Method for detecting persistent organic pollutant tetra-chloro-naphthalene in eel product

Abstract

This invention provides a method for detecting tetrachloronaphthalene, a persistent organic pollutant in eel products. The specific steps include: eel sample preparation, target analyte extraction and matrix purification, followed by analysis using ultra-high performance liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (UPLC-APCI-MS / MS). The measured peak area, fractionation ratio, and other relevant parameters are substituted into a specific formula to calculate the measured value of tetrachloronaphthalene in the test solution. This method fills the gap in my country's technology for detecting tetrachloronaphthalene-like persistent organic pollutants in eel products. It effectively eliminates interference from the complex matrix of eel products, accurately determines four tetrachloronaphthalene isomers in trace amounts, and possesses significant advantages such as high sensitivity, strong anti-interference ability, simple operation, and high detection accuracy.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing and instrumental analysis technology, specifically relating to an analytical method for trace pollutants in complex lipid matrices, particularly a method for determining the residual amounts of four tetrachloronaphthalene (TeCN) isomers in eel products using ultra-high performance liquid chromatography-atmospheric pressure chemical ionization source tandem mass spectrometry (UPLC-APCI-MS / MS). Background Technology

[0002] Tetrachloronaphthalene (TeCN) is a typical persistent organic pollutant (POP) belonging to the polychlorinated naphthalenes (PCNs) family. Due to its recalcitrant nature, bioaccumulation, and potential dioxin-like toxicity, it has been included in the control lists of several international environmental conventions. Tetrachloronaphthalene mainly originates from industrial chemical impurities, wood preservatives, and waste incineration processes.

[0003] As a high-value benthic carnivorous fish, the eel's lifestyle and physiological characteristics make it highly susceptible to accumulating persistent organic pollutants (POPs) in the environment. On the one hand, eels inhabit the bottom of water and frequently come into contact with sediment, which is a major reservoir for hydrophobic organic compounds (such as tetrachloronaphthalene). These pollutants are easily transferred through the "sediment-water-benthic organism" food chain. On the other hand, eels are typically high-fat aquatic products (often 15%-25% or even higher). Tetrachloronaphthalene, a strongly lipophilic compound, readily accumulates and remains in the eel's adipose tissue, posing a potential threat to human health through dietary intake. Therefore, establishing accurate detection methods for tetrachloronaphthalene in eel products is crucial for food safety risk assessment.

[0004] Currently, the detection of polychlorinated naphthalenes mainly relies on gas chromatography-mass spectrometry (GC-MS). However, when analyzing complex biological samples such as eels, existing technologies have the following significant drawbacks: First, severe matrix interference: Eel samples contain a large amount of co-extractants such as triglycerides, wax esters, and pigments. In GC-MS analysis, these high-boiling-point matrix components are prone to remain in the injection port and column, leading to severe matrix effects and interfering with the qualitative and quantitative analysis of trace tetrachloronaphthalenes. Second, cumbersome pretreatment: To adapt to GC analysis, the extract usually requires tedious purification (such as multilayer silica gel column chromatography, gel permeation chromatography, etc.) to remove fats, which is not only time-consuming and labor-intensive but also prone to causing the loss of target analytes.

[0005] Therefore, for eels, a high-fat matrix, existing technologies lack a method that can effectively overcome lipid interference while achieving high sensitivity and high throughput for the detection of tetrachloronaphthalene. This invention aims to solve the aforementioned technical challenges by optimizing the pretreatment extraction strategy and combining ultra-high performance liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry, providing a new technical means for monitoring persistent organic pollutants in eel products.

[0006] Currently, there are no reports on UPLC-APCI-MS / MS detection methods for tetrachloronaphthalene, a persistent organic pollutant in eel products, either domestically or internationally. Furthermore, novelty searches using standards such as ISO, ASTM, GB, and SN have not yielded any relevant standards, and patent searches on patent websites have also failed to find any related patent reports. Therefore, there is an urgent need to develop an accurate and efficient UPLC-APCI-MS / MS quantitative detection technology for tetrachloronaphthalene, a persistent organic pollutant in eel products. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a method for detecting tetrachloronaphthalene, a persistent organic pollutant in eel products. This method can effectively eliminate interference from complex matrices and accurately determine four trace tetrachloronaphthalenes: 1,2,3,4-tetrachloronaphthalene, 1,2,3,5-tetrachloronaphthalene, 1,3,5,7-tetrachloronaphthalene, and 1,4,5,8-tetrachloronaphthalene. It has the advantages of high sensitivity, strong anti-interference ability, simple operation, and high detection accuracy.

[0008] This invention is implemented as follows: A method for detecting tetrachloronaphthalene, a persistent organic pollutant, in eel products, comprising the following steps: Step 1: Sample preparation. Take a representative edible portion from the sample, thoroughly homogenize it with a tissue homogenizer, put it into a clean container, seal it, and freeze it at -18℃ to obtain a uniform sample. Step 2: Extraction and purification. Weigh the sample for extraction. The extract is concentrated, purified, and diluted to a fixed volume to obtain the test solution. Step 3: Instrumental analysis. The test solution is analyzed and detected by ultra-high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry to obtain the response value of tetrachloronaphthalene in the test solution. Based on the response value, the corresponding standard working solution is selected for chromatographic analysis. The standard working solution has 6 concentration gradients including zero point, and the response values ​​of tetrachloronaphthalene in both the standard working solution and the test solution should be within the linear response range of the instrument. A blank control is also set up at the same time. Step 4: Quantitative calculation. Substitute the fractionation ratio obtained from ultra-high performance liquid chromatography-tandem mass spectrometry detection and the peak area of ​​tetrachloronaphthalene into the following formula to calculate the measured value of tetrachloronaphthalene in the test solution. ; in: X — Residual tetrachloronaphthalene content in the sample, μg / kg; A — Peak area of ​​tetrachloronaphthalene in the test solution; As — Peak area of ​​tetrachloronaphthalene in the standard working solution; c — Concentration of tetrachloronaphthalene in the standard working solution, μg / L; V — Final volume of the test solution, mL; m — mass of the sample, in grams; R — the percentage of shares to be distributed.

[0009] Furthermore, the conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry are as follows: A. Ultra-high performance liquid chromatography: Chromatographic column: ACQUITY UPLC® BEH-C18 column, 100 mm × 2.1 mm, 1.7 µm; flow rate: 0.3 mL / min; injection volume: 10 μL; column temperature: 40 ℃; gradient elution program as follows: ; B. Mass spectrometry conditions: Ion source: Atmospheric pressure chemical ionization source (APCI), positive ions; Scanning mode: Multiple reaction monitoring (MRM); Ion source temperature: 400℃; Sprayer current: 3μA; Nebulizing gas, curtain gas, auxiliary heating gas, and collision gas are all high-purity nitrogen; Monitored ion pairs, quantitative ion pairs, declustering voltage, collision gas energy, and collision cell outlet voltage are as follows:

[0010] Note: * indicates quantitative ion pairs.

[0011] Furthermore, the method can simultaneously detect four tetrachloronaphthalene isomers, including: 1,2,3,4-tetrachloronaphthalene (CAS No.: 20020-02-4), 1,2,3,5-tetrachloronaphthalene (CAS No.: 53555-63-8), 1,3,5,7-tetrachloronaphthalene (CAS No.: 53555-64-9), and 1,4,5,8-tetrachloronaphthalene (CAS No.: 3432-57-3).

[0012] Furthermore, the specific operations of steps one and two are as follows: (1) Sample preparation: For fresh eel samples, first thaw (if frozen) and drain, then remove the head, bones and viscera, take 500g of edible part, cut the processed fish meat into small pieces, put it into a tissue homogenizer or meat grinder to grind and homogenize it thoroughly to make a uniform laboratory sample; For roasted eel samples, first remove the sauce and char layer on the surface, then take the edible part and cut it into small pieces, put it into a tissue homogenizer to homogenize at high speed to ensure that the sample is fully mixed. The prepared homogenized sample should be put into a clean, dry sealed container, labeled, and immediately stored in a frozen environment below -18℃ for later use; (2) Sample extraction: Weigh (2±0.05)g of the sample into a 50ml centrifuge tube, accurately add 2ml of water and 10mL of extraction solution, vortex mix for 1min, sonicate for 5min, centrifuge at 6000 r / min for 5min, and transfer the supernatant to another 50mL centrifuge tube; the residue is extracted once again with 10mL of extraction solution, centrifuged at 10000 r / min for 5min, the supernatants are combined, 5g of extraction agent A is added, vortex mix for 1min, centrifuge at 6000 r / min for 5min, and the upper layer solution is taken for later use; (3) Sample purification: Accurately transfer 10 mL of the prepared solution into a 15 mL centrifuge tube, add 300 mg of extractant B, vortex for 1 min, centrifuge at 6000 r / min for 5 min, accurately measure 5 mL of the upper purified solution, blow dry with nitrogen, add 1 mL of 95% methanol aqueous solution, vortex to mix for 1 min, filter through a 0.22 µm organic filter membrane, transfer to a chromatographic sample injection bottle to obtain the test solution for liquid chromatography-tandem mass spectrometry determination.

[0013] Furthermore, the extract is a mixture of methanol, toluene, and nonane in a volume ratio of 1:1:8.

[0014] Furthermore, the extractant A is a 1:4 ratio of sodium chloride and anhydrous sodium sulfate.

[0015] Furthermore, the extractant B is a 2:1 ratio of C18 and PSA.

[0016] The present invention has the following advantages: High sensitivity and anti-interference ability: Using a unique extraction solvent system (methanol-toluene-nonane) and purification reagents (sodium chloride-anhydrous sodium sulfate, C18-PSA), combined with ultra-high performance liquid chromatography separation and atmospheric pressure chemical ionization source (APCI) and multiple reaction monitoring mode, it can effectively eliminate interference from complex matrices and achieve accurate determination of various trace tetrachloronaphthalenes. The method quantitation limit can be as low as 1 μg / kg.

[0017] Accurate quantification: Method validation showed that the recoveries at different spiking levels remained stable between 77.1% and 95.3%, with relative standard deviations of less than 7%, and good linearity, meeting the requirements for accurate quantitative analysis.

[0018] Simple and efficient operation: The pretreatment steps are reasonably designed, easy to operate and standardize in the laboratory, and provide a reliable technical means for environmental monitoring and pollution assessment. Detailed Implementation

[0019] This invention relates to a method for detecting tetrachloronaphthalene, a persistent organic pollutant, in eel products, comprising the following steps: Step 1: Sample Preparation: For fresh eel samples, first clean (thaw frozen samples first) and drain. Then remove the head, bones, and internal organs. Take 500g of the edible portion, cut the processed eel meat into small pieces, and put it into a tissue homogenizer or meat grinder to grind and homogenize it thoroughly to prepare a uniform laboratory sample. For roasted eel samples, first remove the sauce and charred layer from the surface. Then take the edible portion, cut it into small pieces, and put it into a tissue homogenizer for high-speed homogenization to ensure that the sample is thoroughly mixed. The prepared homogenized sample is placed in a clean, dry, sealed container, labeled, and immediately stored in a frozen environment below -18°C for later use. Step 2: Sample Extraction: Weigh (2±0.05) g of sample into a 50 mL centrifuge tube, accurately add 2 mL of water and 10 mL of extraction solution, vortex for 1 min, sonicate for 5 min, centrifuge at 6000 r / min for 5 min, and transfer the supernatant to another 50 mL centrifuge tube; repeat the extraction once with 10 mL of extraction solution, centrifuge at 10000 r / min for 5 min, combine the supernatants, add 5 g of extraction agent A, vortex for 1 min, centrifuge at 6000 r / min for 5 min, and take the upper layer solution for later use; Sample purification: Accurately transfer 10 mL of the prepared solution into a 15 mL centrifuge tube, add 300 mg of extractant B, vortex for 1 min, centrifuge at 6000 r / min for 5 min, accurately measure 5 mL of the supernatant purified solution, dry it with nitrogen, add 1 mL of 95% methanol aqueous solution, vortex to mix for 1 min, filter through a 0.22 µm organic filter membrane, and transfer it into a chromatographic sample vial to obtain the test solution for analysis by liquid chromatography-tandem mass spectrometry.

[0020] The extract is a mixture of methanol, toluene, and nonane in a volume ratio of 1:1:8. Extractant A is a mixture of sodium chloride and anhydrous sodium sulfate in a 1:4 ratio. Extractant B is a mixture of C18 and PSA in a 2:1 ratio.

[0021] Step 3: Instrumental analysis. The test solution is analyzed and detected by ultra-high performance liquid chromatography-atmospheric pressure chemical ionization source mass spectrometry to obtain the response value of tetrachloronaphthalene in the test solution. According to the response value, the corresponding standard working solution is selected for chromatographic analysis. The standard working solution has 6 concentration gradients including zero point. The response values ​​of tetrachloronaphthalene in both the standard working solution and the test solution should be within the linear response range of the instrument. A blank control is also set up at the same time. The conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry are as follows: A. Ultra-high performance liquid chromatography: Chromatographic column: ACQUITY UPLC® BEH-C18 column, 100 mm × 2.1 mm, 1.7 µm; flow rate: 0.3 mL / min; injection volume: 10 μL; column temperature: 40 ℃; gradient elution program as follows:

[0022] B. Mass spectrometry conditions: Ion source: Atmospheric pressure chemical ionization source (APCI), positive ions; Scanning mode: Multiple reaction monitoring (MRM); Ion source temperature: 400℃; Sprayer current: 3μA; Nebulizing gas, curtain gas, auxiliary heating gas, and collision gas are all high-purity nitrogen; Monitored ion pairs, quantitative ion pairs, declustering voltage, collision gas energy, and collision cell outlet voltage are as follows:

[0023] Note: * indicates quantitative ion pairs.

[0024] The method can simultaneously detect four tetrachloronaphthalene isomers, including: 1,2,3,4-tetrachloronaphthalene (CAS No.: 20020-02-4), 1,2,3,5-tetrachloronaphthalene (CAS No.: 53555-63-8), 1,3,5,7-tetrachloronaphthalene (CAS No.: 53555-64-9), and 1,4,5,8-tetrachloronaphthalene (CAS No.: 3432-57-3).

[0025] Step 4: Quantitative calculation. Substitute the fractionation ratio obtained from ultra-high performance liquid chromatography-tandem mass spectrometry detection and the peak area of ​​tetrachloronaphthalene into the following formula to calculate the measured value of tetrachloronaphthalene in the test solution. ; in: X — Residual tetrachloronaphthalene content in the sample, μg / kg; A — Peak area of ​​tetrachloronaphthalene in the test solution; As — Peak area of ​​tetrachloronaphthalene in the standard working solution; c — Concentration of tetrachloronaphthalene in the standard working solution, μg / L; V — Final volume of the test solution, mL; m — mass of the sample, in grams; R — the percentage of shares to be distributed.

[0026] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0027] Example 1: Sample to be tested—eel product (grilled eel) 1.1 Sample preparation: First, remove the sauce and caramelized layer from the surface. Then, cut the edible part into small pieces and put it into a tissue homogenizer for high-speed homogenization to ensure that the sample is fully mixed. The prepared sample should be placed in a clean, dry, sealed container, labeled, and immediately stored in a frozen environment below -18°C for later use.

[0028] 1.2 Standard Curve Construction: Blank samples were added to a standard series (1, 2, 20, 10, 20 μg / kg), and then processed according to the methods described in steps one to three above, followed by sequential injection and analysis. A working curve was plotted using the peak area y of the analyte against its mass concentration x (μg / L). The four tetrachloronaphthalene isomers showed good linearity in the concentration range of 1–20 μg / kg. The linear range, correlation coefficient (r), limit of detection (ILOD, S / N>3), and limit of quantitation (MLOQ, S / N>10) of each analyte are shown in Table 1.

[0029] Table 1. Linear equations, correlation coefficients, limits of detection, and limits of quantitation for the four tetrachloronaphthalene isomers.

[0030] 1.3 Background test: Weigh 2.0 g (accurate to 0.05 g) of the sample obtained in Section 1.1 into each of six 100 mL stoppered centrifuge tubes, and then operate according to the method described in steps one to three above. The actual measured values ​​and average values ​​are shown in Table 2. The average value is the background value.

[0031] Table 2. Background values ​​of tetrachloronaphthalene in eel products.

[0032] 1.4 Verification 1: Adding 1 μg / kg tetrachloronaphthalene Weigh 2.0 g (accurate to 0.01 g) of the sample obtained in Section 1.1 into each of six 100 mL stoppered centrifuge tubes. Then add 0.1 mL of 20 μg / L tetrachloronaphthalene standard solution to each centrifuge tube. Then proceed with the operation as described in steps one to three above. The recovery results are listed in Table 3.

[0033] Table 3 Recovery rate of tetrachloronaphthalene at 1 μg / kg in eel product (grilled eel) samples

[0034] 1.4 Verification 2: Adding 5 μg / kg tetrachloronaphthalene Weigh 2.0 g (accurate to 0.05 g) of the sample obtained in step 1.1 into each of six 100 mL stoppered centrifuge tubes. Then add 0.1 mL of 100 μg / L tetrachloronaphthalene standard solution to each centrifuge tube. Then follow the procedure described in steps one to three above and list the final recovery results in Table 4.

[0035] Table 4. Recovery rate of tetrachloronaphthalene at 5 μg / kg in eel product (grilled eel) samples.

[0036] The experimental results above show that when this invention is applied to detect the content of four tetrachloronaphthalene isomers in eel product samples, the detection limit is 1 μg / kg, the recovery rate is 77.1-95.3%, the standard curve shows good linearity in the range of 1-20 μg / L, and the correlation coefficient r is greater than 0.999.

[0037] The recovery rate and RSD values ​​in the respective verification results indicate that the detection method of the present invention is feasible and highly accurate.

[0038] While specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments described are merely illustrative and not intended to limit the scope of the present invention. Equivalent modifications and variations made by those skilled in the art in accordance with the spirit of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting tetrachloronaphthalene, a persistent organic pollutant, in eel products, characterized in that: The ultra-high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (UHPLC-ATC-MS / MS) technique was employed, including sample preparation, extraction and purification, instrumental analysis, and quantitative calculation steps, as detailed below: Step 1: Sample preparation. Take a portion of eel meat, thoroughly and evenly crush it using a tissue homogenizer, put it into a clean container, seal it, obtain a uniform sample, and freeze it for storage. Step 2: Extraction and purification. Weigh the sample for extraction. The extract is concentrated, purified, and diluted to a fixed volume to obtain the test solution. Step 3: Instrumental analysis. The test solution is analyzed and detected by ultra-high performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry to obtain the response value of tetrachloronaphthalene in the test solution. Based on the response value, the corresponding standard working solution is selected for chromatographic analysis. The standard working solution has 6 concentration gradients including zero point, and the response values ​​of tetrachloronaphthalene in both the standard working solution and the test solution should be within the linear response range of the instrument. A blank control is also set up at the same time. Step 4: Quantitative calculation. Substitute the fractionation ratio obtained from ultra-high performance liquid chromatography-tandem mass spectrometry detection and the peak area of ​​tetrachloronaphthalene into the following formula to calculate the measured value of tetrachloronaphthalene in the test solution. ； in: X — Residual tetrachloronaphthalene content in the sample, μg / kg; A — Peak area of ​​tetrachloronaphthalene in the test solution; As — Peak area of ​​tetrachloronaphthalene in the standard working solution; c — Concentration of tetrachloronaphthalene in the standard working solution, μg / L; V — Final volume of the test solution, mL; m — mass of the sample, in grams; R — the percentage of shares to be distributed.

2. The method for detecting tetrachloronaphthalene, a persistent organic pollutant in eel products according to claim 1, is characterized in that: The conditions for the ultra-high performance liquid chromatography-tandem mass spectrometry are as follows: A. Ultra-high performance liquid chromatography: Chromatographic column: ACQUITY UPLC® BEH-C18 column, 100 mm × 2.1 mm, 1.7 µm; flow rate: 0.3 mL / min; injection volume: 10 μL; column temperature: 40 °C; gradient elution program as follows: ； B. Mass spectrometry conditions: Ion source: Atmospheric pressure chemical ionization source (APCI), positive ions; Scanning mode: Multiple reaction monitoring (MRM); Ion source temperature: 400℃; Sprayer current: 3μA; Nebulizing gas, curtain gas, auxiliary heating gas, and collision gas are all high-purity nitrogen; Monitored ion pairs, quantitative ion pairs, declustering voltage, collision gas energy, and collision cell outlet voltage are as follows: 。 3. The method according to claim 1, characterized in that: The method can simultaneously detect four tetrachloronaphthalene isomers, including: 1,2,3,4-tetrachloronaphthalene, 1,2,3,5-tetrachloronaphthalene, 1,3,5,7-tetrachloronaphthalene, and 1,4,5,8-tetrachloronaphthalene.

4. The method for detecting tetrachloronaphthalene, a persistent organic pollutant in eel products according to claim 1, is characterized in that: The specific operations for steps one and two are as follows: (1) Sample preparation: For fresh eel samples, first clean and drain them, then remove the head, bones and internal organs, take the edible part of the fish meat, cut the processed fish meat into small pieces, put it into a tissue homogenizer or meat grinder to grind and homogenize it thoroughly to make a uniform sample; For roasted eel samples, first remove the sauce and charred layer on the surface, then take the edible part of the fish meat, cut it into small pieces, put it into a tissue homogenizer to homogenize it at high speed to ensure that the sample is fully mixed, put the prepared sample into a sealed container, and immediately place it in a frozen environment below -18℃ for later use; (2) Sample extraction: Weigh (2±0.05)g of the sample into a 50ml centrifuge tube, accurately add 2ml of water and 10mL of extraction solution, vortex mix for 1min, sonicate for 5min, centrifuge at 6000 r / min for 5min, and transfer the supernatant to another 50mL centrifuge tube; the residue is extracted once again with 10mL of extraction solution, centrifuged at 10000 r / min for 5min, the supernatants are combined, 5g of extraction agent A is added, vortex mix for 1min, centrifuge at 6000 r / min for 5min, and the upper layer solution is taken for later use; (3) Sample purification: Accurately transfer 10 mL of the prepared solution into a 15 mL centrifuge tube, add 300 mg of extractant B, vortex for 1 min, centrifuge at 6000 r / min for 5 min, accurately measure 5 mL of the upper purified solution, blow dry with nitrogen, add 1 mL of 95% methanol aqueous solution, vortex to mix for 1 min, filter through a 0.22 µm organic filter membrane, transfer to a chromatographic sample injection bottle to obtain the test solution for liquid chromatography-tandem mass spectrometry determination.

5. The method for detecting tetrachloronaphthalene, a persistent organic pollutant in eel products according to claim 4, is characterized in that: The extract is a mixture of methanol, toluene, and nonane in a volume ratio of 1:1:

8.

6. The method for detecting tetrachloronaphthalene, a persistent organic pollutant in eel products according to claim 4, is characterized in that: The extractant A is sodium chloride and anhydrous sodium sulfate in a 1:4 ratio.

7. The method for detecting tetrachloronaphthalene, a persistent organic pollutant in eel products according to claim 4, is characterized in that: The extractant B is a 2:1 ratio of C18 and PSA.

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