A method for detecting fluoride compounds in squid

Abstract

The application provides a method for detecting fluorine compounds in squid, comprising the following steps: S1, sample preparation, including: taking muscle tissue of squid to be detected to be crushed and homogenized to obtain a sample to be detected; S2, extraction, including: 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 of the organic phase as a liquid to be purified; S3, purification, including: using a solid-phase extraction column to purify the liquid to be purified to obtain a purified liquid; S4, concentration and constant volume, including: concentrating the purified liquid to near dryness, redissolving to obtain a solution to be detected, filtering the solution to be detected to obtain a filtrate; and S5, sample detection, including: using a liquid chromatograph-mass spectrometer to detect the filtrate, and using a standard curve method to qualitatively and quantitatively analyze the fluorine compounds in the squid. The application can simultaneously detect the content of 45 kinds of PFAS compounds with different physical and chemical properties in squid.

Description

Technical Field

[0001] This invention relates to the field of fluorine compound detection technology, and in particular to a method for detecting fluorine compounds in squid. Background Technology

[0002] Per- and poly-fluoroalkyl substances (PFASs) are typical new pollutants in organic compounds where all or part of the hydrogen atoms are replaced by fluorine atoms to form carbon-fluorine (CF) bonds. The carbon-fluorine (CF) bond is the strongest covalent bond in organic chemistry, with an energy level of 488 kJ / mol, giving PFASs significant stability, making them a class of extremely difficult-to-degrade organic pollutants. However, the hydrophobic fluorinated carbon chains and hydrophilic terminal functional groups of PFASs make them useful surfactants and polymers, suitable for various applications such as waterproof fabrics, aqueous film-forming foams, and wire coverings. Therefore, since the last century, PFASs have been widely used in various industrial and consumer chemical products. During production and use, the discharge of industrial wastewater and domestic sewage into water bodies, as well as the indiscriminate disposal and landfilling of waste, have made water bodies and soil the main environmental media for PFAS exposure.

[0003] With the migration of "permanent chemicals" like PFASs in the environment, they are widely distributed globally. PFASs can be detected in surface water, groundwater, activated sludge, air, soil, seawater, and in all animal, plant, and human serum media. The environmental persistence, bioaccumulation, and long-distance migration of these compounds have made them important targets for contaminant detection in squid. However, the differences in the physicochemical properties of different PFASs (such as ionic and non-ionic forms) limit the broad coverage and sensitivity of traditional methods. The squid matrix is ​​complex, containing large amounts of proteins, fats, and pigments. These substances severely interfere with the extraction of PFASs and compete with or bind to target analytes, leading to low or highly fluctuating extraction recoveries. Because different PFASs structures exhibit significantly different retention behaviors on chromatographic columns, it is difficult to simultaneously detect multiple fluorinated compounds in a single analysis. Co-elution can cause mutual interference, affecting quantitative accuracy and resulting in low efficiency for batch detection of squid. In addition, existing technologies mostly use LC-MS for detection, but its sensitivity for detecting certain low-content fluorine-containing compounds, such as perfluorocarboxylic acids, may still be insufficient, making it difficult to meet the needs for detecting trace amounts of fluorine-containing compounds. 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 fluorine compounds in squid, which solves the problems of low detection efficiency, low sensitivity and difficulty in accurate quantification of fluorine compounds in squid in the prior art.

[0005] To achieve the above and other related objectives, the present invention provides a method for detecting fluorine compounds in squid.

[0006] The first aspect of this invention provides a method for detecting fluoride compounds in squid, the method comprising:

[0007] S1. Sample preparation, including: taking the muscle tissue of the squid to be tested, pulverizing and homogenizing it 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 it to obtain the solution to be tested, and filtering the solution to be tested to obtain the filtrate;

[0011] S5. Sample testing, including: using liquid chromatography-mass spectrometry to test the filtrate, and using the standard curve method to perform qualitative and quantitative analysis of fluorine compounds in squid.

[0012] Preferably, the fluorinated compound includes any one or more of perfluoroalkyl carboxylic acids, perfluoroalkyl sulfonic acids, perfluoroalkyl sulfonamides, fluoropolymer sulfonic acids, fluoropolymer alkyl carboxylic acids, perfluoroalkyl ether carboxylic acids, polyfluoroalkyl ether sulfonic acids, and disubstituted polyfluorophosphates.

[0013] More preferably, the perfluoroalkyl carboxylic acid includes any one or more of the following: perfluoropropionic acid, perfluorobutyric acid, perfluorovaleric acid, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluorohexadecanoic acid, and perfluorooctadecanic acid.

[0014] More preferably, the perfluoroalkyl sulfonic acid includes any one or more of the following: perfluoroethane sulfonic acid, perfluorobutane sulfonic acid, perfluoropentane sulfonic acid, perfluorohexane sulfonic acid, perfluoroheptane sulfonic acid, perfluorooctane sulfonic acid, and perfluorodecane sulfonic acid.

[0015] More preferably, the perfluoroalkyl sulfonamide includes any one or more of the following: perfluorobutyl sulfonamide, perfluorohexane sulfonamide, perfluorooctane sulfonamide, N-methylperfluorooctane sulfonamide, N-ethylperfluorooctane sulfonamide, N-methylperfluorooctane sulfonamide ethanol, 2(N-ethylperfluorooctane sulfonamide) ethanol, and perfluorooctane sulfonamide acetic acid;

[0016] More preferably, the fluoropolymer sulfonic acid comprises any one or more of the following: 6:2 fluoropolymer sulfonic acid, 8:2 fluoropolymer sulfonic acid, and 10:2 fluoropolymer sulfonic acid;

[0017] More preferably, the fluoropolymer alkyl carboxylic acid comprises any one or more of the following: 3:3 fluoropolymer carboxylic acid, 5:3 fluoropolymer carboxylic acid, 7:3 fluoropolymer carboxylic acid, 6:2 fluoropolymer carboxylic acid, and 8:2 fluoropolymer carboxylic acid;

[0018] More preferably, the perfluoroalkyl ether carboxylic acid comprises any one or more of the following: 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, and perfluoro-3,6-dioxanone acid;

[0019] More preferably, the polyfluoroalkyl ether sulfonic acid includes any one or two of 1,1,2,2-tetrafluoro-2-(perfluoroethoxy)ethanesulfonic acid and 9-chloroperfluoro-3-nonoxysulfonic acid;

[0020] More preferably, the disubstituted polyfluorophosphate comprises an 8:2 fluoropolymer phosphate diester.

[0021] More preferably, the fluorinated compounds 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, perfluorooctadecanic acid, perfluoroethanesulfonic acid, perfluorobutanesulfonic acid, perfluoropentanesulfonic acid, perfluorohexanesulfonic acid, perfluoroheptanesulfonic acid, perfluorooctanesulfonic acid, perfluorodecanesulfonic acid, perfluorobutylsulfonamide, perfluorohexanesulfonamide, perfluorooctanesulfonamide, N-methylperfluorooctanesulfonamide, N-ethylperfluorooctanesulfonamide, N-methylperfluorooctanesulfonamide ethanol, 2(N-ethylperfluorooctanesulfonamido)ethanol, perfluorooctanesulfonamide acetic acid, 6:2 fluoropolymer sulfonic acid, 8:2 fluoropolymer sulfonic acid, 10:2 fluoropolymer sulfonic acid, 3:3 Fluorinated telomer carboxylic acids, 7:3 fluorotelomer carboxylic acids, 6:2 fluorotelomer carboxylic acids, 8:2 fluorotelomer carboxylic acids, 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 fluorotelomer phosphate diester.

[0022] Preferably, in step S1, a crusher is used to crush and homogenize the squid.

[0023] Preferably, in step S2, the amount of water added is 1~5mL, based on 1g of the sample to be tested.

[0024] More preferably, in step S2, based on 1g of the sample to be tested, the amount of water added is 1~3mL; including but not limited to 1mL, 1.5mL, 2mL, 2.5mL or 3mL.

[0025] 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 0.5~1.5g based on 1mL of water; for example, it can be 0.5g, 0.6g, 0.8g, 1.0g, 1.2g, 1.4g or 1.5g.

[0026] More preferably, the salt is sodium chloride.

[0027] Preferably, the volume ratio of water to organic solvent in step S2 is 1:(1~5).

[0028] More 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.

[0029] 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 the organic solvent.

[0030] Preferably, the organic solvent is acetonitrile.

[0031] More preferably, the hydrochloric acid aqueous solution has a mass fraction of 36% to 38%.

[0032] More preferably, in step S2, before adding salt, the solution containing the sample, internal standard, and water is vortexed for 1-5 minutes.

[0033] More preferably, step S2 further includes adding an organic solvent after vortexing and shaking for 5-10 minutes.

[0034] More preferably, step S2 further includes adding salt after shaking and shaking for 5-10 minutes.

[0035] 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.

[0036] 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 2~20ng; for example, it can be 2ng, 5ng, 10ng, 15ng or 20ng.

[0037] 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).

[0038] 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.

[0039] Preferably, 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:(0.5~2); for example, it can be 1:0.5, 1:1, 1:1.5 or 1:2.

[0040] 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.

[0041] 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.

[0042] Preferably, the mass of the stationary phase in the solid phase extraction column is 100~200 mg.

[0043] More 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.

[0044] Preferably, the volume of the solid-phase extraction column is 5-10 mL; for example, it can be 5 mL, 6 mL, 7 mL, 8 mL, 9 mL or 10 mL.

[0045] More preferably, the volume of the solid-phase extraction column is 5-7 mL.

[0046] 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.

[0047] Preferably, in step S3, the purification process sequentially includes solid-phase extraction column activation, sample loading, rinsing, and elution.

[0048] More preferably, the activation is performed sequentially using 0.05%~0.5% by volume of ammonia-methanol, methanol, and water.

[0049] The percentage refers to the volume percentage of ammonia in the ammonia-methanol solution.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] More preferably, the rinsing solution is ammonium acetate with a concentration of 10-50 mmol / L.

[0054] More preferably, the rinsing solution 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.

[0055] 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.

[0056] More preferably, in step S3, the rinsing process further includes vacuuming the solid-phase extraction column for 1-3 minutes followed by elution.

[0057] More preferably, the elution is performed sequentially using methanol and 0.05%~0.5% by volume of ammonia-methanol solution.

[0058] 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.

[0059] 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.

[0060] More preferably, the elution flow rate is 1~3 s / drop.

[0061] Preferably, in step S4, the concentration is carried out at 30~50°C using nitrogen blowing concentration.

[0062] Preferably, in step S4, the resolution is performed by resolution of the concentrate with methanol.

[0063] Preferably, in step S4, the filtration is performed using a filter membrane with a pore size of 0.1~0.3μm.

[0064] More preferably, the pore size of the filter membrane can be 0.1 μm, 0.2 μm or 0.3 μm.

[0065] Preferably, in step S5, the chromatographic conditions include:

[0066] 1) The chromatographic column is a C18 column;

[0067] 2) Column temperature is 35~45℃;

[0068] 3) The injection volume is 1~5 μL;

[0069] 4) The flow rate is 0.1~0.5 mL / min;

[0070] 5) Mobile phase: Phase A: 1~3mM ammonium acetate aqueous solution; Phase B: methanol;

[0071] 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.

[0072] Preferably, in step S5, the conditions for the mass spectrometry include:

[0073] 1) Electrospray ion source, negative ion mode;

[0074] 2) Air curtain pressure 30~40 psi;

[0075] 3) Spray voltage -4000~-5000 V;

[0076] 4) Atomization temperature: 450~550℃;

[0077] 5) Atomizing pressure: 45~55 psi;

[0078] 6) Auxiliary gas pressure 45~55 psi.

[0079] Preferably, in step S5, the concentration of the standard solution selected for establishing the standard curve in the standard curve method is 0.005~100μg / L.

[0080] Preferably, in step S5, the establishment of the standard curve in the standard curve method further includes adding an internal standard, and the amount of the internal standard added is 5~15ng.

[0081] A second aspect of the present invention provides the use of the above method in the simultaneous detection of 45 fluorine compounds in squid.

[0082] As described above, the method for detecting fluoride compounds in squid according to the present invention has the following beneficial effects:

[0083] This invention employs a combination of liquid-liquid extraction and solid-phase extraction to effectively purify, refine, and enrich PFASs in squid. Furthermore, UPLC-MS / MS is used for quantitative detection of the purified and enriched substances. Using this detection method, the recoveries of 45 PFASs in squid matrix range from 61% to 139%, with RSDs ranging from 0.4% to 13.8%. The limits of detection (LOD) in squid matrix are 0.0006–0.0572 μg / kg, and the limits of quantitation (LOQ) are 0.0019–0.1908 μg / kg. The correlation coefficient of the standard curve is above 0.999. This invention can simultaneously determine the content of at least 45 PFASs compounds with different physicochemical properties in squid, 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

[0084] Figure 1 The diagram shows a schematic of the detection process for detecting fluorine compounds in squid according to the present invention.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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: 6:2 Cl-PFESA; 3: FOSAA; 4: 8:2 diPAP.

[0089] 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: 1:6:2 FTSA; 2:8:2 FTSA; 3:10:2 FTSA.

[0090] Figure 7 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: 1:3:3 FTCA, 2:6:2 FTCA, 3:7:3 FTCA, and 4:8:2 FTCA, respectively.

[0091] 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.

[0092] Figure 9The image shown is a photograph of the squid sampled in Embodiment 1 of this invention.

[0093] Figure 10 The image shown is a chromatogram obtained by detecting fluorine compounds in squid sample 1 using the detection method of Example 1 of this invention.

[0094] Figure 11 The image shown is a chromatogram obtained by detecting fluorine compounds in squid sample 2 using the detection method of Example 1 of this invention. Detailed Implementation

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] The instrument and equipment information used in the embodiments of this invention is as follows:

[0101] Sciex Qtrap 4500 HPLC-MS / MS (AB SCIEX, USA), AL104 electronic analytical balance (Mettler Toledo Technologies), Synerg® UV ultrapure water system (Milipore, USA), CT14RD benchtop high-speed refrigerated centrifuge (Shanghai Tianmei Scientific Instruments Co., Ltd., China), IKA® VIRTEX 3 vortex mixer (IKA GmbH, Germany), HX-12D solid-phase extraction apparatus (Wuhan Hengxin Century Technology, China), KQ-700VDV dual-frequency digitally controlled ultrasonic cleaner (Kunshan Ultrasonic Instruments Co., Ltd., China), and Model 2695 high-performance liquid chromatograph (UV detector) (Waters Corporation, USA).

[0102] The reagents and standards used in the embodiments of this invention are as follows:

[0103] 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, perfluorooctadecanoic acid, perfluoroethanesulfonic acid, perfluoropentanesulfonic acid, perfluoroheptanesulfonic acid, perfluorodecanesulfonic acid, 1,1,2,2-tetrafluoro-2-(perfluoroethoxy)ethanesulfonic acid, 9-chloroperfluoro-3-nonoxysulfonic acid, perfluorobutylsulfonamide, 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, 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, perfluorovalerate, perfluorohexanoic acid, perfluoropropionic acid, perfluoroheptanoic acid, perfluorohexane sulfonic acid, N-methylperfluorooctane sulfonamide, 2-(N-ethylperfluorooctane sulfonamido)ethanol, trifluoroacetic acid, perfluorohexane sulfonamide, 8:2 Fluoropolymer phosphate diester, perfluorobutyric acid, N-ethylperfluorooctane sulfonamide, N-methylperfluorooctane sulfonamide ethanol (purity >80%, Anpu Cloud Laboratory Supplies Co., Ltd.); perfluorooctane sulfonamide, trifluoromethanesulfonic acid (purity >90%, Manhag Biotechnology Co., Ltd.). WAX packing material (Ontwsep™ WAX, Wendu Chromatography Technology Co., Ltd.); HLB packing material (Ontwsep™ HLB, Wendu Chromatography Technology Co., Ltd.); WAX and HLB mixed solid phase extraction column (150mg / 6mL, Wendu Chromatography Technology Co., Ltd.); Column: Excsep™ SiO2@PFP-C18 column (2.1×100 mm, 1.8 μm, Wendu Chromatography Technology Co., Ltd.).

[0104] In step S1, the squid muscle tissue in this application is obtained after removing the squid bones, squid beak, and ink sac. The muscle is the main edible part, and the data from testing the muscle tissue can be directly used to assess food safety.

[0105] In step S2, adding acid to the extract can purify fluorine compounds 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.

[0106] After extraction with water and organic solvents, salt is added to achieve phase separation and facilitate phase separation.

[0107] 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 the fluorine compounds to be effectively removed from the solution and retained in the packing material during S3 purification, without being washed down with the organic solvent.

[0108] In step S3, the solid-phase extraction column used in this application has a specification of (150 mg / 6 mL), which 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 fluorine compounds to be retained in the column, and only a small number of types of fluorine compounds 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 45 kinds of PFASs compounds and improve the detection efficiency of batch samples.

[0109] 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 fluorine compounds of different polarities.

[0110] In step S4, the concentration to near dryness is defined as visually observing no liquid flow at the bottom of the container.

[0111] 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.

[0112] Example 1

[0113] This embodiment 1 provides a method for detecting 45 PFASs compounds in squid. The names and CAS numbers of the 45 compounds are shown below.

[0114] The 45 PFASs compounds include perfluoroalkyl carboxylic acids (PFCAs), perfluoroalkyl sulfonic acids (PFSAs), perfluoroalkyl sulfonamides (FASAs), fluoropolymer sulfonic acids (FTSAs), fluoropolymer alkyl carboxylic acids (FTCAs), perfluoroalkyl ether carboxylic acids (PFECAs), polyfluoroalkyl ether sulfonic acids (PFESAs), and disubstituted polyfluorophosphates (diPAPs). The names and CAS numbers of the 45 compounds are as follows: Perfluoroalkyl carboxylic acids (PFCAs) include: perfluoropropionic acid (PFPrA) CAS number 422-64-0; perfluorobutyric acid (PFBA) CAS number 375-22-4; perfluorovalerate (PFPeA) CAS number 2706-90-3; perfluorohexanoic acid (PFHxA) CAS number 307-24-4307-24-4; perfluoroheptanoic acid (PFHpA) CAS number 375-85-9; perfluorooctanoic acid (PFOA) CAS number 335-67-1; perfluorononanoic acid (PFNA) CAS number 375-95-1; perfluorodecanoic acid (PFDA) CAS number 335-76-2; perfluoroundecanoic acid (PFUnDA) CAS number 2058-94-8; perfluorododecanic acid (PFDoDACA) CAS number 307-55-1; perfluorotridecanoic acid (PFTrDA) The CAS number is 72629-94-8; the CAS number for perfluorotetradecanoic acid (PFTeDA) is 376-06-7; the CAS number for perfluorohexadecanoic acid (PFHxDA) is 67905-19-5; and the CAS number for perfluorooctadecanoic acid (PFODA) is 16517-11-6. Perfluoroalkyl sulfonic acids (PFSAs) include: perfluoroethane sulfonic acid (PFFEtS) CAS number 354-88-1; perfluorobutane sulfonic acid (PFBS) CAS number 375-73-5; perfluoropentane sulfonic acid (PFPeS) CAS number 2706-91-4; perfluorohexane sulfonic acid (PFHxS) CAS number 355-46-4; perfluoroheptane sulfonic acid (PFHpS) CAS number 375-92-8; perfluorooctane sulfonic acid (PFOS) CAS number 1763-23-1; and perfluorodecane sulfonic acid (PFDS) CAS number 335-77-3. Perfluoroalkyl sulfonamides (FASAs) include: perfluorobutyl sulfonamide (FBSA) CAS number 30334-69-1; perfluorohexane sulfonamide (FHxSA) CAS number 41997-13-1; perfluorooctane sulfonamide (FOSA) CAS number 754-91-6; N-methylperfluorooctane sulfonamide (N-MeFOSA) CAS number 31506-32-8; N-ethylperfluorooctane sulfonamide (N-EtFOSA) CAS number 4151-50-2; 2(N-ethylperfluorooctane sulfonamide) ethanol (N-EtFOSE) CAS number 1691-99-2; N-methylperfluorooctane sulfonamide ethanol (N-MeFOSE) CAS number 24448-09-7; and perfluorooctane sulfonamide acetic acid (FOSAA) CAS number 2806-24-8.Fluorinated sulfonic acid (FTSA) compounds include: 6:2 FTSA (CAS No. 27619-97-2); 8:2 FTSA (CAS No. 39108-34-4); and 10:2 FTSA (CAS No. 120226-60-0). Fluorinated alkyl carboxylic acids (FTCA) compounds include: 3:3 FTCA (CAS No. 356-02-5); 7:3 FTCA (CAS No. 812-70-4); 6:2 FTCA (CAS No. 53826-12-3); and 8:2 FTCA (CAS No. 27854-31-5). Perfluoroalkyl ether carboxylic acids (PFECAs) include: 4,8-dioxa-3H-perfluorononanoic acid (ADONA) CAS No. 919005-14-4; 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propionic acid (HFPO-DA) CAS No. 13252-13-6; perfluoro-2,5-dimethyl-3,6-dioxanonanoic acid (HFPO-DA) CAS No. 13252-14-7; 2,2,3,3-tetrafluoro-3-(trifluoromethoxy)propionic acid (PFMPA) CAS No. 377-73-1; perfluoro-4-methoxybutyric acid (PF5OHxA) CAS No. 863090-89-5; and perfluoro-3,6-dioxoheptanoic acid (3-6-OPFHpA) CAS No. 151772-58-6. Polyfluoroalkyl ether sulfonic acids (PFESAs) include: 1,1,2,2-tetrafluoro-2-(perfluoroethoxy)ethanesulfonic acid (PFEESA), CAS number 113507-82-7; and 9-chloroperfluoro-3-nonoxysulfonic acid (6:2 Cl-PFESA), CAS number 756426-58-1. Disubstituted polyfluorophosphates (diPAPs) include 8:2 fluoropolymerized phosphate diesters (8:2 diPAP), CAS number 678-41-1.

[0115] The specific testing method includes the following steps:

[0116] S1, Sample Preparation

[0117] Take as Figure 9 One squid of the indicated type was used. The squid bones, beak, and ink sac were removed, and the muscle tissue was collected. The sample was cut into small pieces and thoroughly pulverized and mixed using a grinder. The mixture was then sealed and stored at -18°C or below, protected from light, for later use. All tools used during the processing must be cleaned with deionized water to prevent cross-contamination between samples.

[0118] S2, Extraction

[0119] 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℃ water bath until approximately 1 mL remains, and add water to bring the volume to 10 mL. This is the purification solution.

[0120] S3, Enrichment and Purification

[0121] 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 test solution for UPLC-MS / MS analysis.

[0122] S4. Preparation of mixed standard working solutions and plotting of standard curves

[0123] A certain amount of perfluorinated / polyfluorinated compound mixed standard stock solution 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.

[0124] S5. Perform UPLC-MS / MS analysis on the test solution.

[0125] S5.1 Chromatographic conditions

[0126] The chromatographic column used is Excsep TMThe column was SiO2@PFP-C18, the column temperature was 40℃, the injection volume was 2 μL, and the flow rate was 0.3 mL / min. The mobile phase was 2 mM ammonium acetate aqueous solution (A) and methanol (B) for elution. The mobile phase gradient is shown in Table 1.

[0127] Table 1. Mobile phase gradient

[0128]

[0129] S5.2 Mass Spectrometry Conditions

[0130] An electrospray ionization source was selected, in negative ion mode. Ionization parameters were 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. Specific mass spectra are shown in Table 2.

[0131] Table 2. Mass spectrometry parameters of 45 PFASs and their corresponding internal standards

[0132]

[0133]

[0134] 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 3. 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.

[0135] Table 3. Linear range, regression equation, and correlation coefficient of 45 PFASs

[0136]

[0137]

[0138] Figures 2-8 To dissolve the target compound in methanol, a standard solution with a concentration of 20 ng / mL was prepared, and the chromatograms of each standard were detected by UPLC-MS / MS under the above chromatographic conditions.

[0139] Furthermore, the matrix effect (ME), spiked recovery, limit of detection (LOD), and limit of quantitation (LOQ) of the target compound in different squid samples were investigated. Specifically, the standard spiking method was used to assess the potential matrix effect of the squid 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: 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 4.

[0140] Table 4. Recovery rates, RSD (n=3), ME, LOD and LOQ of 45 PFASs in squid samples

[0141]

[0142]

[0143]

[0144] The data in Table 4 show that PFTeDA, PFHxDA, PFODA, FHxSA, FOSA, and N-MeFOSA have a strong matrix effect in squid. In actual quantitative testing, matrix calibration curves are needed to calibrate the quantitative results. When the spiked concentration is 2.5 μg / kg or 25 μg / kg, the recoveries of all PFASs are within the acceptable range (60%~140%), with RSD of 0.4~11.0%, which can meet the basic requirements of quantitative analysis. The LOD is 0.0006~0.0572 μg / kg, and the LOQ is 0.0019~0.1908 μg / kg.

[0145] Furthermore, the established detection method was applied to the determination of two squid samples, both purchased from a retail market in a city in Zhejiang Province. The quantitative results are shown in Table 5, and the detection chromatograms are shown below. Figures 10-11 As shown.

[0146] Table 5. Concentrations of PFASs (μg / kg) in squid samples 1 and 2 from the Zhejiang coast.

[0147]

[0148]

[0149] Note: ND means not detected, <LOQ means less than the limit of quantitation.

[0150] According to Table 5 and Figures 10-11The results show that: in squid sample 1, 18 fluorinated compounds were detected, among which the contents of PFPrA, PFOA, PFNA, PFDA, PFUnDA, PFTrDA, PFOS and 8:2 diPAP exceeded the limit of quantification; in squid sample 2, 19 fluorinated compounds were detected, among which the contents of PFBA, PFOA, PFDA, PFUnDA, PFTeDA, 10:2FTSA and 8:2 diPAP exceeded the limit of quantification. PFBA showed a peak between 1 and 2 minutes, and the peak after 5 minutes was an impurity peak.

[0151] In summary, the detection method of this invention is suitable for trace detection of perfluorinated / polyfluorinated compounds (PFAS) in squid, providing a feasible sample processing method for small sample sizes. The extraction, purification, and enrichment methods of this invention are simple, detect a wide range of fluorinated compounds, are time-efficient, have good recovery rates, and low limits of detection and quantitation. Monitoring the PFAS content in squid is of great significance for ensuring food safety and public health, assessing ecological and environmental risks, and tracing pollution sources.

[0152] 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 fluorine compounds in squid, characterized by, The method comprises: S1, sample preparation, comprising: taking the muscle tissue of the squid to be detected for crushing and homogenizing to obtain a sample to be detected; S2, extraction, comprising: using water and acetonitrile to extract the sample to be detected, retaining the 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, and obtaining a detection solution after redissolving, and filtering the detection solution to obtain a filtrate; S5, sample detection, comprising: using a liquid chromatograph-mass spectrometer to detect the filtrate, using a standard curve method to qualitatively and quantitatively analyze fluorine compounds in the squid; The fluorine compounds include: 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, perfluorooctadecanoic acid, perfluoroethanesulfonic acid, perfluorobutanesulfonic acid, perfluoropentanesulfonic acid, perfluorohexanesulfonic acid, perfluoroheptanesulfonic acid, perfluorooctanesulfonic acid, perfluorodecanesulfonic acid, perfluorobutyl sulfonamide, perfluorohexanesulfonamide, perfluorooctanesulfonamide, N-methyl perfluorooctanesulfonamide, N-ethyl perfluorooctanesulfonamide, N-methyl perfluorooctanesulfonamide ethanol, 2(N-ethyl perfluorooctanesulfonamido) ethanol, perfluorooctanesulfonamide 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-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-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 phosphate diester; The step S2 further comprises adding salt to the water and acetonitrile after extraction to make the two phases separate; and the step S2 further adopts a 30%-40% hydrochloric acid aqueous solution, and the amount of the hydrochloric acid aqueous solution is 0.1%-0.5% of the volume of acetonitrile; In step S3, the purification comprises activation, sample loading, elution and elution of the solid phase extraction column in sequence; the stationary phase of the solid phase extraction column in step S3 is a WAX and HLB mixture, wherein the mass ratio of WAX to HLB is 1:(0.5-2); the activation is performed by using 0.05%-0.5% ammonia methanol, methanol and water in sequence; the elution liquid of the elution is ammonium acetate with a concentration of 10-50 mmol / L; and the elution is performed by using methanol and 0.05%-0.5% ammonia methanol in sequence.

2. The detection method according to claim 1, characterized in that, In step S2, the amount of water added is 1-5 mL based on 1 g of the sample to be detected; And / or, in step S2, the amount of salt added is 0.5-1.5 g based on 1 mL of water; And / or, in step S2, the volume ratio of water to acetonitrile is 1:(1-5); And / or, in step S2, an internal standard is added to the sample to be detected before extraction, and the amount of the internal standard added is 2-50 ng based on 1 g of the sample to be detected, and the internal standard is any one or more selected from M4PFBA, M5PFHxA, M8PFOA, MPFDoDA, M8PFOS, M2-8:2FTS, and M3HFPO-DA.

3. The method of claim 1, wherein The mass of the stationary phase of the solid-phase extraction column is 100-200 mg; And / or, the volume of the solid-phase extraction column is 5-10 mL; And / or, the volume ratio of the mass of the stationary phase to the sample to be purified is 6 mg:(5-15) mL.

4. The method of claim 1, wherein, In step S4, the concentration is performed at 30-50°C by nitrogen blowing; And / or, in step S4, the redissolving is performed by redissolving the concentrate with methanol; And / or, in step S4, the filtration is performed by using a filter membrane with a pore size of 0.1-0.3 μm.

5. The method of claim 1, wherein In step S5, the conditions of the chromatography include: 1) The chromatographic column is a C18 column; 2) The column temperature is 35-45°C; 3) The injection volume is 1-5 μL; 4) The flow rate is 0.1-0.5 mL / min; 5) The mobile phase: A phase: 1-3 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 is linearly increased 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 is linearly decreased to 20-25%; column equilibration stage: 16.01 min-20 min, 20-25% B phase is maintained.

6. The method of claim 1, wherein In step S5, the conditions of the mass spectrometry include: 1) Electrospray ion source, negative ion mode; 2) Gas curtain gas pressure 30-40 psi; 3) Spray voltage -4000 to -5000 V; 4) Atomization temperature 450-550°C; 5) Atomization gas pressure 45-55 psi; 6) Auxiliary gas pressure 45-55 psi; And / or, in step S5, the concentration of the standard solution selected for establishing the standard curve in the standard curve method is 0.005-100 μg / L; And / or, in step S5, the standard curve method for establishing the standard curve further includes adding an internal standard, and the amount of the internal standard added is 5-15 ng.

7. Use of a method according to any one of claims 1 to 6, characterized in that The method is used for simultaneously detecting 45 fluorinated compounds in squid.

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