High performance liquid chromatography detection method for ethinylestradiol and nonyl phenol in soil

Abstract

The invention discloses a high performance liquid chromatography detection method for ethinylestradiol and nonyl phenol in soil. The method comprises the steps of S1, sample pretreatment, S2, sample extraction, S3, solid phase extraction and purification, S4, high performance liquid chromatography detection and S5, quantitative analysis. The detection method overcomes the defects of a method for synchronously and quantitatively detecting two typical endocrine disruptors in different types of farmland soil in the prior art by aiming at the industrial technical pain points of complex soil matrix, large polarity difference between ethinylestradiol and nonyl phenol and high trace enrichment difficulty; and a stable and reliable technical support is provided for pollution characteristic research, environment fate analysis and ecological risk assessment of ethinylestradiol and nonyl phenol in soil.

Description

Technical Field

[0001] This invention relates to the field of environmental sample detection technology, specifically a high-performance liquid chromatography method for the detection of ethinylestradiol and nonylphenol in soil. Background Technology

[0002] Endocrine disruption has become a major potential threat to the global ecological environment and public health. Currently, identifying the environmental behavior and toxicological effects of typical pollutants is a key strategy for assessing and controlling their impact on ecosystems and human health. Our laboratory previously conducted systematic research on ethinylestradiol and nonylphenol, which are widely detected in the environment. Ethinylestradiol is a synthetic estrogen, while nonylphenol is an industrial chemical with estrogenic activity; both are typical endocrine disruptors. Studies have shown that even at extremely low environmental concentrations (ng / L), these two pollutants can interfere with normal hormone signaling pathways in organisms, leading to ecological effects such as feminization in male fish, abnormal gonadal development, and population decline. Further analysis revealed that compared to natural estrogens, ethinylestradiol is metabolized slowly and is more persistent in the environment, while nonylphenol has a significant bioaccumulation effect and estrogenic activity. Both can be transmitted through the food chain, posing a potential threat to human reproductive health. Environmental behavior studies indicate that these pollutants readily undergo adsorption and desorption processes in soil and water, with a significant lag in desorption, potentially becoming long-term "chemical time bombs." Therefore, studying the fate and ecotoxicological effects of ethinylestradiol and nonylphenol in the environment is of great scientific value and practical significance for revealing their pollution characteristics, assessing ecological risks, improving water quality standards, and formulating pollution control strategies.

[0003] Currently, research on ethinylestradiol and nonylphenol largely focuses on aquatic media, with limited understanding of their environmental behavior, fate, and ecological effects in soil environments. In fact, soil, as a major sink for organic pollutants and a source of migration to groundwater and organisms, plays a crucial role in the environmental geochemical cycle of pollutants. Ethinylestradiol mainly originates from land use of livestock and poultry waste and wastewater irrigation, while nonylphenol enters the soil environment through various pathways, including industrial emissions, agricultural use of sludge, and atmospheric deposition. Once in the soil, these two types of pollutants can adsorb and bind with soil organic matter, exhibiting retention characteristics and migration and transformation patterns different from those in aquatic environments. However, existing research lacks a systematic understanding of the adsorption and desorption mechanisms, degradation and transformation pathways, bioavailability, and potential risks of ethinylestradiol and nonylphenol transmission along the food chain in soil. Studies have shown that these pollutants have long degradation half-lives in soil, and the desorption process exhibits a significant hysteresis effect, potentially leading to soil becoming a long-term "secondary pollution source," continuously releasing pollutants that threaten groundwater and crop safety.

[0004] Therefore, accurate quantitative research on ethinylestradiol and nonylphenol in the soil environment is fundamental to subsequent studies on pollution characteristics, migration, transformation, and ecological risks. Developing a precise and effective detection method is of significant scientific and practical value in addressing current research gaps, refining theories of pollutant environmental fate, scientifically assessing farmland safety, and guiding the management of polluted soil. Currently, there are limited methods for detecting ethinylestradiol and nonylphenol in soil, both domestically and internationally. This invention aims to establish a high-performance liquid chromatography (HPLC) method for detecting ethinylestradiol and nonylphenol in soil, providing technical support for determining their exposure levels in soil. Summary of the Invention

[0005] The purpose of this invention is to provide a high-performance liquid chromatography (HPLC) method for the detection of ethinylestradiol and nonylphenol in soil, which can meet the needs of rapid detection of large batches of soil samples and significantly improve detection throughput and efficiency.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A high-performance liquid chromatography method for the detection of ethinylestradiol and nonylphenol in soil includes the following steps: S1. Sample pretreatment: 200~300g of collected soil sample is first placed in an environment of -60~-40℃ for freeze drying until constant weight. Then the soil sample is ground and screened with a 10~20 mesh stainless steel sieve to obtain the soil sample to be tested. After sealing the soil sample to be tested, store it in a light-proof environment at -20~-15℃ for later use. S2. Sample extraction: Weigh 3-5g of the soil sample to be tested and place it in a centrifuge tube. First, add ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solutions. Vortex to mix and ensure that the ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solutions are in full contact with the soil sample and allow to stand for equilibrium. Then, add 20-30mL of extraction reagent, vortex to mix, and extract by ultrasound. Then, centrifuge and collect the supernatant. The supernatant was placed in a 40°C constant temperature water bath and blown with nitrogen until completely dry. Then it was redissolved with methanol and diluted to a volumetric flask with ultrapure water. After shaking well, the sample extract was obtained and set aside for use. S3, Solid-phase extraction purification: First, activate the solid-phase extraction column with chromatographically pure methanol and ultrapure water in sequence. Then, pass the sample extract obtained in S2 through the solid-phase extraction column at a stable flow rate of 5~8 mL / min for sample loading. After sample loading, the solid-phase extraction column was rinsed to remove water-soluble impurities, and the solid-phase extraction column was aspirated until completely dry. The solid-phase extraction column was then eluted with eluent, and all the eluent was collected into a clean pistil bottle. The eluent was placed in a 40°C constant temperature water bath and purged with nitrogen until completely dry. Chromatographic grade methanol was added for vortex redissolution, and the solution was filtered through a polytetrafluoroethylene organic phase filter membrane. The filtrate was collected in a brown liquid chromatography vial to obtain the sample to be tested. S4. High-performance liquid chromatography (HPLC) detection: Qualitative and quantitative detection of the sample is performed using a liquid chromatograph equipped with a fluorescence detector; S5. Quantitative analysis: The matrix-matched standard curve method was used to quantitatively calculate ethinylestradiol and nonylphenol in the test sample.

[0007] Preferably, the soil sample in S1 is any one or more mixed soils of yellow-brown soil, red soil, and black soil.

[0008] Note: Yellow-brown soil, red soil, and black soil are the three core zonal soil types for agricultural land in my country, covering the middle and lower reaches of the Yangtze River, the acidic red soil region in the south, and the black soil region in the northeast, respectively. The three types of soil have significant differences in physicochemical properties such as organic matter content, pH value, clay composition, and cation exchange capacity, and basically cover the main matrix types of farmland soil in my country.

[0009] Preferably, in S2, the ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution is: a mixed solution formed by dissolving ethinylestradiol-D4 and nonylphenol-D9 in chromatographically pure methanol, the amount of ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution added is 100~120μL, the concentration of ethinylestradiol-D4 and nonylphenol-D9 in the ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution is 80~100μg / L, after adding ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution, vortex for 10~15s to mix thoroughly, and let stand at room temperature in the dark for 10~15min to complete the equilibrium; After adding the extractant, vortex for 30-50 seconds to mix thoroughly. Next, microwave and ultrasonic combined treatment was performed for 15-20 minutes, with the temperature controlled at 25-30℃; microwave power was 100-150W, ultrasonic power was 200-300W, and then the mixture was placed in a constant temperature oscillation at a frequency of 180-220r / min and a temperature of 25-28℃ for 15 minutes. Centrifuge at 8000~10000r / min for 8~10min, with centrifugation temperature controlled at 3~5℃, and collect the supernatant into a heart-shaped flask; Add an equal amount of extractant to the centrifuged soil residue, and repeat the above steps of vortexing, microwave, ultrasonic combined treatment, shaking, and centrifugation once. Combine the supernatants from the two extractions and perform water bath heating and resolution in S2.

[0010] Explanation: First, ethinylestradiol-D4 and nonylphenol-D9 isotope internal standards are used as retention time markers. These isotope internal standards have the same physicochemical properties as the target analyte and can undergo the entire extraction, purification, and detection process simultaneously. This effectively eliminates target analyte loss during pretreatment and matrix effects during detection, solving the problem of retention time drift in complex soil matrices. The physicochemical properties of the isotope internal standard are completely consistent with those of the target analyte. The relative retention time is not affected by column aging, fluctuations in the mobile phase ratio, or changes in column temperature. The relative retention time deviation between the target analyte and the corresponding isotope internal standard is specified to be ≤0.5%, which serves as a prerequisite for qualitative determination. Furthermore, by employing a three-stage extraction mode of vortex mixing, ultrasonic-assisted extraction, and isothermal oscillation, combined with the operation of secondary extraction and supernatant merging, the extraction efficiency of trace ethinylestradiol and nonylphenol in soil is significantly improved. Among these, vortex mixing can quickly achieve full contact between soil sample and extractant, ultrasonic extraction can destroy soil aggregate structure through cavitation effect and release target pollutants adsorbed by soil organic matter and clay particles, and isothermal oscillation can further enhance the solid-liquid two-phase mass transfer process.

[0011] Preferably, the extractant in S2 is a ternary composite extractant consisting of methanol, ethyl acetate and a deep eutectic solvent, with a volume ratio of 5:5:1. The eutectic solvent is a mixture of choline chloride and ethylene glycol in a mass ratio of 1:2.

[0012] Note: This extractant is an environmentally friendly green solvent that is both hydrophilic and lipophilic. It can bind to the target substance through hydrogen bonds and hydrophobic interactions, while reducing the adsorption strength of soil organic matter to the target substance. Compared with existing technologies, this extractant reduces the amount of organic reagent used by 15% and improves the extraction selectivity of ethinylestradiol and nonylphenol by more than 25%.

[0013] Preferably, the solid-phase extraction column used in S3 is a modified HLB solid-phase extraction column with a specification of 500 mg / 6 mL and the packing material is a hydrophilic-lipophilic balanced reverse polymer packing material. The HLB solid-phase extraction column modification method is as follows: graphene oxide and sodium montmorillonite in a mass ratio of 1:3 are loaded onto the surface of a hydrophilic-lipophilic balanced inverse polymer filler through in-situ polymerization.

[0014] The eluent was a mixture of chromatographically pure methanol and ethyl acetate, with a volume ratio of methanol to ethyl acetate of 80:20.

[0015] Explanation: A modified HLB solid-phase extraction column was selected. The modified packing material has a specific surface area increased by more than 40%, significantly increasing the adsorption capacity for strongly hydrophobic interfering substances such as humic acid. Simultaneously, it exhibits stronger retention capacity for ethinylestradiol and nonylphenol, with a column breakthrough volume approximately doubled and matrix effect reduced to below 10%. It possesses a wider pH operating range and more stable adsorption and retention performance, combining both hydrophilic and hydrophobic retention mechanisms to achieve efficient enrichment and stable retention of trace target analytes. Furthermore, this scheme optimizes the eluent to a mixture of methanol and ethyl acetate at a volume ratio of 80:20. This system minimizes the elution of strongly hydrophobic matrix interfering substances such as humic acid and oils adsorbed on the column while ensuring complete elution of the target analyte. This effectively reduces the soil matrix effect, avoids impurities interfering with the identification and integration of chromatographic peaks of the target analyte, and significantly improves the accuracy of the detection results.

[0016] Preferably, in step S3, the flow rate during the activation process is controlled at 2-3 mL / min, and the column is kept moist after activation.

[0017] Note: Ensure that methanol and ultrapure water fully wet the column packing material, so that the functional groups of the packing material can fully expand and reach an activation equilibrium state.

[0018] Preferably, in S3, the rinsing method is as follows: after sample loading, the column is rinsed with 5-8 mL of ultrapure water at a flow rate of 2-3 mL / min to remove water-soluble impurities. After rinsing, the column is continuously aspirated for 5-10 min until no liquid drips from the column and it is completely dry.

[0019] Description: This method effectively removes water-soluble inorganic salts, polar organic acids, sugars, and other matrix interfering substances adsorbed on the column, preventing these impurities from entering the subsequent detection system and interfering with the elution and quantification of the target analyte.

[0020] Preferably, in S3, the elution method is as follows: the solid-phase extraction column is eluted in fractions with 15 mL of eluent at a flow rate of 1-2 mL / min; Add 1 mL of chromatographically pure methanol and vortex for 30 s to redissolve. After redissolving, filter through a 0.22 μm polytetrafluoroethylene organic phase filter membrane.

[0021] Note: This method ensures complete elution of the target analytes adsorbed on the column, avoiding insufficient elution and low recovery rates caused by excessively high flow rates; it significantly improves the method's ability to detect trace targets in soil; and the use of a 0.22μm polytetrafluoroethylene organic phase filter membrane thoroughly removes tiny particulate impurities from the complex solution, preventing column clogging, reducing baseline noise, and improving detection stability and target peak resolution.

[0022] Preferably, in S4, the chromatographic conditions are set as follows: The chromatographic column used was a core-shell Inertsil ODS-SP-C18 column with dimensions of 150 mm × 4.6 mm and a packing particle size of 2.7 μm. The mobile phase consisted of chromatographically pure methanol and ultrapure water, with gradient elution mode, temperature control at 40℃ ± 0.5℃, mobile phase flow rate at 0.8 mL / min, and injection volume at 20 μL. The excitation wavelength of the fluorescence detector was set to 280 nm, the emission wavelength was set to 310 nm, and the total detection time for a single sample was 20 min.

[0023] Note: The chromatographic conditions in this study were optimized for the physicochemical properties of ethinylestradiol and nonylphenol. The Inertsil ODS-SP-C18 column exhibits excellent separation performance for weakly polar steroids and alkylphenols, enabling baseline separation of the two target compounds from soil matrix interferences. A methanol-water gradient elution mode was employed, allowing for the gradual elution of target compounds with different retention characteristics by dynamically adjusting the mobile phase polarity. Compared to isocratic elution, this significantly shortens the detection time and improves peak shape, avoiding peak tailing and broadening. The fluorescence detector uses an excitation wavelength of 280 nm and an emission wavelength of 310 nm, the optimal combination for detecting ethinylestradiol and nonylphenol. Compared to UV detectors, this combination offers higher sensitivity and stronger anti-interference capabilities, effectively reducing background interference from the soil matrix and enabling precise quantification of trace target compounds. The total detection time for a single sample is controlled at 20 minutes, meeting the needs for rapid detection of large batches of soil samples and significantly improving throughput and efficiency.

[0024] Compared with the prior art, the beneficial effects of the present invention are reflected in the following aspects: 1. The method of this invention is scientifically and rationally designed. Addressing the industry's technical pain points of complex soil matrix, large polarity difference between ethinylestradiol and nonylphenol, and high difficulty in trace enrichment, it constructs a complete detection system that includes freeze-drying pretreatment, composite solvent synergistic extraction-HLB solid-phase extraction purification, high-performance liquid chromatography, and fluorescence detector quantification. This system can achieve simultaneous, accurate, and efficient detection of ethinylestradiol and nonylphenol in soil, making up for the shortcomings of existing technologies for simultaneous quantitative detection of two typical endocrine disruptors in different types of farmland soil. It provides stable and reliable technical support for the study of pollution characteristics, environmental fate analysis, and ecological risk assessment of ethinylestradiol and nonylphenol in soil. 2. The extraction system and process of this invention are optimized and improved, resulting in high extraction efficiency and wide applicability. The use of a methanol-ethyl acetate composite extractant can simultaneously address the moderately polar ethinylestradiol and the strongly hydrophobic nonylphenol, breaking the binding effect between soil organic matter and the target substance, and achieving simultaneous and efficient extraction of two pollutants with significantly different polarities. The combination of three-stage extraction (vortex, ultrasound, and isothermal oscillation) and secondary extraction processes further enhances the release and recovery of the target substance, increasing the extraction efficiency by more than 20% compared to traditional single extraction methods. 3. The purification and detection system of this invention is highly targeted, and the method exhibits excellent sensitivity, accuracy, and precision. Employing an HLB hydrophilic-lipophilic balanced solid-phase extraction column, coupled with optimized parameters for the entire process of activation, loading, elution, and elution, it can efficiently remove matrix interferences such as humic acid and inorganic salts from soil, while simultaneously achieving stable enrichment of the target analytes. The optimized combination of methanol-water gradient elution chromatographic conditions and fluorescence detection wavelength enables baseline separation of the two target analytes, effectively reducing background noise. The sensitivity, accuracy, and precision of the method fully meet the requirements for the detection of trace organic pollutants in soil.

[0025] 4. This invention is simple and quick to operate, and highly scalable. This method does not require complex pretreatment equipment or high-cost detection instruments such as high-resolution mass spectrometry. The entire detection process can be completed using a standard high-performance liquid chromatography-fluorescence detector commonly used in laboratories. The total detection time for a single sample is only 20 minutes, enabling rapid and batch detection of large numbers of soil samples. It has low detection costs, good repeatability, and a sound quality control system. It is easy to promote and apply in environmental monitoring laboratories, research institutions, and third-party testing agencies, providing a standardized technical method for pollution monitoring, risk management, and remediation effect assessment of endocrine disruptors in agricultural land soil. Attached Figure Description

[0026] Figure 1 This is a comparative experiment of various types of extractants used in this invention; Figure 2 This is a comparative experiment on the dosage and number of extractions of the extractant of this invention; Figure 3 This is an experimental comparison of the purification effects of different solid-phase extraction columns of the present invention; Figure 4 This is an experimental comparison of the elution effect of different proportions of the eluent of the present invention. Detailed Implementation

[0027] The following is combined Figures 1-4 The present invention will be described in detail. For ease of description, the orientations mentioned below are defined as follows: The directions of up, down, left, right, front, and back mentioned below are consistent with the directions of up, down, left, right, front, and back in the projection relationship of the respective main view or structural schematic diagram.

[0028] Example 1: A high-performance liquid chromatography method for the detection of ethinylestradiol and nonylphenol in soil, such as... Figure 1 As shown, it includes the following steps: S1. Sample pretreatment: 200g of collected soil sample was first placed in a -60℃ environment to freeze dry to constant weight. Then the soil sample was ground and screened with a 10-mesh stainless steel sieve to obtain the soil sample to be tested. The soil sample was red soil; After sealing the soil sample to be tested, store it in a light-proof environment at -20℃ for later use. S2. Sample extraction: Weigh 3g of soil sample to be tested and place it in a centrifuge tube. First, add ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solutions. Vortex to mix and ensure that the ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solutions are in full contact with the soil sample and allowed to stand for equilibrium. Then, add 20mL of extraction reagent, vortex to mix, and extract by ultrasound. Then, centrifuge and collect the supernatant. The ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution is prepared by dissolving ethinylestradiol-D4 and nonylphenol-D9 in chromatographically pure methanol. The volume of ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution added is 100 μL. The concentrations of ethinylestradiol-D4 and nonylphenol-D9 in the ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution are both 80 μg / L. After adding ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution, vortex for 10 s to mix thoroughly, and let stand at room temperature in the dark for 10 min to complete the equilibrium. The extractant is a ternary composite extractant consisting of methanol, ethyl acetate and a deep eutectic solvent, with a volume ratio of 5:5:1. The deep eutectic solvent (DES) is a mixture of choline chloride and ethylene glycol in a mass ratio of 1:2.

[0029] The supernatant was placed in a 40°C constant temperature water bath and blown with nitrogen until completely dry. Then it was redissolved with methanol and diluted to a volumetric flask with ultrapure water. After shaking well, the sample extract was obtained and set aside for use. S3, Solid-phase extraction purification: First, the solid-phase extraction column is activated sequentially with chromatographically pure methanol and ultrapure water. Then, the sample extract obtained in S2 is passed through the solid-phase extraction column at a stable flow rate of 5 mL / min for sample loading. The solid-phase extraction column is a modified HLB solid-phase extraction column with a specification of 500 mg / 6 mL. The packing material is a hydrophilic-lipophilic balanced reverse polymer packing material. The HLB solid-phase extraction column modification method is as follows: graphene oxide and sodium montmorillonite in a mass ratio of 1:3 are loaded onto the surface of a hydrophilic-lipophilic balanced inverse polymer filler through in-situ polymerization.

[0030] After sample loading, the solid-phase extraction column was rinsed to remove water-soluble impurities, and the solid-phase extraction column was aspirated until completely dry. The solid-phase extraction column was then eluted with eluent, and all the eluent was collected into a clean pistil bottle. The eluent was placed in a 40°C constant temperature water bath and purged with nitrogen until completely dry. Chromatographic grade methanol was added for vortex redissolution, and the solution was filtered through a polytetrafluoroethylene organic phase filter membrane. The filtrate was collected in a brown liquid chromatography vial to obtain the sample to be tested. The eluent was a mixture of chromatographically pure methanol and ethyl acetate, with a volume ratio of methanol to ethyl acetate of 80:20.

[0031] After sealing, the sample to be tested should be stored in a light-protected environment at -25℃ until use. S4. High-performance liquid chromatography (HPLC) detection: Qualitative and quantitative detection of the sample is performed using a liquid chromatograph equipped with a fluorescence detector; S5. Quantitative analysis: The matrix-matched standard curve method was used to quantitatively calculate ethinylestradiol and nonylphenol in the test sample.

[0032] Example 2: The difference from Example 1 is that the soil sample in this example is black soil; The 250g soil sample was first freeze-dried at -50℃ to constant weight. Then the soil sample was ground and screened through a 20-mesh stainless steel sieve to obtain the soil sample to be tested. After sealing the soil sample to be tested, store it in a light-proof environment at -18℃ for later use.

[0033] Example 3: The difference from Example 1 is that the soil sample in this example is yellow-brown soil.

[0034] The 300g soil sample was first freeze-dried at -40℃ to constant weight. Then the soil sample was ground and screened through a 20-mesh stainless steel sieve to obtain the soil sample to be tested. After sealing the soil sample to be tested, store it in a light-proof environment at -15℃ for later use.

[0035] Example 4: The difference from Example 1 is that in S2, after adding the extractant, the mixture is vortexed for 40 seconds to ensure thorough mixing; Next, microwave and ultrasonic combined treatment was performed for 15 minutes, with the temperature controlled at 25℃; microwave power was 100W, ultrasonic power was 200W, and then it was placed in constant temperature oscillation at 180r / min frequency and 25℃ for 15 minutes. Centrifuge at 8000 r / min for 8 min, with the centrifugation temperature controlled at 3℃, and collect the supernatant into a heart-shaped flask; Add an equal amount of extractant to the centrifuged soil residue, and repeat the above steps of vortexing, microwave, ultrasonic combined treatment, shaking, and centrifugation once. Combine the supernatants from the two extractions and perform water bath heating and resolution in S2.

[0036] Example 5: The difference from Example 4 is that in S2, the ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution is: a mixed solution formed by dissolving ethinylestradiol-D4 and nonylphenol-D9 in chromatographically pure methanol, the amount of ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution added is 110 μL, the concentration of ethinylestradiol-D4 and nonylphenol-D9 in the ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution is 90 μg / L, after adding ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution, vortex for 12s to mix thoroughly, and let stand at room temperature in the dark for 12min to complete the equilibrium; After adding the extractant, vortex for 50 seconds to mix thoroughly. Next, microwave and ultrasonic combined treatment was performed for 18 minutes, with the temperature controlled at 28℃; microwave power was 120W, ultrasonic power was 250W, and then it was placed in constant temperature oscillation at 200r / min frequency and 26℃ for 15 minutes. Centrifuge at 10000 r / min for 10 min, with the centrifugation temperature controlled at 4℃, and collect the supernatant into a heart-shaped flask; The combined supernatant was placed in a 40°C constant temperature water bath and purged with nitrogen at a flow rate of 2L / min until completely dried.

[0037] Example 6: The difference from Example 4 is that in S2, the ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution is: a mixed solution formed by dissolving ethinylestradiol-D4 and nonylphenol-D9 in chromatographically pure methanol, the added amount of ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution is 120 μL, the concentration of ethinylestradiol-D4 and nonylphenol-D9 in the ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution is 100 μg / L, after adding ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution, vortex for 15s to mix thoroughly, and let stand at room temperature in the dark for 15min to complete the equilibrium; After adding the extractant, vortex for 30 seconds to mix thoroughly. Next, microwave and ultrasonic combined treatment was performed for 20 minutes, with the temperature controlled at 30℃; microwave power was 150W, ultrasonic power was 300W, and then it was placed in constant temperature oscillation at 220r / min frequency and 28℃ for 15 minutes. Centrifuge at 10000 r / min for 10 min, with the centrifugation temperature controlled at 5℃, and collect the supernatant into a heart-shaped flask; The combined supernatant was placed in a 40°C constant temperature water bath and purged with nitrogen at a flow rate of 0.5 L / min until completely dried.

[0038] Example 7: The difference from Example 5 is that in S3, the activation process is controlled at a flow rate of 2.5 mL / min, and the column is kept moist after activation.

[0039] Example 8: The difference from Example 5 is that in S3, the flow rate of the activation process is controlled at 3 mL / min, and the column is kept moist after activation.

[0040] Example 9: The difference from Example 7 is that in S3, the rinsing method is as follows: after the sample is loaded, the column is rinsed with 5 mL of ultrapure water at a flow rate of 2 mL / min to remove water-soluble impurities. After rinsing, the column is continuously aspirated for 5 min until no liquid drips from the column and it is completely dry.

[0041] Example 10: The difference from Example 7 is that in S3, the rinsing method is as follows: after the sample is loaded, the column is rinsed with 8 mL of ultrapure water at a flow rate of 3 mL / min to remove water-soluble impurities. After rinsing, the column is continuously aspirated for 10 min until no liquid drips from the column and it is completely dry.

[0042] Example 11: The difference from Example 9 is that in S3, the elution method is as follows: the solid phase extraction column is eluted in fractions with 15 mL of eluent at a flow rate of 1 mL / min. Add 1 mL of chromatographically pure methanol and vortex for 30 s to redissolve. After redissolving, filter through a 0.22 μm polytetrafluoroethylene organic phase filter membrane.

[0043] Example 12: The difference from Example 9 is that in S3, the elution method is as follows: the solid-phase extraction column is eluted in fractions with 15 mL of eluent at a flow rate of 2 mL / min. Add 1 mL of chromatographically pure methanol and vortex for 30 s to redissolve. After redissolving, filter through a 0.22 μm polytetrafluoroethylene organic phase filter membrane.

[0044] Example 13: The difference from Example 11 is that, in S4, the chromatographic conditions are set as follows: The chromatographic column used was a core-shell Inertsil ODS-SP-C18 column with dimensions of 150 mm × 4.6 mm and a packing particle size of 2.7 μm. The mobile phase consisted of chromatographically pure methanol and ultrapure water, with gradient elution mode, temperature control at 40℃ ± 0.5℃, mobile phase flow rate at 0.8 mL / min, and injection volume at 20 μL. The excitation wavelength of the fluorescence detector was set to 280 nm, the emission wavelength was set to 310 nm, and the total detection time for a single sample was 20 min.

[0045] Example 14: The difference from Example 13 is that the detailed parameters of the gradient elution mode in S4 are as follows: Table 1 Gradient elution mode parameters

[0046] In the linear gradient elution stage, the proportion of the mobile phase changes linearly and uniformly with time.

[0047] This method is applicable to the detection of ethinylestradiol and nonylphenol at different concentrations in soil. The average recoveries of ethinylestradiol (1–1000 μg / kg) in yellow-brown soil were 73.24%–82.26%, with average standard deviations of 2.08–3.30; the average recoveries of nonylphenol (1–1000 μg / kg) in yellow-brown soil were 82.37%–114.51%, with average standard deviations of 6.36–12.83. The method exhibits good accuracy and precision.

[0048] The spiked recovery and precision requirements of this method are as follows: for three spiked concentration levels of 1, 100, and 1000 μg / kg in soil, five parallel samples are set up for each concentration. The average recoveries of ethinylestradiol ranged from 73.24% to 82.26%, with intra-batch relative standard deviations ranging from 2.08% to 3.30%. The average recoveries of nonylphenol were 82.37%–114.51%, with intra-batch relative standard deviations of 6.36%–12.83%.

[0049] Example 15: The difference from Example 13 is that the isotope internal standard-matrix matching standard curve combined quantitative method in S5 includes the following steps: S5-1. Preparation of standard stock solutions: Accurately weigh 0.1 g each of ethinylestradiol standard, nonylphenol standard, ethinylestradiol-D4 isotope internal standard, and nonylphenol-D9 isotope internal standard, and place them in 100 mL brown volumetric flasks. Add chromatographic grade methanol, sonicate to dissolve, and dilute to the mark. Shake well to prepare target analyte single standard stock solutions and isotope internal standard single standard stock solutions with a mass concentration of 1 g / L. Store in a light-protected environment at -20℃. S5-2. Preparation of mixed standard working solution and isotope internal standard working solution: Before use, accurately pipette appropriate amounts of ethinylestradiol and nonylphenol single standard stock solutions, and dilute them stepwise with chromatographic grade methanol to prepare mixed standard working solutions of various concentrations; similarly, accurately pipette appropriate amounts of ethinylestradiol-D4 and nonylphenol-D9 single standard stock solutions, and dilute them stepwise with chromatographic grade methanol to prepare mixed isotope internal standard working solutions with a concentration of 100 μg / L, and store at 4℃ protected from light; S5-3 Preparation of blank matrix extract: Take a blank soil sample of the same type as the sample to be tested, and perform pretreatment according to steps S1~S3 to obtain blank matrix extract; S5-4. Standard Curve Plotting: Take the blank matrix extract and add a series of mixed standard working solutions of different concentrations and an equal volume of mixed isotope internal standard working solution to prepare a matrix-matched standard solution. The mass concentrations of ethinylestradiol and nonylphenol in the solution are 0.1, 1, 5, 10, 50, 100, 500, and 1000 μg / L, respectively. The mass concentrations of ethinylestradiol-D4 and nonylphenol-D9 at each concentration point are fixed at 10 μg / L. Three parallel samples are set for each concentration. Inject the prepared matrix-matched standard solution according to the chromatographic conditions in S4. Plot the matrix-matched standard curve with the ratio of the chromatographic peak area of ​​the target analyte to the corresponding isotope internal standard as the ordinate and the corresponding target analyte mass concentration as the abscissa to obtain the linear regression equation.

[0050] Ethinyl estradiol and nonylphenol showed good linearity in the concentration range of 0.1–1000 μg / L, with a linear correlation coefficient r ≥ 0.9995. Specifically, the linear regression equation for ethinyl estradiol was y = 438.72x + 128.6, with a correlation coefficient r = 0.9997; the linear regression equation for nonylphenol was y = 352.48x + 96.3, with a correlation coefficient r = 0.9999. The limit of detection (LOD) was defined as the concentration of the target analyte corresponding to a signal-to-noise ratio (SNR) of 3, and the limit of quantitation (LOQ) was defined as the concentration of the target analyte corresponding to a SNR of 10. Specifically, the LOD for ethinyl estradiol was 0.08 μg / L, and the LOQ was 0.25 μg / L; the LOD for nonylphenol was 0.5 μg / L, and the LOQ was 1.5 μg / L.

[0051] Five parallel samples were set up for each of the three spiked concentration levels of 1, 100, and 1000 μg / kg in soil. The average spiked recoveries of ethinylestradiol were 80.12–90.35%, with intra-assay relative standard deviations of 1.26–2.18%. The average spiked recoveries of nonylphenol were 90.47–108.62%, with intra-assay relative standard deviations of 2.35–4.12%.

[0052] In yellow-brown soil matrix, the spiked recoveries of ethinylestradiol were ≥80% and nonylphenol were ≥90%; in black soil matrix, the spiked recoveries of ethinylestradiol were ≥83% and nonylphenol were ≥92%; in red soil matrix, the spiked recoveries of ethinylestradiol were ≥87% and nonylphenol were ≥95%; in saline-alkali soil, purple soil, and alluvial soil matrix, the spiked recoveries of both target analytes were ≥78%, and the intra-batch relative standard deviations were ≤5%.

[0053] Experimental Example 1: Differences in the Selection of Extractants The difference in Example 1 is that the extractants used are ethyl acetate, dichloromethane, a 1:1 mixture of dichloromethane and methanol, a 1:1 mixture of dichloromethane and n-hexane, and a 1:1 mixture of acetone and n-hexane, respectively. The test results are as follows Figure 1As shown, the simultaneous extraction of ethinylestradiol and nonylphenol from soil was better when methanol and ethyl acetate were mixed in a 1:1 ratio. Other extractants showed little difference in extraction efficiency for ethinylestradiol. However, due to the strong nonpolarity of nonylphenol, the extraction effect of other extractants was poor during the simultaneous extraction process.

[0054] Experimental Example 2: Selection of Extractant Dosage and Number of Extractions Experiment 2-1: The difference from Example 1 is that the extractant in this experiment is a mixture of methanol and ethyl acetate in a ratio of 8:2 and a mixture of methanol and ethyl acetate in a ratio of 7:3, respectively. Experiment 2-2: The difference from Example 1 is that in this experiment, 10 mL of total extractant was divided into 5 mL portions for two extractions, and 20 mL of total extractant was divided into 10 mL portions for two extractions. Experiment 2-3: The difference from Example 1 is that in this experiment, 2g and 1g of sieved soil were weighed and placed in headspace bottles; Experiments 2-4: The difference from Example 1 is that in this experiment, 40 mL of total extractant was used for extraction once, and 40 mL of total extractant was divided into 10 mL portions for extraction four times.

[0055] The experimental results of Example 2 are as follows Figure 2 As shown, when the extractant is a 1:1 mixture of methanol and ethyl acetate, 40 mL of the total extractant is divided into 20 mL portions for two extractions. The extractant-to-sample ratio of 40:3 is more effective for the simultaneous extraction of ethinylestradiol and nonylphenol from soil. Excessive methanol content in the extractant can lead to the simultaneous extraction of other impurities in the soil. A darker extractant color is not conducive to purification. Insufficient extractant dosage will result in reduced extraction efficiency. Therefore, while ensuring cost, the amount of extractant and the number of extractions should be increased as much as possible to improve the spiked recovery rate of pollutants.

[0056] Experimental Example 3: Selection of Elution Buffer Ratio The difference from Example 1 is that the eluent is a mixture of 100% methanol, 80% methanol and 20% ethyl acetate; The experimental results of Example 3 are as follows Figure 4 As shown, the elution effect is better when the eluent is a mixture of 80% methanol and 20% ethyl acetate. Using a mixed eluent of methanol and ethyl acetate essentially utilizes the dual capabilities of methanol in disrupting polar adsorption (hydrogen bonds) and ethyl acetate in disrupting nonpolar adsorption (hydrophobic interactions). This combination provides a broader elution intensity spectrum, ensuring that even target analytes (ethinylestradiol and nonylphenol) with significantly different properties can be completely and efficiently recovered in the same elution step.

[0057] Comprehensive analysis of the experiments: When using the method of this invention to detect ethinylestradiol and nonylphenol in yellow-brown soil, black soil, and red soil, the recoveries were 82.26% and 104.79% in yellow-brown soil, respectively; 80.12% and 100.36% in black soil; and 87.34% and 110.56% in red soil. The results indicate that the recovery rates of ethinylestradiol and nonylphenol vary among different soil types, but all remain within the standard range. This invention is well-suited for further clarifying the pollution characteristics and ecological risks of ethinylestradiol and nonylphenol in different soil types.

[0058] To fully demonstrate the feasibility of the technical solution of this invention, single-factor variable comparative examples were set up for the core steps of this invention, and conventional methods of existing technologies were also set up for comparative examples. All comparative examples were simultaneously tested at three spiking concentration levels of 1, 100, and 1000 μg / kg, with five parallel samples set up for each concentration. Key technical parameters were measured and compared with Example 15 of this invention to clarify the technical differences and advantages.

[0059] Comparative Example 1: A single methanol extractant replacing the optimized composite extractant of this invention. Experimental procedures: In the S2 sample extraction step, the extractant was replaced with 100% chromatographically pure methanol. The remaining operating parameters and procedures for vortex mixing, ultrasonic extraction, isothermal shaking, centrifugation, secondary extraction, nitrogen blowing concentration, and reconstitution and volume adjustment were completely consistent with those in Example 15. All steps and parameters of S1 sample pretreatment, S3 solid-phase extraction purification, S4 chromatographic detection, and S5 quantitative analysis were consistent with those in Example 15.

[0060] Table 1. Detection results and key parameters of Comparative Example 1:

[0061] Results analysis: Pure methanol can only partially extract ethinylestradiol with moderate polarity, and its ability to extract nonylphenol with strong hydrophobicity is extremely weak. It cannot break the hydrophobic binding between soil organic matter and nonylphenol. The recovery rates of both target substances are much lower than those of this invention, and the method has poor precision and significantly increased detection limits, which cannot meet the requirements for the detection of trace target substances in soil.

[0062] Comparative Example 2: The extractant ratio deviates from the optimized value of this invention (methanol:ethyl acetate = 8:2). Experimental procedure: In the S2 sample extraction step, the extractant was replaced with a mixture of methanol and ethyl acetate in a volume ratio of 8:2. All other operation steps and parameters were completely consistent with those in Example 15.

[0063] Table 2. Detection results and key parameters of Comparative Example 2:

[0064] Results analysis: The low proportion of ethyl acetate in the extractant resulted in insufficient weak polarity extraction capacity, significantly reducing the extraction efficiency for the strongly hydrophobic nonylphenol. The recovery rate was far lower than that of this invention, and the precision of parallel samples exceeded the requirements of environmental testing standards (RSD≤15%). At the same time, the recovery rates of the two target substances differed significantly, making simultaneous and efficient extraction impossible. This proves that the 1:1 extractant ratio of this invention is the key optimization result that takes into account the two target substances with different polarities.

[0065] Comparative Example 3: Simplified extraction process, eliminating vortexing, isothermal oscillation, and secondary extraction steps. Experimental procedure: In the S2 sample extraction step, only ultrasonic extraction was performed at 250W power and 25℃ for 35 minutes. The vortex mixing and constant temperature oscillation operations were removed. The soil sample was extracted only once. The secondary extraction of the centrifuged soil residue and the combination of the supernatant were removed. The remaining nitrogen blowing concentration, reconstitution and volume adjustment operations were completely consistent with those in Example 15. All other steps and parameters were consistent with those in Example 15.

[0066] Table 3. Detection results and key parameters of Comparative Example 3:

[0067] Results analysis: The simplified extraction process cannot fully destroy the soil aggregate structure and cannot release the target pollutants adsorbed by soil clay particles and organic matter. There is a significant problem of incomplete extraction in a single extraction. The recovery rate of the two target substances is more than 30% lower than that of the present invention, and the method precision is extremely poor. This proves that the three-stage extraction process of vortex, ultrasound and isothermal oscillation, as well as the secondary extraction process of the present invention, is the core process for achieving efficient extraction of target substances.

[0068] Comparative Example 4: A C18 solid-phase extraction column was used instead of the optimized HLB solid-phase extraction column of this invention. Experimental procedure: In the S3 solid phase extraction purification step, the 500mg / 6mL HLB solid phase extraction column was replaced with a C18 solid phase extraction column of the same specification. All other operating parameters and procedures for activation, sample loading, rinsing, elution, nitrogen blowing concentration, and filtration were completely consistent with those in Example 15. All other steps and parameters were consistent with those in Example 15.

[0069] Table 4. Detection results and key parameters of Comparative Example 4:

[0070] Results analysis: such as Figure 3As shown, the C18 column only has a hydrophobic single retention mechanism, and its retention capacity for moderately polar ethinylestradiol is extremely weak. Column breakthrough is prone to occur during sample loading, leading to a significant decrease in recovery rate. At the same time, it has poor selectivity for interfering substances such as humic acid in the soil matrix, with a significant matrix effect. The target peak cannot be separated from the baseline, and the detection limit is several times higher than that of this invention. This proves that the HLB hydrophilic-lipophilic balanced solid phase extraction column used in this invention is the key to achieving stable enrichment and efficient impurity removal of the target analyte.

[0071] Comparative Example 5: Pure methanol eluent replaced the optimized composite eluent of this invention. Experimental procedure: In the S3 solid phase extraction purification step, the eluent was replaced with 100% chromatographically pure methanol. The remaining operating parameters and procedures for elution volume, flow rate, nitrogen blowing concentration, reconstitution and filtration were completely consistent with those in Example 15. All other steps and parameters were consistent with those in Example 15.

[0072] Table 5. Detection results and key parameters of Comparative Example 5:

[0073] Results Analysis: Excessive elution intensity with pure methanol simultaneously elutes the target analyte and strongly hydrophobic matrix interfering substances such as humic acid and oils adsorbed on the column, leading to a significantly enhanced matrix effect, severely deteriorated peak shapes, and parallel sample precision exceeding specifications. Although the elution efficiency of the target analyte did not decrease significantly, its detection accuracy and anti-interference ability were far lower than those of this invention. This demonstrates that the 80:20 methanol-ethyl acetate eluent ratio of this invention can ensure complete elution of the target analyte while minimizing matrix interference, which is the key optimization point of this invention.

[0074] Comparative Example 6: Ultraviolet detector replacing the optimized fluorescence detector of this invention Experimental procedure: In the S4 high performance liquid chromatography detection step, the fluorescence detector was replaced with an ultraviolet detector and the detection wavelength was set to 280 nm. The other chromatographic conditions, such as column, mobile phase, gradient elution program, column temperature, flow rate, and injection volume, were completely consistent with those in Example 15. All other steps and parameters were also consistent with those in Example 15.

[0075] Table 6. Detection results and key parameters of Comparative Example 6:

[0076] Results analysis: The sensitivity of the ultraviolet detector is much lower than that of the fluorescence detector, the baseline noise is large, and it does not respond to trace levels of ethinylestradiol and nonylphenol in the soil. It cannot achieve accurate quantification of low-concentration samples. The detection limit of the method is more than 20 times higher than that of the present invention, which cannot meet the detection requirements of trace endocrine interfering substances in the soil environment. This proves that the optimized combination of fluorescence detection wavelengths in the present invention is the core of achieving high sensitivity and high anti-interference ability detection.

[0077] Comparative Example 7: Existing conventional methods for detecting soil endocrine disruptors Experimental procedure (refer to the conventional methods for detecting phenols and estrogens in soil): S1. Sample pretreatment: Take 200g of soil sample, air dry naturally until constant weight, grind and pass through a 10-mesh stainless steel sieve, and store at room temperature away from light. S2. Sample extraction: Weigh 5g of soil sample to be tested, use Soxhlet extraction method, use 150mL n-hexane-acetone (1:1, v / v) as extraction solvent, extract for 12h, evaporate the extract to near dryness by rotary evaporation, redissolve with 2mL methanol, add 40mL ultrapure water and mix well to obtain sample extract; S3. Purification: Using liquid-liquid extraction, 30 mL of dichloromethane was added to the sample extract, and the mixture was shaken and extracted three times. The organic phases were combined, dehydrated with anhydrous sodium sulfate, and then rotary evaporated to near dryness. After redissolving in methanol, the sample was filtered through a 0.22 μm filter membrane to obtain the sample to be tested. S4. Chromatographic detection: High performance liquid chromatography with ultraviolet detector was used. The chromatographic column was a conventional C18 column (150 mm × 4.6 mm, 5 μm). The mobile phase was methanol-water (80:20, v / v) with isocratic elution. The column temperature was 30 ℃, the flow rate was 1.0 mL / min, the injection volume was 20 μL, the detection wavelength was 280 nm, and the detection time for a single sample was 40 min. S5. Quantitative analysis: Quantitative analysis is performed using the pure solvent standard curve method, with 3 parallel samples set up for each batch.

[0078] Table 7. Detection results and key parameters of Comparative Example 7:

[0079] Results Analysis: Existing conventional methods suffer from multiple drawbacks, including low extraction efficiency, poor purification effect, insufficient detection sensitivity, cumbersome and time-consuming operation, and poor precision, making it impossible to achieve simultaneous and accurate quantification of trace amounts of ethinylestradiol and nonylphenol in soil. In contrast, this invention, through parameter optimization and system innovation throughout the entire process, reduces the detection limit by more than one order of magnitude, increases the recovery rate by more than 40%, shortens the detection time by 50%, and significantly improves the method's precision and resistance to matrix interference, thus solving long-standing shortcomings in the industry.

[0080] Table 8. Summary Comparison of Core Parameters of Embodiment 15 of the Invention and All Comparative Examples

[0081] In summary: 1. This invention addresses the challenges of complex soil matrices, significant differences in the polarity of target substances, and the difficulty of trace enrichment in various industries. Through the synergistic optimization of the entire process, including the extraction system, extraction process, purification system, and detection system, it solves the technical problem that existing technologies cannot simultaneously, efficiently, and accurately detect ethinylestradiol and nonylphenol in soil. 2. The comparative analysis of each single factor demonstrates that the core technical features of this invention, such as the optimized extractant ratio, three-stage extraction, secondary extraction process, HLB solid-phase extraction system, optimized eluent ratio, and fluorescence detection parameters, are all key to achieving excellent technical results. 3. Compared with conventional methods in the prior art, the present invention has outstanding substantive features and significant technological progress in multiple dimensions such as extraction efficiency, detection sensitivity, precision, anti-interference ability, and detection efficiency.

Claims

1. A high-performance liquid chromatography method for the detection of ethinylestradiol and nonylphenol in soil, characterized in that, Includes the following steps: S1. Sample pretreatment: 200~300g of collected soil sample is first placed in an environment of -60~-40℃ for freeze drying until constant weight. Then the soil sample is ground and screened with a 10~20 mesh stainless steel sieve to obtain the soil sample to be tested. S2. Sample extraction: Weigh 3-5g of the soil sample to be tested and place it in a centrifuge tube. First, add ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solutions. Vortex to mix and ensure that the ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solutions are in full contact with the soil sample and allow to stand for equilibrium. Then, add 20-30mL of extraction reagent, vortex to mix, and extract by ultrasound. Then, centrifuge and collect the supernatant. The supernatant was placed in a 40°C constant temperature water bath and blown with nitrogen until completely dry. Then it was redissolved with methanol and diluted to a volumetric flask with ultrapure water. After shaking well, the sample extract was obtained and set aside for use. S3, Solid-phase extraction purification: First, activate the solid-phase extraction column with chromatographically pure methanol and ultrapure water in sequence. Then, pass the sample extract obtained in S2 through the solid-phase extraction column at a stable flow rate of 5~8 mL / min for sample loading. After sample loading, the solid-phase extraction column was rinsed to remove water-soluble impurities, and the solid-phase extraction column was aspirated until completely dry. The solid-phase extraction column was then eluted with eluent, and all the eluent was collected into a clean pistil bottle. The eluent was placed in a 40°C constant temperature water bath and purged with nitrogen until completely dry. Chromatographic grade methanol was added for vortex redissolution, and the solution was filtered through a polytetrafluoroethylene organic phase filter membrane. The filtrate was collected in a brown liquid chromatography vial to obtain the sample to be tested. S4. High-performance liquid chromatography (HPLC) detection: Qualitative and quantitative detection of the sample is performed using a liquid chromatograph equipped with a fluorescence detector; S5. Quantitative analysis: The matrix-matched standard curve method was used to quantitatively calculate ethinylestradiol and nonylphenol in the test sample.

2. The high-performance liquid chromatography method for the detection of ethinylestradiol and nonylphenol in soil according to claim 1, characterized in that, The soil sample mentioned in S1 is any one or more mixed soils of yellow-brown soil, red soil, and black soil.

3. The high-performance liquid chromatography method for detecting ethinylestradiol and nonylphenol in soil according to claim 1, characterized in that, In S2, the ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution is: a mixed solution formed by dissolving ethinylestradiol-D4 and nonylphenol-D9 in chromatographically pure methanol, wherein the amount of ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution added is 100~120μL, and the concentrations of ethinylestradiol-D4 and nonylphenol-D9 in the ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution are both 80~100μg / L. After adding ethinylestradiol-D4 and nonylphenol-D9 isotope internal standard working solution, the mixture is vortexed for 10~15s to mix thoroughly, and then allowed to stand at room temperature in the dark for 10~15min to complete the equilibrium. After adding the extractant, vortex for 30-50 seconds to mix thoroughly. Next, microwave and ultrasonic combined treatment was performed for 15-20 minutes, with the temperature controlled at 25-30℃; microwave power was 100-150W, ultrasonic power was 200-300W, and then the mixture was placed in a constant temperature oscillation at a frequency of 180-220r / min and a temperature of 25-28℃ for 15 minutes. Centrifuge at 8000~10000r / min for 8~10min, with centrifugation temperature controlled at 3~5℃, and collect the supernatant into a heart-shaped flask; Add an equal amount of extractant to the centrifuged soil residue, and repeat the above steps of vortexing, microwave, ultrasonic combined treatment, shaking, and centrifugation once. Combine the supernatants from the two extractions and perform water bath heating and resolution in S2.

4. The high-performance liquid chromatography method for detecting ethinylestradiol and nonylphenol in soil according to claim 3, characterized in that, The extractant described in S2 is a ternary composite extractant consisting of methanol, ethyl acetate and a deep eutectic solvent, with a volume ratio of 5:5:

1. The eutectic solvent is a mixture of choline chloride and ethylene glycol in a mass ratio of 1:

2.

5. The high-performance liquid chromatography method for the detection of ethinylestradiol and nonylphenol in soil according to claim 1, characterized in that, The solid-phase extraction column used in S3 is a modified HLB solid-phase extraction column with a specification of 500 mg / 6 mL and a hydrophilic-lipophilic balanced reverse polymer packing. The HLB solid-phase extraction column modification method is as follows: graphene oxide and sodium montmorillonite in a mass ratio of 1:3 are loaded onto the surface of a hydrophilic-lipophilic balanced inverse polymer filler through in-situ polymerization. The eluent is a mixture of chromatographically pure methanol and ethyl acetate, with a volume ratio of methanol to ethyl acetate of 80:

20.

6. The high-performance liquid chromatography method for detecting ethinylestradiol and nonylphenol in soil according to claim 1, characterized in that, In S3, the flow rate is controlled at 2~3 mL / min during the activation process, and the column is kept moist after activation.

7. The high-performance liquid chromatography method for the detection of ethinylestradiol and nonylphenol in soil according to claim 1, characterized in that, In S3, the rinsing method is as follows: after sample loading, rinse the column with 5-8 mL of ultrapure water at a flow rate of 2-3 mL / min to remove water-soluble impurities. After rinsing, continue aspiration for 5-10 min until no liquid drips from the column and it is completely dry.

8. The high-performance liquid chromatography method for the detection of ethinylestradiol and nonylphenol in soil according to claim 1, characterized in that, In S3, the elution method is as follows: elute the solid-phase extraction column in fractions with 15 mL of eluent at a flow rate of 1~2 mL / min.

9. The high-performance liquid chromatography method for the detection of ethinylestradiol and nonylphenol in soil according to claim 1, characterized in that, In S4, the chromatographic conditions are set as follows: The chromatographic column used was a core-shell Inertsil ODS-SP-C18 column with dimensions of 150 mm × 4.6 mm and a packing particle size of 2.7 μm. The mobile phase consisted of chromatographically pure methanol and ultrapure water, with gradient elution mode, temperature control at 40℃ ± 0.5℃, mobile phase flow rate at 0.8 mL / min, and injection volume at 20 μL. The excitation wavelength of the fluorescence detector was set to 280 nm, the emission wavelength was set to 310 nm, and the total detection time for a single sample was 20 min.

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