CN-121994971-A - High performance liquid chromatography detection method for ethinyl estradiol and nonylphenol in soil

CN121994971ACN 121994971 ACN121994971 ACN 121994971ACN-121994971-A

Abstract

The invention discloses a high performance liquid chromatography detection method for ethinyl estradiol and nonylphenol in soil, which comprises the steps of S1, sample pretreatment, S2, sample extraction, S3, solid phase extraction and purification, S4, high performance liquid chromatography detection, S5 and quantitative analysis, wherein the detection method is used for solving the technical pain points of complex soil matrix, large polarity difference of ethinyl estradiol and nonylphenol and high trace enrichment difficulty in the industry, overcomes the defects of the synchronous quantitative detection method for two typical endocrine disruptors in different types of farmland soil in the prior art, and provides a stable and reliable technical support for pollution characteristic research, environmental return analysis and ecological risk assessment of the ethinyl estradiol and nonylphenol in the soil.

Inventors

GAO YANZHENG
WANG JUNYUE
QIN CHAO
LING WANTING

Assignees

南京农业大学

Dates

Publication Date: 20260508
Application Date: 20260319

Claims (9)

1. The high performance liquid chromatography detection method for ethinyl estradiol and nonylphenol in soil is characterized by comprising the following steps of: S1, sample pretreatment, namely firstly placing 200-300 g of collected soil samples in an environment of minus 60 ℃ to minus 40 ℃ to be freeze-dried to constant weight, then grinding the soil samples, and screening the soil samples by a stainless steel screen with 10-20 meshes to obtain soil samples to be tested; s2, sample extraction, namely weighing 3-5 g of soil sample to be detected, placing the soil sample into a centrifuge tube, adding ethinyl estradiol-D4 and nonylphenol-D9 isotope internal standard working solutions, uniformly mixing by vortex to enable the ethinyl estradiol-D4 and nonylphenol-D9 isotope internal standard working solutions to fully contact the soil sample, standing and balancing, adding 20-30 mL of extractant, uniformly mixing by vortex, extracting by ultrasonic, centrifuging and collecting supernatant; Placing the supernatant in a 40 ℃ constant-temperature water bath, blowing nitrogen to completely dry, then re-dissolving with methanol, fixing the volume in a 40mL volumetric flask with ultrapure water, and shaking uniformly to obtain a sample extracting solution for later use; S3, solid phase extraction and purification, namely sequentially activating a solid phase extraction column by using chromatographic pure methanol and ultrapure water, and then completely passing the sample extracting solution obtained in the S2 through the solid phase extraction column at a stable flow rate of 5-8 mL/min for sample loading treatment; Eluting the solid phase extraction column to remove water-soluble impurities after sample loading is completed, and sucking the solid phase extraction column until the solid phase extraction column is completely dried, eluting the solid phase extraction column by using eluent, collecting all the eluent into a clean chicken heart bottle, placing the eluent into a 40 ℃ constant-temperature water bath, blowing nitrogen into the chicken heart bottle until the chicken heart bottle is completely dried, adding chromatographic pure methanol, vortex re-dissolving, filtering by using a polytetrafluoroethylene organic phase filter membrane, and collecting filtrate into a brown liquid phase vial to obtain a sample to be tested; S4, high performance liquid chromatography detection, namely carrying out qualitative and quantitative detection on the sample to be detected by adopting a liquid chromatograph provided with a fluorescence detector; S5, quantitatively analyzing, namely quantitatively calculating ethinyl estradiol and nonylphenol in the sample to be detected by adopting a matrix matching standard curve method.
2. The method for high performance liquid chromatography detection of ethinyl estradiol and nonylphenol in soil according to claim 1, wherein the soil sample in S1 is any one or more of mixed soil of yellow brown soil, red soil and black soil.
3. The high performance liquid chromatography detection method for the ethinyl estradiol and the nonylphenol in the soil is characterized in that in S2, the isotope internal standard working fluids of the ethinyl estradiol-D4 and the nonylphenol-D9 are mixed solutions formed by dissolving the ethinyl estradiol-D4 and the nonylphenol-D9 in chromatographic pure methanol, the addition amount of the isotope internal standard working fluids of the ethinyl estradiol-D4 and the nonylphenol-D9 is 100-120 mu L, the concentrations of the ethinyl estradiol-D4 and the nonylphenol-D9 in the isotope internal standard working fluids of the ethinyl estradiol-D4 and the nonylphenol-D9 are 80-100 mu g/L, and after the isotope internal standard working fluids of the ethinyl estradiol-D4 and the nonylphenol-D9 are added, vortex 10-15S are fully mixed uniformly, and the mixture is kept away from light for 10-15 min at room temperature to complete balance; adding extractant, and swirling fully and uniformly mixing for 30-50 s; then, carrying out cooperative treatment by adopting microwaves and ultrasound for 15-20 min, controlling the temperature to be 25-30 ℃, carrying out constant-temperature oscillation for 15min at the frequency of 180-220 r/min and the temperature of 25-28 ℃ under the microwave power of 100-150W and the ultrasonic power of 200-300W; centrifuging for 8-10 min at 8000-10000 r/min, controlling the centrifuging temperature to be 3-5 ℃, and collecting supernatant into a heart-shaped bottle; Adding the extractant which is equal to the extractant in the previous step into the centrifuged soil residue again, repeating the steps of vortex, microwave and ultrasonic cooperative treatment, oscillation and centrifugation for 1 time, and combining the supernatants extracted from the two steps together to perform water bath heating and re-dissolving operation of S2.
4. The method for detecting ethinyl estradiol and nonylphenol in soil by high performance liquid chromatography according to claim 3, wherein the extractant in S2 is a ternary composite extractant composed of methanol, ethyl acetate and deep eutectic solvent, and the volume ratio of the three is 5:5:1 in sequence; wherein the deep eutectic solvent is a mixture of choline chloride and ethylene glycol according to a mass ratio of 1:2.
5. The method for detecting ethinyl estradiol and nonylphenol in soil by high performance liquid chromatography according to claim 1, wherein the solid phase extraction column used in the step S3 is a modified HLB solid phase extraction column, the specification of the HLB solid phase extraction column is 500mg/6mL, and the filler is hydrophilic-lipophilic balance type reversed phase polymer filler; the HLB solid phase extraction column modification method comprises the step of loading graphene oxide and sodium montmorillonite with the mass ratio of 1:3 on the surface of a hydrophilic-lipophilic balance type reversed-phase polymer filler through in-situ polymerization. The eluent is a mixed solution of chromatographic pure methanol and ethyl acetate, and the volume ratio of the methanol to the ethyl acetate in the mixed solution is 80:20.
6. The method for detecting ethinyl estradiol and nonylphenol in soil by high performance liquid chromatography according to claim 1, wherein in S3, the flow rate is controlled to be 2-3 mL/min in the activation process, and the column is kept moist after the activation is completed.
7. The high performance liquid chromatography detection method for ethinyl estradiol and nonylphenol in soil according to claim 1, wherein in S3, after the sample loading is completed, the column is leached with 5-8 mL of ultrapure water at a flow rate of 2-3 mL/min, water-soluble impurities are removed, and suction is continuously carried out for 5-10 min after leaching is completed until the column is free from liquid dripping and is completely dried.
8. The method for detecting ethinyl estradiol and nonylphenol in soil by high performance liquid chromatography according to claim 1, wherein in S3, the eluting method is to elute the solid phase extraction column with 15mL of eluent at a flow rate of 1-2 mL/min.
9. The method for high performance liquid chromatography detection of ethinyl estradiol and nonylphenol in soil according to claim 1, wherein in S4, the chromatographic conditions are set as follows: the chromatographic column adopts core-shell type Inertsil ODS-SP-C18 chromatographic column with specification of 150mm×4.6mm and filler particle diameter of 2.7 μm, wherein the mobile phase is chromatographic pure methanol and ultrapure water, the temperature is controlled to 40+ -0.5 ℃ by adopting a gradient elution mode, the flow rate of the mobile phase is 0.8mL/min, and the sample injection amount is 20 μL; The excitation wavelength of the fluorescence detector was set to 280nm, the emission wavelength was set to 310nm, and the total detection duration of the individual samples was 20min.

Description

High performance liquid chromatography detection method for ethinyl estradiol and nonylphenol in soil Technical Field The invention relates to the technical field of environmental sample detection, in particular to a high performance liquid chromatography detection method for ethinyl estradiol and nonylphenol in soil. Background Endocrine disruption has become a major potential threat to the global ecological environment and public health. Currently, the identification of environmental behavior and toxicological effects of typical pollutants is a key strategy to evaluate and control their impact on ecosystems and human health. Systematic studies were carried out in the early stage of the laboratory on ethinyl estradiol and nonylphenol which are widely detected in the environment. Ethinyl estradiol is an artificially synthesized estrogen, while nonylphenol is an industrial chemical with pseudo-estrogenic activity, both of which are typical environmental endocrine disruptors. Research shows that under the condition of extremely low environmental concentration (ng/L), the two pollutants can cause ecological effects such as female sex, gonad dysplasia, population decay and the like of male fishes by interfering a normal hormone signal path of organisms. Further analysis shows that ethinyl estradiol is slowly metabolized in the environment and is strong in durability compared with natural estrogens, while nonylphenol has remarkable bioaccumulation effect and pseudo-estrogenic activity, and both can be transmitted through a food chain, thus potentially threatening human reproductive health. Environmental behavior research shows that the pollutants are easy to be adsorbed and desorbed in soil and water, and desorption has obvious lag, so that the pollutants can become a long-term chemical timing bomb. Therefore, research on the trend and the ecotoxicological effect of ethinyl estradiol and nonylphenol in the environment has important scientific value and practical significance for revealing the pollution characteristics, evaluating the ecological risk, perfecting the water quality standard and formulating the pollution control strategy. At present, the research on ethinyl estradiol and nonylphenol is concentrated on water environment medium, and the cognition on the environmental behavior, the trend and the ecological effect of the ethinyl estradiol and nonylphenol in soil environment is still very limited. In fact, soil acts as the primary "sink" of organic contaminants, and is also the "source" of migration to groundwater and organisms, playing a key role in the geochemical cycling of the contaminant environment. Ethinyl estradiol mainly originates from land utilization and sewage irrigation of livestock and poultry breeding wastes, while nonylphenol enters the soil environment through various ways such as industrial emission, sludge agriculture, atmospheric sedimentation and the like. After entering the soil, the two pollutants can be adsorbed and combined with soil organic matters, and the retention characteristics and migration and transformation rules different from the water environment are shown. However, prior studies have not systematically recognized the mechanisms of adsorption and desorption of ethinyl estradiol and nonylphenol, the degradation transformation pathways, the bioavailability, and the potential risks of delivery along the food chain in the soil. Research shows that the degradation half-life of the pollutants in the soil is longer, and obvious hysteresis effect exists in the desorption process, so that the soil can become a long-term secondary pollution source, and the sustained release of the pollutants threatens the safety of underground water and crops. Therefore, the accurate quantitative research of ethinyl estradiol and nonylphenol in the soil environment is the basis for developing the subsequent pollution characteristics, migration transformation and ecological risks, and the establishment of an accurate and effective detection method has important scientific value and practical significance for making up the defects of the current research, perfecting the pollutant environment trend theory, scientifically evaluating the agricultural land safety and guiding the pollution soil management and control. At present, the detection methods of ethinyl estradiol and nonylphenol in soil at home and abroad are limited. The invention aims to establish a high performance liquid chromatography method for detecting ethinyl estradiol and nonylphenol in soil, and provides technical support for detecting exposure of ethinyl estradiol and nonylphenol in soil. Disclosure of Invention The invention aims to provide a high performance liquid chromatography detection method for ethinyl estradiol and nonylphenol in soil, which can meet the rapid detection requirement of a large number of soil samples and remarkably improve the detection flux and efficiency. In order to achieve the above purpose, the presen