Method for synchronously determining full-chain-length perfluoro and polyfluoroalkyl compounds in biological matrix by two-dimensional liquid chromatography-mass spectrometry

Abstract

The invention relates to the technical field of biological detection, in particular to a method for synchronously determining full-chain-length perfluoro and polyfluoroalkyl compounds in a biological matrix by two-dimensional liquid chromatography-mass spectrometry, which comprises the following steps: pretreatment: removing impurities from the biological matrix by extraction and low-temperature freezing to obtain a supernatant, and carrying out nitrogen blow-drying and redissolving to obtain a supernatant; a to-be-detected sample is obtained; two-dimensional liquid chromatography-mass spectrometry: separating out a part of ultra-short chain PFAS with strong polarity in a sample to be detected through one-dimensional liquid chromatography, trapping the rest target PFAS (mainly medium-long chain PFAS) by a trapping column, supplementing liquid to the target PFAS, separating the trapped target PFAS by two-dimensional liquid chromatography, and carrying out qualitative and quantitative analysis through mass spectrometry. Through the two-dimensional liquid chromatography-mass spectrometry detection method, the synchronous detection process of 43 kinds of PFAS from ultrashort chain to long chain is realized, and stable and reliable analysis results are obtained.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, and particularly relates to a method for synchronously determining full-chain-length perfluoroalkyl and polyfluoroalkyl compounds in biological matrixes by two-dimensional liquid chromatography-mass spectrometry. BACKGROUND

[0002] Perfluoroalkyl and polyfluoroalkyl compounds (PFAS) are a class of structurally diverse, artificially synthesized fluorine-containing organic compounds, which are widely used in industrial production and consumer product fields. Traditionally, PFAS research and management are mainly focused on long-chain compounds, such as perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), perfluorohexane sulfonate (PFHxS), long-chain perfluorocarboxylic acids (PFCAs), and their salts and related substances have been gradually regulated and eliminated. Compared with long-chain PFAS, ultra-short-chain PFAS with three or fewer carbon atoms have weaker bioaccumulation due to their small molecular weight, strong polarity and high water solubility. However, recent studies have shown that the detection frequency and concentration of ultra-short-chain PFAS in the environment and biological samples are increasing significantly, and their load in some samples even exceeds that of long-chain PFAS, highlighting their long-underrated pollution load and health risks, and thus have become a new focus of current PFAS pollution research. Limited population monitoring data shows that ultra-short-chain PFAS, especially trifluoroacetic acid (TFA) and perfluoropropionic acid (PFPrA), dominate in biological samples such as serum and urine.

[0003] However, current PFAS detection methods based on reversed-phase chromatographic columns are mainly established for medium and long-chain PFAS, and have poor retention capacity for ultra-short-chain PFAS with strong polarity, making it difficult to effectively separate and quantitatively analyze ultra-short-chain to long-chain PFAS under the same chromatographic conditions. In addition, reversed-phase chromatographic columns may not effectively separate PFCA (C2-C5) from its analytical interferents, resulting in overestimation of the concentration. If hydrophilic interaction chromatography, ion exchange chromatography and other alternative separation modes are used to specifically analyze ultra-short-chain PFAS, two or more independent analyses of the same biological sample are required, resulting in a long process and increased detection cost. On the other hand, biological matrixes are complex, and the presence of various matrix interferents makes pretreatment technology a necessary step before chromatographic injection, further increasing the complexity of the overall detection. The commonly used weak anion exchange solid-phase extraction method internationally is tedious and time-consuming, and background pollution is easily introduced during the extraction process, especially for ultra-short-chain PFAS such as TFA, which is widely present in the environment, and its pollution control is particularly difficult.

[0004] Therefore, there is an urgent need in the art to develop a simple and universal pretreatment method, and to establish a two-dimensional liquid chromatography-mass spectrometry detection method matched therewith, to realize the synchronous detection of ultra-short-chain to long-chain PFAS in multiple biological matrixes in a single analysis. SUMMARY

[0005] In view of the above deficiencies in the prior art, the purpose of the present application is to provide a method for synchronously determining full-chain-length perfluoro and polyfluoro alkyl compounds in a biological matrix by two-dimensional liquid chromatography-mass spectrometry.

[0006] To achieve the above-mentioned purpose of the application, the technical solution adopted by the present application is: The present application provides a method for synchronously determining full-chain-length perfluoro and polyfluoro alkyl compounds in a biological matrix by two-dimensional liquid chromatography-mass spectrometry, comprising the following steps: Pre-treatment: impurities are removed from the biological matrix by extraction and low-temperature freezing, and the supernatant is obtained after nitrogen blowing and redissolving to obtain the sample to be tested; Two-dimensional liquid chromatography-mass spectrometry: the part of the sample to be tested with strong polarity is separated by one-dimensional liquid chromatography, the remaining target PFAS is captured by a capture column and is supplemented with liquid, and the captured target PFAS is separated by two-dimensional liquid chromatography and is subjected to qualitative and quantitative analysis by mass spectrometry; The PFAS includes: TFA, PFPrA, PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnDA, PFDoDA, Ntf2, PFMeS, PFEtS, PFPrS, PFBS, PFPeS, PFHxS, PFHpS, PFOS, PF5OHxA, PFEESA, 3,6-OPFHpA, ADONA, HFPO-DA, 6:2 Cl-PFESA, 8:2 Cl-PFESA, 4:2 FTSA, 6:2 FTSA, 8:2 FTSA, PFMOAA, PFO2HxA, PFO3OA, PFO4DA, PFO5DoDA, C7 HFPO-TA, C8 HFPO-TA, C9 HFPO-TA, HFPO-TeA, Genx-CF3, PFO2OA, PFO3DA, H-PFMO2OSA.

[0007] Preferably, the pre-treatment method specifically comprises the following steps: (1) The biological matrix sample is added to a centrifugal tube, and an isotopic internal standard solution is added; (2) Acetonitrile is added, and after oscillation and ultrasonic treatment, the centrifugal tube is frozen and placed; (3) The mixed liquid treated in step (2) is centrifuged, and the supernatant is taken to a new centrifugal tube for nitrogen blowing treatment; (4) After the nitrogen blowing treatment is completed, methanol-water solution is added and mixed, and then centrifuged, and the supernatant is taken to a sample vial for testing.

[0008] Preferably, the biological matrix includes serum, urine and breast milk.

[0009] Preferably, the method of step (2) is specifically as follows: Add three times the volume of acetonitrile to the biological matrix sample, shake the mixture in a multi-tube vortex at 900 rpm for 15 min, and ultrasonic at room temperature for 15 min. Then, place the centrifuge tube containing the mixture in a freezer at -40°C for 6-8 hours.

[0010] Preferably, the method of step (3) is specifically as follows: Centrifuge at 4400 rpm for 15 min, take the supernatant to a new centrifuge tube, and then place it in a nitrogen blow at 25°C.

[0011] Preferably, the method of step (4) is specifically as follows: Add a 9:1 volume ratio of methanol and water solution to the centrifuge tube blown dry in step (3), shake the mixture in a multi-tube vortex at 900 rpm for 5 min, and then centrifuge at 12000 rpm for 15 min. Take the supernatant to a sample injection vial for testing.

[0012] Preferably, the one-dimensional liquid chromatography conditions are as follows: The chromatographic column is a hydrophilic interaction and ion exchange composite filler chromatographic column; the flow rate is 0.2 mL / min, the column temperature is 40°C, and the injection volume is 10 μL; the mobile phase A is 10 mM ammonium formate and 0.1% formic acid in water, the mobile phase B is 0.1% formic acid in acetonitrile, and the isocratic elution program is 10% A and 90% B for 0-30 min; The two-dimensional liquid chromatography conditions are as follows: The chromatographic column is a C18 packing chromatographic column; the flow rate is 0-0.5 mL / min, and the column temperature is 40 DEG C; the mobile phase C is 5 mM ammonium acetate aqueous solution, and the mobile phase D is pure methanol, and the gradient elution procedure is as follows: initially 98% C, 2% D, and the flow rate is 0.3 mL / min; 0.0-1.0 min, 98% C, 2% D, and the flow rate is 0.3 mL / min; 1.0-1.1 min, 98%-100% C, 2%-0% D, and the flow rate is 0.3 mL / min; 1.1-7.0 min, 100% C, 0% D, and the flow rate is 0.5 mL / min; 7.0-11.0 min, 100%-98% C, 0%-2% D, and the flow rate is 0 mL / min; 11.0-11.5 min, 98%-90% C, 2%-10% D, and the flow rate is 0 mL / min; 11.5-16.5 min, 90%-45% C, 10%-55% D, and the flow rate is 0.3 mL / min; 16.5-17.5 min, 45%-30% C, 55%-70% D, and the flow rate is 0.3 mL / min; 17.5-20.0 min, 30%-10% C, 70%-90% D, and the flow rate is 0.3 mL / min; 20.0-28.0 min, 10%-0% C, 90%-100% D, and the flow rate is 0.3 mL / min; 28.0-29.0 min, 0% C, 100% D, and the flow rate is 0.3 mL / min; 29.0-29.1 min, 0%-98% C, 100%-2% D, and the flow rate is 0.3 mL / min; 29.1-30.0 min, 98% C, 2% D, and the flow rate is 0.3 mL / min.

[0013] Preferably, the trapping conditions are as follows: The trapping column is Polaris 180A C18-A; the replenishing solution is water as the mobile phase C; the column temperature is 40 DEG C; the replenishing time is 1.1-7.0 min; and the replenishing flow rate is 0.5 L / min.

[0014] Preferably, the mass spectrometry conditions are as follows: The ion source is electrospray ion source, and the determination is under the condition of selecting multiple reaction monitoring and scanning mode of negative ion scanning; the specific parameter settings are as follows: dry gas temperature 100 DEG C, sheath gas temperature 200 DEG C, dry gas flow rate 11 L / min, sheath gas flow rate 11 L / min, atomization gas voltage 25 psi, capillary voltage 2500 V, and collision cell acceleration voltage 5 V.

[0015] Preferably, the two-dimensional liquid chromatography-mass spectrometry system comprises two valves, and different combinations of the columns are achieved by position switching of the two valves; the valve switching program is as follows: 0.0-7.0 min, at this time, the sample outlet of the one-dimensional column is connected to the trapping column and then flows into the mass spectrometer; 7.0-11.0 min, at this time, the sample outlet of the one-dimensional column directly flows into the mass spectrometer; 11.0-30.0 min, at this time, the trapping column is connected to the two-dimensional column and then flows into the mass spectrometer.

[0016] Compared with the conventional solid phase extraction method and the detection method based on the reversed-phase chromatographic column, the advantages of the present application are that: (1) simple operation process: the traditional solid phase extraction is abandoned, and the steps of activation, sample loading and elution are omitted, and good extraction effect can be achieved by single extraction of acetonitrile and freezing step; (2) reduced analysis cost: no need to use expensive solid phase extraction column and related supporting devices, and the required consumables are cheap; (3) wide detection range: the established two-dimensional liquid chromatography-mass spectrometry detection method can cover the quantification of high-polarity ultra-short-chain PFAS to high-hydrophobic long-chain PFAS in a single sample injection, and has the detection ability of more PFAS species; (4) low TFA background interference: the one-dimensional hydrophilic interaction chromatography separates the strong polarity TFA from other components, and the TFA is eluted in the first dimension liquid phase by valve switching, so that it is detected by mass spectrometry before entering the two-dimensional liquid phase, which significantly reduces the continuous background interference caused by TFA in the mobile phase; (5) reduce false positives: through two chromatographic columns with different separation mechanisms, the separation degree of PFCA (especially C2-C5) is effectively enhanced, the target PFAS is separated from interfering compounds, and the risk of false positives is effectively reduced; (6) high sensitivity: the method has high sensitivity, and the limit of quantification of ultra-short-chain TFA, PFPrA, etc. in serum is better than that of other reported methods. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a schematic diagram of the combination mode of the two eight-way valves in the two-dimensional liquid chromatography system in the present application; Figure 2 It is a two-dimensional LC-MS / MS chromatogram of 43 kinds of PFAS in serum of the embodiment; Reference signs: Figure 2Medium 1 : PFMeS; 2: PFEtS; 3: PFPrA; 4: PFMOAA; 5: PFBA; 6: TFA; 7: Ntf2; 8-9: C7 HFPO-TA, PFPrS; 10-12: PFO2HxA, PFPeA, PFBS; 13-14: PF5OHxA, PFEESA; 15-16: 4:2 FTSA, 3,6-OPFHpA; 17-19: PFHxA, PFPeS, PFO3OA; 20-21: HFPO-DA, PFO2OA; 22: H-PFMO2OSA; 23-27: PFHxS, PFHpA, ADONA, PFO4DA, Genx-CF3; 28: PFO3DA; 29-31: PFHpS, PFOA, 6:2 FTSA; 32: C8 HFPO-TA; 33: PFO5DoDA; 34-36: PFOS, PFNA, C9 HFPO-TA; 37: 6:2Cl-PFESA; 38-39: PFDA, 8:2 FTSA; 40: PFUnDA; 41: 8:2Cl-PFESA; 42: HFPO-TeA; 43: PFDoDA; Figure 3 Two-dimensional LC-MS / MS chromatogram of 43 PFAS in breast milk of an example; Reference signs: Figure 31: PFMeS; 2: PFEtS; 3: TFA; 4: PFPrA; 5: PFMOAA; 6: PFBA; 7:Ntf2; 8-10: C7 HFPO-TA, PFPrS, PFO2HxA; 11: PFPeA; 12-13: PFBS, PF5OHxA; 15-19: 4: 2 FTSA, 3,6-OPFHpA, PFHxA, PFPeS, PFO3OA; 20-22: HFPO-DA,PFO2OA, H-PFMO2OSA; 23-27: PFHxS, PFHpA, ADONA, PFO4DA, Genx-CF3; 28-31:PFO3DA, PFHpS, PFOA, 6: 2 FTSA; 32-33: C8 HFPO-TA, PFO5DoDA; 34-36: PFOS,PFNA, C9 HFPO-TA; 37: 6: 2Cl-PFESA; 38-39: PFDA, 8: 2 FTSA; 40: PFUnDA; 41-42: 8: 2Cl-PFESA, HFPO-TeA; 43: PFDoDA; Figure 4 Two-dimensional LC-MS / MS chromatogram of 43 PFAS in urine as an example; Figure label: Figure 4Middle 1: PFMeS; 2: PFEtS; 3: PFPrA; 4: PFMOAA; 5: PFBA; 6: TFA; 7:Ntf2; 16: PFO2OA; 17-22: 4:2 FTSA, PFHxA, PFPeS, PFO3OA, HFPO-DA, H-PFMO2OSA; 23: PFHxS; 24-27: PFHpA, ADONA, PFO4DA, Genx-CF3; 28-32: PFO3DA,PFHpS, PFOA, 6:2 FTSA; C8 HFPO-TA; 33-37: PFO5DoDA, PFOS, PFNA, C9 HFPO-TA,6:2Cl-PFESA; 38-39: PFDA, 8:2 FTSA; 40: PFUnDA; 41-42: 8:2Cl-PFESA, HFPO-TeA; 43: PFDoDA. Detailed Implementation

[0018] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0019] Example The instruments and reagents used in this embodiment are as follows: 1. Analytical instruments: Multidimensional liquid chromatography-mass spectrometry analysis system (Agilent Technologies, USA), VM-MT multi-tube vortex mixer (Oxford Instruments, USA), KX-2120QTD ultrasonic cleaner (Beijing Kexi Century Co., Ltd.), Sorvall ST 8 centrifuge (Fisher Scientific, USA), ND200 nitrogen purging device (Hangzhou Ruicheng Instrument Co., Ltd.).

[0020] 2. Experimental reagents: MPFAC-C-ES isotope internal standard mixed liquid (including...) 13 C4-PFBA, 13 C5-PFPeA, 13 C5-PFHxA, 13 C4-PFHpA, 13 C8-PFOA,13 C9-PFNA, 13 C6-PFDA, 13 C7-PFUnDA, 13 C2-PFDoDA, 13 C2-PFTeDA, 13 C3-PFBS, 13 C3-PFHxS, 13 C8-PFOS was purchased from Wellington, Canada; water (LC-MS grade, Fisher Scientific, USA); acetonitrile (LC-MS grade, Fisher Scientific, USA); methanol (HPLC grade, Dima Technology Development Co., Ltd., China); ammonium acetate, ammonium formate, and formic acid were all trace metal grade and purchased from Sigma-Aldrich, USA.

[0021] 3. The target compounds detected in this experiment included the following 43 PFAS: trifluoroacetic acid (TFA), pentafluoropropionate (PFPrA), perfluorobutanoate (PFBA), perfluoropentanoate (PFPeA), perfluorohexanoate (PFHxA), perfluoroheptanoate (PFHpA), perfluorooctanoate (PFOA), perfluorononanoate (PFNA), perfluorodecanoate (PFDA), perfluoroundecanoate (PFUnDA), perfluorododecanoate (PFDoDA), and bis(trifluoromethanesulfonylimide). Ntf2), trifluoromethansulfonate (PFMeS), perfluoroethylsulfonate (PFEtS), perfluoropropanesulfonate (PFPrS), perfluorobutanesulfonate (PFBS), perfluoropentanesulfonate (PFPeS), perfluorohexanesulfonate (PFHxS), perfluoroheptanesulfonate (PFHpS), perfluorooctanesulfonate (PFOS), perfluoro-5-oxahexanoic acid (PF5OHxA), perfluoro(2-ethoxyethane)sulfonate (perfluoro, 2-ethoxyethane sulfonate), PFEESA), perfluoro-3,6-dioxaheptanoic acid (3,6-OPFHpA), dodecafluoro-3H-4,8-dioxanonanoate,ADONA), hexafluoropropylene oxide dimeracid (HFPO-DA), 9-chlorohexadecylfluoro-3-oxanonane-1-sulfonic acid (6:2 chlorinated polyfluoroalkyl ether sulfonic acids, 6:2 Cl-PFESA), 11-chloroeicosicofluoro-3-oxaundecan-1-sulfonic acid (8:2 chlorinated polyfluoroalkyl ether sulfonic acids, 8:2 Cl-PFESA), 1H,1H,2H,2H-perfluorohexanesulfonic acid (4:2 fluorotelomer sulfonates, 4:2 FTSA), 1H,1H,2H,2H-perfluorooctanesulfonic acid (6:2 fluorotelomer sulfonates, 6:2 FTSA), and 1H,1H,2H,2H-perfluorodecanesulfonic acid (8:2 fluorotelomer sulfonates, 8:2 FTSA) were all purchased from Wellington Company, Canada. Perfluoro-2-methoxyacetic acid (PFMOAA), perfluoro-3,5-dioxahexanoic acid (PFO2HxA), perfluoro-3,5,7-trioxaoctanoic acid (PFO3OA), perfluoro-3,5,7,9-tetraoxadecanoic acid (PFO4DA), perfluoro-3,5,7,9,11-pentaoxadodecanoic acid (PFO5DoDA), and perfluoro-2,5-dimethyl-3,6-dioxo-heptanoic acid (C7) HFPO-TA), perfluoro-2,5-dimethyl-3,6-dioxo-octanoic acid (C8 HFPO-TA), and 2,3,3,3-tetrafluoro-2-(1,1,2,3,3,3-hexafluoro-2-(perfluoropropoxy)propoxy)propionic acid (hexafluoropropylene oxide trimer acid)C9 HFPO-TA), perfluoro-(2,5,8-trimethyl-3,6,9-trioxadecanoic acid) (HFPO-TeA), perfluoro-2-isobutoxypropanoic acid (Genx-CF3), perfluoro-2-methyl-3,6-dioxaoctanoic acid (PFO2OA), perfluoro-2-methyl-3,6,8-trioxadecanoic acid (PFO3DA), 7H-perfluoro-4-methyl-3,6-dioxaoctanesulfonic acid. H-PFMO2OSA was synthesized in the laboratory.

[0022] Analysis steps: 1. Pre-processing flow: Take 200 μL of serum (or 2 mL of artificial urine, or 2 mL of breast milk) into a 15 mL centrifuge tube, add 2.5 μL of isotope internal standard solution (MPFAC-C-ES, 100 ng / mL), then add three times the sample volume of acetonitrile. Vortex the mixture at 900 rpm for 15 min, sonicate at room temperature for 15 min, and then freeze the 15 mL centrifuge tube containing the mixture at -40°C for 6 hours. Immediately after freezing, centrifuge at 4400 rpm for 15 min, collect the supernatant into a new 15 mL centrifuge tube, and place it under nitrogen purging at 25°C. After nitrogen purging, add 100 μL of a 9:1 methanol-water solution, vortex at 900 rpm for 5 min, mix well, centrifuge the mixed solution at 12000 rpm for 15 min, and collect the supernatant into a vial for analysis.

[0023] 2. The pretreated sample was subjected to qualitative and quantitative analysis using a two-dimensional liquid chromatography-mass spectrometry (LC-MS) method: the highly polar ultra-short chain PFAS were separated by the first-dimensional liquid chromatography, and the remaining target PFAS (mainly medium and long chain PFAS) were captured by the trapping column and replenished with liquid. The captured target PFAS were separated by the second-dimensional liquid chromatography and qualitative and quantitative analysis was performed by mass spectrometry.

[0024] The PFAS analysis instrument used was an Agilent multidimensional liquid chromatography-mass spectrometry (LC-MS / MS) instrument, and the specific parameter settings are as follows: One-dimensional liquid chromatography conditions: Raptor Polar X column (100 × 2.1 mm, 2.7 μm, Restek, USA); flow rate 0.2 mL / min; column temperature 40℃; injection volume 10 μL; mobile phase A was an aqueous solution of 10 mM ammonium formate and 0.1% formic acid; mobile phase B was an acetonitrile solution of 0.1% formic acid; isocratic elution program was 0–30 min, 10% A, 90% B. Two-dimensional liquid chromatography was performed using a Poroshell 120 EC-C18 column (100 × 4.6 mm, 2.7 μm, Agilent). (USA); Flow rate: 0–0.5 mL / min; Column temperature: 40 °C; Mobile phase C: 5 mM ammonium acetate aqueous solution; Mobile phase D: pure methanol; Gradient elution program: Initial: 98% C, 2% D, flow rate: 0.3 mL / min; 0.0–1.0 min: 98% C, 2% D, flow rate: 0.3 mL / min; 1.0–1.1 min: 98%–100% C, 2%–0% D, flow rate: 0.3 mL / min; 1.1–7.0 min: 100% C, 0% D, flow rate: 0.5 mL / min; 7.0–11.0 min: 100%–98% C, 0%–2% D, flow rate: 0 mL / min; 11.0–11.5 min: 98%–90% C, 2%–10% D, flow rate: 0 mL / min; 11.5–16. 5 min, 90%~45%C, 10%~55%D, flow rate 0.3 mL / min; 16.5~17.5 min, 45%~30%C, 55%~70%D, flow rate 0.3 mL / min; 17.5~20.0 min, 30%~10%C, 70%~90%D, flow rate 0.3 mL / min; 20.0~28.0 min, 10%~ 0%C, 90%~100%D, flow rate 0.3mL / min; 28.0~29.0min, 0%C, 100%D, flow rate 0.3mL / min; 29.0~29.1min, 0%~98%C, 100%~2%D, flow rate 0.3mL / min; 29.1~30.0min, 98%C, 2%D, flow rate 0.3mL / min.

[0025] The trapping conditions were as follows: the trapping column was Polaris 180A C18-A (4.6 × 50 mm, 5 μm, Agilent, USA); the replenishment solution was water (mobile phase C as described above); the column temperature was 40 °C; the replenishment time was 1.1–7.0 min; and the replenishment flow rate was 0.5 L / min.

[0026] Mass spectrometry conditions were as follows: electrospray ionization source (AJS ESI), multiple reaction monitoring (MRM) was selected, and the scanning mode was negative ion scanning; specific parameter settings were: drying gas temperature 100℃, sheath gas temperature 200℃, drying gas flow rate 11L / min, sheath gas flow rate 11L / min, nebulizer voltage 25psi, capillary voltage 2500V, and collision cell accelerating voltage 5V.

[0027] This two-dimensional liquid chromatography-mass spectrometry (LC-MS) system includes two eight-way valves, allowing for different column combinations by switching the positions of these valves. In this invention, the valve switching program for separating 43 PFAS is as follows: 0.0–7.0 min, valve position P2–P2, during which the sample outlet of the one-dimensional column is connected to the trapping column and flows into the mass spectrometer; 7.0–11.0 min, valve position P1–P2, during which the sample outlet of the one-dimensional column flows directly into the mass spectrometer; 11.0–30.0 min, valve position P1–P1, during which the trapping column is connected to the two-dimensional column and flows into the mass spectrometer. A schematic diagram of the valve combination is shown below. Figure 1 As shown in Table 1, the specific liquid chromatography analysis conditions are shown in Table 1, and the specific instrument acquisition parameters are shown in Table 2. Table 3 shows the method detection limits (MDL) of PFAS in serum, breast milk, and urine. Table 4 shows the spiked recoveries of PFAS in serum, breast milk, and urine. n = 5).

[0028] Table 1 Liquid Chromatography Analysis Conditions

[0029] Table 2. Acquisition parameters of 43 PFAS instruments

[0030]

[0031]

[0032]

[0033] Table 3. Method detection limits for PFAS in serum, breast milk, and urine.

[0034]

[0035] Table 4. Spiked recovery rates of PFAS in serum, breast milk, and urine.

[0036]

[0037] As shown in Tables 3 and 4, the method of this invention can cover the quantification of highly polar ultrashort-chain PFAS to highly hydrophobic long-chain PFAS with a single injection, possessing the ability to detect a wider range of PFAS types. The spiked recoveries of most compounds are stable between 82.7% and 116.0%, demonstrating good analytical accuracy and reliability. This method exhibits excellent sensitivity, especially in the quantification limits of ultrashort chains such as TFA and PFPrA in serum, which are superior to other currently reported methods, making it suitable for trace analysis. These characteristics make the method of this invention of significant application value in the high-throughput and high-sensitivity detection of PFAS in complex biological matrices.

[0038] In summary, the method of this invention removes impurities such as proteins and lipids from biological matrices such as serum, urine, and breast milk through acetonitrile extraction and low-temperature freezing, obtaining an acetonitrile supernatant which is then dried under nitrogen and reconstituted. This invention utilizes a two-dimensional liquid chromatography-mass spectrometry (LC-MS) method to achieve simultaneous detection of 43 PFAS species, ranging from ultrashort to long chains, obtaining stable and reliable analytical results.

[0039] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0040] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for simultaneous determination of full-chain long-chain perfluorinated and polyfluoroalkyl compounds in a biological matrix using two-dimensional liquid chromatography-mass spectrometry, characterized in that, Includes the following steps: Pretreatment: Impurities in the biological matrix were removed by extraction and low-temperature freezing. The supernatant was then dried with nitrogen and reconstituted to obtain the sample to be tested. Two-dimensional liquid chromatography-mass spectrometry: The highly polar ultrashort chain PFAS in the sample are separated by one-dimensional liquid chromatography, and the remaining target PFAS are captured by a trapping column and replenished with liquid. The captured target PFAS are separated by two-dimensional liquid chromatography and qualitative and quantitative analysis is performed by mass spectrometry. The PFAS includes: TFA, PFPrA, PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnDA, PFDoDA, Ntf2, PFMeS, PFEtS, PFPrS, PFBS, PFPeS, PFHxS, PFHpS, PFOS, PF5OHxA, PFEESA, 3,6-OPFHpA, ADONA, HFPO-DA, 6:2 Cl-PFESA, 8:2 Cl-PFESA, 4:2 FTSA, 6:2 FTSA, 8:2 FTSA, PFMOAA, PFO2HxA, PFO3OA, PFO4DA, PFO5DoDA, C7 HFPO-TA, C8 HFPO-TA, C9 HFPO-TA, HFPO-TeA, Genx-CF3, PFO2OA, PFO3DA, H-PFMO2OSA.

2. The method for simultaneous determination of full-chain long-chain perfluorinated and polyfluoroalkyl compounds in a biological matrix using two-dimensional liquid chromatography-mass spectrometry according to claim 1, characterized in that, The preprocessing method specifically includes the following steps: (1) Add the biological matrix sample to a centrifuge tube and add the isotope internal standard solution; (2) Add acetonitrile, shake, sonicate, and then freeze the centrifuge tubes; (3) After centrifuging the mixed liquid treated in step (2), take the supernatant into a new centrifuge tube and perform nitrogen blowing treatment; (4) After nitrogen blowing treatment, add methanol-water solution, mix well, centrifuge, and take the supernatant into the sample vial for testing.

3. The method for simultaneous determination of full-chain long-chain perfluorinated and polyfluoroalkyl compounds in biological matrices using two-dimensional liquid chromatography-mass spectrometry according to claim 2, characterized in that, The biological matrix includes serum, urine, and breast milk.

4. The method for simultaneous determination of full-chain long-chain perfluorinated and polyfluoroalkyl compounds in a biological matrix using two-dimensional liquid chromatography-mass spectrometry according to claim 2, characterized in that, The method of step (2) is as follows: Add acetonitrile in three times the volume of the biological matrix sample, shake the mixture at 900 rpm for 15 min using a multi-tube vortex mixer, sonicate at room temperature for 15 min, and then place the centrifuge tube containing the mixture at -40°C for 6-8 hours.

5. The method for simultaneous determination of full-chain long-chain perfluorinated and polyfluoroalkyl compounds in a biological matrix using two-dimensional liquid chromatography-mass spectrometry according to claim 2, characterized in that, The method of step (3) is as follows: Centrifuge at 4400 rpm for 15 min, transfer the supernatant to a new centrifuge tube, and place it under nitrogen blowing at 25°C.

6. The method for simultaneous determination of full-chain long-chain perfluorinated and polyfluoroalkyl compounds in a biological matrix using two-dimensional liquid chromatography-mass spectrometry according to claim 2, characterized in that, The method of step (4) is as follows: Add a methanol-water solution with a volume ratio of 9:1 to the centrifuge tube dried in step (3), vortex at 900 rpm for 5 min and mix well. Centrifuge the mixed liquid at 12000 rpm for 15 min and take the supernatant into a sample vial for testing.

7. The method for simultaneous determination of full-chain long-chain perfluorinated and polyfluoroalkyl compounds in a biological matrix using two-dimensional liquid chromatography-mass spectrometry according to claim 1, characterized in that, The one-dimensional liquid chromatography conditions are as follows: The chromatographic column is a hydrophilic interaction and ion exchange composite packing column; the flow rate is 0.2 mL / min, the column temperature is 40℃, and the injection volume is 10 μL; mobile phase A is an aqueous solution of 10 mM ammonium formate and 0.1% formic acid, mobile phase B is an acetonitrile solution of 0.1% formic acid, and the isocratic elution program is 0~30 min, 10% A, 90% B; The two-dimensional liquid chromatography conditions are as follows: The chromatographic column was a C18 packed column; the flow rate was 0–0.5 mL / min, and the column temperature was 40 °C; mobile phase C was an aqueous solution of 5 mM ammonium acetate, and mobile phase D was pure methanol. The gradient elution program was as follows: initial 98% C, 2% D, flow rate 0.3 mL / min; 0.0–1.0 min, 98% C, 2% D, flow rate 0.3 mL / min; 1.0–1.1 min, 98%–100% C, 2%–0% D, flow rate 0.3 mL / min; 1.1–7.0 min, 100% C, 0% D, flow rate 0.5 mL / min; 7.0–11.0 min, 100%–98% C, 0%–2% D, flow rate 0 mL / min; 11.0–11.5 min, 98%–90% C, 2%–10% D, flow rate 0 mL / min; 11.5 min… ~16.5 min, 90%~45%C, 10%~55%D, flow rate 0.3 mL / min; 16.5~17.5 min, 45%~30%C, 55%~70%D, flow rate 0.3 mL / min; 17.5~20.0 min, 30%~10%C, 70%~90%D, flow rate 0.3 mL / min; 20.0~28.0 min, 10 28.0~29.0 min, 0%C, 100%D, flow rate 0.3 mL / min; 29.0~29.1 min, 0%~98%C, 100%~2%D, flow rate 0.3 mL / min; 29.1~30.0 min, 98%C, 2%D, flow rate 0.3 mL / min.

8. The method for simultaneous determination of full-chain long-chain perfluorinated and polyfluoroalkyl compounds in a biological matrix using two-dimensional liquid chromatography-mass spectrometry according to claim 1, characterized in that, The conditions for capture are: The trapping column was a Polaris 180A C18-A; the dosing solution was water as the mobile phase C; the column temperature was 40℃; the dosing time was 1.1~7.0 min; and the dosing flow rate was 0.5 L / min.

9. The method for simultaneous determination of full-chain long-chain perfluorinated and polyfluoroalkyl compounds in a biological matrix using two-dimensional liquid chromatography-mass spectrometry according to claim 1, characterized in that, The mass spectrometry conditions are as follows: Electrospray ionization source was used, and the measurement was performed under multiple reaction monitoring (MRM) with negative ion scanning mode. Specific parameter settings were as follows: drying gas temperature 100℃, sheath gas temperature 200℃, drying gas flow rate 11L / min, sheath gas flow rate 11L / min, nebulizer voltage 25psi, capillary voltage 2500V, and collision cell acceleration voltage 5V.

10. The method for simultaneous determination of full-chain long-chain perfluorinated and polyfluoroalkyl compounds in a biological matrix using two-dimensional liquid chromatography-mass spectrometry according to claim 1, characterized in that, The two-dimensional liquid chromatography-mass spectrometry system includes two valves, and different combinations of chromatographic columns can be achieved by switching the positions of the two valves. The valve switching program is as follows: 0.0~7.0 min, during which the sample outlet of the one-dimensional chromatographic column is connected to the trapping column and flows into the mass spectrometer; 7.0~11.0 min, during which the sample outlet of the one-dimensional chromatographic column flows directly into the mass spectrometer. 11.0~30.0 min, at which point the trapping column is connected to the two-dimensional chromatography column and flows into the mass spectrometer.

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