Method for detecting perfluoroalkyl / polyfluoroalkyl substances in fish

Abstract

The invention provides a method for detecting perfluoroalkyl / polyfluoroalkyl substances in fish, which comprises the following steps: S1, sample preparation: crushing and homogenizing to-be-detected fish meat to obtain a to-be-detected sample; s2, extraction: extracting the to-be-detected sample by using water and an organic solvent, retaining an organic phase, concentrating the organic phase, and adding water which is 5-15 times of the volume of the organic phase as a to-be-purified solution; s3, purification: purifying the to-be-purified liquid by using a solid-phase extraction column to obtain a purified liquid; s4, concentration and constant volume: concentrating the purified liquid to be nearly dry, redissolving, and filtering to obtain a to-be-detected solution; and S5, sample detection: detecting the solution to be detected by using a liquid chromatograph-mass spectrometer, and carrying out qualitative and quantitative analysis on the perfluorinated / polyfluoroalkyl substances in the fish by adopting a standard curve method. The method provided by the invention can be used for simultaneously determining the contents of 43 different PFASs compounds in fish.

Description

Technical Field

[0001] This invention relates to the field of detection technology for per- and polyfluoroalkyl substances, and in particular to a method for detecting per- and polyfluoroalkyl substances in fish. Background Technology

[0002] PFASs are organic compounds composed of carbon chains in which hydrogen atoms are replaced by fluorine atoms. They are classified into perfluorinated (completely substituted) and polyfluorinated (partially substituted) compounds. The carbon-fluorine (CF) bond is the strongest covalent bond in organic chemistry, which gives PFASs extremely stable chemical properties and makes them difficult to decompose in the natural environment; therefore, they are called "permanent chemicals." PFASs are difficult to decompose in the environment and organisms, exhibiting strong bioaccumulation. Their persistence and recalcitrant nature allow them to persist in the environment for a long time, migrating long distances through the atmosphere and water bodies, causing pollution to ecosystems. Long-term exposure to PFASs may have various adverse effects on human health, such as liver damage, immunosuppression, and tumor development, and may also increase the risk of cancer metastasis.

[0003] Fish is an important part of the human diet. Detecting PFASs in fish not only helps assess environmental and health risks but also provides crucial support for food safety regulation, pollution control, and scientific research, possessing significant practical application value. However, the differences in the physicochemical properties of different PFASs (such as ionic and non-ionic PFASs) limit the breadth and sensitivity of traditional methods. Fish matrices are complex, containing large amounts of proteins, fats, pigments, etc. These substances severely interfere with PFAS extraction and compete with or bind to target analytes, leading to low or highly fluctuating extraction recoveries. Because different PFAS structures exhibit significantly different retention behaviors on chromatographic columns, it is difficult to simultaneously detect multiple perfluorinated / polyfluoroalkyl substances in a single analysis. Co-elution can cause mutual interference, affecting quantitative accuracy and resulting in low efficiency for batch detection of fish. Furthermore, existing technologies mostly employ LC-MS for detection, but its sensitivity for detecting certain low-level perfluorinated / polyfluoroalkyl substances, such as perfluorocarboxylic acids, may still be insufficient, making it difficult to meet the needs for detecting trace amounts of perfluorinated / polyfluoroalkyl substances. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a method for detecting per- and polyfluoroalkyl substances in fish, which solves the problems of low detection efficiency, low sensitivity, and difficulty in accurate quantification of per- and polyfluoroalkyl substances in fish in the prior art.

[0005] To achieve the above and other related objectives, the present invention provides a method for detecting per- and polyfluoroalkyl substances in fish.

[0006] The first aspect of this invention provides a method for detecting per- and polyfluoroalkyl substances in fish, the method comprising:

[0007] S1. Sample preparation, including: crushing and homogenizing the fish meat to be tested to obtain the sample to be tested;

[0008] S2. Extraction, including: extracting the sample to be tested using water and organic solvent, retaining the organic phase, concentrating the organic phase, and adding 5 to 15 times the volume of water of the organic phase as the purification solution;

[0009] S3. Purification, including: purifying the liquid to be purified using a solid-phase extraction column to obtain a purified liquid;

[0010] S4. Concentration and volume adjustment, including: concentrating the purified liquid to near dryness, redissolving and filtering to obtain the solution to be tested;

[0011] S5. Sample testing, including: using liquid chromatography-mass spectrometry to test the solution to be tested, and using the standard curve method to perform qualitative and quantitative analysis of perfluorinated / polyfluoroalkyl substances in fish.

[0012] Preferably, the perfluoro / polyfluoroalkyl substances include: perfluoropropionic acid, perfluorobutyric acid, perfluoropentanoic acid, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluorohexadecanoic acid, perfluoroethanesulfonic acid, perfluorobutanesulfonic acid, perfluoropentanesulfonic acid, perfluorohexanesulfonic acid, perfluoroheptanesulfonic acid, perfluorooctanesulfonic acid, perfluorodecanesulfonic acid, perfluorobutylsulfonamide, perfluorohexanesulfonamide, N-methylperfluorooctanesulfonamide, N-ethylperfluorooctanesulfonamide, N-methylperfluorooctanesulfonamide ethanol, 2(N-ethylperfluorooctanesulfonamide)ethanol, perfluorooctanesulfonamide acetic acid, 6:2 fluoro Tilomer sulfonic acid, 8:2 fluorotilomer sulfonic acid, 10:2 fluorotilomer sulfonic acid, 3:3 fluorotilomer carboxylic acid, 7:3 fluorotilomer carboxylic acid, 6:2 fluorotilomer carboxylic acid, 8:2 fluorotilomer carboxylic acid, 4,8-dioxa-3H-perfluorononanoic acid, 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propionic acid, perfluoro-2,5-dimethyl-3,6-dioxanonanoic acid, 2,2,3,3-tetrafluoro-3-(trifluoromethoxy)propionic acid, perfluoro-4-methoxybutyric acid, perfluoro-3,6-dioxoheptanoic acid, 1,1,2,2-tetrafluoro-2-(perfluoroethoxy)ethanesulfonic acid, 9-chloroperfluoro-3-nonoxysulfonic acid, and 8:2 fluorotilomer phosphate diester.

[0013] Preferably, the fish species described in this invention include, but are not limited to, pomfret, yellow croaker, tofu fish, grouper, sea bass, flounder, red snapper, silver carp, grass carp, crucian carp, catfish, snakehead, sturgeon, and saury.

[0014] Preferably, a grinder is used to grind and homogenize the fish meat in step S1.

[0015] Preferably, in step S2, the amount of water added is 1 to 3 mL, based on 1 g of the sample to be tested; including but not limited to 1 mL, 1.5 mL, 2 mL, 2.5 mL or 3 mL.

[0016] Preferably, the volume ratio of water to organic solvent in step S2 is 1:(1-3); for example, it can be 1:1, 1:2 or 1:3.

[0017] Preferably, step S2 further includes adding salt to water and organic solvent after extraction to separate the two phases; the amount of salt added is 0.5 to 1.5 g based on 1 mL of water; for example, it can be 0.5 g, 0.6 g, 0.8 g, 1.0 g, 1.2 g, 1.4 g or 1.5 g.

[0018] More preferably, the salt is sodium chloride.

[0019] Preferably, in step S2, a 30% to 40% hydrochloric acid aqueous solution is used for extraction, and the amount of hydrochloric acid aqueous solution is 0.1% to 0.5% of the volume of acetonitrile.

[0020] Preferably, step S2 further includes adding an internal standard to the sample to be tested before extraction, wherein the amount of internal standard added is 2 to 50 ng based on 1 g of sample to be tested.

[0021] More preferably, step S2 further includes vortexing the solution containing the sample, internal standard, and water for 1 to 5 minutes before adding salt.

[0022] More preferably, step S2 further includes adding an organic solvent after vortexing and shaking for 5 to 10 minutes.

[0023] More preferably, step S2 further includes adding salt after shaking and shaking for 5 to 10 minutes.

[0024] More preferably, step S2 further includes centrifuging at 5000-10000 r / min for 5-10 minutes after shaking to separate the water and organic solvent into layers.

[0025] More preferably, step S2 further includes adding an internal standard to the sample to be tested before extraction. Based on 1g of sample to be tested, the amount of internal standard added is 2 to 20ng; for example, it can be 2ng, 5ng, 10ng, 15ng or 20ng.

[0026] More preferably, the internal standard includes perfluorobutyric acid (PFOA). 13 C4(M4PFBA), perfluorohexanoic acid-13 C5(M5PFHxA), perfluorooctanoic acid - 13 C8(M8PFOA), perfluorododecanoic acid- 13 C(MPFDoDA), perfluorooctane sulfonic acid- 13 C8(M8PFOS), 8:2 fluoropolymer sulfonic acid 13 C2(M2-8:2FTS), 2,3,3,3-Tetrafluoro-2-(heptafluoropropoxy)propionic acid- 13 Any one or more of C3(M3HFPO-DA).

[0027] Preferably, in step S2, the volume of the liquid to be purified is 5 to 15 mL; for example, it can be 5 mL, 8 mL, 10 mL, 12 mL or 15 mL.

[0028] Preferably, the stationary phase of the solid-phase extraction column in step S3 is a WAX and HLB mixed column, wherein the mass ratio of WAX to HLB is 1:(0.5 to 1.5); for example, it can be 1:0.5, 1:1, or 1:1.5.

[0029] The WAX ​​is a weak anion exchange packing material, which is a weak anion exchange chromatography packing material with polyvinylpyrrolidone as the matrix and primary / secondary amines bonded in its structure.

[0030] The HLB is a hydrophilic-lipophilic balanced filler, which is a filler made of polystyrene / divinylbenzene bonded with pyrrolidone groups, and its surface has both hydrophilic and lipophilic groups.

[0031] Preferably, the mass of the stationary phase in the solid phase extraction column is 120–170 mg; for example, it can be 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, or 170 mg.

[0032] Preferably, the volume of the solid-phase extraction column is 5 to 10 mL; for example, it can be 5 mL, 6 mL, 7 mL, 8 mL, 9 mL or 10 mL.

[0033] More preferably, the volume of the solid-phase extraction column is 5 to 7 mL.

[0034] Preferably, the mass ratio of the stationary phase to the volume of the purified liquid passing through the column is 6 mg: (5-15) mL.

[0035] More preferably, the mass ratio of the stationary phase to the volume of the purified liquid passing through the column is 6 mg: (8-12) mL.

[0036] Preferably, in step S3, the purification process sequentially includes solid-phase extraction column activation, sample loading, rinsing, and elution.

[0037] More preferably, the activation is performed sequentially using 0.05% to 0.5% ammonia water, methanol, and water.

[0038] The percentage refers to the volume percentage of ammonia in the ammonia-methanol solution.

[0039] More preferably, the volume of ammonia and methanol used during activation is 2 to 10 mL; for example, it can be 2 mL, 4 mL, 5 mL, 6 mL, 8 mL, or 10 mL.

[0040] More preferably, the volume of methanol used during activation is 2 to 10 mL; for example, it can be 2 mL, 4 mL, 5 mL, 6 mL, 8 mL, or 10 mL.

[0041] More preferably, the volume of water used during activation is 2 to 10 mL; for example, it can be 2 mL, 4 mL, 5 mL, 6 mL, 8 mL, or 10 mL.

[0042] Preferably, the eluent is ammonium acetate with a concentration of 20-30 mmol / L; for example, it can be 20 mmol / L, 25 mmol / L or 30 mmol / L.

[0043] More preferably, the volume of the rinsing solution is 2 to 10 mL; for example, it can be 2 mL, 4 mL, 5 mL, 6 mL, 8 mL or 10 mL.

[0044] More preferably, in step S3, the rinsing process further includes vacuuming the solid-phase extraction column for 1 to 3 minutes followed by elution.

[0045] More preferably, the elution is performed sequentially using methanol and 0.05% to 0.5% ammonia-methanol solution.

[0046] More preferably, the volume of methanol used during elution is 1 to 5 mL; for example, it can be 1 mL, 2 mL, 3 mL, 4 mL, or 5 mL.

[0047] More preferably, the volume of ammonia and methanol used in the elution is 2 to 10 mL; for example, it can be 2 mL, 4 mL, 5 mL, 6 mL, 8 mL or 10 mL.

[0048] More preferably, the elution flow rate is 1–3 s / drop.

[0049] Preferably, in step S4, the concentration is carried out at 30–50°C using nitrogen blowing concentration.

[0050] The concentration to near dryness is determined by visual observation that there is no liquid flowing at the bottom of the container.

[0051] Preferably, in step S4, the resolution is performed by resolution of the concentrated purified liquid with methanol.

[0052] Preferably, in step S4, the filtration is performed using a filter membrane with a pore size of 0.1 to 0.3 μm.

[0053] More preferably, the pore size of the filter membrane can be 0.1 μm, 0.2 μm or 0.3 μm.

[0054] Preferably, in step S5, the chromatographic conditions include:

[0055] 1) The chromatographic column is a C18 column;

[0056] 2) The column temperature is 40-45℃; for example, it can be 40℃, 42℃ or 45℃.

[0057] 3) The injection volume is 1 to 3 μL; for example, it can be 1 μL, 2 μL or 3 μL.

[0058] 4) The flow rate is 0.2 to 0.5 mL / min; for example, it can be 0.2 mL / min, 0.3 mL / min, 0.4 mL / min or 0.5 mL / min.

[0059] 5) Mobile phase: Phase A: 1-5 mM ammonium acetate aqueous solution; Phase B: methanol;

[0060] 6) Elution method: Gradient elution, including: Initial solvent composition: 75%-80% A phase and 20-25% B phase; Gradient change phase: 0-14 min, the proportion of B phase increases linearly to 90-95%; Hold phase: 14-16 min, 90-95% B phase is maintained; Column washing phase: 16 min-16.01 min, the proportion of B phase decreases linearly to 20-25%; Column equilibration phase: 16.01 min-20 min, 20-25% B phase is maintained.

[0061] Preferably, in step S5, the mass spectrometry conditions include:

[0062] 1) Electrospray ion source, negative ion mode;

[0063] 2) Air curtain pressure 30-40 psi; for example, it can be 30 pis, 35 pis or 40 pis.

[0064] 3) Spray voltage -4200 to -4800V; for example, it can be -4200V, -4500V or -4800V.

[0065] 4) Atomization temperature 480~520℃; for example, it can be 480℃, 500℃ or 520℃.

[0066] 5) Atomizing pressure 40-55 psi; for example, it can be 45 pis, 50 pis or 55 pis.

[0067] 6) Auxiliary gas pressure 45-55 psi; for example, it can be 45 pis, 50 pis or 55 pis.

[0068] Preferably, in step S5, the concentration of the standard solution selected in the establishment of the standard curve is 0.005 to 100 μg / L.

[0069] Preferably, in step S5, the establishment of the standard curve further includes adding an internal standard, wherein the amount of the internal standard added is 5 to 15 ng.

[0070] A second aspect of the present invention provides the use of the above-described method for simultaneously detecting 43 perfluorinated / polyfluorinated alkyl substances in fish.

[0071] As described above, the method for detecting per- and polyfluoroalkyl substances in fish according to the present invention has the following beneficial effects:

[0072] This invention employs a combination of water and organic solvent extraction and solid-phase extraction to effectively purify, refine, and enrich PFASs in fish. Furthermore, LC-MS / MS is used for quantitative detection of the purified and enriched substances. Using this method, the recoveries of 43 PFASs in fish matrices range from 61% to 139%, with RSDs ranging from 0.4% to 13.8%. The LOD in fish matrices is 0.0012–0.0593 μg / kg; the LOQ is 0.0041–0.1977 μg / kg; and the correlation coefficient of the standard curve is above 0.999. This invention can simultaneously determine the content of at least 43 PFASs compounds with different physicochemical properties in fish, improving the detection efficiency of batch samples. It has advantages such as short processing time, high sensitivity, good accuracy, and strong stability. Attached Figure Description

[0073] Figure 1 The diagram shows a schematic of the detection process for detecting perfluorinated / polyfluoroalkyl substances in fish according to the present invention.

[0074] Figure 2 The chromatogram shown is obtained by UPLC-MS / MS detection of the target compound at a concentration of 20 ng / mL using the chromatographic conditions of Example 1 of this invention. The peaks represent: 1: PFPrA; 2: PFBA; 3: PFPeA; 4: PFHxA; 5: PFHpA; 6: PFOA; 7: PFNA; 8: PFDA; 9: PFUnDA; 10: PFDoDA; 11: PFTrDA; 12: PFTeDA; 13: PFHxDA; 14: PFODA.

[0075] Figure 3 The chromatogram shown is obtained by UPLC-MS / MS detection of the target compound at a concentration of 20 ng / mL using the chromatographic conditions of Example 1 of this invention. The peaks represent: 1: PFEtS; 2: PFBS; 3: PFPeS; 4: PFHxS; 5: PFHpS; 6: PFOS; 7: PFDS.

[0076] Figure 4 The chromatogram shown is obtained by UPLC-MS / MS detection of the target compound at a concentration of 20 ng / mL using the chromatographic conditions of Example 1 of this invention. The peaks represent: 1: PFMPA; 2: PF5OHxA; 3: 3-6-OPFHpA; 4: HPFO-DA; 5: HFPO-TA; 6: ADONA.

[0077] Figure 5 The chromatogram shown is obtained by UPLC-MS / MS detection of the target compound at a concentration of 20 ng / mL using the chromatographic conditions of Example 1 of this invention. The peaks represent: 1: PFEESA; 2: 6:2Cl-PFESA; 3: FOSAA; 4: 8:2diPAP.

[0078] Figure 6 The chromatogram shown is obtained by UPLC-MS / MS detection of the target compound at a concentration of 20 ng / mL using the chromatographic conditions of Example 1 of this invention, wherein each peak represents: 1:6:2FTSA; 2:8:2FTSA; 3:10:2FTSA.

[0079] Figure 7 The image shows a chromatogram obtained by UPLC-MS / MS detection of a target compound with a concentration of 20 ng / mL using the chromatographic conditions of Example 1 of this invention. The peaks represent: 1:3:3FTCA, 2:6:2FTCA, 3:7:3FTCA, and 4:8:2FTCA, respectively.

[0080] Figure 8 The chromatogram shown is obtained by UPLC-MS / MS detection of the target compound at a concentration of 20 ng / mL using the chromatographic conditions of Example 1 of this invention. The peaks represent: 1: FBSA; 2: FHxSA; 3: FOSA; 4: N-MeFOSA; 5: N-MeFOSE; 6: N-EtFOSA; 7: N-EtFOSE.

[0081] Figure 9 The image shows photographs of the fish samples taken, obtained by testing 10 fish samples using the detection method described in Example 1 of this invention.

[0082] Figures 10-19 The images show chromatograms obtained by detecting perfluorinated / polyfluoroalkyl substances in fish samples 1 to 10 using the detection method of Example 1 of this invention; wherein, Figure 10 Fish sample 1; Figure 11 Fish sample 2; Figure 12 Fish sample 3; Figure 13 Fish sample 4; Figure 14 Fish sample 5; Figure 15 Fish sample 6; Figure 16 Fish sample 7; Figure 17 Sample 8; Figure 18 Fish sample 9; Figure 19 For fish sample 10. Detailed Implementation

[0083] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0084] It should be noted that the process equipment or apparatus not specifically mentioned in the following embodiments are all conventional equipment or apparatus in the art.

[0085] Furthermore, it should be understood that the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps, does not preclude the presence of other method steps before or after the combined steps, unless otherwise stated. It should also be understood that the combined connection relationship between one or more devices / apparatus mentioned in this invention does not preclude the existence of other devices / apparatus before or after the combined devices / apparatus, or the insertion of other devices / apparatus between these explicitly mentioned devices / apparatus. Moreover, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or defining the scope of the invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the invention.

[0086] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0087] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0088] The instrument and equipment information used in the embodiments of this invention is as follows:

[0089] Sciex Qtrap 4500 liquid chromatography-mass spectrometry system (AB SCIEX, USA), AL104 electronic analytical balance (Mettler Toledo Technologies). UV ultrapure water system (Milipore, USA), CT14RD benchtop high-speed refrigerated centrifuge (Shanghai Tianmei Scientific Instruments Co., Ltd., China) VIRTEX 3 vortex mixer (IKA GmbH, Germany), HX-12D solid phase extraction device (Wuhan Hengxin Century Technology, China), KQ-700VDV dual-frequency CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., China), and Model 2695 high performance liquid chromatograph (UV detector) (Waters Corporation, USA).

[0090] The reagents and standards used in the embodiments of this invention are as follows:

[0091] Methanol, acetonitrile (chromatographic grade, Shanghai Xingke High Purity Solvent Co., Ltd.); perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanoic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluorohexadecanoic acid, perfluorostachyoctanoic acid, perfluoroethanesulfonic acid, perfluoropentanesulfonic acid, perfluoroheptanesulfonic acid, perfluorodecanesulfonic acid, 1,1,2,2-tetrafluoro-2-(perfluoroethoxy)ethanesulfonic acid, 9-chloroperfluoro-3-nonoxysulfonic acid, perfluorobutylsulfonic acid Amides, perfluorooctanesulfonamide acetic acid, 6:2 fluoropolymer sulfonic acid, 8:2 fluoropolymer sulfonic acid, 10:2 fluoropolymer sulfonic acid, 3:3 fluoropolymer carboxylic acid, 7:3 fluoropolymer carboxylic acid, 6:2 fluoropolymer carboxylic acid, 8:2 fluoropolymer carboxylic acid, 4,8-dioxa-3H-perfluorononanoic acid, 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propionic acid, perfluoro-2,5-dimethyl-3,6-dioxanonanoic acid Acids, 2,2,3,3-tetrafluoro-3-(trifluoromethoxy)propionic acid, perfluoro-4-methoxybutyric acid, perfluoro-3,6-dioxoheptanoic acid (purity greater than 98%, Alta Technology Co., Ltd. official website); perfluorooctane sulfonic acid, perfluorooctanoic acid, perfluorobutane sulfonic acid, perfluorovaleric acid, perfluorohexanoic acid, perfluoropropionic acid, perfluoroheptanoic acid, perfluorohexane sulfonic acid, N-methylperfluorooctane sulfonamide, 2(N-ethylperfluorooctane sulfonamide) ethanol, trifluoroacetic acid, perfluorohexane sulfonamide, 8:2 fluoropolymer phosphate diester, perfluorobutyric acid, N-ethylperfluorooctane sulfonamide, N-methylperfluorooctane sulfonamide ethanol (purity greater than 80%, Anpu Cloud Laboratory Supplies Co., Ltd.); perfluorooctane sulfonamide, trifluoromethanesulfonic acid (purity greater than 90%, Manhag Biotechnology Co., Ltd.); WAX packing material (Wendu Chromatography Technology Co., Ltd., specification: Ontwsep) TM WAX); HLB packing material (Wendu Chromatography Technology Co., Ltd., specification: Ontwsep) TM HLB); WAX and HLB mixed solid-phase extraction column (150 mg / 6 mL, Wendu Chromatography Technology Co., Ltd.). Chromatographic column: Excsep™ SiO2@PFP-C18 column (2.1 × 100 mm, 1.8 μm, Wendu Chromatography Technology Co., Ltd.).

[0092] In step S1, the fish muscle tissue in this application is obtained after removing the fish skin, fish bones, fish viscera and other organs. The muscle is the main edible part, and the data from testing the muscle tissue can be directly used to assess food safety.

[0093] In step S2, adding acid to the extract can purify perfluorinated / polyfluoroalkyl substances in the organic phase, which is beneficial to improving the recovery rate. However, adding too much or too little acid will affect the recovery rate.

[0094] After extraction with water and organic solvents, salt is added to achieve phase separation and facilitate phase separation.

[0095] After extraction, the water and organic phases are separated, and then water is added to form the solution to be purified. The addition of water allows perfluorinated / polyfluoroalkyl substances to be well removed from the solution and retained in the packing material during S3 purification, without being washed down with the organic solvent.

[0096] In step S3, the solid-phase extraction column used in this application has a specification of (150 mg / 6 mL) and is composed of WAX:HLB in a mass ratio of 1:1. The applicant found that when only one type of packing material is used, the single-mechanism packing material makes it impossible for certain types of perfluorinated / polyfluoroalkyl substances to be retained in the column, and only a small number of types of perfluorinated / polyfluoroalkyl substances can be separated. However, when the weak anion exchange packing material and the hydrophilic-lipophilic balanced packing material of this application are mixed and column pass through the column, stronger retention efficiency is provided, which can effectively separate 43 PFASs compounds and improve the detection efficiency of batch samples.

[0097] When purifying samples using a solid-phase extraction column, the steps of activation, loading, rinsing, and elution are performed sequentially. During activation, 0.1% ammonia-methanol, methanol, and water are used sequentially to wet and equilibrate the packing material in the column. During loading, the sample solution is passed through the activated column, allowing the target compound to be selectively retained by the stationary phase. During rinsing, 25 mmol / L ammonium acetate is used to remove weakly adsorbed impurities. During elution, methanol and ammoniated methanol are used sequentially to effectively separate and elute perfluorinated / polyfluoroalkyl substances of different polarities.

[0098] In step S4, the concentration to near dryness is defined as visually observing no liquid flow at the bottom of the container.

[0099] Before HPLC-MS / MS injection, the reconstituted methanol solution needs to be filtered to remove tiny insoluble particles, prevent column clogging, and ensure data quality.

[0100] Example 1

[0101] Example 1 provides a method for detecting PFASs compounds in fish. The names and structural information of 45 compounds are shown in Table 1.

[0102] Table 1. Structure and information of 45 PFASs

[0103]

[0104]

[0105]

[0106]

[0107]

[0108] The specific testing method includes the following steps:

[0109] S1, Sample Preparation

[0110] Take as Figure 9 One fish (sample 1) was used. The skin, bones, and internal organs were removed, leaving only the fish meat. The fish meat was cut into small pieces, thoroughly pulverized and mixed using a food processor, and then sealed. The prepared sample was stored at -18°C or below, protected from light, for later use. All tools used during the process required deionization cleaning to prevent cross-contamination between samples.

[0111] S2, Extraction

[0112] Thaw and homogenize the sample prepared in S1. Weigh 2 g of the sample and place it in a 50 mL polypropylene centrifuge tube. Add 50 μL of internal standard mixed solution (0.2 μg / mL), add 3 mL of water, vortex for 1 min, add 6 mL of acetonitrile and 20 μL of concentrated hydrochloric acid (mass fraction 36%–38%), and shake for 5 min. Add 2 g of sodium chloride, shake again for 5 min, and centrifuge at 8000 r / min for 5 min. Transfer the upper acetonitrile solution to another test tube, purge with nitrogen in a 40 °C water bath until approximately 1 mL remains, and add water to bring the volume to 10 mL. This is the purification solution.

[0113] S3, Enrichment and Purification

[0114] A WAX:HLB (150 mg / 6 mL) solid-phase extraction column with a mass ratio of 1:1 was used to enrich and purify the sample. The solid-phase extraction column was activated sequentially with 4 mL of 0.1% ammonia-methanol solution, 4 mL of methanol, and 4 mL of water, keeping the column moist. Immediately after activation, the sample to be purified was loaded onto the solid-phase extraction column. After loading, the solid-phase extraction column was eluted with 4 mL of 25 mmol / L ammonium acetate solution, and the eluent was discarded. The column was then vacuum-sealed for 2 min until nearly dry. Subsequently, the column was eluted sequentially with 2 mL of methanol and 4 mL of 0.1% ammonia-methanol solution at a flow rate of 1 drop every 2-3 s. The eluent was collected and concentrated to nearly dryness under nitrogen at 40 °C. 1 mL of methanol was accurately added to dissolve the eluent. The solution was filtered through a syringe filter with a 0.22 μm pore size membrane, and the filtrate was used as the analyte for UPLC-MS / MS analysis.

[0115] S4. Preparation of mixed standard working solutions and plotting of standard curves

[0116] A certain amount of mixed standard stock solution of perfluorinated / poly-perfluorinated / poly-fluoroalkyl substances and internal standard solution were diluted with methanol to prepare a series of mixed standard working solutions with concentrations of 0.005, 0.01, 0.05, 0.1, 0.5, 1.0, 5.0, 10.0, 50.0, and 100.0 μg / L (each containing 10 μg / L internal standard). The solutions were then determined by UPLC-MS / MS according to the method established in this paper. A standard curve was plotted with the peak area ratio of 45 PFASs and their corresponding isotopic internal standards as the ordinate (y) and the PFAS concentration as the abscissa (x). Quantitative analysis was performed using the internal standard method.

[0117] S5. Perform UPLC-MS / MS analysis on the test solution.

[0118] S5.1 Chromatographic conditions

[0119] The chromatographic column used is Excsep TM The column was SiO2@PFP-C18, the column temperature was 40℃, the injection volume was 2μL, and the flow rate was 0.3mL / min. The mobile phase consisted of 2mM ammonium acetate aqueous solution (A) and methanol (B) for elution. The mobile phase gradient is shown in Table 2.

[0120] Table 2 Mobile phase gradient

[0121]

[0122] S5.2 Mass Spectrometry Conditions

[0123] An electrospray ionization source was selected, in negative ion mode. The ionization parameters are as follows: curtain gas pressure 35.0 psi; spray voltage -4500 V; atomization temperature 500℃; atomizing gas pressure 50 psi; auxiliary gas pressure 50 psi. The specific mass spectra are shown in Table 3.

[0124] Table 3. Mass spectrometry parameters and corresponding internal standards of 45 PFASs

[0125]

[0126]

[0127]

[0128] The linear equations, correlation coefficients, and linear ranges obtained after determining standard solutions of different concentrations using the above chromatographic and mass spectrometric conditions by UPLC-MS / MS are shown in Table 4. Among them, 7 PFASs showed good linearity in the range of 0.1–100 μg / L, PFDoDA showed good linearity in the range of 0.05–100 μg / L, 27 PFASs showed good linearity in the range of 0.01–100 μg / L, and the remaining 10 PFASs showed good linearity in the range of 0.005–100 μg / L. Their correlation coefficients were all greater than or equal to 0.999.

[0129] Table 4. Linear range, regression equation, and correlation coefficient of 45 PFASs

[0130]

[0131]

[0132] Figures 2-8 To dissolve the target compound in methanol, a standard solution with a concentration of 20 ng / mL was prepared, and the chromatogram was obtained by UPLC-MS / MS detection under the above chromatographic conditions.

[0133] Furthermore, the matrix effect (ME), spiked recovery, limit of detection (LOD), and limit of quantitation (LOQ) of the target compound in fish were investigated. Specifically, the standard spiking method was used to assess the potential matrix effect of the fish matrix, comparing the relative response values ​​of the target compound in the sample matrix with those in the pure solvent. For the spiked recovery, two spiked concentrations were set at 2.5 μg / kg and 25 μg / kg, with three parallel samples for each concentration to verify the accuracy of the method. The LOD value was calculated with a signal-to-noise ratio (S / N) of 3, and the LOQ value was calculated with a S / N of 10. The results are shown in Table 5.

[0134] Table 5. Recovery rates, RSD (n=3), ME, LOD, and LOQ of 45 PFASs in fish samples.

[0135]

[0136]

[0137] The data in Table 5 show that PFHxDA, PFODA, FHxSA, FOSA, and N-MeFOSA have strong matrix effects in fish. In actual quantitative testing, matrix calibration curves are needed to calibrate the quantitative results. Except for PFODA and FOSA, the recoveries of the other PFASs are within the acceptable range (60%–140%), with RSDs of 0.6–13.2%, which can meet the basic requirements for quantitative analysis. The LOD is 0.0012–0.0593 μg / kg, and the LOQ is 0.0041–0.1977 μg / kg. However, the recoveries of PFODA and FOSA are low, indicating that this method is not suitable for the quantitative detection of PFODA and FOSA in fish. This method is suitable for the qualitative and quantitative detection of the 43 PFASs with the above-mentioned recoveries.

[0138] Furthermore, the established detection method was applied to the determination of 43 PFASs in 10 fish samples. Photos of each fish sample are shown below. Figure 9 As shown in Table 6, all samples were purchased from a retail market in a city in Zhejiang Province.

[0139] Table 6. Concentrations of PFASs in fish along the Zhejiang coast (μg / kg)

[0140]

[0141]

[0142] Note: ND means not detected, <LOQ means less than the limit of quantitation.

[0143] According to Table 6 and Figures 10-19 The results show that in fish sample 1 ( Figure 10 Nineteen perfluorinated / polyfluoroalkyl substances could be detected. Among them, the contents of PFHpA, PFOA, PFDA, PFUnDA, PFTrDA, PFEtS, PFBS, PFPeS, PFOS, N-MeFOSE, and 6:2FTCA exceeded the limit of quantitation (LOQ). PFPeS eluted at 5–7 min, and the peak at 11–12 min was a miscellaneous peak. In fish sample 2 ( Figure 11 The assay detected 22 perfluorinated / polyfluoroalkyl substances. Among them, PFDA, PFUnDA, PFTrDA, PFTeDA, PFBS, PFPeS, PFHpS, PFOS, FBSA, N-MeFOSE, and 8:2 diPAP exceeded the limit of quantitation (LOQ). PFDA and FBSA both eluted at approximately 10.5 min, with overlapping peaks. In fish sample 3 (… Figure 12), 26 perfluorinated / polyfluoroalkyl substances were detected, among which PFOA, PFNA, PFDA, PFUnDA, PFTrDA, PFTeDA, PFPeS, PFHpS, PFOS, 6:2Cl-PFESA, FBSA, and 8:2diPAP exceeded the limit of quantitation (LOQ); in fish sample 4 ( Figure 13 ), 22 perfluorinated / polyfluoroalkyl substances were detected, among which PFNA, PFDA, PFUnDA, PFDoDA, PFTrDA, PFTeDA, PFPeS, PFOS, 6:2Cl-PFESA, FBSA, HFPO-TA, and 8:2diPAP exceeded the limit of quantitation (LOQ); in fish sample 5 ( Figure 14 Fourteen perfluorinated / polyfluoroalkyl substances were detected, among which PFNA, PFDA, PFUnDA, PFTrDA, PFTeDA, FBSA, and 8:2diPAP exceeded the limit of quantitation (LOQ). In fish sample 6 ( Figure 15 Eighteen perfluorinated / polyfluoroalkyl substances were detected, among which PFOA, PFNA, PFDA, PFUnDA, PFTrDA, PFTeDA, PFHpS, PFOS, FBSA, and 8:2diPAP exceeded the limit of quantitation (LOQ). In fish sample 7... Figure 16 Thirteen perfluorinated / polyfluoroalkyl substances were detected, among which PFNA, PFDA, PFUnDA, PFTrDA, PFTeDA, and 8:2diPAP exceeded the limit of quantitation (LOQ). In fish sample 8... Figure 17 Nineteen perfluorinated / polyfluoroalkyl substances were detected, among which PFNA, PFDA, PFUnDA, PFTrDA, PFTeDA, PFOS, and 8:2diPAP exceeded the limit of quantitation (LOQ). In fish sample 9 (… Figure 18 Fifteen perfluorinated / polyfluoroalkyl substances were detected, among which PFDA, PFTrDA, PFTeDA, FBSA, and 8:2diPAP exceeded the limit of quantitation (LOQ). In fish sample 10 (… Figure 19 The assay detected 15 per- and polyfluoroalkyl substances (PFAS), among which PFNA, PFDA, PFUnDA, PFTrDA, PFTeDA, PFOS, 6:2Cl-PFESA, FBSA, and 8:2diPAP exceeded the limit of quantitation (LOQ). The results from 10 fish samples showed variations in the types and amounts of PFAS detected, likely due to differences in PFAS concentrations in different fishing areas. Therefore, this detection method, by detecting the PFAS content in fish, can also help determine the fluoride pollution level in marine environments.

[0144] In summary, this invention provides a method for trace detection of at least 43 per- and polyfluoroalkyl substances (PFAS) in fish, exhibiting good recovery rates and low limits of detection. It offers a feasible sample processing method for small sample sizes, improving the efficiency of batch detection. The extraction, purification, and enrichment methods in this invention are simple, it detects a wide range of PFAS, is time-efficient, has good recovery rates, and low limits of detection and quantitation. Monitoring the content of PFAS in fish is of great significance for ensuring food safety and public health, assessing ecological and environmental risks, and tracing pollution sources.

[0145] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for detecting perfluoro / polyfluoroalkyl substances in fish, characterized by, The method comprises: S1, sample preparation, comprising: crushing and homogenizing fish meat to be detected to obtain a sample to be detected; S2, extraction, comprising: using water and an organic solvent to extract the sample to be detected, retaining an organic phase, concentrating the organic phase, adding water with a volume of 5-15 times that of the organic phase as a liquid to be purified; S3, purification, comprising: using a solid-phase extraction column to purify the liquid to be purified to obtain a purified liquid; S4, concentration and constant volume, comprising: concentrating the purified liquid to near dryness, redissolving and filtering to obtain a solution to be detected; S5, sample detection, comprising: using a liquid chromatograph-mass spectrometer to detect the solution to be detected, using a standard curve method to qualitatively and quantitatively analyze perfluoro / polyfluoroalkyl substances in fish.

2. The detection method according to claim 1, characterized in that, The perfluoro / polyfluoroalkyl substances include: perfluoroethane sulfonic acid, perfluorobutane sulfonic acid, perfluoropentane sulfonic acid, perfluorohexane sulfonic acid, perfluoroheptane sulfonic acid, perfluorooctane sulfonic acid, perfluorodecane sulfonic acid, perfluoropropionic acid, perfluorobutyric acid, perfluoropentanoic acid, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanoic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluorohexadecanoic acid, perfluorobutyl sulfonamide, perfluorohexane sulfonamide, N-methyl perfluorooctane sulfonamide, N-ethyl perfluorooctane sulfonamide, N-methyl perfluorooctane sulfonamide ethanol, 2(N-ethyl perfluorooctane sulfonamide) ethanol, perfluorooctane sulfonamide acetic acid, 6:2 fluoroterpolymer sulfonic acid, 8:2 fluoroterpolymer sulfonic acid, 10:2 fluoroterpolymer sulfonic acid, 3:3 fluoroterpolymer carboxylic acid, 7:3 fluoroterpolymer carboxylic acid, 6:2 fluoroterpolymer carboxylic acid, 8:2 fluoroterpolymer carboxylic acid, 4.8-dioxa-3H-perfluorodecanoic acid, 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy) propionic acid, perfluoro-2,5-dimethyl-3,6-dioxanonanoic acid, 2,2,3,3-tetrafluoro-3-(trifluoromethoxy) propionic acid, perfluoro-4-methoxy butyric acid, perfluoro-3,6-dioxoheptanoic acid, 1,1,2,2-tetrafluoro-2-(perfluoroethoxy) ethanesulfonic acid, 9-chloro perfluoro-3-nonyloxy sulfonic acid, and 8:2 fluoroterpolymer phosphoric acid diester.

3. The method of claim 1, wherein, In step S2, the amount of water added is 1-3 mL based on 1 g of the sample to be detected; And / or, the volume ratio of water to organic solvent in step S2 is 1:(1-3); And / or, step S2 further comprises adding salt to water and organic solvent after extraction to make the two phases separate; the amount of salt added is 0.5-1.5 g based on 1 mL of water; And / or, step S2 further comprises using a 35%-40% hydrochloric acid aqueous solution during extraction, and the amount of hydrochloric acid aqueous solution is 0.1%-0.5% of the volume of acetonitrile; And / or, step S2 further comprises adding an internal standard to the sample to be detected before extraction; the amount of internal standard added is 2-50 ng based on 1 g of the sample to be detected; the internal standard is any one or more selected from M4PFBA, M5PFHxA, M8PFOA, MPFDoDA, M8PFOS, M2-8:2FTS, M3HFPO-DA.

4. The method of claim 1, wherein In step S3, the purification comprises, in sequence, activation of the solid-phase extraction column, sample loading, elution and elution; and / or, the stationary phase of the solid-phase extraction column in step S3 is a WAX and HLB mixed column, wherein the mass ratio of WAX to HLB is 1:(0.5-1.5); and / or, the mass of the stationary phase of the solid-phase extraction column is 120-170 mg; and / or, the volume of the solid-phase extraction column is 5-10 mL. and / or, the mass of the stationary phase to the volume of the sample to be purified is 6 mg:(5-15) mL.

5. The detection method according to claim 4, characterized in that, The activation is performed in sequence using 0.05%-0.5% volume of ammonia methanol, methanol and water; and / or, the elution liquid is 20-30 mmol / L ammonium acetate; and / or, the elution is performed in sequence using methanol and 0.05%-0.5% volume of ammonia methanol.

6. The detection method according to claim 5, characterized in that, The volume of ammonia methanol used in the activation is 2-10 mL; and / or, the volume of methanol used in the activation is 2-10 mL; and / or, the volume of water used in the activation is 2-10 mL; and / or, the volume of the elution liquid is 2-10 mL; and / or, in step S3, the elution is further followed by vacuum suction of the solid-phase extraction column for 1-3 min; and / or, the volume of methanol used in the elution is 1-5 mL; and / or, the volume of ammonia methanol used in the elution is 2-10 mL; and / or, the elution flow rate is 1-3 s / drop.

7. The method of claim 1, wherein, In step S4, the concentration is performed at 30-50°C using nitrogen blowing; and / or, in step S4, the redissolution is performed using methanol to redissolve the concentrated product; and / or, in step S4, the filtration is performed using a filter membrane with a pore size of 0.1-0.3 μm.

8. The method of claim 1, wherein, In step S5, the chromatographic conditions include: 1) the chromatographic column is a C18 column; 2) the column temperature is 40-45°C; 3) the sample loading amount is 1-3 μL; 4) the flow rate is 0.2-0.5 mL / min; 5) the mobile phase: A phase: 1-5 mM ammonium acetate aqueous solution, B phase: methanol; 6) elution mode: gradient elution, including: initial solvent composition: 75%-80% A phase and 20-25% B phase, gradient change stage: 0-14 min, the proportion of B phase linearly increases to 90-95%, holding stage: 14-16 min, 90-95% B phase is maintained, column cleaning stage: 16 min-16.01 min, the proportion of B phase linearly decreases to 20-25%; column equilibration stage: 16.01 min-20 min, 20-25% B phase is maintained.

9. The method of claim 1, wherein, In step S5, the mass spectrometry conditions include: 1) electrospray ion source, negative ion mode; 2) gas curtain gas pressure 30-40 psi; 3) spray voltage -4200 to -4800 V; 4) atomization temperature 480-520°C; 5) atomization gas pressure 40-55 psi; 6) auxiliary gas pressure 45-55 psi; and / or, in step S5, the concentration of the standard solution selected in the standard curve establishment is 0.005-100 μg / L; And / or, in step S5, the standard curve establishment further comprises adding an internal standard, and the added amount of the internal standard is 5-15 ng.

10. Use of the method according to any one of claims 1-9 for simultaneously detecting 43 perfluoroalkyl and polyfluoroalkyl substances in fish.

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