Tandem solid-phase extraction and enrichment method for trace organic pollutants based on toxicity effect evaluation

Abstract

The invention discloses a solid-phase extraction method and a detection method for trace organic pollutants in a water body, and belongs to the field of environmental analysis. The method comprises the following steps: sequentially carrying out series solid-phase extraction on a water sample by virtue of activated HLB, WAX and MCX columns, respectively eluting by virtue of acetonitrile, ammonia water methanol and ammonia water methanol, and combining eluents to obtain a mixed extracting solution which is used for detecting trace organic pollutants. According to the method, through a specific column sequence and an elution scheme, the pollutant enrichment range (logKow-1. 47-9.53) is remarkably expanded, the average recovery rate is increased to 80% or above, and the problem of toxicity assessment distortion caused by pre-treatment missing detection and low recovery rate is solved from the source. Finally, the enrichment liquid is used for multi-terminal biotoxicity testing, and quantitative integration is carried out in combination with PCA analysis, so that accurate and quantitative evaluation of the comprehensive toxicity of the trace organic pollutants in the water body is realized, and the accuracy and reliability of the evaluation result are greatly improved.

Description

Technical Field

[0001] This invention belongs to the field of environmental analysis, and more specifically, relates to a tandem solid-phase extraction enrichment method for trace organic pollutants based on toxicity effect assessment. Background Technology

[0002] With the rapid development of industrialization, urbanization, and agricultural modernization, large quantities of chemicals are discharged into water bodies. Many of these pollutants, even at extremely low concentrations (such as ng / L or μg / L), can have serious impacts on ecosystems and human health. These pollutants, existing at low concentrations but still posing potential hazards, are called trace organic pollutants (TOPs). TOPs are diverse, mainly including persistent organic pollutants (POPs), pharmaceuticals, personal care products (PPCPs), pesticides, and herbicides. These pollutants are often characterized by their insidious nature, high toxicity, and difficulty in degradation; even at trace levels, they can cause health risks such as endocrine disruption, carcinogenicity, teratogenicity, and immunotoxicity.

[0003] Due to the extremely low concentrations of trace organic pollutants, traditional detection methods often fail to meet the requirements of sensitivity and selectivity. Therefore, developing highly sensitive, selective, rapid, and low-cost detection technologies has become crucial for environmental monitoring and public health protection. Chinese Patent Publication No. CN1828290A discloses a method for online solid-phase extraction-liquid chromatography (SPE-LC) analysis of trace organic matter in water. Chinese Patent Publication No. CN107389410A discloses a method for the classification, extraction, and detection of trace organic pollutants in water. The solid-phase extraction step in these methods can only enrich pollutants with high log Kow (lipophilic). For strongly acidic and strongly alkaline pollutants with low log Kow (hydrophilic), they are almost completely ionized at any pH and cannot be effectively retained by SPE columns, such as octadecyl-bonded silica columns (C18 columns), leading to serious missed detections. This is one of the reasons for the inaccurate overall toxicity assessment results of trace organic pollutants in water. Another reason is that even if organic pollutants are enriched by solid-phase extraction, they are not completely enriched, resulting in a low recovery rate.

[0004] The high false negative rate and low recovery rate of solid-phase extraction lead to inaccurate assessments of the toxic effects of trace organic pollutants in water. Given the increasingly complex water pollution situation, improving the accuracy of overall toxicity assessments of trace organic pollutants in water is a pressing technical problem that needs to be solved. Summary of the Invention

[0005] 1. The problem to be solved To address the issue that the accuracy of overall toxicity assessment results for trace organic pollutants in existing technologies needs improvement, this invention provides a tandem solid-phase extraction enrichment method for toxicity effect assessment of trace organic pollutants. By expanding the scope of solid-phase extraction enrichment of trace organic pollutants and increasing the recovery rate of trace organic pollutants by solid-phase extraction, the accuracy of overall toxicity assessment results for trace organic pollutants can be improved.

[0006] 2. Technical Solution To solve the above problems, the technical solution adopted by the present invention is as follows: [A Tandem Solid-Phase Extraction Enrichment Method for Trace Organic Pollutants Based on Toxicity Effect Assessment] The first aspect of this invention provides a method for the tandem solid-phase extraction enrichment of trace organic pollutants based on toxicity effect assessment, comprising the following steps: First, water containing trace amounts of organic pollutants was subjected to solid-phase extraction using an activated HLB column, with acetonitrile as the eluent, to obtain eluent A. The water body after HLB column solid-phase extraction is then subjected to solid-phase extraction on an activated WAX column, with methanol containing 1% to 3% ammonia by mass as the eluent to obtain eluent B. Then, the water after solid-phase extraction on the WAX ​​column was subjected to solid-phase extraction on an activated MCX column, with methanol containing 1% to 3% ammonia by mass as the eluent, to obtain eluent C. Finally, eluent A, eluent B and eluent C are mixed to obtain a mixed extract.

[0007] It should be noted that 1% to 3% ammonia methanol is prepared by mixing ammonia water and methanol. Specifically, it can be prepared by mixing 25% ammonia water and methanol.

[0008] This invention utilizes a specific series sequence of solid-phase extraction columns. First, the sample is uniformly regulated under acidic pH conditions, establishing a precise foundation for ion state control throughout the extraction system. Then, an HLB column is used in this pre-acidified environment to preferentially capture all nonpolar and neutral compounds, preventing them from clogging downstream ion exchange columns, saturating their active sites, causing interference or overload, and affecting the retention of target ions by the downstream ion exchange columns. Next, a WAX column is used in the established acidic environment to fully protonate its exchange groups, efficiently capturing acidic compounds in their optimal state, ensuring the selectivity and capacity of weak anion exchange. Finally, an MCX column, in a dedicated acidic environment consistent with its activation state, powerfully captures basic compounds, and its elution mechanism does not interfere with the upstream column, achieving efficient separation and purification of three classes of compounds with vastly different properties.

[0009] Moreover, this tandem solid-phase extraction sequence maximizes the specificity of each SPE column, resolving the core contradiction of conflicting optimal pH conditions required for different types of pollutants, thereby achieving one-time, complete, and highly efficient enrichment of pollutants with a wide polarity and a wide pKa range.

[0010] Furthermore, this invention incorporates the pKa index as a key consideration, utilizing the ion exchange mechanism to select anion exchange columns (WAX columns) and cation exchange columns (MCX columns) to capture strongly polar ionic compounds, avoiding penetration of the solid-phase extraction column and reducing the false detection rate to near zero. Simultaneously, "reverse pH adjustment" based on the pKa index disrupts the ionic bonds between the ion exchange column and the target analyte. Through the attraction between positive and negative ions, the eluent easily and quantitatively elutes the target analyte from the ion exchange column, achieving a high recovery rate. Specifically, when eluting the WAX ​​column, ammonia and methanol are used as the eluent to create a high ionic strength environment, neutralizing the adsorbent with quaternary ammonium functional groups, causing the adsorbent to change from a charged state to neutral, thereby inactivating it and releasing the target pollutant. The high ionic strength environment facilitates elution through competitive exchange sites. When eluting the MCX column, ammonia and methanol are used as the eluent to create a high ionic strength environment, providing OH- - Protonated alkaline pollutants are released from their neutral molecular form, and the high ionic strength environment facilitates elution through competitive exchange sites.

[0011] As a preferred embodiment of any technical solution in the first aspect of the present invention, the pH of the water containing trace organic pollutants is adjusted to 4-6 before solid-phase extraction.

[0012] As a preferred embodiment of any technical solution in the first aspect of the present invention, the activation method of the HLB column is to use dichloromethane and methanol as activation solutions and control the activation flow rate to be 5~15 mL / min. The activation method of the WAX ​​column is to use methanol containing 1% to 3% ammonia by mass as the activation solution, and control the activation flow rate to be 5 to 15 mL / min. The activation method for the MCX column is to use an aqueous solution of acetic acid with a mass percentage of 0.01%~0.9% and methanol as the activation solution, and control the activation flow rate to be 5~15 mL / min.

[0013] The solid-phase extraction method provided by this invention, by setting activation conditions, is beneficial to fully utilize the characteristics of each solid-phase extraction column, thereby expanding the extraction range and recovery rate of trace organic pollutants.

[0014] As a preferred embodiment of any technical solution in the first aspect of the present invention, the solid-phase extraction method using the HLB column includes the following steps: (1) Sample loading: Flow water containing trace amounts of organic pollutants through the HLB column, controlling the flow rate to be 1~10 mL / min; (2) Washing: After the sample loading is completed, add 1~5 mL of ultrapure water to wash the HLB column, and dry it with nitrogen for 10~30 min after washing; (3) Elution: Use 2.5~3.5 mL of acetonitrile as the eluent and elute twice at a flow rate of 0.5~2 mL / min. When eluting HLB columns, acetonitrile is used as the eluent because it has sufficient elution power to break the hydrophobic interaction between the target analyte and the HLB column stationary phase, and it will not cause swelling or contamination of the stationary phase due to excessive elution power. More importantly, acetonitrile has broad-spectrum contaminant compatibility, making it the best choice for HLB column eluent and reducing the false negative rate of HLB columns.

[0015] As a preferred embodiment of any technical solution in the first aspect of the present invention, the solid-phase extraction method using the WAX ​​column includes the following steps: (1) Sample loading: The water after HLB solid phase extraction is passed through a WAX column and the flow rate is controlled at 1~10 mL / min; (2) Washing: After the sample loading is completed, add 1~5 mL of ultrapure water to wash the WAX ​​column, and dry it with nitrogen for 10~30 min after washing; (3) Elution: Use 2.5~3.5 mL of methanol containing 1%~3% ammonia by mass as the eluent and elute twice at a flow rate of 0.5~2 mL / min.

[0016] During the elution of the WAX ​​column, "reverse pH adjustment" is performed based on the pKa index. Ammonia and methanol are used as the eluent to create a high ionic strength environment, which neutralizes the adsorbent with quaternary ammonium salt functional groups, causing the adsorbent to change from a charged state to a neutral state, thereby deactivating it and releasing the target pollutant. At the same time, the high ionic strength environment is conducive to elution through competitive exchange sites, promoting the easy quantitative elution of the target pollutant from the ion exchange column by the eluent, resulting in a high recovery rate.

[0017] As a preferred embodiment of any technical solution in the first aspect of the present invention, the solid-phase extraction method of the MCX column includes the following steps: (1) Sample loading: The water after solid phase extraction on the WAX ​​column is passed through the MCX column and the flow rate is controlled at 1~10 mL / min; (2) Washing: After the sample loading is completed, add 1~5 mL of ultrapure water to wash the MCX column, and dry it with nitrogen for 10~30 min after washing; (3) Elution: Use 2.5~3.5 mL of methanol containing 1%~3% ammonia by mass as the eluent and elute twice at a flow rate of 0.5~2 mL / min.

[0018] During the elution of the MCX column, "reverse pH adjustment" is performed based on the pKa index. Ammonia and methanol are used as the eluent to create a high ionic strength environment, which breaks the ionic bonds between the anion exchange column and the target analyte and provides OH-. - Protonation of alkaline pollutants transforms them from cationic to neutral molecules, thereby causing them to lose their positive charge and inactivating their ion exchange interaction with the MCX column, releasing them from the MCX column. At the same time, the high ion intensity environment facilitates elution through competitive exchange sites, promoting the easy quantitative elution of the target analyte from the ion exchange column by the eluent, resulting in a high recovery rate.

[0019] The above-mentioned tandem solid-phase extraction sequence helps to reduce the influence of different activating and eluent solutions on the three solid-phase extraction columns, reduce the mutual influence of the three solid-phase extraction columns in actual use, and help to achieve one-time, complete, and highly efficient enrichment of pollutants with a wide polarity and a wide pKa range.

[0020] As a preferred embodiment of any technical solution in the first aspect of the present invention, the concentration of trace organic pollutants in the water body is 1 ng / L to 100 μg / L.

[0021] Preferably, the concentration of trace organic pollutants in the water body is 10 ng / L to 1 μg / L, more preferably 40 ng / L to 500 ng / L, and most preferably 50 ng / L to 200 ng / L. For example, it can be 60 ng / L, 70 ng / L, 80 ng / L, 90 ng / L, 110 ng / L, 120 ng / L, 130 ng / L, 140 ng / L, 150 ng / L, 160 ng / L, 170 ng / L, 180 ng / L, or 190 ng / L.

[0022] [Detection methods for assessing the toxic effects of trace organic pollutants] The second aspect of the present invention provides a detection method for assessing the toxic effects of trace organic pollutants. The method uses the solid-phase extraction enrichment method for assessing the overall toxicity of trace organic pollutants in water provided in the first aspect of the present invention to enrich the trace organic pollutants, and uses gas chromatography-mass spectrometry and / or liquid chromatography-mass spectrometry to detect the mixed extract.

[0023] Methods for assessing the toxic effects of trace organic pollutants The third aspect of the present invention provides a method for assessing the toxic effects of trace organic pollutants, which uses the tandem solid-phase extraction enrichment method for trace organic pollutants based on toxicity effect assessment provided in the first aspect of the present invention to enrich the trace organic pollutants and then evaluate them.

[0024] As a preferred embodiment of any technical solution in the third aspect of the present invention, the overall toxicity of trace organic pollutants in the water body is evaluated by testing the mixed extract using the acute toxicity test of luminescent bacteria, the acute toxicity test of CCK-8, and the endocrine disruption toxicity test of yeast. The results of the quantitative toxicity PCA analysis are then integrated to evaluate the comprehensive toxicity of trace organic pollutants in the water body. The quantitative toxicity PCA analysis includes the following steps: A. Define variables in the variable view of SPSS software and enter the data into the data view of SPSS software; B. Perform Z-score standardization preprocessing on the data; C. Perform principal component analysis and calculate the overall toxicity score.

[0025] The method for assessing the toxic effects of trace organic pollutants provided by this invention, compared to most existing studies that only assess recovery rates, truly reflects its practical application value. Furthermore, compared to the ranking and comparison methods for toxicity indicators in the literature, this method is quantitatively persuasive and uses mathematical methods (such as principal component analysis, PCA) to synthesize multidimensional parameters into a comprehensive toxicity score, thus quantitatively demonstrating the advantages of this invention.

[0026] 3. Beneficial effects Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The tandem solid-phase extraction enrichment method for trace organic pollutants based on toxicity effect assessment provided by this invention introduces pKa as a key property to consider, selects specific solid-phase extraction columns, and performs solid-phase extraction enrichment of pollutants in the water body to be tested in a specific order of HLB column, WAX column, and MCX column, thereby expanding the range of solid-phase extraction enrichment of trace organic pollutants, expanding the coverage range of logKow from -1.1~4.26 to -1.47~9.53, significantly expanding the coverage range, and expanding the enriched trace organic pollutants from several simple organic pollutants of the same category in the prior art to 72 different substances such as phenols, amines, nitrobenzenes, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, antibiotics, and perfluorinated compounds, thus expanding the application range, avoiding the missed detection of extreme and polar substances, and improving the average recovery rate of solid-phase extraction for trace organic pollutants to more than 80%.

[0027] (2) In this invention, pollutants are enriched by solid phase extraction of the water body to be tested in a specific order of HLB column, WAX column and MCX column. The average recovery rate is significantly higher than that of two-column SPE column, slightly lower than that of four-column SPE column solid phase extraction, and significantly higher than that of other three-column SPE column. This is beneficial to improve the accuracy of the detection results of the overall toxicity assessment of trace organic pollutants in water.

[0028] (3) The present invention uses acetonitrile to elute the HLB column and ammonia and methanol to elute the WAX ​​and MCX columns. Trace organic pollutants are eluted according to logKow and pKa, and a variety of trace organic pollutants of different polarities are enriched from the water body to be tested, so as to obtain the types and contents of trace organic pollutants in the water body more comprehensively.

[0029] (4) The detection method for assessing the toxic effects of trace organic pollutants provided by the present invention significantly improves the accuracy of the overall toxicity assessment results of trace organic pollutants in water by improving the false negative rate and recovery rate of the solid phase extraction enrichment method for trace organic pollutants in water.

[0030] (5) The method for overall toxicity assessment of trace organic pollutants in water provided by the present invention covers the main risks of new pollutants by selecting a multi-toxic endpoint detection design, using acute toxicity to correspond to industrial accident exposure, using endocrine toxicity to correlate with estrogen-like substances, and using PCA dimensionality reduction analysis and comparison to quantitatively assess the overall toxicity of trace organic pollutants in water. Attached Figure Description

[0031] Figure 1 The bar chart shows the PC1 loading of different elution solutions on the WAX ​​column in Comparative Example 1. Figure 2 The bar chart shows the loading of PC1 with different elution solutions on the MCX column in Comparative Example 2. Figure 3 Bar chart showing the loading of PC1 with different elution solutions on the HLB column in Comparative Example 3; Figure 4 The bar chart shows the average recovery rate of the WAX ​​column eluent at different volumes in Comparative Example 4. Figure 5 The bar graph shows the average recovery rate of the MCX column eluent at different volumes in Comparative Example 5. Figure 6 The bar graph shows the average recovery rate of the HLB column eluent at different volumes in Comparative Example 6. Figure 7 The bar chart shows the average recovery rate of the MIX column at different pH values ​​for the simulated water sample in Comparative Example 7. Figure 8 The polarity distribution of trace organic pollutants enriched in different SPE column systems of Comparative Example 8; Figure 9 This is a graph showing the recovery rate distribution of different trace organic pollutants on the MIX column under different concentrations of trace organic pollutants in Comparative Example 9. Figure 10 This is a bar chart showing the average recovery rates of different tandem SPE columns in Example 5; Figure 11A bar chart showing the inhibition rate of luminescent bacteria in Test Example 1; Figure 12 A bar chart showing the concentration factor of Test Example 2; Figure 13 The E2 standard curve for the two-hybrid yeast dry powder kit in test example 3; Figure 14 A mixed bar graph of E2 equivalent and galactosidase activity in test example 3; Figure 15 A bar chart showing the overall toxicity scores for different SPE column systems.

[0032] Note: Unless otherwise specified, "HLB" in the attached diagram refers to Waters HLB columns, all of which are NDAM-HLB columns. Detailed Implementation

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the term “and / or” as used herein includes any and all combinations of one or more of the associated listed items.

[0034] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0035] As used herein, the term “about” is used to provide for the flexibility and imprecision associated with a given term, measure, or value. Those skilled in the art can readily determine the degree of flexibility for a particular variable.

[0036] Concentration, amount, and other numerical data may be presented in range format herein. It should be understood that such range format is used solely for convenience and brevity and should be flexibly interpreted to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or subranges encompassed within the range, as if each value and subrange were explicitly stated. For example, a range of values ​​from about 1 to about 4.5 should be interpreted to include not only the explicitly stated limits of 1 to 4.5, but also individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges that describe only a single value, such as "less than about 4.5," which should be interpreted to include all the values ​​and ranges described above. Furthermore, this interpretation should apply regardless of the breadth of the range or characteristic described.

[0037] It should be noted that the simulated water samples used in Examples 1-5 and Comparative Examples 1-6, 8, and 9 were prepared by ourselves. The simulated water samples had a pH of 4 and contained 72 pollutants as described in Table 1, with a concentration of 100 ng / L for each pollutant.

[0038] Table 1. Pollutant-Solid Phase Extraction Column Structure-Activity Relationship Database

[0039] It should be noted that the fully automated solid phase extraction instrument used in the embodiments and test examples of this invention is the Autotrace 280.

[0040] The CCK-8 cell proliferation assay kit was purchased from Jiangsu Kaiji Biotechnology Co., Ltd., the ELISA reader (Synergy H4) was manufactured by Bertek Instruments, Inc., USA, and the two-hybrid yeast dry powder kit was purchased from the Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences.

[0041] It should be noted that the gas chromatography-mass spectrometry (GC-LC-MS) instruments used in Examples 4-5 and Comparative Examples 1-9 were triple quadrupole gas chromatography-mass spectrometry systems (Agilent 7890B–7000D). Specific test parameters are shown in Tables 2 and 3. Table 2 Gas Chromatographic Conditions for Different Types of Trace Organic Pollutants

[0042] Table 3 Mass spectrometry conditions for different types of trace organic pollutants

[0043] In Examples 4-5 and Comparative Examples 1-9, the instrument used for HPLC-MS detection was an ultra-high performance liquid chromatography-triple quadrupole mass spectrometer (UPLC-Class & Xevo TQ-MS), and the specific test parameters are as follows: (1) Perfluorinated compounds (PFAS) Chromatographic column: Agilent C18 column (4.6 mm × 150 mm, 4 μm); column temperature: 35℃; mobile phase: A: 2 mmol / L ammonium acetate aqueous solution, B: acetonitrile; flow rate: 0.4 mL / min; mobile phase gradient as follows:

[0044] Mass spectrometry conditions: Ion source: Electrospray negative ion source (ESI-); Monitoring mode: Multiple reaction monitoring (MRM); Mass spectrometry parameters: Negative ion mode; Gas curtain pressure 35.0 psi; Spray voltage -4500 V; Nebulization temperature 550 ℃; Nebulizing gas pressure 55 psi; Auxiliary gas pressure 60 psi.

[0045] (2) Antibiotics Chromatographic column: Agilent C18 column (4.6 mm × 150 mm, 4 μm); column temperature: 35℃; mobile phase: A: 0.1% formic acid aqueous solution, B: methanol; flow rate: 0.4 mL / min; mobile phase gradient as follows:

[0046] Mass spectrometry conditions: Ion source: electrospray negative ion source (ESI-); Monitoring mode: multiple reaction monitoring (MRM); Mass spectrometry parameters: capillary voltage 3kV; nebulizing gas pressure 1 bar; drying gas flow rate 6 mL / min, drying gas temperature 300 ℃, ion transport time: 60 μs.

[0047] (3) Endocrine disruptors (EDs) Chromatographic column: Agilent C18 column (4.6 mm × 150 mm, 4 μm); column temperature: 35℃; mobile phase: A: methanol, B: ultrapure water; flow rate: 0.4 mL / min; mobile phase gradient as follows:

[0048] Mass spectrometry conditions: Ion source: electrospray positive ion source (ESI+); Monitoring mode: multiple reaction monitoring (MRM); Ion source temperature: 500 ℃; Ion source voltage: 5500 V; Collision gas: Medium; Curtain gas: 35 psi; Auxiliary heating gas: 50 psi.

[0049] The present invention will be further described below with reference to specific embodiments.

[0050] Experimental materials The anion exchange column (WAX column) was an NDAM-WAX column, the cation exchange column (MCX column) was an NDAM-MCX column, and the hydrophilic-lipophilic balanced column (HLB column) was an NDAM-HLB column, all purchased from Jiangsu Guochuang Environmental Protection Technology Co., Ltd.; the octadecyl bonded silica column (C18 column) was a Coconut C18 column, and the magnesium silicate solid phase extraction column (Florisil column) was purchased from Waters Corporation; the other hydrophilic-lipophilic balanced column (HLB column) was a Waters HLB column, purchased from Waters Corporation.

[0051] Methanol, 0.1% acetic acid aqueous solution, dichloromethane (DCM), acetonitrile, ethyl acetate (EAC), ultrapure water, and nitrogen were all commercially available. The 2% ammonia-methanol solution was prepared by mixing 25% ammonia with methanol.

[0052] Example 1 This embodiment describes a solid-phase extraction method using an anion exchange column (WAX column), and the specific method includes the following steps: 1. Activation: Using a WAX column as the solid phase extraction column (SPE column) of the fully automated solid phase extraction instrument, 6 mL of 2% ammonia water and methanol and 6 mL of methanol were added sequentially as activation solutions, and the activation flow rate was controlled at 10 mL / min. 2. Sample loading: Flow 1 L of simulated water sample through the SPE column at a constant flow rate of 5 mL / min; 3. Washing: After loading the sample, add 3 mL of ultrapure water to wash the SPE column, and dry it with nitrogen for 20 min after washing. 4. Elution: Use 3 mL of 2% ammonia and methanol as the eluent to elute the compound at a flow rate of 1 mL / min. After elution twice, collect and mix the two eluents.

[0053] 5. Set up three parallel samples.

[0054] Example 2 This embodiment describes a solid-phase extraction method using a cation exchange column (MCX column), and the specific method includes the following steps: 1. Activation: Using an MCX column as the SPE column of an automated solid phase extraction instrument, 6 mL of 0.1% acetic acid aqueous solution and 6 mL of methanol were added sequentially as activation solutions, and the activation flow rate was controlled at 10 mL / min. 2. Sample loading: Flow 1 L of simulated water sample through the SPE column at a constant flow rate of 5 mL / min; 3. Washing: After loading the sample, add 3 mL of ultrapure water to wash the SPE column, and dry it with nitrogen for 20 min after washing. 4. Elution: Use 3 mL of 2% ammonia and methanol as the eluent to elute the compound at a flow rate of 1 mL / min. After elution twice, collect and mix the two eluents.

[0055] 5. Set up three parallel samples.

[0056] Example 3 This embodiment describes a solid-phase extraction method using a hydrophilic-lipophilic equilibrium column (HLB column), which includes the following steps: 1. Activation: Using an NDAM-HLB column as the SPE column of an automated solid phase extraction instrument, 6 mL of dichloromethane (DCM) and 6 mL of methanol were added sequentially as activation solutions, and the activation flow rate was controlled at 10 mL / min. 2. Sample loading: Flow 1 L of simulated water sample through the SPE column at a constant flow rate of 5 mL / min; 3. Washing: After loading the sample, add 3 mL of ultrapure water to wash the SPE column, and dry it with nitrogen for 20 min after washing. 4. Elution: Use 3 mL of acetonitrile as the eluent to elute the compound at a flow rate of 1 mL / min. After elution twice, collect and mix the two eluents.

[0057] 5. Set up three parallel samples.

[0058] Comparative Example 1 This comparative example focuses on the selection of WAX column eluents. The solid-phase extraction method differs from that in Example 1 only in that dichloromethane (DCM), ethyl acetate (EAC), and methanol are used instead of 2% ammonia and methanol as eluents for solid-phase extraction, with three parallel samples prepared for each eluent.

[0059] The mixed eluents obtained after solid-phase extraction using dichloromethane (DCM), ethyl acetate (EAC), methanol, and 2% ammonia-methanol as eluents were analyzed by gas chromatography-mass spectrometry (GC-LC-MS) and high-performance liquid chromatography-mass spectrometry (HPLC-MS), respectively. All data were then analyzed using PCA in Origin software to calculate the average loading of each eluent. The PC1 loading of the WAX ​​column eluents is shown below. Figure 1 As shown, 2% ammonia and methanol are preferred as the eluent for the WAX ​​column.

[0060] Comparative Example 2 This comparative example illustrates the selection of eluent for the MCX column. The solid-phase extraction method differs from that in Example 2 only in that dichloromethane (DCM), ethyl acetate (EAC), and methanol are used instead of 2% ammonia and methanol as eluents for solid-phase extraction, with three parallel samples prepared for each eluent.

[0061] The mixed eluents after solid-phase extraction using dichloromethane (DCM), ethyl acetate (EAC), methanol, and 2% ammonia-methanol as eluents were analyzed by gas chromatography-mass spectrometry (GC-LC-MS) and high-performance liquid chromatography-mass spectrometry (HPLC-MS), respectively. All data were analyzed using PCA in Origin software to calculate the average loading of each eluent. The PC1 loading of the MCX column eluent is shown below. Figure 2 As shown, 2% ammonia-methanol is preferred as the eluent for the MCX column.

[0062] Comparative Example 3 This comparative example illustrates the selection of eluent for the NDAM-HLB column. The solid-phase extraction method differs from that in Example 3 only in that dichloromethane (DCM), ethyl acetate (EAC), and methanol are used instead of acetonitrile for solid-phase extraction, with three parallel samples prepared for each eluent.

[0063] The mixed eluents obtained after solid-phase extraction using dichloromethane (DCM), ethyl acetate (EAC), methanol, and acetonitrile as eluents were analyzed by gas chromatography-mass spectrometry (GC-LC-MS) and high-performance liquid chromatography-mass spectrometry (HPLC-MS), respectively. All data were then analyzed using PCA in Origin software to calculate the average loading of each eluent. The PC1 loading of the WAX ​​column eluent is shown below. Figure 3 As shown, acetonitrile is preferred as the eluent for the HLB column.

[0064] Comparative Example 4 This comparative example is an optimization of the elution volume of the WAX ​​column elution buffer. The solid-phase extraction method in the optimization method differs from that in Example 1 only in that solid-phase extraction is performed twice with elution buffers of 2 mL, 3 mL, 4 mL, and 5 mL, respectively, and three parallel samples are set for each elution buffer volume.

[0065] The mixed eluents after solid-phase extraction of each eluent volume were analyzed by gas chromatography-mass spectrometry (GC-LC-MS) and high-performance liquid chromatography-mass spectrometry (HPLC-MS), and the average recovery rate was calculated. The average recovery rate of the WAX ​​column is as follows: Figure 4 As shown, the optimal elution volume for a WAX column using 2% ammonia and methanol as the eluent is 3 mL × 2.

[0066] Comparative Example 5 This comparative example optimizes the elution volume of the MCX column eluent. The difference between the solid-phase extraction method in the optimized method and that in Example 2 is that solid-phase extraction is performed twice with elution volumes of 2 mL, 3 mL, 4 mL, and 5 mL, respectively, and three parallel samples are set for each eluent volume.

[0067] The mixed eluents after solid-phase extraction of each eluent volume were analyzed by gas chromatography-mass spectrometry (GC-LC-MS) and high-performance liquid chromatography-mass spectrometry (HPLC-MS), and the average recovery rate was calculated. The average recovery rate of the MCX column is as follows: Figure 5 As shown, the optimal elution volume for the MCX column using 2% ammonia and methanol as the eluent is 3 mL × 2.

[0068] Comparative Example 6 This comparative example optimizes the elution volume of the NDAM-HLB column eluent. The solid-phase extraction method in the optimized method differs from that in Example 3 only in that solid-phase extraction is performed twice with elution volumes of 2 mL, 3 mL, 4 mL, and 5 mL, respectively, with three parallel samples for each eluent volume.

[0069] The mixed eluents after solid-phase extraction of each eluent volume were analyzed by gas chromatography-mass spectrometry (GC-LC-MS) and high-performance liquid chromatography-mass spectrometry (HPLC-MS), and the average recovery rate was calculated. The average recovery rate of the HLB column is as follows: Figure 6 As shown, the optimal elution volume for an HLB column using 2% ammonia and methanol as the eluent is 3 mL × 2.

[0070] Example 4 This embodiment describes a tandem SPE column solid-phase extraction method, including the following steps: One L of simulated water sample was continuously passed through an NDAM-HLB column, a WAX column, and an MCX column in sequence (denoted as the MIX column), and solid-phase extraction was performed according to the solid-phase extraction methods of Examples 3, 1, and 2, respectively. The resulting three mixed eluents were mixed again and detected by gas chromatography-mass spectrometry (GC-LC-MS) and liquid chromatography-mass spectrometry (HPLC-MS). The recovery rates of each contaminant and the average recovery rate of the MIX column were calculated.

[0071] Comparative Example 7 This comparative example measures the average recovery rate of the MIX column at different pH values ​​of simulated water samples. The only difference from Example 4 is that 1 mol / L HCl or 1 mol / L NaOH was added dropwise to 1 L of simulated water sample, adjusting the pH of the 1 L simulated water sample to 2, 6, 8, 10, and 12 respectively, to obtain the average recovery rate of the MIX column at different pH values ​​of the simulated water sample. Figure 7 As shown, the optimal pH for the simulated water sample during MIX column solid-phase extraction is 4-6.

[0072] Comparative Example 8 This comparative example demonstrates solid-phase extraction of pollutants from simulated water samples using different SPE columns. The only difference from Example 3 is the use of Coconut C18, Florisil, and Waters HLB columns instead of the NDAM-HLB column for solid-phase extraction. The mixed eluents from different SPE columns were analyzed by gas chromatography-mass spectrometry (GC-LC-MS) and high-performance liquid chromatography-mass spectrometry (HPLC-MS), and the recoveries were calculated. Substances with recoveries greater than or equal to 80% were selected, and their polarities were determined. These results, combined with those from Examples 3 and 4, form a distribution map of the polarity range of pollutants enriched in different SPE column systems. Figure 8 As shown.

[0073] from Figure 8 As can be seen, the triple SPE column (MIX column) solid phase extraction method can enrich more types of pollutants and has a wider polarity range.

[0074] Comparative Example 9 This comparative example demonstrates solid-phase extraction of pollutants at different concentrations using a MIX column. The only difference from Example 4 is that the concentration of each pollutant in the simulated water sample was diluted to 20 ng / L and 50 ng / L, respectively, to obtain the recovery rate and average recovery rate of each pollutant using the MIX column at different concentrations. Figure 9 As shown, when the pollutant concentrations are 20 ng / L, 50 ng / L, and 100 ng / L, the average recoveries of the MIX column can reach 75.55%, 79.00%, and 80.83%, respectively.

[0075] from Figure 9 As can be seen, the higher the concentration of the pollutant to be enriched, the higher the average recovery rate of the MIX column, and the smaller the fluctuation in recovery rate between individual pollutants.

[0076] Example 5 This embodiment describes solid-phase extraction methods using different tandem SPE columns.

[0077] Dual-column solid-phase extraction (HLB+C18): 1 L of simulated water sample was sequentially passed through an NDAM-HLB column and a Coconut C18 column, and solid-phase extraction was performed according to the solid-phase extraction method in Example 3. The resulting two mixed eluents were mixed again and detected by gas chromatography-mass spectrometry (GC-LC-MS) and liquid chromatography-mass spectrometry (HPLC-MS). The recoveries of each pollutant and the average recoveries of the tandem SPE columns were calculated.

[0078] Triple SPE column solid-phase extraction (C18+WAX+MCX): 1 L of simulated water sample was continuously passed through a Coconut C18 column, a WAX column, and an MCX column in sequence, and solid-phase extraction was performed according to the solid-phase extraction methods of Examples 3, 1, and 2, respectively. The resulting three mixed eluents were mixed again and detected by gas chromatography-mass spectrometry (GC-LC-MS) and liquid chromatography-mass spectrometry (HPLC-MS). The recoveries of each pollutant and the average recoveries of the tandem SPE columns were calculated.

[0079] Quadruple SPE column solid-phase extraction (HLB+C18+WAX+MCX): 1 L of simulated water sample was continuously passed through NDAM-HLB column, Coconut C18 column, WAX column, and MCX column in sequence, and solid-phase extraction was performed according to the solid-phase extraction methods of Examples 3, 3, 1, and 2, respectively. The resulting four mixed eluents were mixed again and detected by gas chromatography-mass spectrometry (GC-LC-MS) and liquid chromatography-mass spectrometry (HPLC-MS). The recovery rates of each pollutant and the average recovery rate of the tandem SPE columns were calculated.

[0080] The above test results are compared with the test results of Example 4 in Table 4 and Figure 10As shown.

[0081] Table 4. Recovery and average recovery of solid-phase extraction with different tandem SPE columns

[0082] From Table 2 and Figure 10 It can be seen that the average recovery rate of solid-phase extraction using a triple SPE column (MIX column) is significantly higher than that of a two-column SPE column; while the average recovery rate of solid-phase extraction using a quadruple SPE column is not significantly higher than that of a triple SPE column (MIX column); furthermore, even using a triple SPE column, the average recovery rate of solid-phase extraction using the MIX column is significantly higher than that of solid-phase extraction using a C18+WAX+MCX tandem column. In conclusion, considering cost and experimental accuracy, solid-phase extraction using a triple SPE column (MIX column) is chosen for the enrichment and assessment of toxic pollutants.

[0083] Example 6 A structure-activity relationship (SPR) database of 72 typical pollutants was established, categorized by polarity and ionization characteristics, as shown in Table 1. This database correlates SPE column characteristics (pore size, functional groups, hydrophobicity) with pollutant properties (logKow, pKa) to form optimal matching rules. When trace organic pollutants in water are of a single or concentrated type, the SPR database can provide important reference for selecting suitable SPE columns.

[0084] Test Example 1 This test example is an acute toxicity test of luminescent bacteria in the eluent after solid-phase extraction of different SPE column systems, including blank column, Examples 3 and 4, and Comparative Example 8. The specific test method refers to the "Technical Specification for Wastewater Biotoxicity Monitoring - Acute Toxicity Test of Luminescent Bacteria - Vibrio Fischer Method (Trial)" issued by the Department of Ecological and Environmental Protection of the State Oceanic Administration, and the determination was performed using a Microtox® M500 toxicity analyzer.

[0085] Specific test results are as follows: Figure 11 As shown in the figure, the luminescent bacteria inhibition rate of the eluent from the MIX column solid-phase extraction in Example 4 was the highest, reaching 78.45%, demonstrating a high toxicity effect. This indicates that the solid-phase extraction method using the MIX column is more effective in enriching toxic pollutants.

[0086] Test Example 2 This test example used a CCK-8 cell proliferation assay kit to test the acute toxicity of CCK-8 in the eluents after solid-phase extraction in different SPE column systems of Examples 3, 4, and Comparative Example 8. The specific test methods followed the instructions for the CCK-8 cell proliferation assay kit, and absorbance was measured using a microplate reader to calculate the EC50 value (i.e., the IC50 value in the instructions). 50 ).

[0087] Specific test results are as follows: Figure 12 As shown in the figure, the solid phase extraction method of the MIX column in Example 4 can achieve EC50 at a concentration factor close to 40×, while the concentration factors of other solid phase extraction columns are all higher than those of the MIX column. That is, the MIX column has a better enrichment effect on toxic pollutants and can effectively enrich toxic pollutants in water.

[0088] Test Example 3 This test example uses a two-hybrid yeast dry powder kit to test the yeast endocrine-disrupting toxicity of eluates after solid-phase extraction in different SPE column systems of Examples 3, 4, and Comparative Example 8. The specific test methods followed the operating procedures in the instructions for the two-hybrid yeast dry powder kit. The optical density values ​​were detected using a microplate reader, galactosidase activity (U) was calculated, and an E2 standard curve was established (e.g., ...). Figure 13 As shown, the establishment method is described in the instructions for the Two Hybrid Yeast Dry Powder Kit. At the same time, the galactosidase activity (U) and E2 equivalent (i.e., the 17β-estradiol equivalent concentration in the instructions) were calculated at 10-fold and 100-fold dilutions.

[0089] Specific test results are as follows: Figure 14 As shown in the figure, the eluent of the MIX column solid-phase extraction in Example 4 showed the highest E2 equivalent concentration, indicating that it had the best enrichment efficiency for the target estrogenic substances. The eluent of the MIX column solid-phase extraction in Example 4 also showed the highest galactosidase activity (U), indicating that it contained the most substances with estrogenic effects, further confirming that its eluent had higher biological activity. The MIX column can more accurately enrich and characterize the actual toxic effects of the test samples.

[0090] Test Example 4 This test case presents quantitative PCA analysis of the toxicity of different SPE column systems in Examples 3, 4, and Comparative Example 8. The results of acute toxicity tests on luminescent bacteria, CCK-8 cell acute toxicity tests, and yeast endocrine disruption toxicity tests were integrated into a "comprehensive toxicity score" to compare the overall toxicity enrichment effects of different SPE column systems. The specific method includes the following steps: S1. Data Entry Preparation: Define variables in the "Variable View" of IBM SPSS Statistics (2026). In the first row, under the "Name" column, enter "Sample Name" and set it to "String" in the "Type" column. In the second row, under the "Name" column, enter "Luminescent Bacteria Inhibition Rate". In the third row, under the "Name" column, enter "CCK8_EC50 Factor". In the fourth row, under the "Name" column, enter "Yeast_E2 Equivalent".

[0091] S2. Data entry: Based on the results of test examples 1-3, enter the toxicity data of different SPE column systems into the table in the "Data View".

[0092] S3. Data Standardization Preprocessing: In the SPSS software, click Analyze, Descriptive Statistics, and Description in the top menu bar. In the pop-up window, select the three toxicity variables (luminescent bacteria inhibition rate, CCK8_EC50 fold, and yeast_E2 equivalent) from the left to the "Variables" box on the right. Then check "Save standardized scores as variables," which is crucial for automatic Z-score standardization.

[0093] S4. Perform principal component analysis: In the SPSS software, click Analyze, Dimension Reduction, Factorization in the top menu bar, and select the three newly generated standardized variables (Z luminescent bacteria inhibition rate, ZCCK8_EC50 factor, Z yeast_E2 equivalent) into the "Variables" box on the right. Click the "Description..." button on the right. In the "Statistics" section, check "Initial Solution". In the "Correlation Matrix" section, check "KMO and Bartlett's Test of Sphericity". The KMO and Bartlett's test of sphericity are used to determine whether the data is suitable for PCA. Specifically, the KMO value should be greater than 0.5, and the closer to 1, the more suitable the data is for PCA; the Bartlett's test of sphericity value should be less than 0.05, indicating a significant correlation between the variables, making it suitable for PCA. Click the "Extract..." button on the right. In the "Method" drop-down menu, make sure "Principal Components" is selected. In the "Analysis" section, make sure "Based on Eigenvalues" is selected, and the eigenvalues ​​are greater than "1" (eigenvalues ​​are the default setting, and it will automatically extract principal components with eigenvalues ​​greater than 1). In the "Output" section, check "Unrotated Factor Solutions" and "Scrap Plot". Click the "Score..." button on the right, check "Save as Variable" to generate principal component scores for each sample; in the "Method" section, select "Regression"; check "Show Factor Score Coefficient Matrix" and run the analysis.

[0094] S5. Calculation and Interpretation of Comprehensive Toxicity Score: Explanation of total variance: Look at the "Total" column in the "Initial Eigenvalues" section and note the "Percentage of Variance" for the first principal component (PC1) in the "Initial Eigenvalues" section. This value represents the percentage of total variance that PC1 can explain for the original three toxicity variables. The component matrix table shows the correlation between each original variable and the principal component (PC1). All three values ​​are positive, indicating that PC1 is positively correlated with all toxicity indicators, meaning that PC1 represents "comprehensive toxicity". The component score coefficient matrix table provides the formula coefficients for calculating the PC1 score; Calculate the overall score: Add a new column FAC1_1 in the "Data View" to represent the PC1 score for each sample, which is the overall toxicity score of the SPE column system.

[0095] Specific quantitative PCA analysis results for toxicity are as follows: Figure 15 As shown in the figure, the MIX column has a high overall toxicity score across multiple dimensions, and it is dominant in various biological toxicity indicators, covering more toxic substances in the water.

[0096] The above description provides an illustrative overview of the present invention and its embodiments. This description is not restrictive, and the embodiments shown are merely one example of the invention's implementation. Actual implementations are not limited to these examples. Therefore, if those skilled in the art are inspired by this description and design similar implementations and examples without departing from the spirit of the invention, such designs should fall within the scope of protection of the present invention.

Claims

1. A method for the enrichment of trace organic pollutants based on the evaluation of toxic effects in series with solid phase extraction, characterized by, The method comprises the following steps: First, the water body containing trace organic pollutants is subjected to solid phase extraction through the activated HLB column, acetonitrile is used as the eluent to obtain eluent A; Then, the water body subjected to solid phase extraction through the HLB column is subjected to solid phase extraction through the activated WAX column, methanol containing 1-3% ammonia by mass fraction is used as the eluent to obtain eluent B; Then, the water body subjected to solid phase extraction through the WAX column is subjected to solid phase extraction through the activated MCX column, methanol containing 1-3% ammonia by mass fraction is used as the eluent to obtain eluent C; Finally, the eluent A, the eluent B and the eluent C are mixed to obtain a mixed extraction liquid.

2. The method for trace organic contaminant enrichment by tandem solid phase extraction based on toxicity effect evaluation according to claim 1, characterized in that, The water body containing trace organic pollutants is adjusted to a pH of 4-6 before solid phase extraction.

3. The method for trace organic contaminant enrichment by tandem solid phase extraction based on toxicity effect evaluation according to claim 2, characterized in that, The activation method of the HLB column is to use dichloromethane and methanol as the activation liquid, and the activation flow rate is controlled to be 5-15 mL / min; The activation method of the WAX column is to use methanol containing 1-3% ammonia by mass fraction and methanol as the activation liquid, and the activation flow rate is controlled to be 5-15 mL / min; The activation method of the MCX column is to use 0.01-0.9% acetic acid aqueous solution by mass fraction and methanol as the activation liquid, and the activation flow rate is controlled to be 5-15 mL / min.

4. The method according to claim 2, wherein the solid phase extraction method of the HLB column comprises the following steps: (1) sample loading: the water body containing trace organic pollutants is allowed to flow through the HLB column, and the flow rate is controlled to be 1-10 mL / min; (2) elution: after the sample loading is completed, 1-5 mL of ultrapure water is added to elute the HLB column, and nitrogen is used for drying for 10-30 min after the elution is completed; (3) elution: 2.5-3.5 mL of acetonitrile is used as the eluent to elute twice at a flow rate of 0.5-2 mL / min.

5. The method according to claim 2, wherein the solid phase extraction method of the WAX column comprises the following steps: (1) sample loading: the water body subjected to solid phase extraction through the HLB column is allowed to flow through the WAX column, and the flow rate is controlled to be 1-10 mL / min; (2) elution: after the sample loading is completed, 1-5 mL of ultrapure water is added to elute the WAX column, and nitrogen is used for drying for 10-30 min after the elution is completed; (3) elution: 2.5-3.5 mL of methanol containing 1-3% ammonia by mass fraction is used as the eluent to elute twice at a flow rate of 0.5-2 mL / min. The solid phase extraction method of the MCX column comprises the following steps: (1) sample loading: the water body subjected to solid phase extraction through the WAX column is allowed to flow through the MCX column, and the flow rate is controlled to be 1-10 mL / min; 6. The method for trace organic contaminant tandem solid phase extraction enrichment based on toxicity effect evaluation according to claim 2, characterized in that, (2) elution: after the sample loading is completed, 1-5 mL of ultrapure water is added to elute the MCX column, and nitrogen is used for drying for 10-30 min after the elution is completed; (3) elution: 2.5-3.5 mL of methanol containing 1-3% ammonia by mass fraction is used as the eluent to elute twice at a flow rate of 0.5-2 mL / min. ​ ​ 7. The method for the enrichment of trace organic pollutants in series solid phase extraction based on the evaluation of toxic effect according to any one of claims 1 to 6, characterized in that, The concentration of the trace organic pollutants in the water body is 1 ng / L-100 μg / L.

8. A method for detecting the toxic effect of trace organic pollutants, characterized in that, The trace organic pollutants are enriched by the trace organic pollutants enrichment method based on toxicity effect evaluation in series with solid phase extraction according to claims 1-7, and the mixed extraction liquid is detected by gas chromatography-mass spectrometry and / or liquid chromatography-mass spectrometry.

9. A method for assessing the toxic effects of trace organic contaminants, characterized in that, The trace organic pollutants are evaluated after being enriched by the trace organic pollutants enrichment method based on toxicity effect evaluation in series with solid phase extraction according to claims 1-7.

10. The method for assessing the toxic effects of trace organic contaminants according to claim 9, characterized in that, The overall toxicity of the trace organic pollutants in the water body is evaluated by testing the mixed extraction liquid using the photobacterium acute toxicity test, CCK-8 acute toxicity test and yeast endocrine disruptor toxicity test, and the comprehensive toxicity of the trace organic pollutants in the water body is evaluated by integrating the results of the photobacterium acute toxicity test, CCK-8 acute toxicity test and yeast endocrine disruptor toxicity test through toxicity quantitative PCA analysis, and the toxicity quantitative PCA analysis comprises the following steps: A. Defining variables in the variable view of the SPSS software and entering data into the data view of the SPSS software; B. Preprocessing the data by Z-score standardization; C. Performing principal component analysis and calculating the comprehensive toxicity score.

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