Detection method for enriching and determining multiple endocrine disrupters in water body and sediment

Abstract

The invention discloses a detection method for enriching and determining multiple endocrine disrupters in water and sediments. The detection method comprises the steps of sample pretreatment, enrichment, purification and LC-MS / MS detection. According to the method disclosed by the invention, accurate quantification and accurate qualification of detection of trace endocrine disrupters in water are ensured by adding various isotope internal standards, and the detection capability and detection flux of the endocrine disrupters are improved. The method provided by the invention can realize simultaneous detection of multiple endocrine disrupters in water bodies and sediments, improves the detection efficiency, and reduces the time cost at the same time. Meanwhile, the detection result is reliable in precision, low in detection limit and small in relative standard deviation.

Description

Technical Field

[0001] The invention belongs to the technical field of endocrine disruptor detection in the environment, and specifically relates to a detection method for enriching and measuring multiple endocrine disruptors in water bodies and sediments. Background Art

[0002] At present, new pollutants that are widely concerned at home and abroad mainly include persistent organic pollutants (POPs), endocrine disruptors (EDCs), antibiotics and microplastics controlled by international conventions. Even if new pollutants that are persistent and bioaccumulative meet the standards when discharged and enter the environment at low doses, they will continue to accumulate in organisms and gradually enrich along the food chain, eventually causing harm to environmental organisms and human health.

[0003] Environmental endocrine disruptors (EDCs), also known as environmental hormones, pose a huge threat to human health and ecosystems and have attracted much attention from researchers in recent years. Whether it is lakes, rivers, sediments, groundwater, or sewage treatment plants, studies have reported the detection of environmental hormone pollution and found its toxicity and harm to organisms. Therefore, it is crucial to have a deep understanding of its pollution characteristics in the environment and accurately identify those environmental hormones that may pose risks. Among hundreds of environmental hormones, alkylphenols and steroid hormones are two types of substances that are currently receiving more attention. Alkylphenols have become a type of environmental hormone with more serious pollution due to their extremely wide application in the chemical industry. When its estrogenic effect as an exogenous hormone was discovered, a series of related studies were gradually and orderly carried out. For steroid hormones, they will have negative effects even at extremely low concentrations. After entering the environmental medium, these environmental hormones will undergo a series of complex migration and transformation processes and eventually enter the human body, interfering with the normal endocrine function of humans, thereby causing a series of health hazards.

[0004] At present, the most common method for detecting EDCs is gas chromatography-mass spectrometry, but derivatization is required before on-machine detection, and the experimental steps are relatively cumbersome. Liquid chromatography-mass spectrometry (LC-MS / MS) is widely used in the detection of EDCs in environmental water bodies because it can perform a variety of EDCs analysis and has high sensitivity, and is often used in combination with solid phase extraction. However, most solid phase extraction-high performance liquid chromatography-mass spectrometry tandem detections are currently mostly targeted at a single type of EDCs, and there is no detection method for multiple types of EDCs. The selection of pH, elution solvent and re-dissolution solvent during the pretreatment process has a great influence on the recovery rate of target EDCs in the sample. How to improve the recovery rate of multiple EDCs in water bodies is an issue worth considering. In addition, the content of EDCs in natural water bodies and sediments is low, often existing in water bodies and sediments in trace forms, and the water and sediment matrices are relatively complex. How to avoid the interference of these matrices and accurately characterize and quantify EDCs in water bodies and sediments is also one of the problems that need to be solved.

[0005] Therefore, it is very necessary to develop a detection method for enriching and measuring various endocrine disruptors in water and sediments that can solve the above problems. Summary of the invention

[0006] In order to solve the above technical problems, the purpose of the present invention is to provide a detection method for enriching and determining a variety of endocrine disruptors in water bodies and sediments. Through solid phase extraction-high performance liquid chromatography-mass spectrometry tandem, EDCs in water bodies and sediments can be detected quickly, accurately and simultaneously, which can be applied to actual environmental monitoring to ensure environmental safety.

[0007] The object of the present invention is achieved by comprising the following steps: S1. Sample pretreatment: Water sample pretreatment: After the water sample to be tested passes through a 0.45 μm glass fiber filter membrane, the pH is adjusted to 4.5, and the internal standard mixed solution is added and mixed to obtain the treated sample; Sediment sample pretreatment: After freeze-drying to remove moisture, the sediment was mixed with diatomaceous earth, an internal standard mixed solution was added, and then extracted by rapid solvent extraction. The extraction solvent was ethyl acetate. After extraction, the sediment was dissolved in ultrapure water. After the liquid passed through a 0.45 μm glass fiber filter membrane, the pH was adjusted to 4.5 to obtain the treated sample; S2, enrichment: activating the C18 solid phase extraction column, taking the sample treated in step S1 and enriching it through the solid phase extraction column; S3, purification: Use methanol-water solution to add solid phase extraction column to elute to remove impurities, then use nitrogen to blow dry the solid phase extraction column, and then use ethyl acetate solution to elute the target endocrine disruptors to form an eluent; then use nitrogen to blow the eluent to nearly dryness at room temperature, use 50% methanol-water solution to re-dissolve and make up to 1mL, filter through a 0.22μm filter membrane, and transfer to a liquid phase vial for machine detection; S4, LC-MS / MS detection: Liquid chromatography-tandem mass spectrometry was used to determine the endocrine disruptors, and the concentration of endocrine disruptors in the sample was obtained according to the standard curve; The liquid chromatography conditions were as follows: C18 column, column temperature of 30°C, injection volume of 10 μL; mobile phase A was 0.5% ammonia water, mobile phase B was methanol, and gradient elution was performed at a flow rate of 0.3 mL / min; Gradient elution program: mobile phase A and mobile phase B were used for gradient elution at a flow rate of 0.3 mL / min; The mass spectrometry conditions were as follows: electrospray ion source positive and negative ion modes (ESI+, ESI-); scanning mode was multiple reaction monitoring mode (MRM); DL tube temperature was 200°C; heating module temperature was 450°C; interface temperature was 350°C; interface voltage was 1 kV, nebulizing gas and drying gas were both nitrogen, with flow rates of 3 L / min and 10 L / min, respectively; collision gas was argon, with a pressure of 230 kPa.

[0008] Among them, a single injection of 12 minutes in step S4 can complete the simultaneous analysis of multiple endocrine disruptors and can detect up to 13 types.

[0009] The C18 chromatographic column can specifically be a Shim-pack Scepter C18-120 chromatographic column, and the parameters of the Shim-pack Scepter C18-120 chromatographic column are 100 mm×2.1 mm, 1.9 μm.

[0010] Preferably, the gradient elution program is 0-min-6.0min, 40%-85% of the mobile phase B liquid; 6.0-8.0min, 85%-97% of the mobile phase B liquid, maintained for 4min; 12.0min-12.1min, 97%-40% of the mobile phase B liquid, maintained for 5min.

[0011] Preferably, the endocrine disruptors are bisphenol A (BPA), 4-octylphenol (4-OP), 4-nonylphenol (4-NP), 4-cumylphenol (4-CP), estrone (E1), 17-α-estradiol (17-α-Estradiol, 17-α-E2), 17-β-estradiol (17-β-Estradiol, 17-β-E2), 17-α-ethinylestradiol (17-α-EthinylEstradiol, EE2), estriol (E3), progesterone (PROG), testosterone (TEST), androstenedione (AND) and dihydrotestosterone (DHT), a total of 13 types.

[0012] Preferably, the internal standard mixed solution in step S1 includes bisphenol A-D16 (BPA-d16), 4-nonylphenol-D4 (4-NP-d4) and ethinylestradiol-D4 (EE2-d4) of known concentrations as recovery rate indicators, and testosterone-D3 (TEST-d3), progesterone-D9 (PROG-d9) and estriol-D3 (E3-d3) of known concentrations are added before the machine detection in step S3 as the machine internal standards.

[0013] Preferably, after freeze-drying the sediment in step S1 to remove moisture, 5 g of the sediment is weighed and mixed with diatomaceous earth; after extraction, the sediment is dissolved in 1000 mL of ultrapure water.

[0014] Preferably, in step S1, the extraction is performed at a power of 1500 psi, a temperature of 80° C., a static extraction of 5 min, a flushing volume of 60%, a purge time of 100 s, and is repeated 3 times.

[0015] Preferably, in step S1, the pH is adjusted to 4.5 using a 5 mol / L HCl solution.

[0016] Preferably, in step S2, 1000 ml of pretreated water sample is taken and enriched through a solid phase extraction column at a flow rate of 5 mL / min.

[0017] Preferably, the activation in step S2 is to activate the C18 solid phase extraction column in sequence using 5 mL of ethyl acetate, 5 mL of methanol and three times of 5 mL of deionized water, and the flow rate is controlled at 2 mL / min.

[0018] Preferably, step S3 specifically uses a 10% methanol aqueous solution and blows dry with nitrogen for 5 minutes.

[0019] Preferably, step S3 further includes eluent treatment, specifically, concentrating the eluent to near dryness using nitrogen at room temperature, fixing the volume to 1 mL using 50% methanol aqueous solution, and then transferring it to a liquid phase vial for standby use.

[0020] Preferably, the standard curve of step S4 is prepared by preparing a mixed standard stock solution of all single standards of the endocrine disruptors to be tested, and using a 50% methanol aqueous solution to prepare the mixed standard stock solution into standard working solutions with concentrations of 2.0 μg / L, 5.0 μg / L, 10.0 μg / L, 20.0 μg / L, 50.0 μg / L, and 100.0 μg / L, and measured according to the analytical conditions, with the endocrine disruptor concentration as the abscissa and the ratio of the endocrine disruptor peak area to the internal standard peak area as the ordinate, and linear regression is performed, and the correlation coefficients are all >0.998; The multiple reaction monitoring ion pairs and mass spectrometry related parameters of the compounds are shown in Table 1: Table 1: Multiple reaction monitoring ion pairs and mass spectrometry parameters of endocrine disruptors Table 2: Multiple reaction monitoring ion pairs and mass spectrometry parameters of endocrine disruptor internal standards .

[0021] Among them, the internal standard is selected according to the peak time and properties of different endocrine disruptors on the chromatographic column; the standard curve, internal standard and linear relationship of the compound are shown in Table 3; Table 3: Standard curves, internal standards and linear relationships of compounds Among them, the retention time of the chromatographic peak of endocrine disruptors in the sample should be compared with the retention time of the corresponding standard chromatographic peak, and the variation range should be within ±2.5%.

[0022] In the liquid chromatography conditions of step S4, the total flow rate of mobile phase A + mobile phase B is 0.3 mL / min; the gradient elution is first through 0 min-6 min, 60%-15% mobile phase A + 40%-85% mobile phase B; after 6.0 min-8.0 min, 15%-3% mobile phase A + 85%-97% mobile phase B, maintained for 4 min; finally, after 12.0 min-12.1 min, 3%-60% mobile phase A + 97%-40% mobile phase B, maintained for 5 min.

[0023] Compared with the prior art, the present invention has the following technical effects: 1. The present invention uses a C18 solid phase extraction column for concentration and enrichment, and can simultaneously determine 13 endocrine disruptors, including TEST, PROG, AND, E3, 17-α-E2, 17-β-E2, EE2, E1, BPA, 4-NP, 4-OP, 4-CP and DHT, greatly improving the detection efficiency. At the same time, using a C18 reversed phase liquid chromatography column, only a single injection of 12 minutes is required to complete the simultaneous analysis of 13 endocrine disruptors, reducing the time for machine detection, greatly reducing the time cost, solving the problem of long sample detection cycle and difficulty in simultaneously processing multiple endocrine disruptors in traditional analysis technology, and better meeting the needs of rapid analysis.

[0024] 2. The present invention accurately identifies and quantifies 13 endocrine disruptors in water based on isotope internal standard technology. Six isotope internal standards, BPA-d16, 4-NP-d4, TEST-d3, PROG-d9, E3-d3 and EE2-d4, can be used to ensure the accuracy of quantification in various water bodies. 3. The present invention uses the LC-MS / MS method to accurately quantify 13 endocrine disruptors in water with high sensitivity. The detection limit (LOD) of the 13 endocrine disruptors is 0.06ng / L-1.35ng / L, and the quantification limit (LOQ) is 0.18ng / L-4.51ng / L. The detection limit and quantification limit at low concentration levels ensure the possibility of this analytical method to detect trace EDCs in water environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the process of the present invention; Figure 2 This is the chromatogram for the detection of 13 endocrine disruptors; Figure 3 This is a comparison chart of the recovery rates of endocrine disruptors when using different solid phase extraction cartridges; Figure 4 This is a comparison chart of the recovery rates of endocrine disruptors at different pH values; Figure 5 This is the detection result of endocrine disruptors in actual environment using the method in Example 6. DETAILED DESCRIPTION

[0026] The present invention is further described below in conjunction with the embodiments and drawings, but the present invention is not limited in any way. Any changes or substitutions made based on the teachings of the present invention belong to the protection scope of the present invention.

[0027] Example 1 The detection method for enriching and determining multiple endocrine disrupting chemicals in sediments in this embodiment comprises the following steps: S1. Sample pretreatment: Sediment sample pretreatment: After freeze-drying to remove moisture, the sediment was mixed with 5 g of diatomaceous earth, and internal standards of known concentration (BPA-d16, 4-NP-d4, and EE2-d4) were added as recovery rate indicators. The sediment was then extracted using rapid solvent extraction (extraction power 1500 psi, 80 °C, static extraction 5 min, flushing volume 60%, purge time 100 s, and 3 cycles). The extraction solvent was ethyl acetate. After extraction, the sediment was dissolved in 1000 ml of ultrapure water. After the liquid was filtered through a 0.45 μm glass fiber membrane, the pH was adjusted to 4.5 to obtain the treated sample. S2, enrichment: Activate C18 solid phase extraction column (use 5mL ethyl acetate, 5mL methanol and three times 5mL deionized water to activate C18 solid phase extraction column in sequence, the flow rate is controlled at 2mL / min), take 1000ml sample treated in step S1 and enrich it through solid phase extraction column, the flow rate is 5mL / min; S3, purification: Use 10% methanol aqueous solution to add to the solid phase extraction column for elution to remove impurities, then use nitrogen to blow dry the solid phase extraction column for 5 minutes, then use ethyl acetate solution to elute the target endocrine disruptors to form an eluent, and then use nitrogen to concentrate to near dryness at room temperature, use 50% methanol aqueous solution to make the volume to 1mL, filter through a 0.22μm filter membrane and transfer to a liquid phase vial, add three isotope internal standards of known concentrations, TEST-d3, PROG-d9, and E3-d3, as internal standards for the machine and wait for LC-MS / MS detection; S4, LC-MS / MS detection: Liquid chromatography-tandem mass spectrometry was used to determine the endocrine disruptors, and the concentration of endocrine disruptors in the sample was obtained according to the standard curve; Instrument conditions: The liquid chromatography conditions were as follows: C18 column, column temperature of 30°C, injection volume of 10 μL; mobile phase A was 0.5% ammonia water, mobile phase B was methanol, and gradient elution was performed at a flow rate of 0.3 mL / min; The gradient elution program is 0min-6.0min, 40%-85% of the mobile phase B liquid; 6.0min-8.0min, 85%-97% of the mobile phase B liquid, hold for 4min; 12.0min-12.1min, 97%-40% of the mobile phase B liquid, hold for 5min; The mass spectrometry conditions were as follows: electrospray ion source positive and negative ion modes (ESI+, ESI-); scanning mode was multiple reaction monitoring mode (MRM); DL tube temperature was 200°C; heating module temperature was 450°C; interface temperature was 350°C; interface voltage was 1 kV, nebulizing gas and drying gas were both nitrogen, with flow rates of 3 L / min and 10 L / min, respectively; collision gas was argon, with a pressure of 230 kPa.

[0028] Example 2 The detection method of enriching and determining multiple endocrine disruptors in water in this embodiment is based on Example 1, and the difference from Example 1 is: The sample pretreatment in step S1 is specifically the water sample pretreatment: after the water sample to be tested passes through a 0.45 μm glass fiber filter membrane, the pH is adjusted to 4.5, and an internal standard mixed solution (including internal standards of known concentrations of BPA-d16, 4-NP-d4 and EE2-d4) is added and mixed to obtain a treated sample.

[0029] Example 3: Actual water recovery rate determination verification experiment According to the detection method of Example 2, the actual water recovery rate was determined; Water sample preparation: Take 1000mL of real water sample without endocrine disruptors to be tested, add 13 endocrine disruptors to be tested (TEST, PROG, AND, E3, 17-α-E2, 17-β-E2, EE2, E1, BPA, 4-NP, 4-OP, 4-CP and DHT), the addition amount is 100ng / L, pass the water sample to be tested through a 0.45μm glass fiber filter, adjust the pH to 4.5, add the internal standard mixed solution and mix well, the internal standard concentration is 50ng / L, prepare 5 parallel samples; Experimental results: Table 4: Verification data results of 13 endocrine disruptors in actual water spike recovery experiments The experimental results show that the recovery rate of this experimental method is 54.11%-110.62%, and the accuracy is 1.33%-19.42%. This method can effectively detect trace endocrine disruptors in water bodies.

[0030] Example 4: Sediment recovery rate determination verification experiment According to the detection method of Example 1, the sediment recovery rate was determined; Sediment sample preparation: Sediment samples were collected from a plateau lake in the southwest region. During sample pretreatment, three known concentration internal standards of BPA-d16, 4-NP-d4 and EE2-d4 were added at 20 μg / g as recovery indicators. Experimental results: Table 5: Sediment spike recovery test validation data results The experimental results show that the recovery rate of this experimental method is high and this method can effectively detect trace endocrine disruptors in sediments.

[0031] Example 5: Comparative experiment of C18 solid phase extraction column and HLB solid phase extraction column The experimental methods of C18 solid phase extraction column and HLB solid phase extraction column were the same as those of Example 2, and water samples were tested. The difference was that the comparative example used HLB solid phase extraction column. The experimental results are shown in the attached Figure 3 As shown, it can be seen that the recovery rate when using C18 solid phase extraction column is greater than that using HLB solid phase extraction column.

[0032] Example 6: Comparative experiment at pH 2 and pH 4.5 The experimental methods of C18 solid phase extraction column and HLB solid phase extraction column were the same as those of Example 2, and water samples were tested. The difference was that the pH of the comparative sample was adjusted to 2 during pretreatment. The experimental results are shown in the attached Figure 4 As shown, it can be seen that the recovery rate at pH 4.5 is greater than that at pH 2.

[0033] Example 7 According to the detection method of Example 2, a water sample from a plateau lake basin in the southwest region was detected; Water sample preparation: The sample was taken from a plateau lake in the southwest region. During pretreatment, 100 μg / L of BPA-d16, 4-NP-d4 and EE2-d4 were added as recovery indicators, and TEST-d3, PROG-d9 and E3-d3 were added as internal standards. The experimental results are shown in the attached figure. Figure 5 As shown, except for 4-CP, EE2 and E3, whose concentrations were lower than the instrument detection limit, the other endocrine disruptors were detected, among which BPA and DHT were the main types of endocrine disruptor residues in this area.

Claims

1. A method for enriching and determining a variety of endocrine disruptors in water and sediments, characterized in that The following steps are involved: S1. Sample pretreatment: Water sample pretreatment: After the water sample to be tested passes through a glass fiber filter membrane, the pH is adjusted to 4.5, and the internal standard mixed solution is added and mixed to obtain the treated sample; Sediment sample pretreatment: After freeze-drying to remove moisture, the sediment was mixed with diatomaceous earth, an internal standard mixed solution was added, and then extracted by rapid solvent extraction. The extraction solvent was ethyl acetate. After extraction, the sediment was dissolved in ultrapure water. After the liquid passed through a glass fiber filter membrane, the pH was adjusted to 4.5 to obtain the treated sample. S2, enrichment: activating the C18 solid phase extraction column, taking the sample treated in step S1 and enriching it through the solid phase extraction column; S3, purification: Use methanol-water solution to add solid phase extraction column to elute to remove impurities, then use nitrogen to blow dry the solid phase extraction column, and then use ethyl acetate solution to elute the target endocrine disruptors to form an eluent; then use nitrogen to blow the eluent to nearly dryness at room temperature, use methanol-water solution to redissolve and fix the volume, filter with a filter membrane and wait for machine detection; S4, LC-MS / MS detection: Liquid chromatography-tandem mass spectrometry was used to determine the endocrine disruptors, and the concentration of endocrine disruptors in the sample was obtained according to the standard curve; The liquid chromatography conditions were as follows: C18 column, column temperature of 30°C, injection volume of 10 μL; mobile phase A was 0.5% ammonia water, mobile phase B was methanol, and gradient elution was performed at a flow rate of 0.3 mL / min; The mass spectrometry conditions were as follows: electrospray ion source positive and negative ion modes; scanning mode was multiple reaction monitoring mode; DL tube temperature was 200°C; heating module temperature was 450°C; interface temperature was 350°C; interface voltage was 1 kV, nebulizing gas and drying gas were both nitrogen, with flow rates of 3 L / min and 10 L / min, respectively; The collision gas was argon with a pressure of 230 kPa.

2. The method for enriching and determining a variety of endocrine disruptors in water and sediments according to claim 1, characterized in that The endocrine disruptors are bisphenol A, 4-octylphenol, 4-nonylphenol, cumylphenol, estrone, 17-α-estradiol, 17-β-estradiol, 17-α-ethynyl estradiol, estriol, progesterone, testosterone, androstenedione and dihydrotestosterone.

3. The method for enriching and determining a variety of endocrine disruptors in water and sediments according to claim 1, characterized in that In step S1, the internal standard mixed solution includes bisphenol A-D16, 4-nonylphenol-D4 and ethinylestradiol-D4 of known concentrations. In step S3, testosterone-D3, progesterone-D9 and estriol-D3 of known concentrations are added before the machine is tested.

4. The method for enriching and determining a variety of endocrine disrupting chemicals in water and sediments according to claim 1, characterized in that After freeze-drying to remove moisture from the sediment in step S1, 5 g of the sediment was weighed and mixed with diatomaceous earth; after extraction, the sediment was dissolved in 1000 mL of ultrapure water.

5. The method for enriching and determining a variety of endocrine disrupting chemicals in water and sediments according to claim 1, characterized in that The extraction in step S1 was performed with a power of 1500 psi, a temperature of 80°C, a static extraction of 5 min, a flushing volume of 60%, a purge time of 100 s, and three cycles.

6. The method for enriching and determining a variety of endocrine disruptors in water and sediments according to claim 1, characterized in that In step S2, 1000 ml of pretreated water sample was taken and enriched through a solid phase extraction column at a flow rate of 5 mL / min.

7. The method for enriching and determining a variety of endocrine disruptors in water and sediments according to claim 1, characterized in that Step S3 specifically uses 10% methanol aqueous solution and blows dry with nitrogen for 5 minutes.

8. The method for enriching and determining a variety of endocrine disruptors in water and sediments according to claim 1, characterized in that Step S3 also includes eluent treatment, specifically, concentrating the eluent to near dryness using nitrogen at room temperature, making the volume to 1 mL using 50% methanol aqueous solution, and then transferring it to a liquid phase vial for standby use.

9. The method for enriching and determining a variety of endocrine disruptors in water and sediments according to claim 1, characterized in that The standard curve in step S4 is prepared by preparing a single standard of endocrine disruptors into a mixed standard stock solution, which is then prepared using a 50% methanol aqueous solution into standard working solutions with concentrations of 2.0 μg / L, 5.0 μg / L, 10.0 μg / L, 20.0 μg / L, 50.0 μg / L, and 100.0 μg / L. The standard solution is determined according to the analytical conditions, with the endocrine disruptor concentration as the horizontal axis and the ratio of the endocrine disruptor peak area to the internal standard peak area as the vertical axis. Linear regression is performed, and the correlation coefficients are all >0.998.

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