Method for detecting environmental pollutants by using dispersive liquid-liquid microextraction technology

Abstract

The invention provides a method for detecting environmental pollutants by using a dispersive liquid-liquid microextraction technology. The method is particularly suitable for detecting endocrine disrupters in a urine sample. According to the method, a proper amount of a NaCl solution, a dispersing agent acetonitrile and an extracting agent trichloromethane are added into a urine sample, and target pollutants are rapidly extracted; and after centrifugation and nitrogen blowing drying, redissolving by using a methanol-water mixed solution in a ratio of 1: 1, and finally detecting and quantitatively analyzing through UHPLC (Ultra High Performance Liquid Chromatography). Compared with liquid-liquid extraction and solid-phase extraction, the method has the characteristics of simplicity and convenience in operation, high extraction efficiency and high detection sensitivity, is suitable for detecting bisphenol compounds in various matrixes, conforms to the green chemistry principle, and reduces the use amount of organic solvents. The method can be widely applied to detection and analysis of trace pollutants in environmental samples, and provides reliable technical support for environmental monitoring and pollutant risk assessment.

Description

Technical Field

[0001] The invention relates to the technical field of environmental pollutant detection, and in particular to a method for detecting environmental pollutants using a dispersed liquid-liquid microextraction technology. Background Art

[0002] With the continuous development of industrialization and urbanization, environmental pollution problems are becoming increasingly serious, among which the widespread presence of chemical pollutants has attracted widespread attention. Endocrine disrupting chemicals (EDCs), as an emerging pollutant, have become a research hotspot in the field of environmental science in recent years due to their potential harm to human health and ecosystems. EDCs are a class of chemical substances that can interfere with the normal function of hormones in organisms, which may lead to abnormal reproduction, development, behavior and immune function of organisms, and even cause serious diseases such as cancer. Endocrine disruptors are widely present in environmental media such as air, water, soil, etc., and accumulate into the human body through the food chain, causing a series of health problems.

[0003] Bisphenol compounds are one of the most common endocrine disruptors, especially bisphenol A (BPA), which has been widely detected due to its wide application in the plastics industry. Bisphenol A is mainly used to make polycarbonate plastics and epoxy resins, and is widely present in food packaging materials, beverage bottles, baby bottles, coatings and other products. Bisphenol A and its analogs (such as bisphenol B, bisphenol F, bisphenol S, etc.) can enter the natural environment such as water bodies and soil through environmental media, pollute organisms, especially aquatic organisms, and further affect human health through the food chain. Studies have shown that bisphenol compounds can simulate estrogen, interfere with the endocrine system, and cause human reproductive system disorders, developmental disorders and cancer, especially for children and pregnant women. Therefore, the detection of bisphenol compounds in the environment and their risk assessment have become one of the key contents of current environmental science research.

[0004] Due to the low concentration of bisphenol compounds in the environment and the complex matrix of environmental samples, traditional analytical methods, such as liquid-liquid extraction (LLE) and solid phase extraction (SPE), are difficult to effectively achieve efficient separation and detection of bisphenol compounds. In addition, traditional extraction methods are time-consuming, use a large amount of solvents, and are complex to operate, making it difficult to meet the requirements of rapid, sensitive, and green modern analysis. In order to overcome these shortcomings, dispersive liquid-liquid microextraction (DLLME) technology has been introduced into the field of environmental pollutant detection as a new sample pretreatment technology. DLLME technology combines the high efficiency of liquid-liquid extraction with the simplicity of solid phase extraction. It has the advantages of simple operation, fast extraction speed, high extraction efficiency, and green environmental protection. It is particularly outstanding in the enrichment and detection of trace pollutants.

[0005] The principle of dispersed liquid-liquid microextraction technology is to quickly disperse a small amount of extractant into the water sample through a dispersant to form tiny droplets, which greatly increases the specific surface area of ​​the extractant, thereby improving the extraction efficiency of the target pollutants. By selecting appropriate extractants and dispersants, this technology can complete the extraction and enrichment of trace pollutants in a short time, greatly improving the detection sensitivity. DLLME technology not only effectively reduces the use of solvents and meets the requirements of green chemistry, but also can be used in conjunction with a variety of detection methods, such as gas chromatography (GC), liquid chromatography (LC) and mass spectrometry (MS), etc., to achieve efficient detection of trace pollutants in complex matrices.

[0006] In practical applications, DLLME technology has been widely used in the detection of various pollutants in water, food, soil and biological samples, such as pesticide residues, heavy metals, drug residues, etc. In particular, in the detection of endocrine disruptors in environmental samples, DLLME technology has shown extremely high sensitivity and accuracy. Compared with traditional methods, DLLME technology has obvious advantages in sample pretreatment time, operation complexity, solvent usage and detection sensitivity. However, for different matrix samples and different target pollutants, the operating conditions of DLLME technology (such as the selection of extractants and dispersants, centrifugation time and speed, nitrogen blowing conditions, etc.) need to be optimized to ensure maximum extraction efficiency.

[0007] Based on the above background, the present invention provides a method for detecting endocrine disruptors in the environment using dispersed liquid-liquid microextraction technology combined with ultra-high performance liquid chromatography (UHPLC), which is particularly suitable for trace detection of bisphenol A and its analogs in urine samples. This method greatly improves the detection sensitivity and accuracy of bisphenol compounds by optimizing the ratio of dispersant and extractant, centrifugation conditions and detection parameters, and can effectively enrich and separate low-concentration bisphenol A and its analogs in urine samples to meet the needs of trace pollutant detection. Compared with the prior art, the method of the present invention is not only simple to operate, fast and efficient, but also meets the requirements of green chemistry and has broad application prospects.

[0008] Through the development and application of this method, an efficient, sensitive, green and environmentally friendly solution can be provided for the detection of endocrine disruptors in the environment, which is of great significance to environmental protection, human health monitoring and pollutant risk assessment.

[0009] To this end, we designed a method to detect environmental pollutants using dispersed liquid-liquid microextraction technology to solve the above problems. Summary of the invention

[0010] The purpose of the present invention is to provide a method for detecting environmental pollutants using dispersed liquid-liquid microextraction technology to address the deficiencies of the prior art and to solve the problems raised in the background technology.

[0011] To achieve the above object, the present invention provides the following technical solution: a method for detecting environmental pollutants using dispersed liquid-liquid microextraction technology, comprising the following steps:

[0012] S1: Prepare a NaCl solution with a mass concentration of 10%;

[0013] S2: Prepare chromatographically pure acetonitrile as a dispersant and chloroform as an extractant, and place them in two 10 mL EP tubes for later use;

[0014] S3: Take 0.5 mL of urine sample, add 0.5 mL of 10% NaCl solution obtained from S1, then add 50 μL of acetonitrile obtained from S2 and 50 μL of chloroform obtained from S2, mix in a 1.5 mL EP tube, and shake thoroughly to mix;

[0015] S4: centrifuging the mixed solution obtained in S3 for 5 minutes at a centrifugal speed of 12000 rpm;

[0016] S5: Use a pipette to remove the upper aqueous phase of the solution treated in S4, extract 45 μL of the chloroform extraction phase from the lower layer, and transfer it to a new 1.5 mL EP tube;

[0017] S6: drying the chloroform extracted in S5 by nitrogen blowing;

[0018] S7: Redissolve the dried residue obtained in S6 with 50 μL of a 1:1 methanol-water mixture and transfer the reconstituted solution to a brown injection bottle;

[0019] S8: Using ultra-high performance liquid chromatography to detect the reconstituted sample, and obtaining the chromatographic peak area of ​​bisphenol A and its analogs in urine;

[0020] S9: By using the standard curve method drawn using the bisphenol A standard solution, using the linear equation y=mx+b, where y is the chromatographic peak area measured in the sample, substitute it into the linear equation to calculate the concentration x of bisphenol A and its analogues in the urine sample.

[0021] As a preferred technical solution of the present invention, the analogs are endocrine disruptors, specifically including bisphenol A, bisphenol B, bisphenol F and bisphenol S.

[0022] As a preferred technical solution of the present invention, the dispersant used in the dispersed liquid-liquid microextraction technology is acetonitrile, and the extractant is chloroform, and the ratio of the two is optimized to improve the extraction efficiency.

[0023] As a preferred technical solution of the present invention, in the centrifugation step, the centrifugation time is 3 to 10 minutes and the centrifugation speed is 10,000 to 15,000 rpm to ensure effective separation of the target substance and the matrix.

[0024] As a preferred technical solution of the present invention, in the nitrogen blowing and drying step, the nitrogen flow rate is 1.0 to 2.0 L / min, and the nitrogen blowing time is 2 to 5 minutes to ensure that the chloroform is completely volatilized.

[0025] As a preferred technical solution of the present invention, in the re-dissolution step, the re-dissolving solution used is a mixed solution of methanol and water prepared in a volume ratio of 1:1, with a volume of 50 μL.

[0026] As a preferred technical solution of the present invention, the wavelength of the ultra-high performance liquid chromatography detection is set to 280 nm, and a binary mobile phase is used for gradient elution.

[0027] As a preferred technical solution of the present invention, the standard curve is drawn by using a series of bisphenol A standard solutions of known concentrations, detecting their chromatographic peak areas, drawing a graph of the relationship between concentration and peak area, and obtaining a linear equation through linear fitting.

[0028] As a preferred technical solution of the present invention, the method is suitable for detecting bisphenol A and its analogs as low as 10 ng / mL in urine samples, and has high sensitivity and accuracy.

[0029] As a preferred technical solution of the present invention, the method is suitable for detecting environmental samples and biological samples of various matrices, including environmental water samples, soil extracts, etc. in addition to urine.

[0030] Compared with the prior art, the present invention has the following beneficial effects: the present invention utilizes dispersed liquid-liquid microextraction technology to effectively enrich low-concentration endocrine disruptors, especially bisphenol A and its analogs, in environmental samples (such as urine). By optimizing the ratio of dispersant to extractant, the extractant is quickly dispersed in the sample and fully contacts the target, which greatly improves the extraction efficiency and enrichment effect of the target. Compared with the traditional liquid-liquid extraction method, this method has a short extraction time and high efficiency, and is suitable for the rapid analysis of trace pollutants.

[0031] The present invention combines ultra-high performance liquid chromatography (UHPLC) technology to significantly improve the detection sensitivity of bisphenol compounds, and the detection limit can reach the ng / mL level. The method is easy to operate and only requires a small amount of solvent to complete sample processing, which reduces the amount of solvent used, reduces analysis costs and experimental steps. At the same time, the centrifugation step and nitrogen drying conditions are optimized to ensure the stability and recovery rate of the target in the sample.

[0032] The method complies with the principle of green chemistry, significantly reduces the use of organic solvents, reduces environmental pollution, and is environmentally friendly. In addition, the present invention is not only applicable to the analysis of urine samples, but also to the detection of endocrine disruptors in various matrices such as water samples and soil extracts, and has broad application prospects, and can provide an efficient and sensitive detection method for monitoring and risk assessment of environmental pollutants. DETAILED DESCRIPTION

[0033] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0034] The specific implementation of the present invention is described in detail below in conjunction with multiple embodiments.

[0035] Example 1: Detection of Bisphenol A Based on Urine Samples

[0036] Experimental steps: Sample preparation: Take 0.5 mL of urine sample and add 0.5 mL of 10% NaCl solution to increase the ionic strength and reduce matrix interference.

[0037] Dispersive liquid-liquid microextraction: Add 50 μL of acetonitrile as a dispersant and 50 μL of chloroform as an extractant. Add them to a 1.5 mL EP tube and shake vigorously for 1 minute to ensure that the extractant and dispersant are fully in contact with the sample.

[0038] Centrifugation: The mixed sample was centrifuged at 12,000 rpm for 5 minutes. After centrifugation, the layers were clearly separated. The upper layer was the aqueous phase and the lower layer was the chloroform extraction phase.

[0039] Extraction phase collection: remove the upper aqueous phase with a pipette, extract 45 μL of the lower chloroform extraction phase, and transfer it to a new 1.5 mL EP tube.

[0040] Nitrogen drying: The chloroform extract phase was dried with nitrogen to ensure that the solvent was completely evaporated.

[0041] Reconstitution: Reconstitute the dried residue with 50 μL of a 1:1 methanol-water mixture and transfer to a brown injection vial.

[0042] UHPLC detection: An ultra high performance liquid chromatography (UHPLC) instrument was used with the detection wavelength set at 280 nm. The concentration of bisphenol A in the sample was calculated using a standard curve prepared using a bisphenol A standard solution.

[0043] Result analysis: Table 1 shows the chromatographic peak areas of standard solutions of bisphenol A at different concentrations and the detection results of bisphenol A in actual samples.

[0044]

[0045] Example 2: Effect of different ratios of extractant and dispersant on extraction effect

[0046] Experimental steps: This example studies the effect of changing the amount of acetonitrile and chloroform on the extraction efficiency of bisphenol A. The steps in Example 1 were followed, but the volumes of the dispersant (acetonitrile) and the extractant (chloroform) were changed.

[0047] Experimental conditions: Dispersant / extractant ratio: 50μL / 50μL, 100μL / 50μL, 150μL / 50μL

[0048] Sample: 0.5mL urine sample + 0.5mL 10% NaCl solution

[0049] Result analysis: Table 2 shows the chromatographic peak area and the detection results of bisphenol A under different dispersant to extractant ratios.

[0050]

[0051] The results showed that a ratio of 100 μL acetonitrile to 50 μL chloroform could achieve a higher extraction efficiency.

[0052] Example 3: Effect of different centrifugation times and rotation speeds on extraction results

[0053] Experimental steps: This experiment studies the influence of centrifugal time and speed on the extraction effect by changing the centrifugal time and speed. Other experimental steps and conditions are the same as those in Example 1.

[0054] Experimental variables:

[0055] Centrifugation time: 3 minutes, 5 minutes, 10 minutes

[0056] Centrifugal speed: 10000rpm, 12000rpm, 15000rpm

[0057] Result analysis: Table 3 shows the detection results of bisphenol A under different centrifugation conditions.

[0058]

[0059] The results showed that the best extraction effect was achieved when the centrifugation speed was 12,000 rpm for 5 minutes.

[0060] Example 4: Detection of different pollutants - bisphenol B, bisphenol F and bisphenol S

[0061] Experimental steps: This experiment verifies the wide applicability of the method by detecting other bisphenol compounds (bisphenol B, bisphenol F and bisphenol S). The experimental steps are the same as those in Example 1.

[0062] Preparation of standard curve: Standard curves were prepared using standard solutions (0 to 100 ng / mL) of bisphenol B, bisphenol F, and bisphenol S, respectively.

[0063] Result analysis: Table 4 shows the detection results of bisphenol B, bisphenol F and bisphenol S in urine samples.

[0064]

[0065] This result shows that this method is not only suitable for the detection of bisphenol A, but also for the detection of other bisphenol endocrine disruptors.

[0066] Example 5: Detection in different matrix samples

[0067] Experimental steps: different matrices (urine, environmental water samples and soil extracts) were selected for detection, the method was the same as in Example 1, and the applicability of the research method in different matrices was studied.

[0068] Result analysis: Table 5 shows the detection results of bisphenol A in different matrix samples.

[0069]

[0070] The results showed that this method could effectively detect bisphenol A in different matrix samples and had good matrix adaptability.

[0071] Conclusion: The above examples verify the wide applicability of the detection method based on dispersive liquid-liquid microextraction technology in different conditions, different pollutants and different matrices. The experimental results show that by optimizing the ratio of extractant, dispersant and centrifugation conditions, higher detection sensitivity and stability can be obtained. This method is particularly suitable for the detection of trace bisphenol compounds and can be widely used in a variety of environmental samples and biological samples.

[0072] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in the field. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting environmental pollutants using dispersed liquid-liquid microextraction technology, characterized in that: The steps include: S1: Prepare a NaCl solution with a mass concentration of 10%; S2: Prepare chromatographically pure acetonitrile as a dispersant and chloroform as an extractant, and place them in two 10 mL EP tubes for later use; S3: Take 0.5 mL of urine sample, add 0.5 mL of 10% NaCl solution obtained from S1, then add 50 μL of acetonitrile obtained from S2 and 50 μL of chloroform obtained from S2, mix in a 1.5 mL EP tube, and shake thoroughly to mix; S4: centrifuging the mixed solution obtained in S3 for 5 minutes at a centrifugal speed of 12000 rpm; S5: Use a pipette to remove the upper aqueous phase of the solution treated in S4, extract 45 μL of the chloroform extraction phase from the lower layer, and transfer it to a new 1.5 mL EP tube; S6: drying the chloroform extracted in S5 by nitrogen blowing; S7: Redissolve the dried residue obtained in S6 with 50 μL of a 1:1 methanol-water mixture and transfer the reconstituted solution to a brown injection bottle; S8: Using ultra-high performance liquid chromatography to detect the reconstituted sample, and obtaining the chromatographic peak area of ​​bisphenol A and its analogs in urine; S9: By using the standard curve method drawn using the bisphenol A standard solution, using the linear equation y=mx+b, where y is the chromatographic peak area measured in the sample, substitute it into the linear equation to calculate the concentration x of bisphenol A and its analogues in the urine sample.

2. The method for detecting environmental pollutants using dispersed liquid-liquid microextraction technology according to claim 1, characterized in that: The analogs are endocrine disruptors, specifically including bisphenol A, bisphenol B, bisphenol F and bisphenol S.

3. The method for detecting environmental pollutants using dispersed liquid-liquid microextraction technology according to claim 1, characterized in that: The dispersant used in the dispersed liquid-liquid microextraction technology is acetonitrile, and the extractant is chloroform.

4. The method for detecting environmental pollutants using dispersed liquid-liquid microextraction technology according to claim 1, characterized in that: In the centrifugation step, the centrifugation time is 3 to 10 minutes, and the centrifugation speed is 10000 to 15000 rpm.

5. The method for detecting environmental pollutants using dispersed liquid-liquid microextraction technology according to claim 1, characterized in that: In the nitrogen blowing and drying step, the nitrogen flow rate is 1.0 to 2.0 L / min, and the nitrogen blowing time is 2 to 5 minutes.

6. The method for detecting environmental pollutants using dispersed liquid-liquid microextraction technology according to claim 1, characterized in that: In the re-dissolution step, the re-dissolution solution used is a mixture of methanol and water in a volume ratio of 1:1, with a volume of 50 μL.

7. The method for detecting environmental pollutants using dispersed liquid-liquid microextraction technology according to claim 1, characterized in that: The wavelength of the ultra-high performance liquid chromatography detection was set to 280 nm, and a binary mobile phase was used for gradient elution.

8. The method for detecting environmental pollutants using dispersed liquid-liquid microextraction technology according to claim 1, characterized in that: The standard curve is drawn by using a series of bisphenol A standard solutions with known concentrations, detecting their chromatographic peak areas, drawing a graph of the relationship between concentration and peak area, and obtaining a linear equation through linear fitting.

9. The method for detecting environmental pollutants using dispersed liquid-liquid microextraction technology according to claim 1, characterized in that: This method is suitable for the detection of BPA and its analogs in urine samples at concentrations as low as 10 ng / mL.

10. The method for detecting environmental pollutants using dispersed liquid-liquid microextraction technology according to claim 1, characterized in that: This method is suitable for detecting environmental samples and biological samples in a variety of matrices, including environmental water samples and soil extracts in addition to urine.

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