Method for detecting perfluoroalkyl substances in shellfish

Abstract

The application provides a method for detecting perfluoroalkyl substances in shellfish, characterized in that the method comprises the following steps: S1, sample preparation, including crushing and homogenizing the shellfish to be detected to obtain a sample to be detected; S2, extraction, including adding water and an organic solvent to the sample to be detected for extraction, retaining the organic phase and concentrating, adding water to the concentrated organic phase as a liquid to be purified; S3, purification, including purifying the liquid to be purified by using a solid-phase extraction column to obtain a purified liquid; S4, concentration and constant volume, including concentrating the purified liquid to near dryness, redissolving and filtering to obtain a solution to be detected; and S5, sample detection, including detecting the solution to be detected by using a liquid chromatograph-mass spectrometer, and qualitatively and quantitatively analyzing the perfluoroalkyl substances in the shellfish by using a standard curve method. The method can simultaneously determine the content of 42 different PFAS compounds in the shellfish.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of detection of perfluoro / polyfluoroalkyl substances, and particularly relates to a detection method of perfluoro / polyfluoroalkyl substances in shellfish. BACKGROUND

[0002] PFASs (Perfluoroalkyl and Polyfluoroalkyl Substances) are a class of synthetic organic compounds. Due to their high chemical stability, thermal stability, hydrophobicity and other properties, they are widely used in industry and consumer products. The persistence and non-degradability of PFASs make them persist in the environment for a long time, migrate over long distances through the atmosphere, water bodies and other channels, and pollute the ecological system. Marine organisms (such as shellfish, fish) absorb PFASs from the surrounding water and sediments through gill respiration, feeding and direct contact, and accumulate them in the body, with a concentration much higher than that in the surrounding environment. Eating contaminated seafood is one of the main ways for people in coastal areas and high-consumption groups to be exposed to PFASs. Especially for communities that rely on fishing and people who love to eat seafood, the risk is higher.

[0003] Oysters, clams, mussels and other shellfish are "filter-feeding" organisms that filter a large amount of seawater, resulting in very high concentrations of pollutants in the body. Detecting PFASs in shellfish not only helps to assess environmental and health risks, but also provides important support for food safety supervision, pollution control and scientific research, and has important practical application value. However, the differences in the physicochemical properties of different PFASs (such as ionic, non-ionic) result in limitations of traditional methods in terms of wide coverage and sensitive detection. The shellfish matrix is complex, containing a large amount of protein, fat, pigment and other substances. These substances can seriously interfere with the extraction of PFASs, and compete or bind with the target substances, resulting in low or fluctuating extraction recovery. Due to the large difference in retention behavior of PFASs with different structures on the chromatographic column, it is difficult to simultaneously detect multiple perfluoro / polyfluoroalkyl substances in one analysis. If they are co-eluted, they will interfere with each other, affecting the quantitative accuracy, resulting in low efficiency of batch detection of shellfish. In addition, the existing technology mainly uses LC-MS for detection, but the detection sensitivity of some low-content perfluoro / polyfluoroalkyl substances, such as perfluorocarboxylic acids, may still be insufficient, making it difficult to meet the detection needs of trace perfluoro / polyfluoroalkyl substances. SUMMARY

[0004] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a detection method of perfluoro / polyfluoroalkyl substances in shellfish, which solves the problems of low detection efficiency, low sensitivity and difficulty in accurate quantification of perfluoro / polyfluoroalkyl substances in shellfish in the prior art.

[0005] To achieve the above object and other related objects, the present application provides a method for detecting perfluoroalkyl substances in shellfish.

[0006] The first aspect of the present application provides a method for detecting perfluoroalkyl substances in shellfish, which comprises:

[0007] S1, sample preparation, comprising: crushing and homogenizing the shellfish to be detected to obtain a sample to be detected;

[0008] S2, extraction, comprising: adding water and an organic solvent to the sample to be detected for extraction, retaining the organic phase and concentrating, adding water to the concentrated organic phase as a liquid to be purified;

[0009] S3, purification, comprising: purifying the liquid to be purified using a solid phase extraction column to obtain a purified liquid;

[0010] S4, concentration and constant volume, comprising: concentrating the purified liquid to near dryness, redissolving and filtering to obtain a solution to be detected;

[0011] S5, sample detection, comprising: detecting the solution to be detected using a liquid chromatography-mass spectrometry instrument, and using a standard curve method to qualitatively and quantitatively analyze the perfluoroalkyl substances in the shellfish.

[0012] Preferably, the perfluoroalkyl substances include perfluoropropionic acid, perfluorobutyric acid, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanoic acid, perfluorotridecanoic acid, perfluorohexadecanoic 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, 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, 8:2 fluoroterpolymer phosphate diester, 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-dioxo-3H-perfluorononanoic acid, and 2, 3, 3, 3-tetrafluoro-2- (heptafluoropropoxy) propionic acid.

[0013] Preferably, the shellfish according to the present application include but are not limited to clams, scallops, mussels, oysters, whelks, and cockles.

[0014] Preferably, the crushing and homogenizing of the shellfish meat in step S1 is performed using a crusher.

[0015] Preferably, the volume of the organic phase after concentration in step S2 is 1-5 mL.

[0016] Further preferably, the volume of the organic phase after concentration in step S2 is 1-2 mL; including but not limited to 1 mL, 1.5 mL, and 2 mL.

[0017] Preferably, the amount of water added in step S2 is 1-4 mL based on 1 g of the sample to be detected; including but not limited to 1 mL, 1.5 mL, 2 mL, 2.5 mL, 3 mL, 3.5 mL, or 4 mL.

[0018] Preferably, the volume ratio of water to organic solvent in step S2 is 1:(1-4); for example, it can be 1:1, 1:2, 1:3, or 1:4.

[0019] Preferably, step S2 further includes adding salt to the water and organic solvent after extraction to make the two phases separate; the amount of salt added is 0.5-2 g based on 1 mL of water; for example, it can be 0.5 g, 0.6 g, 0.8 g, 1.0 g, 1.2 g, 1.4 g, 1.5 g, or 2 g.

[0020] Further preferably, the salt is sodium chloride.

[0021] Preferably, a 30%-40% hydrochloric acid aqueous solution is also used in the extraction in step S2, and the amount of hydrochloric acid aqueous solution is 0.1%-0.5% of the volume of acetonitrile.

[0022] Preferably, step S2 further includes adding an internal standard to the sample to be detected before extraction; the amount of internal standard added is 2-20 ng based on 1 g of the sample to be detected; for example, it can be 2 ng, 5 ng, 10 ng, 15 ng, or 20 ng.

[0023] Preferably, the internal standard includes perfluorobutyric acid- 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 fluorotelomer sulfonic acid- 13C2 (M2-8:2 FTS), 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy)propanoic acid 13 Any one or more of C3 (M3HFPO-DA).

[0024] Preferably, in the step S2, the volume ratio of the concentrated organic phase and the added water is 1: (5-15).

[0025] Further preferably, in the step S2, the volume ratio of the concentrated organic phase and the added water is 1: (8-12).

[0026] Further preferably, in the step S2, it further comprises vortex mixing the added sample, internal standard and water solution for 1-5 min before adding salt.

[0027] Further preferably, in the step S2, it further comprises adding organic solvent after vortexing and shaking for 5-10 min.

[0028] Further preferably, in the step S2, it further comprises adding salt after shaking and shaking for 5-10 min.

[0029] Further preferably, in the step S2, it further comprises centrifuging at 5000-10000 r / min for 5-10 min after shaking to separate water and organic solvent.

[0030] Preferably, in the step S2, the volume of the liquid to be purified is 5-15 mL; for example, it can be 5 mL, 8 mL, 10 mL, 12 mL or 15 mL.

[0031] Preferably, in the step S3, the stationary phase of the solid phase extraction column is a mixture of WAX and HLB, 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.

[0032] The WAX is a weak anion exchange filler, which is a weak anion exchange chromatographic filler with polyvinylpyrrolidone as the matrix and bonded primary / secondary amine in the structure.

[0033] The HLB is a hydrophilic-lipophilic balance filler, which is a filler formed by bonding a pyrrolidone group to a polystyrene / divinyl benzene, and its surface has both hydrophilic and lipophilic groups.

[0034] Preferably, the mass of the stationary phase of the solid phase extraction column is 110-180 mg; for example, it can be 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg or 180 mg.

[0035] Preferably, the volume of the solid phase extraction column is 5-8 mL; for example, it can be 5 mL, 6 mL, 7 mL or 8 mL.

[0036] Preferably, the mass of the stationary phase to the volume of the liquid to be purified passing through the column is 6 mg:(8-12) mL.

[0037] Preferably, in step S3, the purification comprises, in sequence, activation of the solid phase extraction column, sample loading, elution and elution.

[0038] Further preferably, the activation is performed in sequence using 0.05%-0.2% ammonia methanol, methanol and water.

[0039] The percentage is the volume percentage of ammonia in the ammonia methanol solution.

[0040] Further preferably, the volume of ammonia methanol used in the activation is 2-10 mL; for example, it can be 2 mL, 4 mL, 5 mL, 6 mL, 8 mL or 10 mL.

[0041] Further preferably, the volume of methanol used in the activation is 2-10 mL; for example, it can be 2 mL, 4 mL, 5 mL, 6 mL, 8 mL or 10 mL.

[0042] Further preferably, the volume of water used in the activation is 2-10 mL; for example, it can be 2 mL, 4 mL, 5 mL, 6 mL, 8 mL or 10 mL.

[0043] Preferably, the elution liquid is ammonium acetate with a concentration of 22-28 mmol / L; for example, it can be 22 mmol / L, 25 mmol / L or 28 mmol / L.

[0044] Further preferably, the volume of the elution liquid is 2-10 mL; for example, it can be 2 mL, 4 mL, 5 mL, 6 mL, 8 mL or 10 mL.

[0045] Further preferably, in step S3, after the elution, the solid phase extraction column is further subjected to vacuum suction for 1-3 min; and then elution is performed.

[0046] Further preferably, the elution is performed in sequence using methanol and 0.05%-0.2% ammonia methanol.

[0047] Further preferably, the volume of methanol used in the elution is 1-5 mL; for example, it can be 1 mL, 2 mL, 3 mL, 4 mL or 5 mL.

[0048] Further preferably, the volume of ammonia methanol used in the elution is 2-10 mL; for example, it can be 2 mL, 4 mL, 5 mL, 6 mL, 8 mL or 10 mL.

[0049] Further preferably, the elution flow rate is 1-3 s / drop.

[0050] Preferably, in step S4, the concentration is performed by nitrogen blowing at 35-50 °C; for example, the concentration temperature can be 35 °C, 40 °C, 55 °C or 50 °C.

[0051] In step S4, the concentration to near dryness is visually observed at the bottom of the container without liquid flow.

[0052] Preferably, in step S4, the reconstitution is performed by reconstituting the concentrate with methanol.

[0053] Preferably, in step S4, the filtration is performed by using a filter membrane with a pore size of 0.1-0.3 pm.

[0054] Further preferably, the pore size of the filter membrane can be 0.1 pm, 0.2 pm or 0.3 pm.

[0055] Preferably, in step S5, the chromatographic conditions include:

[0056] 1) the chromatographic column is a C18 column;

[0057] 2) the column temperature is 35-45 °C; for example, it can be 35 °C, 40 °C or 45 °C.

[0058] 3) the injection volume is 1-3 pL; for example, it can be 1 pL, 2 pL or 3 pL.

[0059] 4) the flow rate is 0.1-0.5 mL / min; for example, it can be 0.1 mL / min, 0.2 mL / min, 0.3 mL / min, 0.4 mL / min or 0.5 mL / min.

[0060] 5) the mobile phase: A phase: 1.5-2.5 mM ammonium acetate aqueous solution, B phase: methanol;

[0061] 6) elution mode: gradient elution, including: initial solvent composition: 75-80% A phase and 20-25% B phase, gradient change stage: the proportion of B phase is linearly increased to 90-95% for 0-14 min, holding stage: 90-95% B phase is maintained for 14-16 min, column cleaning stage: the proportion of B phase is linearly reduced to 20-25% for 16 min-16.01 min; column equilibration stage: 20-25% B phase is maintained for 16.01 min-20 min.

[0062] Preferably, in step S5, the mass spectrometry conditions include:

[0063] 1) Electrospray ion source, negative ion mode;

[0064] 2) Gas curtain pressure 32~38 psi; for example, it can be 32 psi, 35 psi or 38 psi.

[0065] 3) Spray voltage -4300~-4700 V; for example, it can be -4300 V, -4500 V or -4700 V.

[0066] 4) Nebulization temperature 470~520℃; for example, it can be 470℃, 500℃ or 520℃.

[0067] 5) Nebulization gas pressure 48~52 psi; for example, it can be 48 psi, 50 psi or 52 psi.

[0068] 6) Auxiliary gas pressure 48~52 psi; for example, it can be 48 psi, 50 psi or 52 psi.

[0069] Preferably, in step S5, the concentration of the standard solution selected in the standard curve establishment is 0.005~100 μg / L.

[0070] Preferably, in step S5, the standard curve establishment also includes adding an internal standard, and the amount of the internal standard added is 5~15 ng.

[0071] The second aspect of the present application provides a use of the above-mentioned method for simultaneously detecting 42 kinds of perfluoro / polyfluoroalkyl substances in shellfish.

[0072] As described above, the method for detecting perfluoro / polyfluoroalkyl substances in shellfish has the following beneficial effects:

[0073] The present application adopts the method of liquid-liquid extraction combined with solid phase extraction, first extracts the perfluoro / polyfluoroalkyl substances into the organic phase, and then purifies them through the solid phase extraction column, thereby realizing the effective purification, purification and enrichment of PFASs in shellfish, and further combining LC-MS / MS for quantitative detection of the purified and enriched substances; through testing, the recovery rate of 42 kinds of PFASs in shellfish matrix using the detection method of the present application is 61%~139%, and the RSD is 0.4%~13.8%, which can meet the requirements of quantitative analysis. The LOD of the method in the shellfish matrix is 0.0006~0.0718 μg / kg, the LOQ is 0.0019~0.2393 μg / kg, and the correlation coefficient of the standard curve is above 0.999. The present application can simultaneously determine the content of at least 42 kinds of PFAS compounds with different physicochemical properties in shellfish, improve the detection efficiency of batch samples, and has the advantages of short time efficiency, high sensitivity, good accuracy and strong stability. BRIEF DESCRIPTION OF DRAWINGS

[0074] Figure 1 A shows the detection process schematic diagram of detecting perfluoroalkyl substances in shellfish in the present application.

[0075] Figure 2 A shows the chromatogram obtained by UPLC-MS / MS detection of the target compounds with a concentration of 20 ng / mL by using the chromatographic conditions of Example 1 in the present application, wherein each peak represents: 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.

[0076] Figure 3 A shows the chromatogram obtained by UPLC-MS / MS detection of the target compounds with a concentration of 20 ng / mL by using the chromatographic conditions of Example 1 in the present application, wherein each peak represents: 1: PFEtS; 2: PFBS; 3: PFPeS; 4: PFHxS; 5: PFHpS; 6: PFOS; 7: PFDS.

[0077] Figure 3 B shows the chromatogram obtained by UPLC-MS / MS detection of the target compounds with a concentration of 20 ng / mL by using the chromatographic conditions of Example 1 in the present application, wherein each peak represents: 1: PFMPA; 2: PF5OHxA; 3: 3-6-OPFHpA; 4: HPFO-DA; 5: HFPO-TA; 6: ADONA.

[0078] Figure 4 A shows the chromatogram obtained by UPLC-MS / MS detection of the target compounds with a concentration of 20 ng / mL by using the chromatographic conditions of Example 1 in the present application, wherein each peak represents: 1: PFEESA; 2: 6:2 Cl-PFESA; 3: FOSAA; 4: 8:2 diPAP.

[0079] Figure 4 B shows the chromatogram obtained by UPLC-MS / MS detection of the target compounds with a concentration of 20 ng / mL by using the chromatographic conditions of Example 1 in the present application, wherein each peak represents: 1: 6:2 FTSA; 2: 8:2 FTSA; 3: 10:2 FTSA.

[0080] Figure 5A shows the chromatogram obtained by UPLC-MS / MS detection of the target compound with a concentration of 20 ng / mL using the chromatographic conditions of Example 1, wherein each peak represents: 1: 3:3 FTCA 2: 6:2 FTCA; 3: 7:3 FTCA; 4: 8:2 FTCA.

[0081] Figure 5 B shows the chromatogram obtained by UPLC-MS / MS detection of the target compound with a concentration of 20 ng / mL using the chromatographic conditions of Example 1, wherein each peak represents: 1: FBSA; 2: FHxSA; 3: FOSA; 4: N-MeFOSA; 5: N-MeFOSE; 6: N-EtFOSA; 7: N-EtFOSE.

[0082] Figure 6 The detection of 7 shellfish samples using the detection method of Example 1 is shown, and the photos of the sampled shellfish are shown.

[0083] Figures 7-13 The chromatograms obtained by detecting the perfluoro / polyfluoroalkyl substances in shellfish sample 1 to shellfish sample 7 using the detection method of Example 1 are shown, respectively; wherein, Figure 7 is shellfish 1; Figures 8-10 is shellfish 2-4; Figures 11-13 is shellfish 5-7. DETAILED DESCRIPTION

[0084] The embodiments of the present application will be described in detail by specific examples, and those skilled in the art can easily understand other advantages and effects of the present application from the disclosure of the specification. The present application can also be implemented or applied by other different specific embodiments, and each detail in the specification can be modified or changed based on different views and applications without departing from the spirit of the present application.

[0085] It should be noted that the process equipment or device not specifically mentioned in the following examples is the conventional equipment or device in the art.

[0086] Furthermore, it should be understood that the combination of one or more steps of the methods mentioned in the present application does not exclude that other steps of the method can be present before and / or after the mentioned combination of steps or that other steps of the method can be inserted between two explicitly mentioned steps, unless otherwise indicated; it should also be understood that the combination of one or more devices / apparatuses mentioned in the present application does not exclude that other devices / apparatuses can be present before and / or after the mentioned combination of devices / apparatuses or that other devices / apparatuses can be inserted between two explicitly mentioned devices / apparatuses. Moreover, unless otherwise indicated, the numbering of the steps of the methods is merely intended to identify various steps of the method for the convenience of the reader, and is in no way intended to limit the order of arrangement of the steps of the method or to define the scope of the application, and any change of relative position or order of the steps, without substantially altering the technical content, is deemed to be within the scope of the application.

[0087] Before further description of the specific embodiments of the present application, it should be understood that the scope of the present application is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present application are intended to describe specific embodiments and are not intended to limit the scope of the present application; in the specification and claims of the present application, the singular forms "a", "an" and "the" include the plural forms unless the context clearly indicates otherwise.

[0088] When the embodiments give a numerical range, it should be understood that, unless otherwise indicated by the present application, both ends of each numerical range and any number between the two ends can be selected. Unless otherwise defined, all technical and scientific terms used in the present application have the same meaning as generally understood by those skilled in the art. In addition to the specific methods, devices, materials used in the embodiments, any method, device and material of the prior art similar or equivalent to the methods, devices and materials described in the embodiments of the present application can also be used to implement the present application according to the mastery of the prior art by those skilled in the art and the description of the present application.

[0089] The information of the instruments and devices used in the embodiments of the present application is as follows:

[0090] Sciex Qtrap 4500 liquid chromatograph-mass spectrometer (AB SCIEX, USA), AL104 electronic analytical balance (Mettler Toledo), Synerg® UV ultrapure water machine (Milipore, USA), CT14RD desktop high-speed refrigerated centrifuge (Shanghai Tianmei Scientific Instrument Co., Ltd., China), IKA® VIRTEX 3 vortex mixer (IKA, Germany), HX-12D solid phase extraction device (Hengxin Century Technology, Wuhan, China), KQ-700VDV dual-frequency digital ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd., China), 2695 type high performance liquid chromatograph (ultraviolet detector) (Waters, USA).

[0091] The reagents and standard information used in the embodiments of the present application are as follows:

[0092] Methanol, acetonitrile (chromatographic grade, Shanghai Starke 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-nonyloxy sulfonic acid, perfluorobutyl sulfonamide, 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)propanoic acid, perfluoro-2,5-dimethyl-3,6-dioxanonanoic acid, 2,2,3,3-tetrafluoro-3-(trifluoromethoxy)propanoic acid, perfluoro-4-methoxybutyric acid, perfluoro-3,6-dioxoheptanoic acid (purity greater than 98%, Alfa Aesar Technology Co., Ltd. website); perfluorooctanesulfonic acid, perfluorooctanoic acid, perfluorobutanesulfonic acid, perfluoropentanoic acid, perfluorohexanoic acid, perfluoropropionic acid, perfluoroheptanoic acid, perfluorohexanesulfonic acid, N-methyl perfluorooctanesulfonamide, 2(N-ethyl perfluorooctanesulfonamido)ethanol, trifluoroacetic acid, perfluorohexanesulfonamide, 8:2 fluoroterpolymer phosphate diester, perfluorobutyric acid, N-ethyl perfluorooctanesulfonamide, N-methyl perfluorooctanesulfonamide ethanol (purity greater than 80%, Anfengyun Laboratory Supplies Co., Ltd.); perfluorooctanesulfonamide, trifluoromethanesulfonic acid (purity greater than 90%, Manhag Biological Technology Co., Ltd.); WAX filler (Xinmu Chromatography Technology Co., Ltd., specification: Ontwsep™ WAX); HLB filler (Xinmu Chromatography Technology Co., Ltd., specification: Ontwsep™ HLB); WAX and HLB mixed solid-phase extraction column (150 mg / 6 mL, Xinmu Chromatography Technology Co., Ltd.). Chromatographic column: ExcsepTM SiO2@PFP-C18 column (2.1x100 mm, 1.8 μm, Xinmu Chromatography Technology Co., Ltd.).

[0093] In step S1, the shell of the shellfish is removed, and the edible part of the shellfish is detected. The detection of perfluoro / polyfluoroalkyl substances in the shellfish can be directly used to evaluate food safety.

[0094] In step S2, the addition of acid in the extraction solution can purify the perfluoro / polyfluoroalkyl substances in the organic phase, which is beneficial to improve the recovery rate. The amount of acid added needs to be within a certain range, and too high or too low will affect the recovery rate.

[0095] After water and organic solvent extraction, salt is added to realize layering to facilitate phase separation.

[0096] After extraction, the water and organic phase are separated, and then further water is added to form the liquid to be purified. The addition of water allows the perfluoro / polyfluoroalkyl substances to be well removed from the solution and retained in the packing during the purification in S3, and not to be washed down with the organic solvent.

[0097] In step S3, the solid phase extraction column used in the application is (150 mg / 6 mL), which is composed of WAX:HLB with a mass ratio of 1:1; the applicant found that when only one kind of packing was used, the single mechanism of the packing could not retain some kinds of perfluoro / polyfluoroalkyl substances in the column, and only a small amount of perfluoro / polyfluoroalkyl substances could be separated out. The use of the weak anion exchange packing and the hydrophilic-lipophilic balance packing mixed in the application provides stronger retention power, and can effectively separate 42 kinds of PFASs compounds, improving the detection efficiency of batch samples.

[0098] When using a solid phase extraction column for sample purification, the steps of activation, sample loading, elution and elution are carried out in turn; during activation, 0.1% ammonia methanol, methanol and water are used in turn to wet and balance the packing in the column; during sample loading, the sample solution passes through the activated column, so that the target compounds are selectively retained by the stationary phase; during elution, 25 mmol / L ammonium acetate can remove weakly adsorbed impurities; during elution, methanol and ammoniated methanol can be used to effectively separate and elute perfluoro / polyfluoroalkyl substances of different polarity.

[0099] In step S4, the concentration to near dryness is to visually observe no liquid flow at the bottom of the container.

[0100] Before UPLC-MS / MS injection, the reconstituted methanol solution needs to be filtered to remove small insoluble particles, prevent the chromatographic column from being blocked, and ensure data quality.

[0101] Example 1

[0102] This example 1 provides a detection method for PFASs compounds in shellfish, and the names and structure information of 45 compounds are shown in Table 1.

[0103] Table 1 Structure and information of 45 PFASs

[0104]

[0105]

[0106]

[0107]

[0108]

[0109] The specific detection method comprises the following steps:

[0110] S1, sample preparation

[0111] Take several clams of the clam as shown in the figure 1, remove the shell, leave the clam meat, cut the clam meat into small pieces, use a crusher to fully crush, mix and seal the clam meat, and prepare the test sample. The prepared test sample is stored at-18℃ in the dark, and is ready for use. During the processing, all tools used need to be cleaned with deionized water to prevent mutual contamination between samples. Figure 6

[0112] S2, extraction

[0113] The sample prepared in S1 is thawed and homogenized, 2 g of the test sample is weighed and placed in a 50 mL polypropylene centrifuge tube, 50 μL of internal standard mixed solution (0.2 μg / mL) is added, 3 mL of water is added, vortex mixing is performed for 1 min, 6 mL of acetonitrile and 20 μL of concentrated hydrochloric acid (mass fraction of 36%-38%) are added, and shaking is performed for 5 min. 2 g of sodium chloride is added, and shaking is performed for 5 min again. Centrifugation is performed at 8000 r / min for 5 min. The upper acetonitrile solution is transferred to another test tube, and is blown to about 1 mL in a 40℃ water bath with nitrogen. Water is added to 10 mL, and is used as a to-be-purified liquid.

[0114] S3, enrichment and purification

[0115] A solid phase extraction column with a mass ratio of 1:1 WAX:HLB (150 mg / 6 mL) is selected to enrich and purify the sample. The solid phase extraction column is activated with 4 mL of 0.1% ammonia methanol, 4 mL of methanol and 4 mL of water in sequence, and the column body is kept wet. The to-be-purified liquid is immediately loaded onto the solid phase extraction column after activation. After the sample is loaded, the solid phase extraction column is eluted with 4 mL of 25 mmol / L ammonium acetate, and the eluate is discarded. Vacuum suction is performed for 2 min until the column body is nearly dry. Then 2 mL of methanol and 4 mL of 0.1% ammonia methanol solution are used in sequence, the flow rate is 2 s-3 s 1 drop, the eluate is collected, and is concentrated to near dryness at 40℃ with nitrogen. 1 mL of methanol is accurately added for dissolution. The solution is filtered with a needle cylinder filter and a filter membrane with a pore size of 0.22 μm. The filtrate is used as a test solution for UPLC-MS / MS analysis.

[0116] S4, preparation of mixed standard working solution and drawing of standard curve

[0117] ​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, 100.0 μg / L (containing 10 μg / L of internal standard, respectively) were prepared by diluting a certain amount of perfluoro / polyfluoroalkyl substance mixed standard stock solution and internal standard solution with methanol. The peak area ratio of 45 PFASs and their corresponding isotopic internal standards was used as the ordinate (y), and the concentration of PFASs was used as the abscissa (x) to draw the standard curve. The internal standard method was used for quantitative analysis.

[0118] S5. UPLC-MS / MS analysis of the test solution

[0119] S5.1. Chromatographic conditions

[0120] The chromatographic column was Excsep TM SiO2@PFP-C18 column, column temperature 40℃, injection volume 2 μL, flow rate 0.3 mL / min. The mobile phase was 2 mM ammonium acetate aqueous solution (A) and methanol (B) for elution, and the mobile phase gradient is shown in Table 2.

[0121] Table 2. Mobile phase gradient

[0122]

[0123] S5.2. Mass spectrometry conditions

[0124] The electrospray ion source was selected in negative ion mode. The ionization parameters were as follows: gas curtain pressure 35.0 psi; spray voltage -4500 V; atomization temperature 500℃; atomization gas pressure 50 psi; auxiliary gas pressure 50 psi. The specific mass spectrum is shown in Table 3.

[0125] Table 3. Mass spectrometry parameters of 45 PFASs and corresponding internal standards

[0126]

[0127]

[0128]

[0129] The linear equation, correlation coefficient and linear range obtained by UPLC-MS / MS method for determining different concentrations of standard solution under the above chromatographic and mass spectrometric conditions are shown in Table 4. Among them, 7 kinds of PFASs have good linear relationship in the range of 0.1-100 μg / L, PFDoDA has good linear relationship in the range of 0.05-100 μg / L, 27 kinds of PFASs have good linear relationship in the range of 0.01-100 μg / L, and the remaining 10 kinds of PFASs have good linear relationship in the range of 0.005-100 μg / L, and their correlation coefficients are greater than or equal to 0.999.

[0130] Table 4 Linear range, regression equation and correlation coefficient of 45 kinds of PFASs

[0131]

[0132]

[0133]

[0134] Figures 2-5 To dissolve the standard solution of the target compound in methanol, a standard solution with a concentration of 20 ng / mL is prepared, and the chromatogram obtained by UPLC-MS / MS detection under the above chromatographic conditions is shown in the following figure.

[0135] Further, the matrix effect (ME) of the target compound in shellfish, the recovery rate, the detection limit (LOD) and the quantification limit (LOQ) are detected. Specifically: the standard addition method is used to evaluate the matrix effect that may be produced by the shellfish matrix, and the relative response value of the target compound added in the sample matrix and the relative response value of the target compound in the pure solvent are compared; the recovery rate is set at two levels of 2.5 μg / kg and 25 μg / kg, and three parallel samples are set at each concentration to verify the accuracy of the method; the LOD value is calculated according to the signal-to-noise ratio S / N=3, and the LOQ value is calculated according to the signal-to-noise ratio S / N=10; the results are shown in Table 5.

[0136] Table 5 Recovery rate, RSD (n=3), ME, LOD and LOQ of 45 kinds of PFASs in shellfish samples

[0137]

[0138]

[0139]

[0140] It can be seen from the data in Table 5 that PFHxDA, PFODA, FHxSA, FOSA, N-MeFOSA and N-EtFOSA have strong matrix effects in shellfish, and matrix correction curves need to be used to calibrate the quantitative results in actual quantitative test; when the spiked concentration is 2.5 μg / kg, the recoveries of PFTeDA and PFODA exceed the acceptable range, when the spiked concentration is 25 μg / kg, the recoveries of PFPeA and PFTeDA exceed the acceptable range, the recoveries of the rest of PFASs are within the acceptable range (60%~140%), RSD is 0.7~13.8%, which can meet the basic requirements of quantitative analysis, LOD is 0.0006~0.0718 μg / kg, and LOQ is 0.0019~0.2393 μg / kg. It is shown that the method is not suitable for quantitative detection of PFTeDA, PFODA and PFPeA in shellfish, and the method is suitable for qualitative and quantitative detection of the above 42 kinds of PFASs with recoveries meeting the requirements.

[0141] Further, the established detection method is applied to the determination of 42 kinds of PFASs in 7 shellfish samples, the photos of the shellfish samples are shown in Figure 6 , the samples are purchased from the retail market of a city in Zhejiang Province, and the detection results are shown in Table 6.

[0142] Table 6 Concentrations of PFASs in shellfish along the coast of Zhejiang Province (μg / kg)

[0143]

[0144]

[0145] Note: ND is not detected, < LOQ is less than the limit of quantification

[0146] According to the results in Table 6 and Figures 7-13 , it can be seen that in shellfish sample 1 ( Figure 7 ), 30 kinds of perfluoro / polyfluoroalkyl substances can be detected, among which the contents of PFPrA, PFBA, PFOA, PFNA, PFDA, PFTrDA, PFHxDA, PFPeS, PFHpS, PFOS, FBSA, N-MeFOSE, 6:2 FTCA, ADONA, HFPO-TA, PFMPA, PF5OHxA and 8:2 diPAP exceed the limit of quantification LOQ; in shellfish sample 2 ( Figure 8), 26 PFASs were detected, and the contents of PFPrA, PFBA, PFOA, PFNA, PFDA, PFUnDA, PFTrDA, PFOS, 6:2Cl-PFESA, FBSA, HFPO-TA, PFMPA and 8:2 diPAP exceeded the limit of quantification (LOQ); in shellfish sample 3 ( Figure 9 ), 21 PFASs were detected, and the contents of PFPrA, PFBA, PFOA, PFDA, PFOS, 6:2Cl-PFESA, ADONA, HFPO-DA, HFPO-TA, PFMPA, PF5OHxA and 8:2 diPAP exceeded the limit of quantification (LOQ); in shellfish sample 4 ( Figure 10 ), 17 PFASs were detected, and the contents of PFPrA, PFBA, PFOA, PFDA, PFOS, 6:2Cl-PFESA and PFMPA exceeded the limit of quantification (LOQ); in shellfish sample 5 ( Figure 11 ), 16 PFASs were detected, and the contents of PFPrA, PFBA, PFOA, PFDA, PFTrDA, ADONA and PFMPA exceeded the limit of quantification (LOQ); in shellfish sample 6 ( Figure 12 ), 14 PFASs were detected, and the contents of PFPrA, PFBA, PFOA, PFDA, PFTrDA, FOSA, ADONA and PFMPA exceeded the limit of quantification (LOQ); in shellfish sample 7 ( Figure 13 ), 16 PFASs were detected, and the contents of PFPrA, PFBA, PFOA, PFDA, PFTrDA, FOSA, ADONA and PFMPA exceeded the limit of quantification (LOQ). It can be seen from the detection results of the seven shellfish samples that the types and contents of PFASs detected in the seven shellfish samples are different, which may be caused by the different contents of PFASs in different fishing sea areas. Therefore, the detection method can also assist in judging the fluorine pollution situation of the sea area by detecting the content of PFASs in shellfish.

[0147] In summary, the detection method is suitable for trace detection of at least 42 PFASs in shellfish, has good recovery rate and low detection limit, provides a feasible sample processing method when the sample amount is small, and improves batch detection efficiency. The extraction, purification and enrichment method in the method is simple, the types of PFASs detected are relatively complete, the time consumption is short, the recovery rate is good, the detection limit and the limit of quantification are low, and monitoring the content of PFASs in shellfish has important significance for ensuring food safety and public health, evaluating ecological environmental risk and tracing pollution sources.

[0148] The above merely describes the preferred embodiments of the present application, and is not intended to limit the present application in any form or in essence. It should be noted that those skilled in the art can make some improvements and supplements without departing from the method of the present application, and these improvements and supplements should also be considered as the protection scope of the present application. For those skilled in the art, some slight changes, modifications and equivalent changes made by using the disclosed technical content without departing from the spirit and scope of the present application are equivalent embodiments of the present application; meanwhile, any equivalent changes, modifications and evolution made according to the essential technology of the above embodiments are still within the scope of the technical solutions of the present application.

Claims

1. A method for detecting perfluoroalkyl substances in shellfish, characterized by, The method comprises: S1, sample preparation, comprising: crushing and homogenizing the shellfish to be detected to obtain a sample to be detected; S2, extraction, comprising: adding water and acetonitrile to the sample to be detected for extraction, retaining the organic phase and concentrating, adding water to the concentrated organic phase as a liquid to be purified; S3, purification, comprising: purifying the liquid to be purified using a solid phase extraction column to obtain a purified liquid; S4, concentration and constant volume, comprising: concentrating the purified liquid to near dryness, redissolving and filtering to obtain a solution to be detected; S5, sample detection, comprising: detecting the solution to be detected using a liquid chromatograph-mass spectrometer, and using a standard curve method to qualitatively and quantitatively analyze perfluoro / polyfluoroalkyl substances in shellfish; The perfluoro / polyfluoroalkyl substances include: perfluoropropionic acid, perfluorobutyric acid, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanoic acid, perfluorotridecanoic acid, perfluorohexadecanoic 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, 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, 8:2 fluorotelomer phosphate diester, perfluorooctanesulfonamide acetic acid, 6:2 fluorotelomer sulfonic acid, 8:2 fluorotelomer sulfonic acid, 10:2 fluorotelomer sulfonic acid, 3:3 fluorotelomer carboxylic acid, 7:3 fluorotelomer carboxylic acid, 6:2 fluorotelomer carboxylic acid, 8:2 fluorotelomer carboxylic acid, 4.8-dioxa-3H-perfluorononanoic acid, and 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy) propionic acid; In step S2, salt is added to the water and organic solvent after extraction to make the two phases separate; In step S2, 35%-40% hydrochloric acid aqueous solution is used for extraction, and the amount of 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 in sequence; 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); The activation is carried out in sequence using 0.05%-0.2% ammonia methanol, methanol and water; The elution liquid is 22-28 mmol / L ammonium acetate; The elution is carried out in sequence using methanol and 0.05%-0.2% ammonia methanol.

2. The detection method according to claim 1, characterized in that, In step S2, the amount of water added is 1-4 mL based on 1 g of the sample to be detected; And / or, the volume of the organic phase after concentration in step S2 is 1-5 mL; And / or, the volume ratio of water to organic solvent in step S2 is 1:(1-4); And / or, the volume ratio of the organic phase after concentration to water in step S2 is 1:(5-15); And / or, in step S2, the amount of salt added is 0.5-2 g based on 1 mL of water; And / or, in step S2, an internal standard is added to the sample to be detected before extraction, and the amount of internal standard added is 2-50 ng based on 1 g of sample to be detected; the internal standard is any one or more selected from M4PFBA, M5PFHxA, M8PFOA, MPFDoDA, M8PFOS, M2-8:2FTS, M3HFPO-DA.

3. The method of claim 1, wherein, The mass of the stationary phase of the solid phase extraction column is 110-180 mg; And / or, the volume of the solid phase extraction column is 5-8 mL; And / or, the volume ratio of the mass of the stationary phase to the volume of the liquid to be purified through the column is 6 mg:(8-12) mL.

4. The method of claim 1, wherein The volume of the eluent is 2-10 mL; And / or, in step S3, the solid phase extraction column is vacuumed for 1-3 min after elution; And / or, the volume of methanol used in elution is 1-5 mL; And / or, the volume of ammonia methanol used in elution is 2-10 mL; And / or, the elution flow rate is 1-3 s / drop.

5. The method of claim 1, wherein In step S4, the concentration is carried out at 35-50°C using nitrogen blowing; And / or, in step S4, the reconstitution is carried out by reconstituting the concentrate with methanol; And / or, in step S4, the filtration is carried out using a filter membrane with a pore size of 0.1-0.3 μm.

6. The method of claim 1, wherein In step S5, the chromatographic conditions include: 1) The chromatographic column is a C18 column; 2) The column temperature is 35-45°C; 3) The sample size is 1-3 μL; 4) The flow rate is 0.1-0.5 mL / min; 5) The mobile phase: A phase: 1.5-2.5 mM ammonium acetate aqueous solution, B phase: methanol; 6) Elution mode: gradient elution, including: initial solvent composition: 75%-80% A phase and 20-25% B phase, gradient change stage: 0-14 min, the proportion of B phase linearly increases to 90-95%, holding stage: 14-16 min, 90-95% B phase is maintained, column cleaning stage: 16 min-16.01 min, the proportion of B phase linearly decreases to 20-25%; column equilibration stage: 16.01 min-20 min, 20-25% B phase is maintained.

7. The method of claim 1, wherein, In step S5, the mass spectrometry conditions include: 1) Electrospray ion source, negative ion mode; 2) Gas curtain gas pressure 32-38 psi; 3) Spray voltage -4300 to -4700 V; 4) Atomization temperature 470-520°C; 5) Atomization gas pressure 48-52 psi; 6) Auxiliary gas pressure 48-52 psi; And / or, in step S5, the concentration of the standard solution selected in the standard curve establishment is 0.005-100 μg / L; And / or, in step S5, the standard curve establishment further comprises adding an internal standard, and the added amount of the internal standard is 5-15 ng.

8. Use of the method according to any one of claims 1-7 for simultaneously detecting 42 perfluoroalkyl and polyfluoroalkyl substances in shellfish.

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