Method for analyzing novel plasticizer pollutants in brain tissues of East Asian Jiangguinea pigs
By combining ultrasonic extraction and C18 purification agent with GC-MS/MS, the problem of rapid and accurate quantitative screening of novel plasticizer pollutants in the brain tissue of East Asian finless porpoises has been solved, achieving simple, low-cost, and efficient analysis, and improving the accuracy and sensitivity of detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-09
- Publication Date
- 2026-04-14
AI Technical Summary
Current technologies lack highly sensitive and selective methods for detecting novel plasticizer pollutants in the brain tissue of East Asian finless porpoises, especially in complex biological matrices such as marine mammals, where analytical methods are still imperfect.
Ultrasonic extraction combined with C18 purifier and gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) was used for sample pretreatment and detection. Quantitative analysis was achieved by deuterated standard mixing and internal standard methods, which simplified the pretreatment process and reduced equipment requirements.
It enables efficient and convenient analysis of novel plasticizer pollutants under conventional experimental conditions, reduces equipment costs, improves analytical accuracy and sensitivity, and keeps matrix effects within an acceptable range.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of environmental chemical pollutant analysis technology, specifically relating to an analytical method for novel plasticizer pollutants in the brain tissue of East Asian finless porpoises. Through the synergistic optimization of sample pretreatment steps and instrument detection conditions, the method achieves accurate and efficient detection of novel plasticizer pollutants in brain tissue. Background Technology
[0002] Novel plasticizers are a class of compounds developed in recent years to replace traditional phthalate plasticizers. Traditional phthalate plasticizers have been restricted in many countries and regions for their endocrine-disrupting toxicity, reproductive and developmental toxicity, and potential carcinogenic risks, and are used in food packaging, medical devices, and children's products. To address this issue, novel plasticizers have been gradually developed and are being promoted for application. These mainly include epoxy fatty acid esters, citrate esters (such as ATBC), terephthalates (such as DOTP), bio-based plasticizers (such as vegetable oil derivatives), and composite environmentally friendly plasticizers. These substances maintain their plasticizing effect while emphasizing biodegradability, low migration rate, low ecotoxicity, and renewable raw materials, aligning with the development direction of green chemistry.
[0003] Although novel plasticizers are designed as safer alternatives, their migration, transformation, and accumulation effects in the environment cannot be ignored. Studies have shown that some novel plasticizers, such as epoxidized soybean oil and tributyl acetyl citrate, may still accumulate in organisms and potentially be transferred through the food chain, posing a potential exposure risk to higher organisms. Particularly in marine environments, plasticizers can enter ecosystems through water, suspended particulate matter, and plastic waste, accumulating in high-trophic-level marine mammals such as the Yangtze finless porpoise. Because the Yangtze finless porpoise has a high lipid content in its brain tissue and the blood-brain barrier is somewhat permeable to hydrophobic organic pollutants, novel plasticizers may have a potential impact on its nervous system health. Currently, analytical methods for novel plasticizers in biological samples are still imperfect, especially lacking highly sensitive and selective detection methods for complex biological matrices such as those used by marine mammals.
[0004] Currently, the main methods for extracting biological samples are ultrasound-assisted extraction and accelerated solvent extraction. 。 Ultrasonic-assisted extraction (UART) is widely used in most sample preparation laboratories due to its simplicity and ease of implementation. This technique mainly utilizes the vibration of ultrasound to accelerate and oscillate molecules within the sample, increasing intermolecular collisions and diffusion, thereby promoting the release and dissolution of the target component.
[0005] Accelerated solvent extraction (CSE) technology works by increasing the temperature and pressure during the extraction process, thereby increasing the solubility and diffusion rate of the target compound and significantly improving extraction efficiency. CSE is known for its low organic solvent consumption, high extraction efficiency, and high degree of automation; however, this method often places high demands on the equipment used.
[0006] Currently, there are two main methods for purifying marine mammal tissue samples. One of them is gel permeation chromatography (GPC), a widely used method in sample pretreatment that separates substances based on differences in molecular weight. This technique can efficiently separate substances with relative molecular weights below 10. 2 Up to 10 7 Gel permeation chromatography (GPC) is increasingly widely used in environmental monitoring and food safety fields due to its short analysis cycle and good reproducibility. It can effectively remove high molecular weight fats and certain endogenous hormones from samples, thereby improving the accuracy and reliability of the analysis. In the literature "Science of The Total Environment, 2015, 527–528: 306–312," Balmer et al. used dichloromethane as the extractant to extract persistent organic pollutants from bottlenose dolphin fat using accelerated solvent extraction technology. Sample purification utilized silica / alumina column chromatography and GPC to remove polar substances and large molecular impurities such as fats from the sample, and gas chromatography-mass spectrometry (GC-MS) was used for detection. No significant matrix effect interference was observed in the obtained GC-MS chromatograms, and the detected compounds showed good peak shapes, indicating high detection efficiency. However, this technique requires sophisticated equipment, and the pretreatment process is relatively cumbersome and time-consuming.
[0007] Another widely used method for purifying marine mammal tissue samples is dispersion solid-phase extraction (SPME), which uses adsorbents such as PSA (N-propylethylenediamine) and C18 (octadecyl silica gel) to remove impurities from the sample. SPME is widely used in the purification of marine mammal tissue samples due to its simplicity and high purification efficiency. This method effectively removes impurities such as fats, proteins, and pigments from the sample by using highly efficient adsorbents such as PSA and C18. After purification, the purity of the sample is significantly improved, providing more accurate and reliable results for subsequent analysis of persistent organic pollutants. Furthermore, SPME also has advantages such as large sample throughput, low solvent consumption, and environmental friendliness, making its application prospects in marine environmental monitoring and biological sample analysis even broader.
[0008] In the paper "Science & Justice, 2015, 55(5): 307–315", Luzardo et al. constructed an analytical and quantitative method for multiple pesticides in human blood in multiple reaction monitoring mode (MRM mode) of GC-MS / MS. According to the quantitative results, it has a lower detection limit and improved detection sensitivity compared with single ion monitoring (SIM mode). Moreover, the method has been successfully applied in actual cases, verifying its effectiveness and reliability in practical applications.
[0009] Currently, although there is considerable research on pretreatment and analytical methods for marine mammal tissues, there is still a lack of novel pretreatment and instrumental analysis methods for plasticizer compounds in the brain tissue of the East Asian finless porpoise. Since different species of marine mammals often exhibit significant differences in physiological parameters such as water content and lipid content, it is necessary to systematically optimize the pretreatment process and analytical conditions to establish a novel, specific analytical method for plasticizers in the brain tissue of the East Asian finless porpoise. Summary of the Invention
[0010] The technical problem to be solved by this invention is the rapid and accurate quantitative screening of novel plasticizers in the brain tissue of the Yangtze finless porpoise. A method for analyzing novel plasticizer pollutants in the brain tissue of the Yangtze finless porpoise is proposed.
[0011] The technical solution of the present invention is as follows:
[0012] An analytical method for novel plasticizer pollutants in the brain tissue of East Asian finless porpoises, the specific steps of which are as follows:
[0013] Step (1) Dissect the dead East Asian finless porpoise to obtain its brain tissue, freeze-dry it to make a dried sample, and then grind it into a uniform powder;
[0014] Step (2) Take the sample powder, add the deuterated standard mixture and acetonitrile solvent, vortex and ultrasonically extract, add the extraction salts anhydrous sodium sulfate and sodium chloride, vortex, centrifuge and take the supernatant, then add C18 purifying agent to the supernatant, vortex and centrifuge, and take the supernatant.
[0015] Step (3) After the supernatant was concentrated by nitrogen blowing, it was diluted to volume with ethyl acetate and an internal standard was added. The gas chromatography-triple quadrupole mass spectrometry (GC-MS / MS) was used for detection, and the internal standard method was used for quantitative analysis.
[0016] The novel plasticizer contaminants include methyl salicylate, diethyl adipate, dibutyl maleate, dimethyl sebacate, triethyl citrate, dibutyl adipate, N-butylbenzenesulfonamide, dibutyl terephthalate, dibutyl sebacate, tributyl citrate, acetylated tributyl citrate, diethylene glycol dibenzoate, dioctyl isophthalate, dioctyl terephthalate, dioctyl sebacate, and trioctyl trimellitate.
[0017] Further, in step (2), the sample powder is weighed and acetonitrile solvent is added, with 5 mL of acetonitrile added per 0.2 g sample; then, a deuterated standard mixture is added, including: DMP-D4, BHT-D2, DEP-D4, TBP-D27, BP-D10, DIBP-D4, DBP-D4, BMPP-D4, DEEP-D4, DPP-D4, DHXP-D4, BBP-D4, TPHP-D15, DBEP-D4, DCHP-D4, DEHP-D4, DPHP-D4, DNOP-D4, and DNP-D4. After vortexing and ultrasonic extraction for 20-40 min, the same amount of acetonitrile solvent as in the above step is added, followed by vortexing and ultrasonic extraction.
[0018] Further, in step (2), the mass ratios of anhydrous sodium sulfate and sodium chloride to the sample powder are 10:1 and 5:1, respectively. After adding the extraction salt, the mixture is vortexed, and then centrifuged to collect the supernatant.
[0019] Furthermore, in step (2), the mass ratio of C18 to sample powder is 1:1. After adding C18, vortex, centrifuge, and collect the supernatant;
[0020] Further, in step (3), the supernatant before nitrogen blowing is filtered through a polytetrafluoroethylene filter membrane with a pore size of 0.22 μm, and the filtrate is collected into a nitrogen blowing tube for nitrogen blowing. High-purity nitrogen is used for nitrogen blowing during the nitrogen blowing process. After blowing to near dryness, ethyl acetate solvent is added to the nitrogen blowing tube for redissolution. After vortexing, it is transferred to a brown liquid phase vial, and hexamethylbenzene and diazinon-d10 internal standard solutions are added respectively. Then, the volume is adjusted with ethyl acetate.
[0021] Furthermore, the operating conditions for GC-MS / MS include:
[0022] The chromatographic column was an HP-5MS UI, 30 m × 0.250 mm × 0.25 μm;
[0023] The heating program is as follows: 60℃ held for 1 min, heated to 220℃ at a heating rate of 10℃ / min and held for 1 min, heated to 250℃ at a heating rate of 5℃ / min and held for 1 min, and heated to 300℃ at a heating rate of 10℃ / min and held for 8 min.
[0024] Carrier gas: Helium, purity ≥ 99.999%, constant pressure mode;
[0025] The injection port temperature is 300℃, the injection volume is 1μL, and the splitless injection mode is used.
[0026] Ion source: Electron ionization source EI, ion source temperature 280℃;
[0027] Scanning mode: Selective ion monitoring (MRM) mode;
[0028] The standard solutions used for the markings had concentrations of 5, 20, 50, 100, 200, 500, and 1000 ppb, respectively.
[0029] The beneficial effects of this invention are:
[0030] (1) Simple method, low cost and low equipment requirements: It realizes efficient and convenient analysis of novel plasticizer pollutants in brain tissue under conventional experimental conditions, reduces the dependence on special equipment and high costs, and facilitates promotion and application.
[0031] (2) High accuracy and reliability of analysis: After methodological verification, the recovery rate of deuterated products reached 71.26%–104.05%, and the matrix effect of 16 new plasticizers could be controlled between -18.8%–46.8%, which effectively reduced matrix interference and ensured the accuracy of quantitative results.
[0032] (3) Good sensitivity and linearity: The method quantitation limit is between 0.77 and 144.00 ng / g (ww). In the detection by gas chromatography-triple quadrupole mass spectrometry, the linear correlation coefficient R² of the standard curve of the 16 target compounds is higher than 0.99. Attached Figure Description
[0033] Figure 1 This is a flowchart of the brain tissue pretreatment process for East Asian finless porpoises.
[0034] Figure 2 This is a graph showing the recovery rate of deuterated compounds in the brain tissue of the East Asian finless porpoise. Detailed Implementation
[0035] The specific implementation of this invention is described in detail through the following examples. The experimental subjects were four East Asian finless porpoises that died from stranding. Their basic information is listed in Table 1, and the specific operation process is as follows: Figure 1 As shown.
[0036] First, the stranded and dead finless porpoises were dissected, and their whole brain tissue was taken as an experimental sample. The obtained brain tissue was freeze-dried to make a dry sample, and then ground into a uniform powder for later use.
[0037] In the experiment, 0.2 g of dried brain tissue powder was weighed and placed in centrifuge tube 1. 50 μL of a 1 ppm deuterated standard solution was added, and the mixture was allowed to stand for 30 min to allow for complete infiltration. Then, 5 mL of acetonitrile was added to centrifuge tube 1, vortexed for 2 min, and then sonicated for 20 min. The above extraction steps were repeated once, i.e., another 5 mL of acetonitrile was added, vortexed for 2 min, and then sonicated for 20 min.
[0038] After extraction, 2 g of anhydrous sodium sulfate (Na2SO4) and 1 g of sodium chloride (NaCl) were added to centrifuge tube 1. After vortexing for 2 min, the mixture was centrifuged at 3000 r / min for 5 min. The supernatant was transferred to another centrifuge tube 2, and 200 mg of C18 adsorbent was added for purification. After vortexing for 5 min, the mixture was centrifuged at 3000 r / min for 5 min.
[0039] Collect the supernatant and filter it through a 0.22 μm PTFE membrane. Collect the filtrate in a nitrogen blow-off tube. Blow the filtrate to near dryness with high-purity nitrogen. Then, redissolve the filtrate with 200 μL of ethyl acetate solution, vortex to mix, and transfer to a liquid chromatography vial. Rinse the nitrogen blow-off tube with 100 μL of ethyl acetate, vortex, and combine the rinsings with the rinsed filtrate in the same vial. Finally, add 50 μL of 1 ppm hexamethylbenzene and 50 μL of 1 ppm diazinon-D10 as internal standards, and bring the volume to 500 μL with ethyl acetate for analysis.
[0040] Table 1 Basic Information of East Asian Finless Porpoise Samples
[0041]
[0042] Sample analysis: Quantitative screening of samples was performed using the Multiple Reaction Monitoring (MRM) mode on a Thermo Fisher Scientific Gas Chromatography-Triple Quadrupole Mass Spectrometer (TSQ9610). The chromatographic method was as follows: an HP-5MS UI column (30m × 0.250mm × 0.25μm) was used, with a stable helium flow rate of 1.0mL / min. The sample injection volume was 1μL, and splitless mode was employed. The gas chromatograph column oven temperature program is shown in Table 2, with a total run time of 38min. The transfer line temperature was 280℃, the ion source temperature was 280℃, and the injection port temperature was 300℃. The instrumental methods and limits of quantitation for novel plasticizer target compounds are shown in Table 3, and the instrumental methods for deuterated compounds are shown in Table 4.
[0043] Table 2 Chromatographic conditions and parameters for targeted quantitative screening
[0044]
[0045] Table 3 Instrumental methods and limits of quantitation for novel plasticizer target compounds
[0046]
[0047] Table 4 Instrumental Methods for Deuterated Substances
[0048]
[0049] Quantitative analysis was performed using the internal standard method. The standard curve was set with seven concentration gradient points: 5 ppb, 20 ppb, 50 ppb, 100 ppb, 200 ppb, 500 ppb, and 1000 ppb. The raw data was quantitatively analyzed using an Xcalibur data processing workstation. The specific procedure included: importing the sample data file; setting the qualitative ion, quantitative ion, retention time window, and internal standard for each target component in the quantification method; then, in the calibration interface, selecting the linear regression equation and inputting the standard concentrations for the seven concentration gradients; the software automatically generated the standard curve and regression equation; finally, batch processing of unknown samples was performed to calculate the content of novel plasticizer compounds. Detailed quantitative results are shown in Table 5, and the deuterated compound recovery rate is shown in [Table 5 missing]. Figure 2 .
[0050] Table 5. Test results of batch samples (unit: ng / g(ww)) (nd indicates not detected)
[0051]
Claims
1. A method for analyzing novel plasticizer pollutants in the brain tissue of the East Asian finless porpoise, characterized in that, The specific steps are as follows: Step (1) Dissect the dead East Asian finless porpoise to obtain its brain tissue, freeze-dry it to make a dried sample, and then grind it into a uniform powder; Step (2) Take the sample powder, add the deuterated standard mixture and acetonitrile solvent, vortex and ultrasonically extract, add the extraction salts anhydrous sodium sulfate and sodium chloride, vortex, centrifuge and take the supernatant, then add C18 purifying agent to the supernatant, vortex and centrifuge, and take the supernatant. Step (3) After concentrating the supernatant by nitrogen blowing, dilute to volume with ethyl acetate and add internal standard. Detect the solution using gas chromatography-triple quadrupole mass spectrometry (GC-MS / MS) and perform quantitative analysis using the internal standard method.
2. The analytical method for novel plasticizer pollutants in the brain tissue of the East Asian finless porpoise according to claim 1, characterized in that, The novel plasticizer contaminants include methyl salicylate, diethyl adipate, dibutyl maleate, dimethyl sebacate, triethyl citrate, dibutyl adipate, N-butylbenzenesulfonamide, dibutyl terephthalate, dibutyl sebacate, tributyl citrate, acetylated tributyl citrate, diethylene glycol dibenzoate, dioctyl isophthalate, dioctyl terephthalate, dioctyl sebacate, and trioctyl trimellitate.
3. The analytical method for novel plasticizer pollutants in the brain tissue of the East Asian finless porpoise according to claim 1, characterized in that, In step (2), weigh the sample powder and add acetonitrile solvent. The amount of acetonitrile added to each 0.2g sample is 5mL. Then add the deuterated standard mixture. Vortex and ultrasonically extract for 20-40 min. Then add the same amount of acetonitrile solvent as in the above step, vortex, and ultrasonically extract.
4. The analytical method for novel plasticizer pollutants in the brain tissue of the East Asian finless porpoise according to claim 3, characterized in that, The deuterated product is one or a mixture of two or more of the following: DMP-D4, BHT-D2, DEP-D4, TBP-D27, BP-D10, DIBP-D4, DBP-D4, BMPP-D4, DEEP-D4, DPP-D4, DHXP-D4, BBP-D4, TPHP-D15, DBEP-D4, DCHP-D4, DEHP-D4, DPHP-D4, DNOP-D4, and DNP-D4.
5. The analytical method for novel plasticizer pollutants in the brain tissue of the East Asian finless porpoise according to claim 1, characterized in that, In step (2), the mass ratios of anhydrous sodium sulfate and sodium chloride to the sample powder are 10:1 and 5:1, respectively; after adding the extraction salt, the mixture is vortexed and then centrifuged to collect the supernatant.
6. The analytical method for novel plasticizer pollutants in the brain tissue of the East Asian finless porpoise according to claim 1, characterized in that, In step (2), the mass ratio of C18 to sample powder is 1:1; after adding C18, vortex, centrifuge, and take the supernatant.
7. The analytical method for novel plasticizer pollutants in the brain tissue of the East Asian finless porpoise according to claim 1, characterized in that, In step (3), the supernatant before nitrogen blowing is filtered through a polytetrafluoroethylene filter membrane with a pore size of 0.22 μm, and the filtrate is collected into a nitrogen blowing tube for nitrogen blowing. High-purity nitrogen is used for nitrogen blowing during the nitrogen blowing process. After blowing to near dryness, ethyl acetate solvent is added to the nitrogen blowing tube for redissolution. After vortexing, it is transferred to a brown liquid phase vial, and hexamethylbenzene and diazinon-d10 internal standard solutions are added respectively. Then, the volume is adjusted with ethyl acetate.
8. The analytical method for novel plasticizer pollutants in the brain tissue of the East Asian finless porpoise according to claim 1, characterized in that, In step (3), the operating conditions for GC-MS / MS are as follows: The chromatographic column was an HP-5MS UI, 30 m × 0.250 mm × 0.25 μm; The heating program is as follows: 60℃ held for 1 min, heated to 220℃ at a heating rate of 10℃ / min and held for 1 min, heated to 250℃ at a heating rate of 5℃ / min and held for 1 min, and heated to 300℃ at a heating rate of 10℃ / min and held for 8 min. Carrier gas: Helium, purity ≥ 99.999%, constant pressure mode; The injection port temperature is 300℃, the injection volume is 1μL, and the splitless injection mode is used. Ion source: Electron ionization source EI, ion source temperature 280℃; Scanning mode: Selective ion monitoring mode; The standard solutions used for the markings had concentrations of 5, 20, 50, 100, 200, 500, and 1000 ppb, respectively.