Method for detecting fluorine compounds in crabs

Abstract

The invention provides a method for detecting fluorine compounds in crabs, which comprises the following steps: S1, sample preparation: removing shells of to-be-detected crabs, and crushing and homogenizing edible parts of the to-be-detected crabs to obtain a to-be-detected sample; s2, extraction: extracting a sample to be detected, retaining an organic phase, and concentrating to obtain a concentrated solution; then adding water which is 5-15 times of the concentrated solution for dilution to obtain a to-be-purified solution; the extraction solvent is an aqueous solution of an organic solvent; 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 filtrate; and S5, sample detection: detecting the filtrate by using a liquid chromatograph-mass spectrometer, and carrying out qualitative and quantitative analysis on the fluorine compounds in the crabs by adopting a standard curve method. The method can be used for simultaneously determining the contents of 41 PFASs compounds with different physicochemical properties in crabs.

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 crabs. 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−1, 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. As PFASs, these "permanent chemicals," migrate through the environment, they are widely distributed globally. PFASs can be detected in surface water, groundwater, activated sludge, air, soil, seawater, and in the serum of all animals, plants, and humans.

[0003] Crabs are a popular and common food due to their high nutritional value and sweet taste. However, their growing environment makes them highly susceptible to the influence of permanent fluorinated compounds (PFASs). The environmental persistence, bioaccumulation, and long-distance migration of these compounds have made them important targets for contaminant detection in crabs. However, the differences in the physicochemical properties of different PFASs (e.g., ionic and non-ionic) limit the breadth and sensitivity of traditional methods. Crab matrices are complex, containing large amounts of proteins, fats, and pigments. These substances severely interfere with PFAS extraction and compete with or bind to target compounds, leading to low or highly fluctuating extraction recoveries. Because different PFAS structures exhibit significantly different retention behaviors on chromatographic columns, simultaneous detection of multiple fluorinated compounds in a single analysis is difficult. Co-elution can cause interference, affecting quantitative accuracy and resulting in low efficiency for batch detection of crabs. Furthermore, current techniques often employ LC-MS for detection, but its sensitivity for low-level fluorinated compounds, such as perfluorocarboxylic acids, may still be insufficient, failing to meet the needs for trace fluorinated compound detection. 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 crabs, which solves the problems of low detection efficiency, low sensitivity and difficulty in accurate quantification of fluorine compounds in crabs in the prior art.

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

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

[0007] S1. Sample preparation, including: removing the shell of the crab to be tested, crushing and homogenizing its edible parts to obtain the sample to be tested;

[0008] S2. Extraction, including: extracting the sample to be tested, retaining the organic phase and concentrating it to obtain a concentrated solution; then diluting the concentrated solution with 5 to 15 times the amount of water to obtain a purified solution; the solvent for extraction is an aqueous solution of an organic solvent;

[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, reconstituted and then filtered to obtain 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 crabs.

[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, and perfluorohexadecanoic 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, and perfluorooctane sulfonic acid.

[0015] More preferably, the perfluoroalkyl sulfonamide comprises any one or more of the following: perfluorobutyl sulfonamide, perfluorohexane sulfonamide, N-methyl-N-methylperfluorooctane sulfonamide ethanol, N-ethylperfluorooctane sulfonamide, 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 includes any one or more of the following: 3:3 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: 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.

[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 compound includes: 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, perfluorobutylsulfonamide, perfluorohexanesulfonamide, N-methylperfluorooctanesulfonamide, N-methylperfluorooctanesulfonamide ethanol, N-ethylperfluorooctanesulfonamide, 2(N-ethylperfluorooctanesulfonamido)ethanol, 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 Fluorinated telomer carboxylic acids, 8:2 fluorotelomer carboxylic acids, 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 diesters.

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

[0023] Preferably, in step S2, the amount of water added is 1~10mL, 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 0.5~2.1g based on 1mL of water; for example, it can be 0.5g~0.6g, 0.6g~0.8g, 0.8g~1.0g, 1.0g~1.2g, 1.2g~1.4g, 1.4g~1.6g, 1.6g~1.8g, or 1.8g~2g.

[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, the organic solvent in step S2 is acetonitrile, and a 30% to 40% hydrochloric acid aqueous solution is also used during extraction, with the amount of hydrochloric acid aqueous solution being 0.1% to 0.5% of the volume of acetonitrile.

[0030] More preferably, the hydrochloric acid aqueous solution has a mass fraction of 36% to 38%.

[0031] More preferably, in step S2, before adding salt, the solution containing the sample, internal standard, and water is vortexed for 1-5 minutes.

[0032] More preferably, step S2 further includes adding an organic solvent after vortexing and shaking for 5-10 minutes.

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

[0034] 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.

[0035] 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.

[0036] 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).

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] Preferably, the mass of the stationary phase in the solid phase extraction column is 100~200 mg.

[0042] 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.

[0043] 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.

[0044] More preferably, the volume of the solid-phase extraction column is 5-7 mL.

[0045] 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.

[0046] Preferably, in step S3, the purification process sequentially includes solid-phase extraction column activation, sample loading, rinsing, and elution.

[0047] More preferably, the activation is performed sequentially using 0.05%~0.5% by volume of ammonia-methanol, methanol, and water.

[0048] The percentage refers to the volume percentage of ammonia in the ammonia-methanol solution.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] More preferably, the eluent is ammonium acetate with a concentration of 10-50 mmol / L.

[0053] More 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.

[0054] More preferably, the volume of the eluent is 2-10 mL; for example, it can be 2 mL, 4 mL, 5 mL, 6 mL, 8 mL, or 10 mL. More preferably, in step S3, after elution, the solid-phase extraction column is subjected to vacuum extraction for 1-3 minutes, followed by elution.

[0055] More preferably, the elution is performed sequentially using methanol and 0.05%~0.5% by volume of ammonia-methanol solution.

[0056] 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.

[0057] 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.

[0058] More preferably, the elution flow rate is 1~3 s / drop.

[0059] Preferably, in step S4, the concentration is carried out at 30~50°C using nitrogen blowing concentration.

[0060] Preferably, in step S4, the resolution is performed by resolution of the concentrate with methanol.

[0061] Preferably, in step S4, the filtration is performed using a filter membrane with a pore size of 0.1~0.3μm.

[0062] More preferably, the pore size of the filter membrane can be 0.1 μm, 0.2 μm or 0.3 μm.

[0063] Preferably, in step S5, the chromatographic conditions include:

[0064] 1) The chromatographic column is a C18 column;

[0065] 2) Column temperature is 35~45℃;

[0066] 3) The injection volume is 1~5 μL;

[0067] 4) The flow rate is 0.1~0.5 mL / min;

[0068] 5) Mobile phase: Phase A: 1~3mM ammonium acetate aqueous solution; Phase B: methanol;

[0069] 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.

[0070] Preferably, in step S5, the mass spectrometry conditions include:

[0071] 1) Electrospray ion source, negative ion mode;

[0072] 2) Air curtain pressure 30~40 psi;

[0073] 3) Spray voltage -4000~-5000 V;

[0074] 4) Atomization temperature: 450~550℃;

[0075] 5) Atomizing pressure: 45~55 psi;

[0076] 6) Auxiliary gas pressure 45~55 psi.

[0077] Preferably, in step S5, the concentration of the standard solution selected in the establishment of the standard curve is 0.005~100μg / L.

[0078] 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 5~15ng.

[0079] A second aspect of the present invention provides the use of the above method in the simultaneous detection of 41 fluorine compounds in crabs.

[0080] It should be noted that the acceptable recovery rate in this application is 60-140%. Recovery rates outside this range cannot accurately reflect the actual content of the test target. Therefore, substances with recovery rates exceeding 60-140% cannot be quantitatively detected.

[0081] As described above, the method for detecting fluoride compounds in crabs according to the present invention has the following beneficial effects:

[0082] This invention employs a combination of liquid-liquid extraction and solid-phase extraction to effectively purify, refine, and enrich PFASs in crab matrices. Further UPLC-MS / MS is used for quantitative detection of the purified and enriched substances. Using this method, the recoveries of 41 PFASs in crab matrices range from 60% to 140%, with RSDs ranging from 0.7% to 13.9%. The limits of detection (LOD) in crab matrices are 0.0011–0.1591 μg / kg, and the limits of quantitation (LOQ) are 0.0036–0.5302 μg / kg. The correlation coefficient of the standard curve is above 0.999. This invention can simultaneously determine the content of at least 41 PFASs compounds with different physicochemical properties in crabs, 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

[0083] Figure 1 The diagram shows a schematic of the detection process for detecting fluorine compounds in crabs according to the present invention.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] Figure 5The 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] Figure 9 The image shown is a photograph of a crab sample taken in Example 1 of this invention.

[0092] Figure 10 The image shown is a chromatogram obtained by detecting fluorine compounds in crab sample 1 using the detection method of Example 1 of this invention.

[0093] Figure 11 The image shown is a chromatogram obtained by detecting fluorine compounds in crab sample 2 using the detection method of Example 1 of this invention. Detailed Implementation

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] The instrument and equipment information used in the embodiments of this invention is as follows:

[0100] 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).

[0101] The reagents and standards used in the embodiments of this invention are as follows:

[0102] 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.).

[0103] Figure 1The diagram shows a schematic of the detection process for detecting fluorine compounds in crabs according to the present invention. The detection process includes: Step S1: The edible part of the crab to be tested is crushed and homogenized to obtain the sample to be tested; Step S2: The sample to be tested is extracted, the organic phase is retained and concentrated to obtain a concentrated solution; then, 5 to 15 times the amount of water is added to dilute the concentrated solution to obtain a purified solution; the solvent for extraction is an aqueous solution of an organic solvent; Step S3: The purified solution is purified using a solid-phase extraction column to obtain a purified solution; Step S4: The purified solution is concentrated to near dryness, redissolved and filtered to obtain a filtrate; Step S5: The filtrate is detected using liquid chromatography-mass spectrometry, and the standard curve method is used to perform qualitative and quantitative analysis of fluorine compounds in crabs.

[0104] In step S1, the edible part of the crab in this application is obtained by removing the shell and inedible parts of the crab. The data from testing the edible part 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 41 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 this application, the target compound is the aforementioned 45 PFASs compounds. 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 (PFDoDA) 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 (PFHxDACAS) 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.

[0111] In step S4, the concentration to near dryness is defined as visually observing no liquid flow at the bottom of the container. Before injection into UPLC-MS / MS, the reconstituted methanol solution needs to be filtered to remove small insoluble particles, prevent column clogging, and ensure data quality.

[0112] Example 1

[0113] This embodiment 1 provides a method for detecting 41 PFASs compounds in crabs. The difference between these 41 PFASs compounds and the 45 PFASs compounds mentioned above is that they do not include perfluorooctanoic acid (PFODA, CAS number 16517-11-6); perfluorodecane sulfonic acid (PFDS, CAS number 335-77-3); perfluorooctane sulfonamide (FOSA, CAS number 754-91-6); and 4,8-dioxa-3H-perfluorononanoic acid (ADONA, CAS number 919005-14-4). The specific detection method includes the following steps:

[0114] S1, Sample Preparation

[0115] Take as Figure 9 One crab of the indicated type was used. The shell was removed, and the edible parts were taken. The sample was cut into small pieces, thoroughly pulverized and mixed using a grinder, then sealed. The prepared sample was stored at -18°C or below, protected from light, for later use. All tools used during the process must be cleaned with deionized water to prevent cross-contamination between samples.

[0116] S2, Extraction

[0117] 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.

[0118] S3, Enrichment and Purification

[0119] 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.

[0120] S4. Preparation of mixed standard working solutions and plotting of standard curves

[0121] 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). These solutions were then analyzed by UPLC-MS / MS according to the method established in this paper. A standard curve was plotted with the peak area ratio of the 45 PFASs and their corresponding isotopic internal standards as the ordinate (y) and the PFASs concentration as the abscissa (x). Quantitative analysis was performed using the internal standard method. The solute in the perfluorinated / polyfluorinated compound mixed standard stock solution was the aforementioned 45 PFASs compounds, and the solvent was methanol. The concentrations of the 45 PFASs compounds were consistent in the perfluorinated / polyfluorinated compound mixed standard stock solution. The solvent of the internal standard solution was methanol, and the solutes, as shown in Table 2, were all of consistent concentration.

[0122] S5. Perform UPLC-MS / MS analysis on the test solution.

[0123] S5.1 Chromatographic conditions

[0124] The chromatographic column used is Excsep TM The 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.

[0125] Table 1. Mobile phase gradient

[0126]

[0127] S5.2 Mass Spectrometry Conditions

[0128] 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.

[0129] Table 2. Mass spectrometry parameters of 45 PFASs and their corresponding internal standards

[0130]

[0131]

[0132]

[0133] 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.

[0134] Table 3. Linear range, regression equation, and correlation coefficient of 45 PFASs

[0135]

[0136]

[0137] 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 obtained by UPLC-MS / MS under the above chromatographic conditions.

[0138] Furthermore, the matrix effect (ME), spiked recovery, limit of detection (LOD), and limit of quantitation (LOQ) of the target compound in different crab species were investigated. Specifically, the standard spiking method was used to assess the potential matrix effect of the crab 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 4.

[0139] Table 4. Recovery rates, RSD (n=3), ME, LOD and LOQ of 45 PFASs in crab samples

[0140]

[0141]

[0142] Table 4 shows that PFTeDA, PFODA, FHxSA, FOSA, N-MeFOSA, and N-EtFOSA exhibit strong matrix effects in crabs. Therefore, matrix calibration curves are needed to calibrate the quantitative results during actual quantitative analysis. At a spike concentration of 2.5 μg / kg, the recovery rate of FOSA exceeds the acceptable recovery range. At a spike concentration of 25 μg / kg, the recoveries of PFODA, PFDS, FOSA, and ADONA also exceed the acceptable recovery range. Therefore, these four PFASs do not meet the basic requirements for quantitative analysis, and their data will not be included in the statistical range of subsequent actual sample tests. The recoveries of the remaining 41 PFASs are all within the acceptable recovery range (60%–140%), with RSDs of 0.7–13.9%, meeting the basic requirements for quantitative analysis. LODs range from 0.0011 to 0.1591 μg / kg, and LOQs range from 0.0036 to 0.5302 μg / kg.

[0143] Furthermore, the established detection method was applied to the determination of two crab samples, where crab samples 1 and 2 were... Figure 9 The crab samples were all 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 in the figure. Figures 10-11 As shown.

[0144] Table 5. Concentrations (μg / kg) of 41 PFASs in crab samples 1-5 from the Zhejiang coast

[0145]

[0146]

[0147] Note: ND means not detected, <LOQ means less than the limit of quantitation.

[0148] According to Table 5 and Figures 10-11The results show that: in crab sample 1, 26 fluorine-containing compounds were detected, among which PFBA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnDA, PFDoDA, PFTrDA, PFTeDA, PFBS, PFPeS, PFOS, FBSA, FOSAA, HFPO-TA, PF5OHxA, and 8:2 diPAP exceeded the limit of quantification; in crab sample 2, 26 fluorine-containing compounds were detected, among which PFBA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnDA, PFDoDA, PFTrDA, PFTeDA, PFBS, PFOS, FBSA, HFPO-DA, HFPO-TA, and 8:2 diPAP exceeded the limit of quantification.

[0149] In summary, the detection method of this invention is suitable for trace detection of perfluorinated / polyfluorinated compounds (PFAS) in crabs, 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 exhibit low limits of detection and quantitation. Monitoring the PFAS content in crabs is of great significance for ensuring food safety and public health, assessing ecological and environmental risks, and tracing pollution sources.

[0150] 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 crabs, characterized in that, The method includes: S1. Sample preparation, including: removing the shell of the crab to be tested, crushing and homogenizing its edible parts to obtain the sample to be tested; S2. Extraction, including: extracting the sample to be tested, retaining the organic phase and concentrating it to obtain a concentrated solution; then diluting the concentrated solution with 5 to 15 times the amount of water to obtain a purified solution; the solvent for extraction is an aqueous solution of an organic solvent; S3. Purification, including: purifying the liquid to be purified using a solid-phase extraction column to obtain a purified liquid; S4. Concentration and volume adjustment, including: concentrating the purified liquid to near dryness, reconstituted and then filtered to obtain filtrate; 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 crabs.

2. The detection method according to claim 1, characterized in that, The fluorinated compounds include 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.

3. The detection method according to claim 2, characterized in that, The perfluoroalkyl carboxylic acids include any one or more of the following: perfluoropropionic acid, perfluorobutyric acid, perfluoropentanoic acid, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, and perfluorohexadecanoic acid; and / or, the perfluoroalkyl sulfonic acids include any one or more of the following: perfluoroethane sulfonic acid, perfluorobutane sulfonic acid, perfluoropentane sulfonic acid, perfluorohexane sulfonic acid, perfluoroheptane sulfonic acid, and perfluorooctane sulfonic acid; And / or, the perfluoroalkyl sulfonamides include any one or more of the following: perfluorobutyl sulfonamide, perfluorohexane sulfonamide, N-methylperfluorooctane sulfonamide, N-methylperfluorooctane sulfonamide ethanol, N-ethylperfluorooctane sulfonamide, 2(N-ethylperfluorooctane sulfonamide) ethanol, and perfluorooctane sulfonamide acetic acid; And / or, 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; And / or, the fluoropolymer alkyl carboxylic acid includes any one or more of the following: 3:3 fluoropolymer carboxylic acid, 7:3 fluoropolymer carboxylic acid, 6:2 fluoropolymer carboxylic acid, and 8:2 fluoropolymer carboxylic acid; And / or, the perfluoroalkyl ether carboxylic acid comprises any one or more of the following: 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; And / or, 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; And / or, the disubstituted polyfluorophosphates include 8:2 fluoropolymer phosphate diesters.

4. The detection method according to claim 1, characterized in that, In step S2, based on 1g of the sample to be tested, the amount of water added is 1~10mL; And / or, step S2 further includes adding salt to water and organic solvent after extraction to cause the two phases to separate; the amount of salt added is 0.5~2.1g based on 1mL of water; And / or, in step S2, the volume ratio of water to organic solvent is 1:(1~5); And / or, in step S2, the organic solvent is acetonitrile, and a 30%~40% hydrochloric acid aqueous solution is also used during extraction, with the amount of hydrochloric acid aqueous solution being 0.1%~0.5% of the volume of acetonitrile; And / or, 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 2~50ng, and the internal standard is any one or more selected from M4PFBA, M5PFHxA, M8PFOA, MPFDoDA, M8PFOS, M2-8:2FTS, and M3HFPO-DA.

5. The detection method according to claim 1, characterized in that, In step S3, the purification process sequentially includes solid-phase extraction column activation, sample loading, rinsing, and elution. And / or, the stationary phase of the solid-phase extraction column in step S3 is a mixture of WAX and HLB, wherein the mass ratio of WAX to HLB is 1:(0.5~2). And / or, the mass of the stationary phase in 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 mass ratio of the stationary phase to the volume of the purified liquid passing through the column is 6 mg: (5~15) mL.

6. The detection method according to claim 5, characterized in that, The activation process involves sequentially activating the mixture with 0.05%~0.5% by volume of ammonia-methanol, methanol, and water. And / or, the eluent is ammonium acetate with a concentration of 10-50 mmol / L; And / or, the elution is performed sequentially with methanol and 0.05%~0.5% by volume ammonia-methanol solution.

7. The detection method according to claim 1, characterized in that, In step S4, the concentration is carried out at 30~50°C using nitrogen blowing concentration; And / or, in step S4, the resolution is performed by resolution of the concentrate with methanol; And / or, in step S4, the filtration is performed using a filter membrane with a pore size of 0.1~0.3μm.

8. The detection method according to claim 1, characterized in that, In step S5, the chromatographic conditions include: 1) The chromatographic column is a C18 column; 2) Column temperature is 35~45℃; 3) The injection volume is 1~5 μL; 4) The flow rate is 0.1~0.5 mL / min; 5) Mobile phase: Phase A: 1~3mM ammonium acetate aqueous solution; Phase B: methanol; 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.

9. The detection method according to claim 1, characterized in that, In step S5, the mass spectrometry conditions include: 1) Electrospray ion source, negative ion mode; 2) Air curtain pressure 30~40 psi; 3) Spray voltage -4000~-5000 V; 4) Atomization temperature: 450~550℃; 5) Atomizing pressure: 45~55 psi; 6) Auxiliary gas pressure 45~55 psi; And / or, in step S5, the concentration of the standard solution selected in the establishment of the standard curve is 0.005~100μg / L; And / or, in step S5, the establishment of the standard curve also includes adding an internal standard, wherein the amount of the internal standard added is 5~15ng.

10. Use of the method described in any one of claims 1 to 9 for the simultaneous detection of 41 fluorine compounds in crabs.

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