.

[0059] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for extracting microcystin from water, characterized in that, Includes the following steps: S1. Water sample collection: After collecting the water sample, pass it through a filter membrane to remove suspended particles and collect the filtrate; place the resulting filter residue in water, centrifuge it, extract the supernatant, and combine it with the filtrate; S2. Enzymatic hydrolysis: Add protease and pH buffer to the filtrate obtained in S1, adjust the pH to 7.0-8.0, shake for 3-4 hours, and then inactivate at 60-70℃ to obtain the sample to be extracted; S3. Solid-phase extraction: Before use, the cationic modified solid-phase extraction column is activated with methanol and ultrapure water in sequence. The sample to be extracted obtained in S2 is passed through the cationic modified solid-phase extraction column, then washed with ultrapure water, and finally eluted with eluent. The eluent is concentrated and then diluted with ultrapure water. After passing through the filter membrane, the extraction is completed and the test solution is obtained. The cationic modified solid-phase extraction column is packed with a cationic solid adsorbent; the preparation method of the cationic solid adsorbent is as follows: (1) Preparation of quaternary ammonium salt cationic compound: 1 eq of 3,5-difluoropyridine was dispersed in acetonitrile, and then at least 1.5 eq of iodomethane was added. The mixture was reacted by microwave at a temperature of 100-120°C for at least 2 h. After cooling to room temperature, the mixture was concentrated, tetrahydrofuran was added, the mixture was filtered, the solid was collected, and washed with tetrahydrofuran at least twice. After drying, the quaternary ammonium salt cationic compound was obtained. (2) Preparation of cationic solid adsorbent: Under inert gas protection, 1 eq of cyclodextrin, 2-4 eq of the quaternary ammonium salt cationic compound obtained in step (1) and 8-12 eq of potassium carbonate were weighed into a reaction vessel. A mixed solvent of tetrahydrofuran and N,N-dimethylformamide was added. After reacting at 80-90℃ for 1-2 days, the mixture was cooled and filtered. The solid was washed successively with 1-2 mol / L hydrochloric acid solution, water and tetrahydrofuran. After drying, the cationic solid adsorbent was obtained.

2. The method for extracting microcystin from water as described in claim 1, characterized in that, The filter membrane in step S1 is a 0.45μm microporous filter membrane; the filter membrane in step S3 is a 0.22μm microporous filter membrane.

3. The method for extracting microcystin from water as described in claim 1, characterized in that, The content of the protease is 1-2 mg / mL.

4. The method for extracting microcystin from water as described in claim 1, characterized in that, The protease in step S2 is proteinase K, and the pH buffer is phosphate buffer.

5. The method for extracting microcystin from water as described in claim 1, characterized in that, The cyclodextrin is one or more of α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin.

6. The method for extracting microcystin from water as described in claim 1, characterized in that, In step S3, the column flow rate of the sample to be extracted is 0.5–1.0 mL / min; the volume ratio of methanol to ultrapure water is 1:1, the flow rate is 1.0–1.5 mL / min, and the activation time is 4–8 min.

7. The method for extracting microcystin from water as described in claim 1, characterized in that, In step S3, the volume of ultrapure water used for rinsing is 3-8 mL; the eluent is a methanol solution containing ammonium hydroxide, the mass fraction of the ammonium hydroxide is 25-35 wt.%, the elution flow rate is 0.2-0.4 mL / min, and the elution time is 10-15 min.

8. The method for extracting microcystin from water as described in claim 1, characterized in that, In step S3, the mass of the cationic solid adsorbent in the cationic modified solid phase extraction column is (1 / 500) to (1 / 1500) of the mass of the water sample.

9. A method for detecting microcystin in water, characterized in that, The test solution was analyzed and detected by high performance liquid chromatography; the test solution was obtained by the extraction method of microcystin in water according to any one of claims 1 to 8.

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