A method for simultaneous determination of 19 chlorophenol compounds in aquatic products

Abstract

The invention discloses a method for simultaneously determining 19 chlorophenol compounds in aquatic products, and belongs to the technical field of food safety detection. The method of the invention is based on UPLC-MS method using QuEChERSEMR-Lipid pretreatment technology, and BEH C 18 Chromatographic column separation, acetonitrile-ammonium formate as the mobile phase gradient elution, and atmospheric pressure chemical ionization source ionization were used to detect 19 chlorophenol compounds in aquatic products. This method has high separation, accuracy, rapidity and good repeatability, and provides a practical and reliable technical means for the high-throughput rapid detection of the contents of 19 CPs in aquatic products.

Description

Technical Field

[0001] The invention relates to a method for simultaneously determining 19 chlorophenol compounds in aquatic products, belonging to the technical field of food safety detection. Background Art

[0002] Chlorophenols (CPs) are a class of organic pollutants formed when the hydrogen atoms at the active sites of the benzene ring are replaced by chlorine atoms. According to the number of chlorine atoms replaced, they can be divided into 19 isomers, including monochlorophenol (CP), dichlorophenol (DCP), trichlorophenol (TrCP), tetrachlorophenol (TeCP) and pentachlorophenol (PCP). There are 6 isomers of dichlorophenol, namely 2-CP, 3-CP, and 4-CP; there are 6 isomers of dichlorophenol, namely 2,3-DCP, 2,4-DCP, 2,5-DCP, 2,6-DCP, 3,4-DCP, and 3,5-DCP; there are also 6 isomers of trichlorophenol, namely 2,3,4-TCP, 2,3,5-TCP, 2,3,6-TCP, 2,4,5-TCP, 2,4,6-TCP, and 3,4,5-TCP; there are 3 isomers of tetrachlorophenol, namely 2,3,4,5-TeCP, 2,3,4,6-TeCP, and 2,3,5,6-TeCP. Pentachlorophenol has no isomers. CPs mainly come from the use of pesticides, herbicides, preservatives, and disinfectants, as well as by-products of chlorination disinfection in aquaculture. CPs are a class of extremely stable and difficult-to-degrade compounds that can remain in the environment for a long time. They can cause harm to aquatic products and then to human health through enrichment in the food chain. CPs have the three toxic effects of biotoxicity, bioaccumulation and "teratogenicity, carcinogenicity and mutagenicity". The toxicity increases with the number of chlorine atoms, and the toxicity of the meta-chlorine substituent is higher than that of the ortho-chlorine substituent. Long-term contact or ingestion of chlorinated phenol compounds can cause dizziness, skin itching, anemia and various nervous system diseases. Excessive ingestion can lead to acute poisoning. Therefore, many countries have introduced relevant laws and policies to restrict the use of chlorinated compounds. For example, the U.S. Environmental Protection Agency (EPA) and the Human Environmental Health Assessment Group classified 2,4,5-TCP as a Class D carcinogen based on research on its possible tumorigenic effects. In addition, 2-CP, 2,4-DCP, 2,4,6-TCP and PCP have been listed as priority controlled pollutants by the EPA. Therefore, it is necessary to monitor the water environment and the organisms in it. However, the current detection standards for chlorophenols in organisms are limited to the determination of PCP and its sodium salt. There are no method standards and limit standards covering the simultaneous detection of 19 CPs in aquatic organisms at home and abroad.

[0003] Most of the methods for determining CPs reported in the existing literature are chromatographic methods, mainly gas chromatography (GC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography (LC), and liquid chromatography-mass spectrometer (LC-MS). Both GC and GC-MS require CPs to be derivatized before analysis, and some isomers of CPs cannot be separated. LC and LC-MS do not require derivatization and have faster detection. At present, there are literature reports on methods for the simultaneous determination of multiple CPs in environmental samples, textiles, leather products, and paper products. For example, Wang Chengyun et al. established a method for the determination of 19 CPs in paper and paper products by GC-MS, and the quantitative limit of each component was 2 μg / kg. Mo Xianke et al. established a method for the determination of 19 CPs in leather by GC-MS. The pretreatment method process is relatively complicated, but it can also meet the daily detection of CPs in leather.

[0004] At present, the detection of chlorophenols in aquatic products is mostly the determination of trichlorophenol, tetrachlorophenol, pentachlorophenol or sodium pentachlorophenol, while there are few reports on the simultaneous detection of 19 CPs. Zhong Huiying et al. established a GC-MS method for the determination of 19 CPs in large yellow croaker and grass carp. The pretreatment used liquid-liquid extraction after sulfuric acid digestion. The pretreatment was relatively complicated, consumed a large amount of organic solvent, and the retention times of 2,5-DCP and 3,5-DCP overlapped and were not separated. In recent years, the rapid development of QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) simplifies the sample processing process and has less environmental pollution. It has the advantages of high efficiency, economy, speed and simplicity, and is widely used in the pretreatment of organic pollutant residue detection. As in our previous article, we used QuEChERS pretreatment combined with GC-MS to detect 19 CPs in fish. Although the pretreatment of this method is simple and the detection limit is low, the pretreatment requires derivatization and the detection time is long, and 3,5-DCP and 2,6-DCP are not separated. Huang Jianfang used QuEChERS combined with HPLC-MS / MS to determine the content of PCP in large yellow croaker, with a detection limit of 0.1μg / kg. However, this method only detected PCP and did not analyze the isomers of other chlorophenol compounds. Different from the existing methods, the QuEChERS EMR-Lipid used in this paper is a new QuEChERS purifier that effectively removes lipids based on the structural characteristics of oils and fats (such as free fatty acids, phospholipids, triglycerides, etc.), which can reduce the adsorption interference of lipid matrix on target compounds. This technology has been applied to veterinary drug detection in farmed aquatic products, but has not yet been applied to the detection of chlorophenol residues in aquatic products.

[0005] Given that there are currently few methods for the simultaneous detection of 19 CPs in aquatic products, existing studies have complex pre-treatment or can only detect a few CPs. In addition, since the 19 CPs have extremely similar material structures and exist as isomers, the GC-MS method is usually difficult to separate some of them well, resulting in the inability to accurately identify and quantify some substances. Summary of the invention

[0006] In order to solve the technical defects of the prior art, the present invention provides a method for simultaneously determining 19 CPs in aquatic products based on QuEChERS EMR-Lipid combined with UPLC-MS. The samples are pretreated with improved QuEChERS EMR-Lipid technology and then purified by BEH C 18The chromatographic column (100 mm × 2.1 mm, 1.7 μm) was used for separation, acetonitrile-1 mmol / L ammonium formate was used as the mobile phase for gradient elution, the flow rate was 0.4 mL / min, the column temperature was 30 ℃, atmospheric pressure chemical ionization (APCI) was used for ionization, negative ion detection, and full scan mode for screening and quantification. The method has high separation, accuracy, rapidity, and good reproducibility, and provides a practical and reliable technical means for the high-throughput rapid detection of 19 CPs in aquatic products. The established method can separate the 19 CPs well, does not require derivatization treatment to reduce analysis time and solvent consumption, reduce environmental pollution, and improve detection efficiency. It can be applied to the rapid detection of 19 CPs in aquatic products.

[0007] The object of the present invention is to provide a method for simultaneously determining 19 chlorophenol compounds in aquatic products, the method comprising the following steps:

[0008] (1) Sample extraction

[0009] Weigh the aquatic product sample into a centrifuge tube, add ethyl acetate-acetonitrile, anhydrous MgSO4 and NaCl, homogenize, sonicate, centrifuge, and take out all the organic phases; then transfer the organic phase to the QuEChERSEMR-Lipid purification tube, vortex and oscillate, centrifuge again, take out the supernatant, blow nitrogen to dryness, and then re-dissolve it with acetonitrile-water. After passing the reconstituted solution through an organic filter membrane, the sample extract to be tested is obtained;

[0010] (2) Preparation of matrix standard solution

[0011] Using the blank sample extract obtained by the same method as step (1) as the matrix solution, prepare a series of concentrations of standard solutions of 19 chlorophenol compound mixed standards;

[0012] (3) Content determination

[0013] The standard solutions of the mixed standard of 19 chlorophenol compounds with a series of concentrations prepared in step (2) and the sample extract prepared in step (1) are measured by UPLC-MS, and a quantitative relationship model is constructed with the concentration of the mixed standard of 19 chlorophenol compounds as the horizontal axis and the peak area of ​​each target analyte as the vertical axis; the content of chlorophenol compounds in the sample to be tested is calculated based on the quantitative relationship model and the peak area of ​​the sample extract.

[0014] In one embodiment, the aquatic product in step (1) includes one or more of grass carp, crucian carp, river shrimp, and clam.

[0015] In one embodiment, the 19 chlorophenol compounds in step (2) include 2-chlorophenol, 4-chlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, 3,5-dichlorophenol, 2,4,6-trichlorophenol, 2,4,5-trichlorophenol, 3,4,5-trichlorophenol, 2,3,5,6-tetrachlorophenol, 2,3,4,6-tetrachlorophenol, 2,3,4,5-tetrachlorophenol, pentachlorophenol; 3-chlorophenol, 2,3-dichlorophenol, 2,5-dichlorophenol, 3,4-dichlorophenol, 2,3,4-trichlorophenol, 2,3,5-trichlorophenol, 2,3,6-trichlorophenol.

[0016] In one embodiment, the liquid chromatography conditions for the UPLC-MS determination in step (3) are: chromatographic column: ACQUITY-UPLC-BEH-C18 (100 mm × 2.1 mm, 1.7 μm), column temperature 30 ~ 35 ° C; flow rate: 0.4 ~ 0.6 mL / min; mobile phase: A is ammonium formate aqueous solution, B is acetonitrile; gradient elution, injection volume: 3 μL.

[0017] In one embodiment, the concentration of ammonium formate is 1-5 mmol / L, preferably 1 mmol / L.

[0018] In one embodiment, the gradient elution program is: 0-10 min, 20%-50% acetonitrile phase, 10-11 min, 50%-95% acetonitrile phase, 11-15 min, 95% acetonitrile phase, 15-16 min, 95%-20% acetonitrile phase, 16-18 min, 20% acetonitrile phase.

[0019] In one embodiment, the mass spectrometry conditions during the UPLC-MS determination in step (3) are: scanning mode: atmospheric pressure chemical ionization source negative ion mode (APCI - ); capillary voltage 5 kV; cone voltage 20 V; ion source temperature 150 ℃; ion source default voltage 80 V; desolvation gas temperature 600 ℃; desolvation nitrogen flow rate 900 L / h; cone gas flow rate 50 L / h; continuous scanning; recording range 50~600.

[0020] In one embodiment, the centrifugation conditions in step (1) are: 4000-5000 r / min, and the time is 5-10 min.

[0021] In one embodiment, the re-centrifugation conditions in step (1) are: 8000-10000 r / min, and the time is 10-15 min.

[0022] In one embodiment, the volume ratio of ethyl acetate to acetonitrile in step (1) is 1:1.

[0023] In one embodiment, the mass volume ratio of the water product to ethyl acetate-acetonitrile in step (1) is 1:2, g / mL.

[0024] In one embodiment, the mass ratio of anhydrous MgSO4 and NaCl in step (1) is 3:2; wherein the total mass ratio of the water product to the anhydrous MgSO4 and NaCl is 1:1.

[0025] In one embodiment, the QuEChERS EMR-Lipid purification tube is pre-activated with 5 mL of 5 mmol ammonium acetate aqueous solution.

[0026] In one embodiment, the volume ratio of acetonitrile to water in step (1) is 2:8, and the volume is 1-2 mL.

[0027] Another object of the present invention is to provide an application of the above-mentioned method in the field of food safety detection.

[0028] Beneficial effects of the present invention:

[0029] The present invention can separate 19 isomers with similar properties, realize the simultaneous detection of 19 chlorophenol compounds, and compared with the GC-MS method, the present method does not need to be derivatized, simplifies the sample pretreatment steps, greatly shortens the pretreatment time, and the 19 chlorophenols all have peaks within 12 minutes, reducing the detection time. The establishment of the present method can realize the rapid detection of 19 chlorophenol compounds in different aquatic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is the total ion current chromatogram of 19 chlorophenols in Example 1 of the present invention; wherein, 1: 2-CP; 2: 4-CP; 3: 3-CP; 4: 2,6-DCP; 5: 2,3-DCP; 6: 2,5-DCP; 7: 2,4-DCP; 8: 3,4-DCP; 9: 3,5-DCP; 10: 2,3,6--TCP; 11: 2,4,6--TCP; 12: 2,3,4-TCP; 13: 2,4,5-TCP; 14: 2,3,5-TCP; 15: 3,4,5-TCP; 16: 2,3,5,6-TeCP; 17: 2,3,4,6-TeCP; 18: 2,3,4,5-TeCP; 19: PCP;

[0031] Figure 2 This is a total ion current chromatogram of 14 chlorophenols when a C8 chromatographic column is used in Example 2 of the present invention;

[0032] Figure 3 This is the total ion current chromatogram of 13 chlorophenols when using the T3 chromatographic column in Example 2 of the present invention;

[0033] Figure 4 This is a total ion current chromatogram of eight chlorophenols when a methanol-water mobile phase is used in Example 2 of the present invention;

[0034] Figure 5 This is a total ion current chromatogram of 19 chlorophenols when acetonitrile-1 mmol / L ammonium formate aqueous solution is used as the mobile phase in Example 2 of the present invention;

[0035] Figure 6 The total ion current chromatogram of 17 chlorophenols when acetonitrile-5 mmol / L ammonium formate aqueous solution is used as the mobile phase in Example 2 of the present invention;

[0036] Figure 7 Data diagram showing the effects of different extractants on the recovery rates of 19 chlorophenol compounds;

[0037] Figure 8 Data graph showing the effects of different purification methods of the present invention on the recovery rates of 19 chlorophenol compounds. DETAILED DESCRIPTION

[0038] The present invention is described below in conjunction with specific embodiments. It should be noted that the embodiments are only used to further illustrate the present invention and do not represent the protection scope of the present invention. Non-essential modifications and adjustments made by others based on the present invention still fall within the protection scope of the present invention.

[0039] 1. Instruments, reagents and materials

[0040] Xevo TQ-XS triple quadrupole mass spectrometer, BEH C 18 Chromatographic column (50 mm×2.1 mm, 1.7 μm), HSS T3 chromatographic column (50 mm×2.1 mm, 1.8 μm) (Waters, USA); Heraeus Multifuge X1R high-speed refrigerated centrifuge (Ningbo Noli Electronic Technology Co., Ltd.); KS-300EI ultrasonic cleaning machine (Ningbo Kesheng Equipment Co., Ltd.); Vortex 3 automatic vortex mixer (IKA, Germany); Reeko AH-20 fully automatic homogenizer (Reeko Group, China).

[0041] Acetonitrile, methanol, ethyl acetate, chromatographically pure reagents, were purchased from Merck, Germany; dichloromethane and n-hexane (chromatographically pure) were purchased from Jiangsu Yonghua Chemical Technology Co., Ltd., China; anhydrous magnesium sulfate and sodium chloride (analytical pure) were purchased from Sinopharm Chemical Reagent Co., Ltd., China;

[0042] 19 chlorophenol single-label standards: 2-chlorophenol (2-CP), 4-chlorophenol (4-CP), 2,4-dichlorophenol (2,4-DCP), 2,6-dichlorophenol (2,6-DCP), 3,5-dichlorophenol (3,5-DCP), 2,4,6-trichlorophenol (2,4,6-Trichlorophenol ophenol, 2,4,6-TCP), 2,4,5-trichlorophenol (2,4,5-Trichlorophenol, 2,4,5-TCP), 3,4,5-trichlorophenol (3,4,5-Trichlorophenol, 3,4,5-TCP), 2,3,5,6-tetrachlorophenol (2,3,5,6-Tetrachlorophenol, 2,3,5,6-TeCP), 2,3,4,6-tetrachlorophenol (2,3 ,4,6-TeCP), 2,3,4,5-Tetrachlorophenol (2,3,4,5-TeCP), and pentachlorophenol (Pentachlorophenol, PCP) were purchased from Shanghai Anpu Cuishi Standard Technology Service Co., Ltd.; 3-Chlorophenol (3-CP), 2,3-dichlorophenol (2,3-DCP), 2,5-dichlorophenol (2,5-Dichlorophenol, 2,5-DCP), 3,4-dichlorophenol (3,4-DCP), 2,3,4-trichlorophenol (2,3,4-Trichlorophenol, 2,3,4-TCP), 2,3,5-trichlorophenol (2,3,5-Trichlorophenol, 2,3,5-TCP), 2,3,6-trichlorophenol (2,3,6-Trichlorophenol, 2,3,6-TCP), with a purity greater than 99.7%, were purchased from Beijing Zhenxiang Technology Co., Ltd.

[0043] 19 chlorophenol mixed standards: 1000 μg / mL, purchased from Shanghai Anpu Laboratory Technology Co., Ltd. Enhanced degreasing dispersion purification tube (Agilent Bond Elut dSPE Enhanced Matrix Removal (EMR) -Lipid).

[0044] The experimental subjects were river shrimp, crucian carp, and clam.

[0045] Example 1

[0046] The method of the present invention is based on QuEChERSEMR-Lipid combined with UPLC-MS method to simultaneously determine 19 chlorophenol compounds in aquatic products, which specifically includes the following steps:

[0047] 1. Preparation of standard solution

[0048] Single-label standard stock solution: Accurately weigh 10.0 mg (accurate to 0.0001 g) of 19 phenol standards into a 10 mL volumetric flask, dissolve and dilute to volume with methanol to prepare a 1 mg / mL single-label standard stock solution, and store it in a sealed container at -20 °C.

[0049] Mixed standard intermediate solution: Accurately pipette 100 μL of 19 chlorophenol mixed standards (1000 μg / mL) into a 10 mL volumetric flask, dilute to the mark with methanol to prepare a 10 μg / mL mixed standard intermediate solution, and store it in a sealed container at -20 ℃ for later use.

[0050] 2. Sample pretreatment

[0051] Fresh aquatic samples are processed, edible muscle tissue is extracted, and then minced for later use.

[0052] Extraction: Accurately weigh 5 g (accurate to 0.01 g) of aquatic product sample into a 50 mL centrifuge tube, add 10 mL of ethyl acetate-acetonitrile (v:v=1:1), 3 g of anhydrous MgSO4 and 2 g of NaCl, homogenize (12000 r / m, 5 min), ultrasonicate (500 W, 15 min), and then centrifuge at 4500 r / min for 5 min to remove all the organic phase.

[0053] Purification: The collected organic phase was transferred to a QuEChERSEMR-Lipid purification tube activated with 5 mL of 5 mmol ammonium acetate aqueous solution, vortexed for 20 min, and centrifuged at 9500 r / min for 10 min. All the supernatant was removed, blown to dryness with nitrogen, and then re-dissolved with 1 mL of acetonitrile-water (v:v=2:8). The reconstituted solution was filtered through a 0.22 μm organic filter membrane and used for UPLC-MS detection.

[0054] 3. Liquid chromatography conditions

[0055] Column: ACQUITY-UPLC-BEH-C 18(100 mm × 2.1 mm, 1.7 μm), column temperature 30 ℃; flow rate: 0.4 mL / min; mobile phase: A is 1 mmol / L ammonium formate aqueous solution, B is acetonitrile; gradient elution, elution program (expressed in volume fraction of mobile phase A) is shown in Table 1; injection volume: 3 μL.

[0056] Table 1 Gradient elution program

[0057]

[0058] 4. Mass spectrometry conditions

[0059] Scan mode: atmospheric pressure chemical ionization source negative ion mode (APCI - ); capillary voltage 5 kV; cone voltage 20 V; ion source temperature 150 ℃; ion source default voltage 80 V; desolvation gas temperature 600 ℃; desolvation nitrogen flow rate 900 L / h; cone gas flow rate 50 L / h; continuous scanning; recording range 50~600.

[0060] Table 2 APCI - Retention time and mass spectrometry parameters of 19 chlorophenol compounds in this mode

[0061]

[0062] 5. Determination of the method linear range, detection limit and quantification limit

[0063] The blank matrix was used to prepare matrix-matched mixed standard working solutions at six gradient levels (10, 20, 50, 100, 200, 400, and 500 μg / L). The quantitative working curve was drawn with the concentration (x, μg / L) as the abscissa and the peak area (y) of each target analyte as the ordinate. The correlation coefficient R of the linear regression equation of 19 chlorophenols was obtained. 2 The linear relationship was good in the range of 10-500 μg / L. The detection limit (LOD) was 1.5-4.0 μg / / kg and the quantitation limit (LOQ) was 5.0-13 μg / / kg, calculated by 3 times the signal-to-noise ratio (S / N). The specific results are shown in Table 3.

[0064] Table 3 Linear equations, linear ranges, detection limits and quantification limits of 19 chlorophenol compounds

[0065]

[0066] 6. Recovery and precision determination

[0067] In order to further evaluate the accuracy and precision of the method, spike recovery experiments were carried out on river shrimp, crucian carp and clam. Each compound was added at three concentration levels, and the concentration was determined at 1, 2, and 10 times the quantification limit of each chlorophenol compound (low: 5.0 μg / / k; medium: 10 μg / / k; high: 50 μg / / k). Six parallel samples were made at each level and then the sample pretreatment was performed according to 1.2. The results are shown in Table 3. The average recoveries of 19 CPs at the three addition levels of low, medium and high were between 72.5% and 115%, and the RSD was less than 2.8% to 9.8%, indicating that the method has good accuracy and precision.

[0068] Table 4 Recovery and precision of 19 chlorophenol compounds in fish meat

[0069]

[0070] 7. Actual sample testing

[0071] In order to verify the application effect of this method in actual samples, 10 different aquatic products (grass carp, crucian carp, river shrimp, clam, etc.) were tested according to this method. The results showed that pentachlorophenol was detected in two samples, one for grass carp and one for river shrimp, with contents of 6.5 μg / / kg and 7.3 μg / / kg respectively. No chlorophenol compounds were detected in the other aquatic samples.

[0072] Example 2 Selection of chromatographic column

[0073] The 19 CPs of the present invention have four pairs of isomers, and the isomers usually have the same parent ion and daughter ion, and can be accurately identified and quantified only when the chromatographic peaks are completely separated; and when using chromatographic columns of different models or specifications, the response values ​​and separation conditions are also different.

[0074] Therefore, BEH C 18 Column (100 mm × 2.1 mm × 1.7 μm), BEH C8 column (100 mm × 2.1 mm × 1.7 μm) and HSS T3 column (100 mm × 2.1 mm × 3 μm) were used to analyze 19 CPs. Figure 1 , Figure 2 , Figure 3 As shown;

[0075] The results showed that when using the BEH C8 column, there were 6 isomers of dichlorophenol and trichlorophenol, but only two compounds were detected for dichlorophenol, and only one compound of trichlorophenol had a good response and peak shape, such as Figure 2 As shown; when using HSS T3 column for detection, the situation is the same as that of BEH C8 column, such as Figure 3As shown in the figure, the peak times of all compounds on this column were delayed; while using BEH C 18 The 19 isomers can be separated by chromatographic column, and the response and peak shape are good. The peak order and retention time of each isomer are shown in Figure 1 and Table 2.

[0076] Example 3 Selection of mobile phase

[0077] The composition and ratio of the mobile phase not only affect the chromatographic separation of the compounds, but also affect the ionization efficiency of the target compounds, thereby affecting the detection sensitivity. 18 The column was used for analysis, and other conditions were the same as those in Example 1. The effects of mobile phases such as methanol-water and acetonitrile-ammonium formate aqueous solutions with different concentrations (1 and 5 mmol / L) on the retention behavior of the target compounds were compared.

[0078] The results show that the response values ​​of each compound are higher when using methanol-water, but the separation is poor. Figure 4 As shown in the figure, only 8 of the 19 chlorophenol compounds were separated and detected (only two of the three isomers of monochlorophenol were separated; only two of the six isomers of dichlorophenol were separated; only one of the six isomers of trichlorophenol was separated; and only two of the three isomers of tetrachlorophenol were separated). When acetonitrile-1 mmol / L ammonium formate aqueous solution was used as the mobile phase, the separation of each target compound was good, but the response value was reduced. Figure 5 As shown in the figure; when the concentration of ammonium formate increases to 5 mmol / L, the separation degree of trichlorophenol is not good, the peak shape is not good, and the overall peak time is delayed; as shown in the figure Figure 6 shown.

[0079] Example 4 Optimization of mass spectrometry conditions

[0080] The purpose of optimizing mass spectrometry detection conditions is to ensure that the measured ions have good sensitivity and stability.

[0081] The single-label solution of each target compound was used as the measurement object, and the MS Scan acquisition mode of UPLC-MS was used for scanning. The parent ions of each compound were determined by optimizing the cone voltage, and the additive form of the primary parent ions was inferred. The ion characteristics in the positive and negative ion modes of atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) were compared. The results showed that the 19 CPs had higher response intensity and sharper peak shape in the negative ion mode of atmospheric pressure chemical ionization; therefore, the ionization mode was selected as (APCI - ).

[0082] Example 5 Optimization of extraction solvent

[0083] In UPLC-MS analysis, the single extraction solvents of dichloromethane, n-hexane, and acetonitrile had low ionization efficiency for 19 chlorophenol compounds, resulting in low response values; based on Example 1, the extraction solvents were replaced with ethyl acetate, respectively. It was found that the matrix effect of individual compounds was strong, resulting in a recovery rate of more than 120%, which was inconsistent with the actual situation.

[0084] The extraction was performed using ethyl acetate-n-hexane (v:v=1:1), ethyl acetate-acetonitrile (v:v=1:1), and ethyl acetate-dichloromethane (v:v=1:1) as extraction solvents. The experimental results are shown in Figure 7 As shown;

[0085] When ethyl acetate-n-hexane (1:1) was used, the extract had more turbidity and a large amount of oil was extracted, the subsequent purification operation was more complicated, and the recovery rate of each substance was low; when ethyl acetate-dichloromethane (v:v=1:1) was used, the recovery rate of monochlorophenol was low, and the peak shape of 3,4-DCP was poor; when ethyl acetate-acetonitrile (v:v=1:1) was used, the peak shape of each compound was the best, and the recovery rate was also the best. In addition, when ethyl acetate was used to extract river shrimp, the stratification was not good. When pure acetonitrile was used for extraction, the protein was quickly precipitated to wrap the target compound, resulting in a low recovery rate of 19 chlorophenol compounds.

[0086] Example 6 Optimization of purification method

[0087] Aquatic products contain particularly high levels of protein, and lipid interference is a major challenge in the determination of trace residues in food or biological matrices, which not only results in reduced analyte sensitivity but also increases the need for MS maintenance, thereby shortening instrument and column life.

[0088] This paper compares the purification effects of traditional QuEChERS and the new enhanced lipid removal product QuEChERS EMR-Lipid on samples. The experimental results are as follows: Figure 8 As shown in the figure, the number of recoveries between 80% and 100% after QuEChERS purification is the same as that of QuEChERS EMR-Lipid purification method, but the recovery of QuEChERS EMR-Lipid purification method is higher than that of QuEChERS purification method when the recovery is between 90% and 110%. In addition, when using QuEChERS EMR-Lipid, there is no need to weigh the purification agent additionally, which saves pretreatment time and is more convenient and convenient. The degreasing effect is better, and the purification solution is clear and transparent, without damaging the chromatographic column.

[0089] Example 7 Optimization of the complex solution

[0090] After purification, the extract needs to be re-dissolved and passed through a membrane before it can be tested on the machine; acetonitrile-water (v:v=2:8), acetonitrile-water (v:v=1:1), and methanol were investigated. The experimental results showed that when acetonitrile-water (v:v=2:8) was used as the re-solvent, the response of the 19 target compounds was the highest and the peak shape was better.

[0091] Example 8 Matrix Effect

[0092] The present invention uses the slope of a mixed standard solution prepared with blank matrix extracts of crucian carp, river shrimp and clam and a mixed standard solution prepared with methanol to evaluate the matrix effect;

[0093] The calculation formula is: ME =[(slope of matrix-matched standard curve / slope of solvent standard curve)-1]×100%. ME Negative values ​​indicate matrix inhibition, and positive values ​​indicate matrix enhancement. The absolute value is used as the basis for judgment. The larger the absolute value, the stronger the matrix effect.

[0094] When ME ∣<20%, ​​weak matrix effect, can be ignored;

[0095] When 20%≤∣ ME ∣≤50%, medium-intensity matrix effect;

[0096] When ME ∣>50%, indicating a strong matrix effect.

[0097] The results of the determination are shown in Table 5. In crucian carp, except for 4-CP, 2,6-DCP, and 2,4-DCP, which showed strong matrix effects, other compounds showed weak to moderate matrix effects. In river shrimp, except for 3,4-DCP, 3,5-DCP, and 2,3,6-TCP, which showed matrix enhancement effects, other compounds showed weak to moderate matrix effects. Clams showed weak to moderate matrix effects. Therefore, in order to eliminate the influence of matrix effects on the determination of target compound content to a certain extent, the present invention adopts blank matrix matching standard curve to offset the matrix effect.

[0098] Table 5 Matrix effects of 19 CPs in different aquatic products

[0099]

Claims

1. A method for simultaneously determining 19 chlorophenol compounds in aquatic products, characterized in that: The method comprises the following steps: (1) Sample extraction Weigh the aquatic product sample into a centrifuge tube, add ethyl acetate-acetonitrile, anhydrous MgSO4 and NaCl, homogenize, sonicate, centrifuge, and take out all the organic phases; then transfer the organic phase to a QuEChERSEMR-Lipid purification tube, vortex oscillate, centrifuge again, take out the supernatant, blow nitrogen to dryness, and then re-dissolve it with acetonitrile-water. After passing the re-dissolved solution through an organic filter membrane, an extract of the sample to be tested is obtained; the volume ratio of ethyl acetate to acetonitrile is 1:1; (2) Preparation of matrix standard solution Using the blank sample extract obtained by the same method as step (1) as the matrix solution, prepare a series of concentrations of standard solutions of 19 chlorophenol compound mixed standards; (3) Content determination The standard solutions of the mixed standard products of 19 chlorophenol compounds with a series of concentrations prepared in step (2) and the sample extract prepared in step (1) are measured by UPLC-MS, and a quantitative relationship model is constructed with the concentration of the mixed standard products of 19 chlorophenol compounds as the horizontal axis and the peak area of ​​each target analyte as the vertical axis; the content of the chlorophenol compound in the sample to be tested is calculated based on the quantitative relationship model and the peak area of ​​the sample extract to be tested; The liquid chromatography conditions for the UPLC-MS determination are as follows: chromatographic column: ACQUITY-UPLC-BEH-C18, column temperature 30-35°C; flow rate: 0.4-0.6 mL / min; mobile phase: A is 1 mmol / L ammonium formate aqueous solution, B is acetonitrile; gradient elution, injection volume: 3 μL; The gradient elution program is: 0-10 min, 20%-50% acetonitrile phase, 10-11 min, 50%-95% acetonitrile phase, 11-15 min, 95% acetonitrile phase, 15-16 min, 95%-20% acetonitrile phase, 16-18 min, 20% acetonitrile phase.

2. The method according to claim 1, characterized in that The mass spectrometry conditions during the UPLC-MS determination in step (3) are as follows: Scanning mode: atmospheric pressure chemical ionization source negative ion mode (APCI - ); capillary voltage 5 kV; cone voltage 20 V; ion source temperature 150 ℃; ion source default voltage 80 V; desolvation gas temperature 600 ℃; desolvation nitrogen flow rate 900 L / h; cone gas flow rate 50 L / h; continuous scanning; recording range 50~600.

3. The method according to claim 1, characterized in that: The mass ratio of anhydrous MgSO4 and NaCl in step (1) is 3:2; wherein the total mass ratio of the water product to anhydrous MgSO4 and NaCl is 1:

1.

4. The method according to claim 1, characterized in that: The mass volume ratio of the water product and ethyl acetate-acetonitrile in step (1) is 1:2, g / mL.

5. The method according to claim 1, characterized in that The volume ratio of acetonitrile to water in step (1) is 2:8, and the volume is 1-2 mL.

6. Application of the method according to any one of claims 1 to 5 in the field of food safety testing.

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