A method for high-throughput extraction, purification and separation of aryl hydrocarbon receptor agonists in environmental samples
Patent Information
- Application Number
- CN202510506949.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-22
- Publication Date
- 2026-10-23
AI Technical Summary
[0004]针对现有技术存在的缺陷，即环境样品分离与制备方法通量低、溶剂用量大、耗时久，无法满足多学科交叉研究中对大量环境样品进行效应导向分析的需求，难以全面准确地识别环境样品中的关键效应物质，本发明旨在解决这些问题，构建一种高通量、高效且准确的环境样品中芳香烃受体激动剂的提取、净化与分离方法
[0025]1)高通量：优化后的方法实际通量达到72样/6.5h(见图1)，相较于传统方法(如ASE+GPC+多维色谱+半制备柱组合方法的24样/天和分离通量2样/天)，通量提升显著，能够满足多学科对大量环境样品进行效应导向分析的需求，减少因样品量不足导致的研究偏差，更全面地揭示环境样品的毒性效应。
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of environmental analytical chemistry, specifically relating to a high-throughput extraction, purification and separation method for aromatic hydrocarbon receptor (AhR) agonists in environmental samples. This method can be applied to the analysis of the toxic effects of environmental samples in environmental science research, toxicology research and multidisciplinary fields. Background Technology
[0002] Toxicological studies have shown that many adverse health outcomes, such as inflammation, neurodevelopmental abnormalities, tumors, and cancer, are associated with the activation of aryl hydrocarbon receptor (AhR)-mediated signaling pathways. AhR is an important target for the toxic effects of exogenous pollutants on organisms. Pollutants in the environment that bind to AhR and activate downstream signaling pathways are called aryl hydrocarbon receptor agonists. Identifying key AhR agonists in environmental samples is crucial for revealing the mechanisms underlying the toxic effects of environmental samples.
[0003] Currently, environmental sample separation and preparation methods are mainly based on semi-preparative chromatography. However, semi-preparative chromatography suffers from problems such as high column flow rates and low column efficiency, resulting in long collection and concentration times for each fraction, large solvent consumption, and limited throughput in practical applications. In practice, strategies such as mixing samples or selecting a small number of representative samples are often used to address the throughput limitation, but these strategies can mask the heterogeneity of environmental samples, and some key effector substances may not be identified. As environmental research increasingly emphasizes interdisciplinary collaboration, effect-oriented analysis techniques need to be widely applied in geosciences, medicine, and other disciplines. Therefore, developing high-throughput sample separation and preparation methods has become a pressing challenge for effect-oriented analysis. Summary of the Invention
[0004] To address the shortcomings of existing technologies, namely low throughput, large solvent consumption, and long processing time in environmental sample separation and preparation methods, which cannot meet the needs of effect-oriented analysis of large numbers of environmental samples in multidisciplinary research and make it difficult to comprehensively and accurately identify key effect substances in environmental samples, this invention aims to solve these problems and construct a high-throughput, efficient, and accurate method for the extraction, purification, and separation of aromatic hydrocarbon receptor agonists in environmental samples.
[0005] The present invention provides a method for the extraction, purification, and separation of aromatic hydrocarbon receptor agonists from environmental samples, comprising the following steps:
[0006] 1) Sample extraction: Environmental samples containing aryl hydrocarbon receptor agonists were extracted using ultrasonic extraction, and the extract containing aryl hydrocarbon receptor agonists was collected;
[0007] 2) Sample purification: The extract containing the aromatic hydrocarbon receptor agonist obtained in step 1) was purified using the QuEChERS method, and the purified solution was collected.
[0008] 3) Sample separation: The purified solution obtained in step 2) was separated into components using a Thermo UltiMate 3000 dual ternary liquid chromatograph, and the fraction containing the aromatic hydrocarbon receptor agonist was collected.
[0009] Furthermore, the method further includes: nitrogen blowing concentration of the fraction containing the aromatic hydrocarbon receptor agonist for subsequent analysis of the aromatic hydrocarbon receptor agonist.
[0010] In step 1) of the above method, the extraction solvent for ultrasonic extraction is an organic solvent, which may be selected from at least one of the following: n-hexane, acetone, or a mixture of n-hexane and acetone. According to an embodiment of the present invention, a mixture of n-hexane and acetone in a volume ratio of 1:1 may be used.
[0011] In step 1) of the above method, the ultrasonic extraction conditions are: power 400-600W, extraction time 20-30 min. In step 1) of the above method, the environmental sample is taken as a PM2.5 filter membrane or 10g of lake sediment, to which 10mL of n-hexane:acetone (1:1, V / V) is added, and ultrasonic extraction is performed for 30 min. Ultrasonic extraction utilizes ultrasound to accelerate the equilibrium between organic pollutants in the environmental matrix and the extraction solvent. Compared to ASE, which can only extract one sample at a time, and microwave extraction, which is highly dangerous and has limited throughput, the throughput of ultrasonic extraction is only limited by the size of the instrument, and theoretically, there is no limit to the throughput.
[0012] In step 2) of the above method, the specific steps for purifying the extract containing the aromatic hydrocarbon receptor agonist obtained in step 1) using the QuEChERS method are as follows:
[0013] S1. Concentrate the extract containing the aromatic hydrocarbon receptor agonist by nitrogen blowing to obtain a concentrate; add an appropriate amount of acetone to the concentrate, then add QuEChERS purification packing material, vortex and centrifuge, and collect the supernatant.
[0014] S2. Filter the supernatant through a hydrophobic polytetrafluoroethylene (PTFE) filter membrane and collect the filtrate; concentrate the filtrate to near dryness by nitrogen blowing and then redissolve it with n-hexane to obtain the purified extract.
[0015] In step S1 above, the volume ratio of the concentrate to acetone can be 1:3-3.2.
[0016] In step S1 above, the QuEChERS purification packing material can be composed of copper powder, silica gel and anhydrous sodium sulfate, with the mass ratio of the three being 1:(2-2.5):(1.5-2), preferably 1:2:2.
[0017] In step S1 above, the vortex duration can be 20s-40s.
[0018] In step S2 above, the pore size of the hydrophobic polytetrafluoroethylene (PTFE) filter membrane can be 0.4μm-0.5μm.
[0019] According to an embodiment of the present invention, 0.5 mL of the concentrate is mixed with 1.5 mL of acetone, and then QuEChERS purification packing (100 mg copper powder, 200 mg silica gel, and 200 mg anhydrous sodium sulfate) is added to the concentrate. The mixture is vortexed, then filtered through a hydrophobic PTFE filter membrane, and finally concentrated to near dryness by nitrogen blowing and then redissolved in n-hexane to 1.0 mL to obtain the extract.
[0020] The QuEChERS method can effectively remove matrix interference in environmental sample extracts, meeting the needs of high-throughput sample preparation.
[0021] In step 3) of the above method, the component separation was performed using a Thermo UltiMate 3000 dual ternary liquid chromatograph. The chromatographic conditions were as follows: a CNW Athena Silica column (4.6×150mm, 5μm) was used, the injection volume was set to 950μL (actual injection volume 900μL), the column temperature was 25℃, and the column flow rate was 2.0mL / min.
[0022] A gradient elution program was used: 0–1.5 min with 100% n-hexane, 1.5–1.75 min linearization to 100% dichloromethane, 1.75–2.0 min with 100% dichloromethane, 2.0–5.5 min with 100% acetone, and 5.5–6.5 min equilibration with 100% n-hexane.
[0023] The eluent is collected in the fraction collector for 1.0–5.5 min, of which F1 is the fraction for 1.0–1.75 min (1.5 mL), F2 is the fraction for 1.75–3.7 min (3.9 mL), and F3 is the fraction for 3.7–5.5 min (3.6 mL). The three components are concentrated by nitrogen blowing for subsequent analysis. According to chromatographic theory, the separation throughput is related to the theoretical plate number. In analytical liquid chromatography, at the same theoretical plate number, by selecting a suitable stationary phase packing particle size (such as the 5 μm particle size used in this invention), the optimal column flow rate can be reduced, thereby achieving high-throughput separation.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] 1) High throughput: The optimized method achieves an actual throughput of 72 samples / 6.5 hours (see...). Figure 1Compared to traditional methods (such as the ASE+GPC+multidimensional chromatography+semi-preparative column combination method with a throughput of 24 samples / day and a separation throughput of 2 samples / day), the throughput is significantly improved, which can meet the needs of multidisciplinary groups for effect-oriented analysis of a large number of environmental samples, reduce research bias caused by insufficient sample quantity, and more comprehensively reveal the toxic effects of environmental samples.
[0026] 2) Solvent savings: The amount of solvent used is significantly reduced during sample preparation and separation. Taking the processing of a PM2.5 filter membrane with dimensions of 12cm in length and 5cm in width as an example, the amount of solvent used during preparation is reduced from more than 100mL to 20mL, and during separation it is reduced from 3000mL to 16mL, resulting in a total solvent saving of 99.5%, which reduces experimental costs and minimizes environmental pollution from organic solvents.
[0027] 3) Reduced time: The extraction time for processing a PM2.5 filter membrane with a size of 12cm×5cm was reduced from more than 30min to 30 / Nmin (N is the number of samples that the instrument can process at the same time). The concentration time was also significantly reduced during the preparation and separation processes. The total time was reduced by 95%, which improved experimental efficiency and accelerated the research process.
[0028] 4) High accuracy and reliability: The accuracy and reliability of the method were validated by monitoring the effect recovery rate and determining the explanatory power of the AhR agonist in the F2 component for total AhR activity. The mean effect recovery rate for all PM2.5 samples was 85.89 ± 37.3%, and the median was 94.30%. Figure 2 The results indicate that the loss of AhR agonist effect during component separation is minimal; the AhR agonist in the F2 component explains 111.5% of the total AhR activity of the sample, with a median of 91%. 57.4% (31 / 54) of the sample explanations fall within the reasonable range of 50%-150%, proving that aromatic hydrocarbon receptor agonists are enriched in the F2 component, effectively identifying key effector substances. Attached Figure Description
[0029] Figure 1 The component separation record shows the time required for component separation.
[0030] Figure 2 The recovery rate represents the loss of the AhR activating effect during component separation.
[0031] Figure 3 The explanatory power of the AhR agonist in the F2 component for the total AhR activity of the sample demonstrates the enrichment of AhR agonists by this method. Detailed Implementation
[0032] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0034] Example 1
[0035] (I) Sample Extraction
[0036] Seventy-two PM2.5 filter membranes (12cm long x 5cm wide) were used as the sample source. Each PM2.5 filter membrane was placed in a suitable container, and 10mL of a hexane:acetone (1:1, v / v) mixture was added. The container was then placed in an ultrasonic extraction device, with the ultrasonic power set to 500W and the extraction time set to 30min. During ultrasonic extraction, the mechanical vibration and cavitation effect of ultrasound accelerate the transfer of organic pollutants from the matrix to the extraction solvent, achieving efficient extraction.
[0037] (II) Sample Cleaning
[0038] (1) Transfer the supernatant after ultrasonic extraction to a nitrogen blowing device and dry and concentrate it to 0.5 mL by blowing with nitrogen.
[0039] (2) Add 1.5 mL of acetone to the concentrated solution and mix thoroughly.
[0040] (3) Next, add QuEChERS purification packing material, which contains 100mg of copper powder, 200mg of silica gel and 200mg of anhydrous sodium sulfate. After adding, vortex for 30s to ensure that the purification packing material is in full contact with the solution and remove impurities and interfering substances.
[0041] (4) The mixture is filtered through a 0.45μm hydrophobic PTFE filter membrane to further remove solid particles and other impurities.
[0042] (5) Finally, the filtered solution was concentrated again by nitrogen blowing until it was nearly dry, and then redissolved in n-hexane to 1.0 mL to obtain the extract for subsequent analysis.
[0043] (III) Sample Separation
[0044] (1) Prepare a Thermo UltiMate 3000 dual ternary liquid chromatograph and install a CNW Athena Silica column (4.6×150mm, 5μm).
[0045] (2) Set the instrument parameters: injection volume of 950 μL, column temperature of 25℃, and column flow rate of 2.0 mL / min.
[0046] (3) Elute according to the set gradient elution program: 0–1.5 min maintain 100% n-hexane, 1.5–1.75 min linearly change to 100% dichloromethane, 1.75–2.0 min maintain 100% dichloromethane, 2.0–5.5 min use 100% acetone, and 5.5–6.5 min equilibrate with 100% n-hexane.
[0047] (4) Collect the eluent from 1.0–5.5 min using a fraction collector to obtain three fractions: F1 (fraction from 1.0–1.75 min, 1.5 mL), F2 (fraction from 1.75–3.7 min, 3.9 mL), and F3 (fraction from 3.7–5.5 min, 3.6 mL). Add the same sample fractions F1, F2, and F3 to each vial in volumes of 150 μL, 390 μL, and 360 μL, respectively. Then add 90 μL of DMSO solution and purge with nitrogen for 8–10 min to obtain the reconstituted solution.
[0048] (5) The obtained reconstituted solution was used for subsequent analysis of aromatic hydrocarbon receptor agonists.
[0049] Figure 1 The component separation record shows the time required for component separation. Figure 1 It can be seen that the time for separating the components of a sample is 6-8 seconds.
[0050] The recovery rate of the effect was determined, and the specific method is as follows:
[0051] (1) Cell plating and exposure
[0052] One dish of CBG2.8D cells in the logarithmic growth phase was collected by digestion and resuspended in exposed culture medium (Pronosa MEM medium, composition: MEM + 10% FBS + 1% P / S + 1% GlutaMax). Cell counts were then performed, and the cell concentration was adjusted to 1×10⁻⁶ cells based on the count results. 5 Approximately 100 μL of cells per mL. Cells were seeded into both bottom-permeable and fully permeable 96-well plates, with 100 μL of cells added to each well, and the plates were sealed with PBS.
[0053] Sample preparation: First, replace the solvent in the extract of each sample with an equal volume of dimethyl sulfoxide (DMSO). Then, perform serial dilutions of the solvent-replaced extract (using exposed culture medium as the diluent). The dilution gradient is as follows: the original extract is diluted three-fold, i.e., original extract—0.33—0.11—0.037—0.012…, for a total of 9 concentrations. A 1% DMSO solution (using exposed culture medium as the solvent) is used as a blank control.
[0054] The reconstructed solutions of each sample were serially diluted (dilution gradient was the same as that of the extract), and 1% DMSO solution (solvent was the exposed culture medium) was used as a blank control.
[0055] Before exposure, discard the culture medium in the well plate, add 100 μL of the prepared sample, and use serially diluted 2,3,7,8-TCDD as a positive control (the dilution concentrations of 2,3,7,8-TCDD are 10 nmol / ml—3.33 nmol / ml—1.11 nmol / ml—0.37 nmol / ml…, for a total of 9 concentrations). Seal the plate with PBS and expose it for 24 hours.
[0056] (2) AhR agonist effect detection
[0057] The AhR agonist effect was tested using a luciferase reporter gene assay in CBG2.8D cell lines with high AhR expression. The test solution was serially diluted to concentrations with no significant cytotoxicity before the assay. The AhR agonist effect was expressed as relative fluorescence intensity, with serially diluted 2,3,7,8-TCDD serving as a positive control.
[0058] (3) Toxicity equivalent calculation: The EC20 value of the positive reference 2,3,7,8-TCDD (EC20) was used. 2,3,7,8-TCDD Using as a reference, the AhR agonist effect of each component is expressed as the toxicity equivalent TEQbio, as shown in the following formula:
[0059]
[0060] (4) Calculation of effect recovery rate: The toxicity equivalents of the extract and the reconstituted solution are denoted as TEQbioP and TEQbioR, respectively. The effect recovery rate is TEQbioR / TEQbioP×100%. When the average effect recovery rate of all samples is greater than 80%, we consider that the AhR effect loss in the component separation process is very small.
[0061] Figure 2 The recovery rate represents the loss of the AhR activating effect during component separation. Figure 2The mean recovery rate of all PM2.5 samples was 85.89 ± 37.3%, and the median was 94.30%, indicating that the loss of the AhR activating effect during the component separation process was very small.
[0062] The explanatory power of the AhR agonist in the F2 fraction for the total AhR activity of the sample was also determined. The specific determination method is as follows:
[0063] (1) Screening of suspected targets: Screening for potential AhR agonists was performed using an Agilent 8890GC-7000E TQ system. The injection port temperature was set to 280℃. 1 μL was injected in splitless pulse mode at a pressure of 70 psi and a purge time of 0.50 min. The purge flow rate was set to 100 mL·min⁻¹, and gas-saving mode was activated 2 min after injection with a flow rate of 20 mL·min⁻¹. The carrier gas was controlled using programmed upflow mode, with an initial flow rate of 0.300 mL·min⁻¹ for 3.00 min, followed by an increase to 0.400 mL·min⁻¹ at a rate of 0.50 mL·min⁻², held for 10.0 min, and then increased to 0.7 mL·min⁻¹ at a rate of 0.50 mL·min⁻², held until the end. The column oven was initially set at 65°C and held for 1.0 min. The temperature was then increased to 130°C at a rate of 40°C / min and held for 1.5 min, followed by an increase to 150°C at 45°C / min and held for 1.5 min. Next, the temperature was increased to 200°C at 40°C / min and held for 1.0 min, then increased to 230°C at 35°C / min and held for 1.0 min, followed by an increase to 250°C at 35°C / min and held for 1.0 min. Finally, the temperature was increased to 305°C at 30°C / min and held for 2.4 min. The mass spectrometer was operated in MS1 scan mode with a solvent delay time of 2.8 min. The temperatures of the transfer line, ion source, and quadrupole were set to 300°C, 230°C, and 150°C, respectively. Automatic baseline subtraction and time filtering were enabled (peak width 0.8 s). The scan range was set to 45 to 450 m / z, and the dwell time was 100 ms.
[0064] After screening, potential AhR agonists were identified using Agilent MassHunter Unknown Analysis software (version 10.2). First, the acquired data files were imported into the software. Then, peak detection was performed using the deconvolution method with the default retention time window ratio. Target matching was performed using the National Institute of Standards and Technology (NIST23.L) database. Retention time matching factors were enabled, and a Gaussian penalty function was used with a standard deviation set to 60 s. The retention index (RI) was calculated using n-alkanes (C12 to C37) under the same gas chromatographic conditions, with a minimum matching factor set to 60.
[0065] Based on the key AhR effect pollutants identified through screening Figure 2 The assay method determines the toxicity equivalent and, based on the 20% lethal concentration of the key effector pollutant, i.e., EC20. chem Calculate its toxicity equivalent factor ReP. The formula is as follows:
[0066]
[0067] Total AhR activity (∑TEQ) of key effect pollutants chem The concentration (Ci) of each pollutant is multiplied by its corresponding toxicity equivalent factor (RePi) and then summed. The formula is as follows:
[0068]
[0069] To assess the explanatory power of the enrichment of key effect pollutants in F2 on the total AhR activity, C was introduced. chem (%) indicator. , C chem (%) represents the total AhR activity (∑TEQ) of key effect pollutants. chem ) and the AhR agonist effect obtained by biodetection (TEQ) bio The ratio between () is given by the following formula. C chem The higher the percentage, the greater the explanatory power of the key effect pollutants identified by EDA on the total AhR activity.
[0070]
[0071] Figure 3 The explanatory power of AhR agonists in the F2 fraction for the total AhR activity of the samples demonstrates the enrichment of AhR agonists by this method. The mean explanatory power of AhR agonists in the F2 fraction for the total AhR activity of the samples was 111.5%, with a median of 91%. 57.4% (31 / 54) of the sample explanatory power fell within the reasonable range of 50%–150%, proving that aromatic hydrocarbon receptor agonists are enriched in the F2 fraction, effectively identifying key effector substances.
[0072] Through the above specific implementation steps, high-throughput extraction, purification and separation of aromatic hydrocarbon receptor agonists in environmental samples can be achieved. Experimental verification shows that this method has significant advantages in terms of throughput, solvent consumption, time consumption, accuracy and reliability.
[0073] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.
Claims
1. A method for extracting, purifying, and separating aromatic hydrocarbon receptor agonists from environmental samples, comprising the following steps: 1) Sample extraction: Environmental samples containing aryl hydrocarbon receptor agonists were extracted using ultrasonic extraction, and the extract containing aryl hydrocarbon receptor agonists was collected; 2) Sample purification: The extract containing the aromatic hydrocarbon receptor agonist obtained in step 1) was purified using the QuEChERS method, and the purified solution was collected. 3) Sample separation: The purified solution obtained in step 2) was separated into components using a Thermo UltiMate 3000 dual ternary liquid chromatograph, and the fraction containing the aromatic hydrocarbon receptor agonist was collected.
2. The method according to claim 1, characterized in that: In step 1), the extraction solvent for ultrasonic extraction is an organic solvent, which may be selected from at least one of the following: n-hexane and acetone.
3. The method according to claim 2, characterized in that: In step 1), the extraction solvent for ultrasonic extraction is a mixture of hexane and acetone, with a volume ratio of 1:1-1.
2.
4. The method according to any one of claims 1-3, characterized in that: In step 1), the conditions for ultrasonic extraction are: power 400-600w, extraction time 20-30min.
5. The method according to any one of claims 1-4, characterized in that: In step 2), the specific steps for purifying the extract containing the aromatic hydrocarbon receptor agonist obtained in step 1) using the QuEChERS method are as follows: S1. Concentrate the extract containing the aromatic hydrocarbon receptor agonist by nitrogen blowing to obtain a concentrate; add an appropriate amount of acetone to the concentrate, then add QuEChERS purification packing material, vortex and centrifuge, and collect the supernatant. S2. Filter the supernatant through a hydrophobic polytetrafluoroethylene (PTFE) filter membrane and collect the filtrate; concentrate the filtrate to near dryness by nitrogen blowing and then redissolve it with n-hexane to obtain the purified extract.
6. The method according to claim 5, characterized in that: In step S1, the volume ratio of the concentrate to acetone is 1:3-3.
2. And / or, in step S1, the QuEChERS purification packing is composed of copper powder, silica gel and anhydrous sodium sulfate, with the mass ratio of the three being 1:(2-2.5):(1.5-2), preferably 1:2:
2.
7. The method according to claim 5 or 6, characterized in that: The vortex time in step S1 is 20s-40s; And / or, in step S2, the pore size of the hydrophobic polytetrafluoroethylene (PTFE) filter membrane is 0.4μm-0.5μm.
8. The method according to any one of claims 1-7, characterized in that: In step 3), the ThermoUltiMate 3000 dual ternary liquid chromatograph was used for component separation. The chromatographic conditions were as follows: CNW Athena Silica column, size: 4.6×150mm, 5μm; injection volume set to 950μL; column temperature 25℃; column flow rate 2.0mL / min. A gradient elution program was used: 0–1.5 min with 100% n-hexane, 1.5–1.75 min linearization to 100% dichloromethane, 1.75–2.0 min with 100% dichloromethane, 2.0–5.5 min with 100% acetone, and 5.5–6.5 min equilibration with 100% n-hexane.
9. The method according to any one of claims 1-8, characterized in that: The fraction collector collects the eluent for 1.0–5.5 min, of which F1 is the fraction for 1.0–1.75 min, F2 is the fraction for 1.75–3.7 min, and F3 is the fraction for 3.7–5.5 min.
10. The method according to any one of claims 1-9, characterized in that: The method further includes: nitrogen blowing concentration of the fraction containing the aromatic hydrocarbon receptor agonist for subsequent analysis of the aromatic hydrocarbon receptor agonist.