A method for simultaneous determination of 19 chlorophenols in fish based on UPLC-Q-TOF-MS

Abstract

The present invention discloses a method for simultaneously determining 19 chlorophenol compounds in fish meat based on UPLC-Q-TOF-MS method, which belongs to the technical field of food safety detection. The method of the present invention for simultaneously determining 19 chlorophenol compounds in fish meat based on UPLC-Q-TOF-MS method comprises the following steps: pre-treating the fish meat with ethyl acetate, and then subjecting the fish meat to C 18 Chromatographic separation was performed using an acetonitrile-ammonium formate mobile phase with gradient elution, atmospheric pressure chemical ionization (APCI), and negative ion detection. This method is accurate, rapid, and reproducible, with limits of detection (LODs) of 0.006 to 0.010 mg / kg for 19 chlorophenols and recoveries of 61.68% to 90.17%. It is suitable for the qualitative and quantitative determination of 19 chlorophenols in fish, providing a practical and reliable technical means for the high-throughput, rapid detection of 19 chlorophenols in fish.

Description

Technical Field

[0001] The present invention relates to a method for simultaneously determining 19 chlorophenol compounds in fish meat based on a UPLC-Q-TOF-MS method, belonging to the technical field of food safety detection. Background Art

[0002] Chlorophenols (CPs) are widely used as pesticides, organic solvents, and pharmaceuticals. There are 19 common CPs, all formed by chlorine atoms replacing some of the hydrogen atoms on the benzene ring. They can be classified into monochlorophenol, dichlorophenol, trichlorophenol, tetrachlorophenol, and pentachlorophenol based on the number of chlorine atoms replaced. CPs are commonly found in industrial wastewater and domestic sewage from oil refining, coking, coal gas, and pulping. They are extremely stable and difficult to degrade, persisting in the environment for long periods and accumulating in organisms through the food chain. Long-term exposure to or ingestion of chlorophenol-containing compounds can cause dizziness, itchy skin, anemia, and various neurological disorders. Excessive ingestion can lead to acute poisoning.

[0003] Ministry of Agriculture Announcement No. 250 prohibits the use of sodium pentachlorophenate in all food-producing animals and prohibits its detection in animal-derived foods. To further standardize food safety supervision and random inspections, the "National Food Safety Supervision and Random Inspection Implementation Rules (2022 Edition)" explicitly stipulates the testing of sodium pentachlorophenate in livestock and poultry meat and their by-products.

[0004] Since current domestic and international laws and regulations are mostly limited to textiles, leather and livestock and poultry products, and the phenol limit standards are mostly aimed at pentachlorophenol and its salt compounds, there are no relevant standards for the limit of chlorophenols in aquatic products.

[0005] As one of the three major animal foods, alongside livestock and poultry meat and eggs, aquatic products account for approximately 30% of human animal protein intake. Compared to livestock and poultry meat, aquatic products not only offer unique nutritional and health benefits, but also offer better economic and ecological benefits in production. Therefore, monitoring the CPs content in aquatic products is particularly important.

[0006] Currently, methods for detecting CPs include gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS / MS). For example, Zhong Huiying et al. established a method combining liquid-liquid extraction with GC-MS to determine the content of 19 CPs in aquatic products. The average recovery rate of this method was 55.2%-125%, and the detection limit was 0.2-0.4 μg / kg. Wang Chengyun et al. used solid-phase extraction combined with GC-MS to establish a method for detecting 19 chlorophenols in paper and paper products. The average recovery rate of this method was 81.35%-93.48%, and the limit of quantification was 2 μg / kg. Zhou Min et al. established a liquid-liquid extraction-LC-MS / MS method for the determination of sodium pentachlorophenate in yellow throat. The detection limit of this method was 0.2 μg / kg, and the sample recovery rate was 80.3%-91.7%. Wang Lianzhu et al. used ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS) to determine pentachlorophenol and its sodium salt in animal-derived foods, achieving spiked recoveries of 73.2%-108.4% and a limit of quantification of 1.0 μg / kg. The accuracy and precision of this method meet the requirements for pollutant analysis, but the need for derivatization before GC-MS detection undoubtedly increases the complexity of the experimental process and operation. Furthermore, because the 19 phenolic compounds are structurally very similar and have isomers, this method often struggles to separate some of them, resulting in inaccurate qualitative and quantitative analysis of some substances and low sensitivity.

[0007] Based on this, the present invention establishes a method for the simultaneous and rapid detection of 19 CPs in fish meat based on QuEChERS pretreatment and ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The method can well separate the 19 phenolic substances. The method is accurate, rapid, and reproducible, providing a practical and reliable technical means for the high-throughput rapid detection of the content of 19 chlorophenols in fish meat. Summary of the Invention

[0008] In order to solve the technical defects of the prior art, the present invention provides a method for simultaneously determining 19 chlorophenol compounds in fish meat based on UPLC-Q-TOF-MS. The fish meat samples were pretreated by QuEChERS method and BEH C 18 Separation was performed on a 100 mm × 2.1 mm, 1.7 μm column, using a gradient elution system consisting of acetonitrile and 1 mM ammonium formate at a flow rate of 0.4 mL / min and a column temperature of 30°C. Screening and quantification were performed using atmospheric pressure chemical ionization (APCI) ionization with negative ion detection in full-scan mode. This method demonstrated high resolution, accuracy, rapidity, and excellent reproducibility, providing a practical and reliable means for the high-throughput rapid detection of 19 chlorophenols in fish.

[0009] The object of the present invention is to provide a method for simultaneously determining 19 chlorophenol compounds in fish based on UPLC-Q-TOF-MS, the method comprising the following steps:

[0010] (1) Sample extraction

[0011] Weigh the fish sample into a centrifuge tube, add ethyl acetate, homogenize, sonicate, centrifuge, and collect the upper organic phase; then transfer the organic phase to a centrifuge tube containing C18 and MgSO4, vortex, sonicate again, and centrifuge; collect the supernatant, blow nitrogen to dryness, and reconstitute with acetonitrile-water. Pass the reconstituted solution through an organic filter membrane to obtain the sample extract;

[0012] (2) Preparation of fish matrix standard solution

[0013] Using the blank fish extract extracted in the same manner as step (1) as the matrix solution, a series of concentrations of 19 chlorophenol compound mixed standard solutions were prepared;

[0014] (3) Content determination

[0015] The standard solutions of the mixed standard substances of 19 chlorophenol compounds with a series of concentrations prepared in step (2) and the extract of the sample to be tested prepared in step (1) are measured by UPLC-Q-TOF-MS, with the concentration of the mixed standard substances of 19 chlorophenols as the abscissa and the peak area of ​​each target analyte as the ordinate, to construct a quantitative relationship model; based on the quantitative relationship model and the peak area of ​​the extract of the sample to be tested, the content of the chlorophenol compounds in the sample to be tested is calculated.

[0016] In one embodiment, the liquid chromatography conditions during the UPLC-Q-TOF-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 0.5 to 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-Q-TOF-MS determination in step (3) are as follows: scan mode: atmospheric pressure chemical ionization source negative ion mode (APCI-); capillary voltage 5 kV; cone voltage 20 V; ion source temperature 150° C.; ion source default voltage 80 V; desolvation gas temperature 600° C.; desolvation nitrogen flow rate 900 L / h; cone gas flow rate 50 L / h; continuous scan; recording range 50 to 600; calibration solution 200 pg / μL leucine enkephalin, 0.5 mmol / L sodium formate.

[0020] In one embodiment, the vortexing time in step (1) is 10 to 30 minutes, preferably 20 minutes.

[0021] In one embodiment, the centrifugal conditions in step (1) are: 4000-5000 r / min, and the time is 5-10 min.

[0022] In one embodiment, the re-centrifugation conditions in step (1) are: 8000-10000 r / min, and the time is 10-15 min.

[0023] In one embodiment, the C 18 The mass ratio of MgSO4 is 5:1.

[0024] In one embodiment, the acetonitrile-water volume ratio in step (1) is 1:1.

[0025] In one embodiment, the mass volume ratio of the fish sample and ethyl acetate in step (1) is 1:2, g / mL.

[0026] In one embodiment, the homogenization in step (1) is carried out at 12000-15000 r / min for 5-10 minutes.

[0027] In one embodiment, the ultrasonication in step (1) is performed at 300-500W for 10-15 minutes.

[0028] Another object of the present invention is to provide an application of the above-described method in food safety testing.

[0029] Beneficial effects of the present invention:

[0030] The present invention can separate 19 isomers with similar properties, enabling the simultaneous detection of 19 chlorophenol compounds. Compared with the gas chromatography-mass spectrometry method, the present invention significantly shortens the detection time. The spectral library established by this method can screen 19 chlorophenol compounds in different fish species without adding spikes. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The ion current chromatogram of nine chlorophenol compounds separated when the mobile phase is methanol-water in Example 2 of the present invention;

[0032] Figure 2 This is an ion current chromatogram of 17 chlorophenol compounds separated when the mobile phase is acetonitrile-0.1% ammonia water in Example 2 of the present invention;

[0033] Figure 3 This is an ion current chromatogram of 16 chlorophenol compounds separated when the mobile phase is acetonitrile-0.05% ammonia water in Example 2 of the present invention;

[0034] Figure 4 This is an ion current chromatogram of 19 chlorophenol compounds when the mobile phase is acetonitrile-0.5 mmol / L ammonium formate in Example 2 of the present invention;

[0035] Figure 5 This is an ion current chromatogram of 19 chlorophenol compounds when the mobile phase is acetonitrile-1 mmol / L ammonium formate in Example 2 of the present invention;

[0036] Figure 6 ion current chromatograms of 18 chlorophenol compounds when the mobile phase is acetonitrile-2 mmol / L ammonium formate in Example 2 of the present invention;

[0037] Figure 7 This is an ion current chromatogram of 18 chlorophenol compounds when the mobile phase is acetonitrile-5 mmol / L ammonium formate in Example 2 of the present invention;

[0038] Figure 8 This is the ion current chromatogram of 9 chlorophenol compounds separated when the chromatographic column is a C8 column;

[0039] Figure 9 Eight chlorophenol compounds were separated using the Phenyl column; only one of the three monochlorophenols was separated; only two of the six dichlorophenols were separated; and only one of the six trichlorophenols was separated.

[0040] Figure 10 The ion current chromatograms of 10 chlorophenol compounds separated by ESI ionization mode; 1: 4-CP; 2: 3-CP; 3: 3,4-DCP; 4: 3,5-DCP; 5: 2,4,5-TCP; 6: 2,3,5,6-TeCP; 7: 2,3,4,6-TeCP; 8: 2,3,4,5-TeCP; 9: PCP;

[0041] Figure 11 This is a total ion current chromatogram of 19 chlorophenol compounds measured in Example 1 of the present invention;

[0042] Figure 12This is a graph showing the effect of different water addition amounts on the recovery rates of 19 chlorophenols during the extraction process of Example 5 of the present invention;

[0043] Figure 13 This is a graph showing the effects of different extraction methods on the recovery rates of 19 chlorophenols in Example 6 of the present invention;

[0044] Figure 14 This is a graph showing the effect of different shaking times on the recovery rates of 19 chlorophenols in Example 7 of the present invention. DETAILED DESCRIPTION

[0045] The present invention will be described below with reference to specific embodiments. It should be noted that the embodiments are only used to further illustrate the present invention and do not represent the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the present invention still fall within the scope of protection of the present invention.

[0046] 1. Instruments, reagents, and materials

[0047] SYNAPT G2-Si ultra-performance liquid chromatography-quadrupole-time of flight-mass spectrometry (UPLC-Q-TOF-MS, Waters, USA); ME204E electronic balance (Beijing Sartorius Scientific Instrument Co., Ltd.); Heraeus Multifuge X1R high-speed refrigerated centrifuge (Ningbo Noli Electronic Technology Co., Ltd.); KS-300EI ultrasonic cleaner (Ningbo Kesheng Equipment Co., Ltd.); Vortex 3 automatic vortex mixer (IKA, Germany); Reeko AH-20 fully automatic homogenizer (China Ruike Group).

[0048] Acetonitrile, methanol, ethyl acetate, and chromatographically pure reagents were purchased from Merck, Germany; ammonium formate (≥99.99%), sodium formate (≥99.99%), and leucine enkephalin were purchased from Shanghai McLean Biochemical Technology Co., Ltd.

[0049] 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-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), and 2,3,6-trichlorophenol (2,3,6-Trichlorophenol, 2,3,6-TCP), all with a purity greater than 99.7%, were purchased from Beijing Zhenxiang Technology Co., Ltd.

[0050] 19 chlorophenol mixed standards: 1000 μg / mL, purchased from Shanghai Anpu Laboratory Technology Co., Ltd.

[0051] 2. Experimental treatment objects: Hairtail

[0052] Example 1

[0053] The present invention provides a method for simultaneously determining 19 chlorophenol compounds in fish meat based on UPLC-Q-TOF-MS, which specifically comprises the following steps:

[0054] 1. Preparation of standard solution

[0055] Single-label standard stock solution: Accurately weigh 10.0 mg (accurate to 0.0001 g) of each of the 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 in a sealed container at -20°C.

[0056] 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 make a 10 μg / mL mixed standard intermediate solution, and store in a sealed container at -20°C for future use.

[0057] 2. Sample pretreatment

[0058] The fresh fish is processed by removing the head, bones and internal organs, and then minced for use.

[0059] Extraction: Accurately weigh 5 g (accurate to 0.01 g) of fish sample into a 50 mL centrifuge tube, add 10 mL of ethyl acetate, homogenize at 12000 rpm for 5 min, ultrasonicate at 500 W for 15 min, and then centrifuge at 4500 rpm for 5 min to remove all the organic phase.

[0060] Purification: The collected organic phase was transferred to a 18 and 100 mg MgSO4 in a centrifuge tube, vortex and oscillate for 20 minutes, sonicate for 5 minutes, and then centrifuge at 9500 r / min for 10 minutes. Remove all the supernatant, blow nitrogen to near dryness, and then re-dissolve with 1 mL acetonitrile-water (v:v = 1:1). The reconstituted solution is filtered through a 0.22 μm organic filter membrane and used for UPLC-Q-TOF-MS detection.

[0061] 3. Liquid chromatography conditions

[0062] Chromatographic column: ACQUITY-UPLC-BEH-C18 (100 mm × 2.1 mm, 1.7 μm), column temperature: 30°C; flow rate: 0.4 mL / min; mobile phase: A: 1 mmol / L ammonium formate aqueous solution, B: acetonitrile; gradient elution, elution program (expressed as volume fraction of mobile phase A) is shown in Table 1; injection volume: 3 μL.

[0063] Table 1 Gradient elution program

[0064]

[0065] 4. Mass spectrometry conditions

[0066] Scan mode: atmospheric pressure chemical ionization source negative ion mode (APCI-); capillary voltage 5 kV; cone voltage 20 V; ion source temperature 150°C; ion source default voltage 80 V; desolvation gas temperature 600°C; desolvation nitrogen flow rate 900 L / h; cone gas flow rate 50 L / h; continuous scan; recording range 50-600. The calibration solution was 200 pg / μL leucine enkephalin (LE) ([MH] - =554.2615), 0.5 mM sodium formate.

[0067] 5. Determination of method linear range, detection limit and quantification limit

[0068] Matrix-matched mixed standard working solutions with nine gradient levels (10, 20, 50, 100, 200, 400, 500, 1000, and 1500 μg / L) were prepared using blank hairtail matrix. A quantitative working curve was drawn with concentration (x, μg / L) as the abscissa and peak area (y) of each target analyte as the ordinate. The correlation coefficient R of the linear regression equation for 19 chlorophenols was obtained. 2 The linearity was 0.991–0.999, indicating good linearity over the range of 10–1500 μg / L. Calculated using a signal-to-noise ratio (S / N) of 3, the limit of detection (LOD) was 0.006–0.010 mg / kg, and the limit of quantitation (LOQ) was 0.018–0.030 mg / kg. Detailed results are shown in Table 2.

[0069] Table 2 Linear equations, linear ranges, detection limits, and quantification limits of 19 chlorophenol compounds

[0070]

[0071]

[0072] 6. Recovery and precision determination

[0073] Three different concentrations of chlorophenol standard solutions were spiked into the blank sample matrix at concentrations of 1, 2, and 10 times the limit of quantification (LOQ) for each chlorophenol compound (low: 0.018 / 0.030 mg / kg; medium: 0.036 / 0.060 mg / kg; high: 0.180 / 0.300 mg / kg). Pretreatment was performed according to 1.3 and the assay was run on the instrument. Both intra-day experiments (repeated six times) and inter-day experiments (repeated three times per day for three consecutive days) were performed for each spike level. The results are shown in Table 3. The recoveries for each compound ranged from 61.68% to 90.17%, with intra-day precision (RSDr) ranging from 4.3% to 9.5% and inter-day precision (RSDwR) ranging from 5.1% to 9.3%. All results met the methodological requirements.

[0074] Table 3 Recovery and precision of 19 chlorophenol compounds in fish meat

[0075]

[0076]

[0077] 7. Actual sample testing

[0078] To validate the effectiveness of this method in actual samples, 10 different fish species (hairtail, carp, snakehead carp, and grass carp) were tested using this method. The results showed that pentachlorophenol was detected in two samples—one from grass carp and one from carp—at levels of 0.016 mg / kg and 0.015 mg / kg, respectively. No chlorophenols were detected in the remaining fish samples.

[0079] Example 2 Selection of mobile phase

[0080] The peak shapes and sensitivities of 19 CPs were analyzed using different mobile phase systems. The chromatographic behavior and sensitivity of the target compounds were analyzed in mobile phase systems containing methanol-water, acetonitrile-water, and systems supplemented with different concentrations of ammonia (0.05%, 0.1%) and ammonium formate (0.5mmol / L, 1mmol / L, 2mmol / L, and 5mmol / L).

[0081] The results showed that the peak shape was better when methanol-water was used as the mobile phase, but only 9 target compounds were separated (monochlorophenol had three isomers but only two were separated; dichlorophenol had six isomers but only two were separated; trichlorophenol had six isomers but only one was separated). Figure 1 As shown;

[0082] When acetonitrile-different amounts of ammonia water were used as the mobile phase, the 19 target compounds could not be separated simultaneously. When an acetonitrile-water mobile phase system was used and ammonium formate was added to the aqueous phase, the peak shape and separation effect of the 19 chlorophenol compounds were better than those of other mobile phase systems. The specific results are shown in Table 4:

[0083] Table 4 Separation effect of different mobile phases

[0084]

[0085]

[0086] Further investigation of the amount of ammonium formate added revealed that when the ammonium formate concentration was greater than 2 mmol / L, only five isomers of trichlorophenol were separated. When the ammonium formate concentration was less than 2 mmol / L, all 19 isomers could be separated. However, as the ammonium formate concentration decreased, the separation effect and peak shape of the 19 chlorophenol compounds gradually deteriorated. When the ammonium formate concentration was 1 mmol / L, the peak shape and separation of the 19 CPs were better, and the sensitivity was higher.

[0087] Therefore, an aqueous solution containing 1 mmol / L ammonium formate and acetonitrile were finally used as the mobile phase in this experiment to achieve good separation effect and sensitivity for all 19 chlorophenol compounds.

[0088] Example 3 Optimization of chromatographic column

[0089] Key factors to consider when optimizing chromatographic separations include coelution separation and acquisition time. To achieve optimal separation and minimize separation time, and based on the structural characteristics of 19 chlorophenols, a mixed standard solution was used to experimentally compare the separation performance of 19 chlorophenols using common columns (100 mm × 2.1 mm, 1.7 μm)—ACQUITY-UPLC-BEH-C18, ACQUITY-UPLC-BEH-C8, and ACQUITY-UPLC-BEH-Phenyl.

[0090] The results are as follows Figure 8 、 Figure 9 、 Figure 11 As shown, Figure 11 The separation effect of 19 chlorophenol compounds measured in the present invention in the C18 chromatographic column is good, and the peak shape is more symmetrical. Under the same conditions, 8 and 9 target compounds (such as Figure 9 and Figure 8The isomers of monochlorophenol, dichlorophenol, and trichlorophenol were not completely separated. Furthermore, the peak of pentachlorophenol on the C8 column was wide and had poor peak shape. The retention times of the various phenols also shifted backward, increasing the detection time. Therefore, after considering factors such as column retention characteristics and separation performance, a 100 mm C18 column was ultimately selected.

[0091] Example 4 Optimization of mass spectrometry conditions

[0092] Standard solutions of 19 chlorophenol compounds (300 ng / mL) were prepared and analyzed by UPLC-Q-TOF-MS. - In this mode, the MS Scan acquisition mode is used for scanning analysis. The accurate mass number and retention time of the parent ion of the analyte are determined by the first-level full scan. E Using the MSMS acquisition mode, we varied the energy consumption to capture characteristic fragments of phenols. We found that chlorophenols had fewer characteristic fragments. Given that chlorophenols all contain chlorine, we ultimately employed primary scanning for quantitative and qualitative analysis, using their isotopes as the primary qualitative basis. By summarizing the retention time and accurate mass of each compound's primary precursor ion, combined with basic information such as the compound's name, molecular formula, and structural formula (mol file), we imported the mass spectral data for each compound into UNIFI software, where we constructed a screening library for 19 chlorophenol compounds.

[0093] The basic information, precursor ion information, and mass deviations of the 19 compounds are given in Table 5. Matching parameters were set in UNIFI software to establish qualitative and quantitative analysis methods.

[0094] The ion characteristics of 19 chlorophenol compounds were investigated in the negative ion mode of electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). Figure 10 It shows that in the ESI-ionization mode, 9 target compounds were separated, namely 4-CP, 3-CP, 3,4-DCP, 3,5-DCP, 2,4,5-TCP, 2,3,5,6-TeCP, 2,3,4,6-TeCP, 2,3,4,5-TeCP, and PCP. Among them, except for 2,3,4,5-TeCP which had a tailing peak, the other 8 chlorophenol compounds had high sensitivity and good response, but the remaining 10 chlorophenols had extremely low responses and were not detected.

[0095] In APCI-ionization mode, the sensitivity of 2-CP, 3-CP, 4-CP, PCP, etc. is somewhat reduced, but the response intensity of the 19 chlorophenol compounds is basically at the same level; APCI-ionization mode was finally selected for acquisition; the final total ion current chromatogram of the 19 chlorophenol compounds after optimization is shown in the attached figure. Figure 11 .

[0096] Figure 11 Middle 1: 2-CP; 2: 4-CP; 3: 3-CP; 4: 2,3,5,6-Tecp; 5: 2,6-DCP; 6: 2,3,6-TCP; 7: 3,4,5-TCP; 8: 2,3,4,6-Tecp; 9: PCP; 10: 2,3-DCP; 11:2 ,5-DCP; 12: 2,4,6-TCP; 13: 2,4-DCP; 14: 3,4-DCP; 15: 3,5-DCP; 16: 2,3,4-TCP; 17: 2,3,5-TCP; 18: 2,4,5-TCP; 19: 2,3,4,5-TeCP.

[0097] Table 5 Qualitative and quantitative mass spectrometry data of 19 chlorophenol compounds

[0098]

[0099] Example 5 Optimization of the amount of water added during the extraction process

[0100] Considering that if a certain amount of water is added during the pretreatment, a corresponding dehydrating agent needs to be added. At the same time, in order to better separate the aqueous phase and the organic extractant, a certain amount of salting-out agent needs to be added, which undoubtedly increases the complexity of the pretreatment experiment. In response to this situation, the present invention investigates the effect of adding water or not on the recovery rate of 19 chlorophenols in fish meat.

[0101] The specific operation is to add 0, 2, and 5g of dehydrating agent, i.e., anhydrous Na2SO4, respectively without adding water. In addition, when adding water, 1, 3, and 5g of water were selected to investigate the effect on the extraction effect of the target substance. The type and dosage of the salting-out agent were 3g of sodium chloride and 5g of magnesium sulfate, respectively.

[0102] The results showed that the recovery rates of the 19 chlorophenols were 60.46% to 79.28% when neither water nor desiccant was added. When 2g and 5g of desiccant were added without water, the recoveries were 57.87% to 76.77% and 56.88% to 75.34%, respectively. When 1g, 3g, and 5g of water were added, the recoveries were 51.34% to 69.78%, 43.56% to 75.74%, and 45.67% to 70.76%, respectively.

[0103] The above results show that when neither water nor desiccant was added, the recoveries of the 19 chlorophenols were all above 60%, while in other cases, the recoveries of some chlorophenols were only 40% to 50%. For example, the recovery of 2,5-DCP was only 43.56% to 52.18% when water was added. In addition, although the highest recoveries of 2,3,5,6-TeCP, 2,3,4,6-TeCP, and PCP were 72.69%, 69.47%, and 69.55%, respectively, when 3g of water, 3g of sodium chloride, and 5g of magnesium sulfate were added, the recoveries of the other phenols were either lower or slightly similar to those of the phenols when neither water nor desiccant was added. Detailed data are attached. Figure 12 Therefore, considering the recovery rates of 19 chlorophenols, this experiment chose not to add water or desiccant during the extraction process.

[0104] Example 6 Optimization of extraction method

[0105] Commonly used extraction methods for chlorophenol compounds include ultrasonic-assisted extraction, Soxhlet extraction, solid-liquid oscillation extraction, and accelerated solvent extraction. Among them, Soxhlet extraction has a high efficiency, but the process is cumbersome, the extraction time is long, and the actual consumption is large, so it is generally not recommended. Accelerated solvent extraction equipment is expensive and has a low penetration rate. Therefore, based on the popularity and operability of the method, and in order to ensure sufficient mixing between the extraction reagent and the sample, the present invention investigates the effects of an extraction method combining homogenization (8000r / min, 30s), vortex dispersion (3000r / min, 60s) and ultrasonic extraction (40°C, 15min) on the extraction effects of 19 chlorophenols.

[0106] The results are as follows Figure 13 As shown in the results, when vortex oscillation and ultrasonic water bath extraction were used to extract 19 chlorophenols from fish, the recoveries of 2-CP and 2,3,6-TCP were the lowest, at 38.12% and 42.16%, respectively. The recoveries of the remaining chlorophenols ranged from 50% to 60%. However, when homogenization combined with ultrasonic extraction was used, the recoveries of all 19 chlorophenols reached over 60%. Therefore, this study ultimately chose a combination of homogenization and ultrasonic-assisted extraction to extract the 19 chlorophenols from fish.

[0107] Example 7 Optimization of purification time

[0108] In the early experiments, the oscillation time during the purification process was set to 30 min. Considering that the oscillation time was too long in the detection of a large number of samples, affecting the detection efficiency, and it was not optimized in the early experiments, the present invention optimized and investigated different oscillation times (10, 20, and 30 min).

[0109] The results are as follows Figure 14As shown, the recovery rates of the samples after 10 minutes of oscillation were generally low, ranging from 30.35% to 46.87%. As the oscillation time increased, the recovery rates of the 19 chlorophenols in the samples showed a gradual upward trend. However, generally speaking, there was little difference in the recovery rate results of the samples under 20-minute and 30-minute oscillations.

[0110] Therefore, from the perspectives of energy conservation and detection efficiency improvement, the oscillation time selected for this experiment was 20 minutes. In addition, the recovery rates of chlorophenol compounds under different ultrasonic times (5, 10, 15 minutes) were also investigated in this experiment. The results showed that the influence of different ultrasonic times on the recovery rate was not obvious. Therefore, considering the comprehensive factors of not affecting the detection effect and saving the detection cost, the ultrasonic time of 5 minutes was adopted in this invention for the experiment.

[0111] Example 8 Matrix Effect Optimization

[0112] Matrix effect (ME) is the influence caused by the competition for ionization between the target compound and the co-extracted interferents in the matrix. For high-fat and high-protein complex matrix samples, its matrix effect will affect the ionization of phenolic pollutants, causing signal enhancement or suppression, thus affecting the accuracy of the results. Generally speaking, when ME = 1.0, it indicates that there is no matrix effect; 0.8 < ME < 1.2 is a weak matrix effect, which can generally be ignored; 0.5 < ME < 0.8 or 1.2 < ME < 1.5 is a medium matrix effect; ME < 0.5 or ME > 1.5 is a strong matrix effect. When ME is between 0.8 and 1.2, the matrix effect can be basically ignored.

[0113] Matrix-matched calibration solutions and standard solutions with the same concentration were prepared using blank matrix extraction solution and pure solvent. The matrix effect was reflected by the ratio of the response value of the average peak area of the negative sample (B) to the response value of the average peak area of the pure solvent (A), and was calculated by the following formula:

[0114]

[0115] In this invention, blank fish samples were used for matrix effect evaluation. The results showed that the ME of the 19 chlorophenols was 0.72 - 2.24. Except for 3 monochlorophenols, 2,4,6-TCP, and 2,3,4-TCP having matrix enhancement effects, the remaining phenols had medium matrix suppression effects. Considering that the matrix effect cannot be completely eliminated, only corresponding measures can be taken to reduce its influence on compound analysis.

[0116] Therefore, the present invention adopts blank matrix extract to prepare standard curve to achieve the purpose of eliminating matrix effect to a certain extent. In addition, the matrix effect corresponding to different types of fish has also been investigated, and it is found that the matrix effect of the same phenolic compound between different fish is not much different. Specific results are shown in Table 6. Therefore, in the actual detection process, in the face of more fish samples of variety, a fish sample can be randomly selected to prepare a matrix standard curve to improve detection efficiency.

[0117] Table 6 Matrix effects of 19 chlorophenols in different fish species

[0118]

[0119]

Claims

1. A method for simultaneously determining 19 chlorophenols in fish based on UPLC-Q-TOF-MS, characterized in that: The method comprises the following steps: (1) Sample extraction Weigh the fish sample in a centrifuge tube, add ethyl acetate, homogenize, sonicate, centrifuge, and take the upper organic phase; then transfer the organic phase to a centrifuge tube containing C18 and MgSO4, vortex, sonicate, centrifuge, take the supernatant, blow nitrogen to dryness, and re-dissolve with acetonitrile-water. After the reconstituted solution is passed through an organic filter membrane, the sample extract to be tested is obtained; the vortex oscillation time is 20-30 minutes; the C 18 The mass ratio of MgSO4 is 5:1; (2) Preparation of fish matrix standard solution Using the blank fish extract extracted in the same manner as step (1) as the matrix solution, a series of concentrations of 19 chlorophenol mixed standard solutions were prepared; (3) Determination of chlorophenol content The standard solutions of the 19 chlorophenol mixed standard substances with a series of concentrations prepared in step (2) and the sample extract prepared in step (1) are measured by UPLC-Q-TOF-MS, with the concentration of the 19 chlorophenol mixed standard substances as the abscissa and the peak area of ​​each target analyte as the ordinate, to construct a quantitative relationship model; based on the quantitative relationship model and the peak area of ​​the sample extract, the content of chlorophenol compounds in the sample is calculated; The liquid chromatography conditions for the UPLC-Q-TOF-MS determination were as follows: 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: ammonium formate aqueous solution, B: acetonitrile; gradient elution, injection volume: 3 μL; the concentration of ammonium formate was 1 mmol / L. The mass spectrometry conditions for the UPLC-Q-TOF-MS determination were as follows: scan mode: atmospheric pressure chemical ionization source negative ion mode APCI-; capillary voltage 5 kV; cone voltage 20 V; ion source temperature 150°C; ion source default voltage 80 V; desolvation gas temperature 600°C; desolvation nitrogen flow rate 900 L / h; cone gas flow rate 50 L / h; continuous scan; recording range 50-600; The gradient elution procedure was as follows: 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; The 19 chlorophenol compounds are 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 and 2,3,6-trichlorophenol.

2. The method according to claim 1, characterized in that The acetonitrile-water volume ratio in step (1) is 1:

1.

3. The method according to claim 1, characterized in that The mass volume ratio of the fish sample and ethyl acetate in step (1) is 1:2, g / mL.

4. Use of the method according to any one of claims 1 to 3 in food safety testing.

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