Fractionation method of endocrine disrupter in environmental water body

Abstract

The invention discloses a fractionation method of an endocrine disrupter in an environmental water body, and belongs to the technical field of water body pollutant analysis. The fractionation method of the endocrine disrupter in the environmental water body comprises the following steps: S1, enriching and concentrating a water sample to obtain a to-be-fractionated liquid; s2, fractionating the to-be-fractionated liquid by adopting an analytical liquid chromatography system and a C18 semi-preparative chromatographic column to obtain fractions. According to the method, high-resolution synchronous fractionation of the endocrine disrupter in a wide polarity range (Log Kw:-0.19-6.62) is realized, the defects of low fractionation efficiency and high solvent consumption of the traditional semi-preparative liquid chromatography are effectively overcome by optimizing a gradient program of an analytical liquid chromatography system, the time and economic cost are saved, and the method is suitable for industrial production. And the fraction can be directly compatible with biological activity test after salting-out-extraction treatment.

Description

Technical Field

[0001] This invention belongs to the field of water pollutant analysis technology, specifically relating to a fractionation method for endocrine disruptors in environmental water. Background Technology

[0002] Endocrine-disrupting chemicals (EDCs) can interfere with the synthesis, secretion, transport, binding, reaction, or elimination of endogenous signaling molecules in the human body at extremely low concentrations, significantly impacting human reproductive and developmental behaviors and thus attracting widespread attention. EDCs in environmental water bodies have a wide range of sources, including pesticides, preservatives in artificial care products, additives, and raw materials for industrial chemicals. However, as of 2019, the number of chemicals produced and used internationally had reached 350,000, the vast majority of which were released into the environment without endocrine-disrupting effects assessment. Therefore, identifying and assessing endocrine-disrupting chemicals in the environment has become a major challenge.

[0003] To address these issues, the European Union proposed Effect-Directed Analysis (EDA), which combines bioactivity testing with chemical separation analysis. In a typical EDA process, high-performance liquid chromatography (HPLC) fractionation is a key step, aiming to separate extracts from complex environments into several fractions based on polarity differences. This simplifies the mixture and aids in subsequent identification of bioactive target fractions and their constituent compounds.

[0004] However, current liquid chromatography fractionation methods used in EDA processes still face the following limitations: (1) Limited instrument applicability: It generally relies on semi-preparative liquid chromatography for fractionation, but the semi-preparative liquid chromatography has a long single fractionation time and a large solvent consumption, which greatly reduces the overall efficiency of the EDA process.

[0005] (2) Limitations of fractionation resolution and sensitivity: Since EDCs typically have a wide polarity range, current fractionation methods often struggle to achieve both high-resolution fractionation and high-throughput analysis when separating such mixtures. Co-elution is an unavoidable commonality of traditional fractionation methods, resulting in lower resolution and a greater number of compounds in the same fraction, making the composition of the bioactive compounds in that fraction extremely complex and difficult to analyze. At the same time, due to low fractionation efficiency, the concentration of active substances in the fraction may be too low to meet the sensitivity of the instrument. Endocrine disruptors can interfere with normal human signaling pathways even at extremely low concentrations, making it difficult for current fractionation methods to accurately identify trace levels of active substances, leading to failure in activity localization. Summary of the Invention

[0006] To address the problems existing in the prior art, the primary objective of this invention is to provide a method for fractionating endocrine disruptors in environmental water.

[0007] Another objective of this invention is to provide a method for quantitative analysis of endocrine disruptors in environmental water.

[0008] Another objective of this invention is to provide a method for testing the bioactivity of endocrine disruptors in environmental water.

[0009] Another objective of this invention is to provide an effect-oriented analysis method.

[0010] To achieve the above objectives, the present invention provides the following technical solution: A method for fractionating endocrine disruptors in environmental water includes the following steps: S1. The water sample is enriched and concentrated to obtain the liquid to be fractionated; S2. The distillate to be fractionated was obtained by using an analytical liquid chromatography system and a C18 semi-preparative chromatographic column; The flow rate was 4-5 mL / min, and the wavelength of the UV detector was 254 nm. Pure water was used as mobile phase A, and pure acetonitrile was used as mobile phase B. The gradient of mobile phase B was as follows: 0-6 min, mobile phase B was 20%; 6-12 min, mobile phase B increased from 20% to 30%; 12-18 min, mobile phase B increased from 30% to 100%; 18-25 min, mobile phase B was 100%; 25-29 min, mobile phase B decreased to 20% and reached equilibrium.

[0011] This invention utilizes an analytical liquid chromatography system and a semi-preparative chromatographic column to replace traditional fractionation methods. Through optimized chromatographic conditions and post-fractionation processes, it successfully performs simultaneous high-resolution fractionation of endocrine disruptors with a wide polarity range and diverse types. This solves the problem of insufficient coverage in traditional methods, effectively ensures the integrity of the fractionated products, greatly reduces the risk of missed detection of activity in subsequent biological tests, and significantly improves fractionation efficiency.

[0012] Preferably, the analytical liquid chromatography system includes a binary pump, an autosampler, a low-pressure gradient unit, an ultraviolet detector, and a fraction collector.

[0013] Preferably, the Log K of the endocrine disruptor ow The value ranges from -0.19 to 6.62.

[0014] Preferably, the endocrine disruptors include oxime ester, chlorfenapyr, azoxystrobin, n-heptyl-p-hydroxybenzoate, bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether, (Z)-phenoxystrobin, isobutylparaben, bisphenol A (3-chloro-2-hydroxypropyl)(2,3-dihydroxypropyl) ether, isopropylparaben, azoxystrobin, fluopyram, pyraclostrobin, acetamiprid, and dinotefuran.

[0015] Preferably, step S1 specifically involves: injecting the water sample into a solid-phase extraction column for enrichment, then eluting with an organic solvent, and finally concentrating and redissolving to obtain the distillate to be fractionated.

[0016] More preferably, the solid phase extraction column is an HLB solid phase extraction column.

[0017] More preferably, the organic solvent is methanol, a methanol / methyl tert-butyl ether mixture, or dichloromethane.

[0018] Preferably, the fractionation method further includes post-fractionation of the fraction.

[0019] Post-processing of distillates is beneficial for quantitative analysis and bioactivity testing of the distillates.

[0020] More preferably, the post-processing of the distillate includes the following steps: salting out, extracting, drying, concentrating, and redissolving the distillate to obtain the distillate to be tested.

[0021] More preferably, the post-processing of the distillate includes the following steps: adding excess NaCl to the distillate, transferring it to an extract in which the upper layer is acetonitrile and the lower layer is water, drying it with anhydrous magnesium sulfate after extraction, concentrating it under a nitrogen stream, and finally resolving it with an organic solvent.

[0022] More preferably, the organic solvent is acetonitrile or dimethyl sulfoxide.

[0023] More preferably, the extraction is performed 2 to 3 times.

[0024] Preferably, the inner diameter of the semi-preparative chromatographic column is ≥6 mm.

[0025] More preferably, the semi-preparative chromatographic column has dimensions of 10 mm × 150 mm and 10 μm.

[0026] A quantitative analysis method for endocrine disruptors in environmental water is proposed, which uses ultra-high performance liquid chromatography-triple quadrupole mass spectrometry to determine the fractions obtained by the above fractionation method and calculates the recovery rate.

[0027] A method for testing the bioactivity of endocrine disruptors in environmental water uses recombinant yeast-anti-androgen effect detection to test the activity of fractions obtained by the above fractionation method.

[0028] An effect-oriented analysis method, including the fractionation method described above.

[0029] Compared with the prior art, the present invention has the following beneficial effects: This invention utilizes an analytical liquid chromatography system and a semi-preparative chromatographic column to replace traditional fractionation methods, achieving high-throughput, high-resolution, and high-sensitivity continuous fractionation of endocrine disruptors in water samples from complex environments. By optimizing the gradient program of the analytical liquid chromatography system, this invention achieves high sensitivity across a wide polarity range (Log K). ow High-resolution synchronous fractionation of endocrine disruptors (-0.19~6.62) effectively solves the defects of low fractionation efficiency and high solvent consumption in traditional semi-preparative liquid chromatography, saving time and economic costs. Moreover, the fraction can be directly compatible with bioactivity testing after salting out and extraction.

[0030] This invention achieves high-resolution fractionation of 14 representative EDCs, overcoming the insufficient coverage problem of traditional methods. The recovery rates of all target compounds are higher than 60%, with 11 exceeding 80%, effectively ensuring the integrity of the fractions and significantly reducing the risk of missed activity detection in subsequent bioassays. This invention requires only 29 minutes for a single fractionation and can continuously obtain 14 fractions. Compared with traditional semi-preparative chromatography (typically taking ≥60 minutes), the efficiency is significantly improved, greatly enhancing the overall efficiency of effect-directed analytical methods.

[0031] The fractionation method of this invention has high sensitivity and can capture and separate trace levels (ppb level) of fractionated products. By coupling with ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, accurate qualitative and quantitative analysis of trace levels (ppb level) of fractionated products is achieved, thus providing reliable technical support for the identification of trace active substances in complex systems.

[0032] The fractionation method of this invention can achieve precise activity localization in water samples with high and low matrix interference, and exhibits excellent matrix adaptability, stability and stable activity distribution in real environmental water samples. Attached Figure Description

[0033] Figure 1 This is a flowchart of the detection process for the analytical liquid chromatography system and the semi-preparative chromatography column combined with ultra-high performance liquid chromatography-triple quadrupole mass spectrometry in Example 1.

[0034] Figure 2 The reversed-phase high-performance liquid chromatography (RP-HPLC) separation spectra of 14 endocrine disruptors are shown.

[0035] Figure 3 The flowcharts for the analytical liquid chromatography system and semi-preparative chromatography column combined with bioactivity testing in Examples 2 and 3 are shown.

[0036] Figure 4 This is a reversed-phase high-performance liquid chromatography (RP-HPLC) separation spectrum of the influent from the NX wastewater treatment plant.

[0037] Figure 5 This is a reversed-phase high-performance liquid chromatography (RP-HPLC) separation spectrum of the effluent from the NX wastewater treatment plant. Detailed Implementation

[0038] The present invention is further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions in the art or as recommended by the manufacturer; the raw materials and reagents used, unless otherwise specified, are all commercially available from the conventional market. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention are within the scope of protection claimed by the present invention.

[0039] Example 1: Verification of the fractionation effect and recovery rate of mixed standard solutions This embodiment provides a method for fractionating endocrine disruptors in environmental water, including the following steps: S1. Based on the types of endocrine disruptors, 14 representative endocrine disruptors were selected, and their details are shown in Table 1. The applicability of the fractionation method to hydrophilic to lipophilic endocrine disruptors (EDCs) was verified. A mixed standard solution of the 14 endocrine disruptors with a component concentration of 100 μg / L was prepared to simulate low-dose exposure scenarios of endocrine disruptors in environmental water bodies.

[0040] S2. The above-mentioned mixed standard solution is injected into an analytical liquid chromatography system and a semi-preparative column for fractionation. The analytical liquid chromatography system includes an LC-20AD binary pump, a SIL-10AD autosampler, an LCGE-20A low-pressure gradient unit, an SPD-20A ultraviolet detector, and an FRC-10A fraction collector. The C18 semi-preparative column has a length of 150 mm, an inner diameter of 10 mm, and a packing particle size of 10 μm.

[0041] The flow rate was 4.5 mL / min, and the wavelength of the UV detector was 254 nm. Pure water was used as mobile phase A, and pure acetonitrile was used as mobile phase B. The gradient of mobile phase B was as follows: 0-6 min, mobile phase B was 20%; 6-12 min, mobile phase B increased from 20% to 30%; 12-18 min, mobile phase B increased from 30% to 100%; 18-25 min, mobile phase B was 100%; 25-29 min, mobile phase B decreased to 20% and reached equilibrium.

[0042] Using a fraction collector, one fraction was collected every 2 minutes from 0.5 to 28.5 minutes, for a total of 14 fractions.

[0043] Post-distillation treatment: Excess sodium chloride granules were added to the distillate, and the mixture was transferred to acetonitrile. The lower aqueous layer was extracted three times with acetonitrile. The solutions were combined and dried over anhydrous magnesium sulfate to remove water. The combined acetonitrile solution was concentrated to near dryness under a gentle nitrogen stream and finally redissolved with chromatographic grade acetonitrile.

[0044] The concentrations of target analytes in the complex solutions of each fraction were determined using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry (UHPLC-MS / MS), and the recoveries were calculated by comparing the concentrations with the initial injection concentration (100 ppb). The results are shown in Table 2. The chromatographic conditions for the UHPLC-triple quadrupole mass spectrometry system (AB SCIEX Triple Quad 5500+) were as follows: pure water (A) and pure methanol (B) as the mobile phases. An ACQUITY UPLC® HSS T3 column (1.8 μm, 2.1 mm × 100 mm, Waters) was used. The column temperature was 40 °C, and the flow rate was 0.3 mL / min. The injection volume was 5 μL; the mobile phase gradient was set as follows: 0-1 min, mobile phase B was 5%; 1-6 min, mobile phase B increased from 5% to 30%; 6-11 min, mobile phase B increased from 30% to 100%; 11-13 min, mobile phase B was maintained at 100%; 13-13.10 min, mobile phase B decreased back to 5%; 13.1-15 min, mobile phase B was maintained at 5%. The mass spectrometry conditions for the ultra-high performance liquid chromatography-triple quadrupole mass spectrometer were as follows: Positive mode: curtain gas pressure (CUR, N2) 35 psi, ionization voltage (IS) -4500 V, nebulizer gas pressure (GS1, N2) and heating gas pressure (GS2, N2) 55 psi, collision gas pressure (CAD, N2) 8 psi, ion source temperature 500 °C; Negative mode: curtain gas pressure (CUR, N2) 35 psi, ionization voltage (IS) 4500 V, nebulizer gas pressure (GS1, N2) and heating gas pressure (GS2, N2) 55 psi, collision gas pressure (CAD, N2) 8 psi, ion source temperature 500 °C. The standard fractionation and fraction recovery testing procedures are as follows: Figure 1 As shown.

[0045] Concentration calculation: Using the external standard method, a standard curve was plotted against the concentration using the peak area of ​​the target analyte. The peak area of ​​the distillate sample was then substituted into the curve equation to calculate its concentration C. 测 .

[0046] Recovery rate calculation:

[0047] Table 1 14 endocrine disruptors

[0048] Table 2. Recovery rates of 14 endocrine disruptors in different fractions (flow rate 4.5 mL / min)

[0049] Note: Fx refers to the xth fraction.

[0050] Table 2 shows the enrichment of endocrine disruptors with different polarities in different fractions. The recovery rates of all 14 endocrine disruptors were higher than 60%, and 11 of them exceeded 80%. This fully demonstrates that the fractionation method of the present invention can achieve high-resolution fractionation for complex mixtures, and that the fractionation method of the present invention has excellent and stable recovery performance for the target analytes.

[0051] Table 2 shows that endocrine disruptors with recoveries of 1.22–5.56% were found in the second and ninth fractions. The initial injection concentration was 100 ppb, indicating that the analytical liquid chromatography system and semi-preparative column can capture endocrine disruptors of approximately 1.22–5.56 ppb and fractionate them into a single fraction. This demonstrates that the fractionation method of this invention can achieve high-resolution and high-sensitivity continuous fractionation of endocrine disruptors in water samples from complex environments.

[0052] Example 2: Fractionation Treatment and Effect Verification of Actual Environmental Water Samples (Influent of Wastewater Treatment Plant) This embodiment provides a method for fractionating endocrine disruptors in environmental water, including the following steps: S1. Influent samples were collected from a wastewater treatment plant (NX) in Shaoguan City, Guangdong Province, with 16 L collected from each point. Pretreatment was completed within 48 hours after sample collection. The following steps were used for treatment: 2 L of water sample was filtered through a 0.70 μm glass fiber filter (Whatman) to remove suspended particulate matter.

[0053] Solid-phase extraction enrichment: The water sample was enriched using an Oasis® PRiME HLB solid-phase extraction column (6cc, 500mg). The column was activated and equilibrated sequentially with 5 mL of methyl tert-butyl ether (chromatographic grade), 5 mL of methanol (chromatographic grade), and 5 mL of ultrapure water (chromatographic grade) at a flow rate of approximately 1 drop / second. The filtered water sample was then passed through the activated extraction column at a controlled flow rate (1 mL / min) for enrichment.

[0054] Drying: After enrichment, the extraction column is vacuum dried for 30 minutes to remove residual moisture.

[0055] Elution: The target compound was eluted sequentially with 3 mL of methanol (chromatographic grade), 3 mL of methanol / methyl tert-butyl ether mixture (1:1, v / v), and 3 mL of dichloromethane (chromatographic grade).

[0056] Concentration and volume adjustment: Combine the eluents and gently concentrate to near dryness using a nitrogen blower at room temperature. Adjust the volume to 3.0 mL with chromatographic grade acetonitrile to obtain the concentrated test solution of the environmental water sample.

[0057] S2. Fractionation was performed using a Shimadzu high-performance liquid chromatography (HPLC) system (equipped with an LC-20AD binary pump, a SIL-10AD autosampler, an LCGE-20A low-pressure gradient unit, an SPD-20A UV detector, and an FRC-10A fraction collector) and an ECOSIL C18 semi-preparative column (10 μm, 10 × 150 mm). 1 mL of concentrated analyte solution was injected into the analytical HPLC system for fractionation.

[0058] Chromatographic conditions: Column: ECOSIL C18 reversed-phase column (10μm, 10×150mm); Mobile phase: pure water (A), pure acetonitrile (B); The flow rate was 4.5 mL / min, and the wavelength of the UV detector was 254 nm. Pure water was used as mobile phase A, and pure acetonitrile was used as mobile phase B. The gradient of mobile phase B was as follows: 0-6 min, mobile phase B was 20%; 6-12 min, mobile phase B increased from 20% to 30%; 12-18 min, mobile phase B increased from 30% to 100%; 18-25 min, mobile phase B was 100%; 25-29 min, mobile phase B decreased to 20% and reached equilibrium.

[0059] Fraction collection: Using a fraction collector, one fraction was collected every 2 minutes over a period of 0.5–28.5 minutes, for a total of 14 fractions. The volume of each fraction collected was approximately 9 mL. Fraction processing: Excess sodium chloride granules were added to the fraction, which was then transferred to acetonitrile. The lower aqueous layer was extracted three times with acetonitrile. The solutions were combined and dried over anhydrous magnesium sulfate to remove water. The combined acetonitrile solution was concentrated to near dryness under a gentle nitrogen stream and finally redissolved with chromatographic grade dimethyl sulfoxide for subsequent bioassays. The sample fractionation and fraction bioassay procedures are as follows: Figure 3 As shown, the reversed-phase high-performance liquid chromatography (RP-HPLC) separation spectrum of the influent from the NX wastewater treatment plant is as follows: Figure 4 As shown.

[0060] This embodiment provides a bioactivity assay, including the detection of androgen receptor antagonist activity in the above-mentioned fractions using a recombinant yeast-anti-androgen effect assay. The DNA sequence of the human androgen receptor is integrated into a plasmid containing the lacZ reporter gene (encoding β-galactosidase) to construct a recombinant yeast. In the experiment, a certain concentration of exogenous androgen receptor agonist (dihydrotestosterone, DHT) is first added to activate the basal expression of the reporter gene. If an anti-androgen active substance is present in the sample, it can competitively antagonize the binding of the androgen receptor to the agonist, thereby inhibiting the expression of the reporter gene and weakening the β-galactosidase-catalyzed substrate color reaction. By measuring the absorbance value of the colorimetric product, the strength of the anti-androgen activity of each fraction can be quantitatively evaluated.

[0061] β-galactosidase activity calculation:

[0062] Constructing a standard curve for flutamide (FLU): The same experiment was conducted with flutamide concentrations of 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078125, 0.0390625, and 0.01953125 μM. For each standard concentration, the corresponding β-galactosidase activity (Y-axis) was calculated. A dose-response curve was plotted with the logarithm of the flutamide concentration (log[FLU]) as the X-axis and the corresponding enzyme activity as the Y-axis.

[0063] Calculate the flutamide equivalent concentration (FEQ): The enzyme activity value measured in the sample is substituted into the FLU standard curve to obtain the flutamide equivalent concentration of the sample.

[0064] Fraction recovery rate:

[0065] Example 3: Fractionation Treatment and Effect Verification of Actual Environmental Water Samples (Wastewater Treatment Plant Effluent) The difference between this embodiment and Embodiment 2 is that, in step S1, effluent was collected from a wastewater treatment plant (NX) in Shaoguan City, Guangdong Province, with 16 L collected from each point. The remaining fractionation methods and bioactivity testing methods are the same as in Embodiment 2. The reversed-phase high-performance liquid chromatography (RP-HPLC) separation spectrum of the effluent from the NX wastewater treatment plant is shown below. Figure 5 As shown.

[0066] The recovery rates of Example 2 (influent sample) and Example 3 (effluent sample) are shown in Table 3.

[0067] Table 3 Recovery rates (%) of influent and effluent samples and fractions from the NX wastewater treatment plant

[0068] Note: Fx refers to the xth fraction.

[0069] As shown in Table 3, according to the bioassay results, approximately 76% of the active substances in the influent sample of the NX wastewater treatment plant were concentrated in fractions F10, F11, and F12. Approximately 87% of the active substances in the effluent sample of the NX wastewater treatment plant were concentrated in fractions F9, F10, and F11. These results confirm that the fractionation method provided by this invention can effectively overcome the interference of complex environmental matrices, selectively enriching substances with endocrine-disrupting activity in specific fractions based on polarity differences. This greatly simplifies the subsequent identification process of active substances, demonstrating the high applicability of this method to actual environmental samples and significantly simplifying the subsequent identification of active substances.

[0070] Among them, the cumulative recovery rate of the influent sample was less than that of the original sample. This is because the samples with a high original sample effect all showed that the cumulative effect of each fraction was less than that of the original sample. This may be because the matrix effect enhanced the original sample effect, while the additive effect of the matrix compounds in the fraction was small. Some weak effects were also superimposed and did not reach the detection limit when separated into the fraction.

[0071] The cumulative recovery rate of the effluent sample is greater than that of the original sample because the effluent matrix is ​​relatively clean and the matrix effect is weak. At the same time, the fractionation process efficiently removes interfering substances and may enrich substances in the fraction below the effect threshold concentration to reach detectable levels.

[0072] Comparative Example 1 The difference between this comparative example and Example 1 is that the liquid flow rate in step S2 is 1 mL / min, while the rest is the same as in Example 1. The distribution of the recovery results is shown in Table 4 below.

[0073] Table 4. Recovery rates of 14 endocrine disruptors in different fractions (flow rate 1 mL / min)

[0074] Note: Fx refers to the xth fraction.

[0075] As shown in Table 4, among the 14 target endocrine disruptors, only acetamiprid (Log K) was fractionated at a liquid flow rate of 1 mL / min. ow The value is 0.4) and fipronil (Log K) ow (Value -0.19) These two substances were fractionated. The overall recovery rate was not as good as that used in Example 1 at a flow rate of 4.5 mL / min.

[0076] Comparative Example 2 The difference between this comparative example and Example 1 is as follows: In step S2, the flow rate is 4.5 mL / min, the wavelength of the UV detector is 254 nm; pure water is used as mobile phase A, and pure acetonitrile is used as mobile phase B. The gradient change of mobile phase B is as follows: 0~2.27 min, mobile phase B remains at 5%; 2.27~13.61 min, mobile phase B linearly increases from 5% to 30%; 13.61~24.95 min, mobile phase B linearly increases from 30% to 100%; 24.95~29.71 min, mobile phase B remains at 100%; 29.71~34 min, mobile phase B decreases to 5% and remains at equilibrium.

[0077] Using a fraction collector, a fraction was collected every 3 minutes over a period of 0.5–33.5 minutes. The rest of the procedure was the same as in Example 1. The distribution of the recovery results is shown in Table 5 below.

[0078] Table 5. Recovery rates of 14 endocrine disruptors in different fractions

[0079] Note: Fx refers to the xth fraction.

[0080] The data in the table show that, under these chromatographic conditions, the recoveries of four substances—isopropylparaben, azoxystrobin, acetamiprid, and dinotefuran—were all less than 60% during fractionation of the 14 target endocrine disruptors. The overall recovery rate of the remaining substances was not as good as in Example 1. Moreover, the total fractionation time (34 min) was slightly longer than that in Example 1 (29 min).

[0081] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for fractionating endocrine disruptors in environmental water, characterized in that, Includes the following steps: S1. Concentrate and enrich the water sample to obtain the liquid to be fractionated; S2. The distillate to be fractionated was obtained by using an analytical liquid chromatography system and a C18 semi-preparative chromatographic column; The flow rate was 4-5 mL / min, and the wavelength of the UV detector was 254 nm. Pure water was used as mobile phase A, and pure acetonitrile was used as mobile phase B. The gradient of mobile phase B was as follows: 0-6 min, mobile phase B was 20%; 6-12 min, mobile phase B increased from 20% to 30%; 12-18 min, mobile phase B increased from 30% to 100%; 18-25 min, mobile phase B was 100%; 25-29 min, mobile phase B decreased to 20% and reached equilibrium.

2. The fractionation method according to claim 1, characterized in that, Log K of the endocrine disruptor ow The value ranges from -0.19 to 6.

62.

3. The fractionation method according to claim 1, characterized in that, The endocrine disruptors include oxime ester, cymoxanil, fenpropathrin, n-heptyl-p-hydroxybenzoate, bisphenol A (3-chloro-2-hydroxypropyl) glycidyl ether, (Z)-phenoxybenzoic acid, isobutylparaben, bisphenol A (3-chloro-2-hydroxypropyl)(2,3-dihydroxypropyl) ether, isopropylparaben, oxime fenpropathrin, fluopyram, azoxystrobin, acetamiprid, and dinotefuran.

4. The fractionation method according to claim 1, characterized in that, Step S1 specifically involves: injecting the water sample into a solid-phase extraction column for enrichment, then eluting with an organic solvent, and finally concentrating and redissolving to obtain the distillate to be fractionated.

5. The fractionation method according to claim 4, characterized in that, The solid-phase extraction column is an HLB solid-phase extraction column.

6. The fractionation method according to claim 4, characterized in that, The organic solvent is methanol, a methanol / methyl tert-butyl ether mixture, or dichloromethane.

7. The fractionation method according to claim 1, characterized in that, The inner diameter of the semi-preparative chromatographic column is ≥6 mm.

8. A method for quantitative analysis of endocrine disruptors in environmental water, characterized in that, The fractions obtained by any of the fractionation methods described in claims 1 to 7 were determined using ultra-high performance liquid chromatography-triple quadrupole mass spectrometry, and the recovery rates were calculated.

9. A method for testing the bioactivity of endocrine disruptors in environmental water, characterized in that, The activity of the fractions obtained by any of the fractionation methods described in claims 1 to 7 was detected using the recombinant yeast-anti-androgen effect assay.

10. An effect-oriented analysis method, characterized in that, Includes the fractionation method described in any one of claims 1 to 7.

Images