A method for simultaneous detection of per- and polyfluoroalkyl substances in urine

Abstract

The present invention discloses a method for synchronous detection of perfluorinated and polyfluoroalkyl substances in urine. It relates to the technical field of pollutant detection. The above-mentioned method for synchronous detection of perfluorinated and polyfluoroalkyl substances in urine is characterized in that it comprises the following steps: sample pretreatment: S1 adding a stable isotope-labeled internal standard compound to the urine sample to be tested; S2 adding an extractant to the sample of step S1 to extract the target analyte in the sample; S3 repeating step S2 1-3 times, and obtaining the sample to be tested after re-dissolving; detection of the sample to be tested: using ultra-high performance liquid chromatography-tandem mass spectrometry to detect the sample to be tested, so as to analyze at least 40 perfluorinated and polyfluoroalkyl substances in urine. The present invention realizes the synchronous extraction and analysis of at least 40 new and traditional PFASs in urine by improving the sample pretreatment and instrumental analysis methods, which is simple and convenient and saves solvents.

Description

Technical Field

[0001] The invention relates to the technical field of pollutant detection, and in particular to a method for synchronously detecting perfluoro and polyfluoroalkyl substances in urine. Background Art

[0002] Per-and polyfluoroalkyl substances (PFASs) are a class of synthetic chemicals widely used in commerce and industry. They are called "forever chemicals" because of the chemical and thermal stability of their CF bonds. PFASs can be discharged into the environment at every step of their product life cycle. A large number of studies have shown that traditional perfluoroalkyl sulfonic acids (PFSAs) and perfluoroalkyl carboxylic acids (PFCAs) are widely detected in various environmental media and organisms, and have significant mobility, bioaccumulation and high toxicity. Studies have shown that PFASs can enter the human body through food, drinking water, skin contact and breathing, and pose a potential threat to human health.

[0003] PFASs are protein-affinity. After entering the human body, they are mainly concentrated in protein-rich tissues and organs such as the liver and blood, and are mainly excreted from the body through urine. The composition of urine samples is complex, and PFASs exist in trace amounts in the samples. Currently, related research mainly focuses on traditional PFASs and a few alternatives, and there is a lack of systematic research on new PFASs. However, the production and application of new PFASs have developed so rapidly that the structural characteristics of many new alternatives have not been fully studied, making it impossible to accurately evaluate the true level of new PFASs in the population.

[0004] Existing analytical methods mainly target traditional PFASs, and have problems such as large solvent usage and sample consumption, high detection limits, long time consumption, and poor accuracy and repeatability of recovery rates. Since PFASs can be exposed to the human body at low doses for a long time and produce many adverse health effects, it is urgent to establish an analytical method for the simultaneous extraction and analysis of new and traditional PFASs in urine to assess the exposure levels of new and traditional PFASs in the population, and to provide necessary conditions for further exploring the impact of exposure to new PFASs alternatives on human health issues. Summary of the invention

[0005] The technical problems to be solved by the present invention are:

[0006] A method for simultaneous detection of per- and polyfluoroalkyl substances in urine is provided.

[0007] In order to solve the technical problem, the technical solution adopted by the present invention is:

[0008] A method for simultaneous detection of perfluoroalkyl and polyfluoroalkyl substances in urine comprises the following steps:

[0009] Sample pretreatment:

[0010] S1 adds a stable isotope-labeled internal standard compound to the urine sample to be tested;

[0011] S2 adds an extractant to the sample of step S1 to extract the target analyte in the sample;

[0012] S3 repeats step S2 1-3 times to obtain the sample to be tested after re-dissolving;

[0013] Testing of samples to be tested:

[0014] Ultra-high performance liquid chromatography-tandem mass spectrometry was used to detect the samples.

[0015] The recovery rate of each target compound was calculated by the following formula ①;

[0016] Recovery rate (%) = (C ss -C s ) / C st ×100 ①

[0017] Among them, C ss is the concentration of the spiked sample, i.e. the measured value of the spiked sample; C s is the concentration of the unspiked sample, that is, the measured value of the unspiked sample, also known as the matrix blank; C st is the spike concentration.

[0018] According to the embodiments of the present invention, one of the technical solutions has at least one of the following advantages or beneficial effects:

[0019] 1. The present invention provides a method for simultaneous detection of perfluoroalkyl and polyfluoroalkyl substances in urine, which overcomes many shortcomings of existing detection technologies, especially the problem that most existing detection methods focus on traditional PFASs (perfluoroalkyl sulfonic acids (PFSAs) and perfluoroalkyl carboxylic acids (PFCAs)) and a few substitutes. The present invention achieves simultaneous extraction and analysis of at least 40 new and traditional PFASs in urine by improving sample pretreatment and instrumental analysis methods, which is simple and convenient and saves solvents. At the same time, the average recovery rate of the method of the present invention ranges from 70% to 123%, and the intra-day and inter-day relative standard deviations are within 19% and 26%, respectively; the linearity is good, and R 2 Both were greater than 0.99; the sensitivity was high, and the LOQs and LODs of the method were 0.001ng / mL~3ng / mL and 0.0003ng / mL~0.9ng / mL.

[0020] 2. As for the solvent saving, the total consumption of solvent required for pretreatment of the existing solid phase extraction method is at least 16.72mL, while the total consumption of solvent required for pretreatment of the method of the present invention is at most 2mL. Compared with the solid phase extraction method, the amount of solvent required for pretreatment is only 11.96% of the amount of solvent used in the solid phase extraction method. Obviously, the method of the present invention can save more solvent, is simpler and more convenient, does not require solid phase extraction column enrichment and purification, and is more economical and environmentally friendly.

[0021] 3. Regarding the recovery rate, the average recovery rate of the method of the present invention is in the range of 70% to 123%, while the average recovery rate of the existing methods, such as solid phase extraction, is in the range of 4% to 145%, which is much lower than the recovery rate of the present invention.

[0022] 4. The present invention provides a method for the simultaneous detection of perfluorinated and polyfluoroalkyl substances in urine, which overcomes many shortcomings of existing detection technologies, analyzes 40 new and traditional PFASs, simplifies the detection steps, reduces the detection cost, and provides the necessary conditions for further exploring the impact of exposure to new PFASs alternatives on health issues, thereby expanding the application scope of health exposure risk assessment.

[0023] According to one embodiment of the present invention, the concentration of the matrix in the urine to be tested is 0.1-20 ng / mL.

[0024] According to one embodiment of the present invention, the concentration of the matrix in the urine to be tested is 0.1-10 ng / mL.

[0025] According to one embodiment of the present invention, the concentration of the matrix in the urine to be tested is greater than or equal to 0.001 ng / mL.

[0026] According to one embodiment of the present invention, the urine sample to be tested is at least one of human or animal urine.

[0027] According to one embodiment of the present invention, the step of adding a stable isotope-labeled internal standard compound to the urine sample to be tested comprises the following steps:

[0028] Add 0.4-20 ng of the mixed internal standard compound labeled with a stable isotope to 0.2-2 mL of the urine sample to be tested, place it in a 1.5-50 mL centrifuge tube, cover it tightly, and vortex for 0.2-5 min to mix the sample evenly.

[0029] According to one embodiment of the present invention, the ratio of the volume of the urine sample to be tested in step S1 to the volume of the extractant in step S2 is 1-1.5:1-1.5.

[0030] According to one embodiment of the present invention, the extractant comprises acetonitrile.

[0031] According to one embodiment of the present invention, the extractant also includes ethyl acetate, but ethyl acetate can only extract part of the perfluoroalkyl and polyfluoroalkyl substances and cannot completely extract at least 40 perfluoroalkyl and polyfluoroalkyl substances.

[0032] According to one embodiment of the present invention, in step S2, after extracting the target analyte from the sample, the step further includes vortexing, vibrating, adding a phase separation inducer, vortexing, centrifuging, and then extracting the supernatant.

[0033] According to one embodiment of the present invention, the phase separation inducer includes NaCl. The function of the phase separation inducer is to induce phase separation between the organic phase and the aqueous phase, thereby obtaining stratification.

[0034] According to one embodiment of the present invention, the vortexing time is 0.2 to 5 minutes.

[0035] According to one embodiment of the present invention, the vortexing time is 0.5 to 5 minutes.

[0036] According to one embodiment of the present invention, the oscillation time is 0.2 to 3 hours.

[0037] According to one embodiment of the present invention, the oscillation time is 0.5 to 3 hours.

[0038] According to one embodiment of the present invention, the amount of the phase separation inducer is 0.05 to 0.7 g.

[0039] According to one embodiment of the present invention, the amount of the phase separation inducer is 0.05 to 0.1 g.

[0040] According to one embodiment of the present invention, the amount of the phase separation inducer is 0.1 to 0.7 g.

[0041] According to one embodiment of the present invention, the centrifugal rotation speed is 1200-18500 rpm.

[0042] According to one embodiment of the present invention, the centrifugal rotation speed is 1200-8000 rpm.

[0043] According to one embodiment of the present invention, the ratio of the volume of the urine sample to be tested in step S1 to the volume of the solvent required for reconstitution in step S3 is 1-1.5:1-1.5.

[0044] According to one embodiment of the present invention, step S3 is: repeating step S2 1 to 2 times to obtain a sample to be tested after redissolution.

[0045] Repeat step S2 1 to 2 times, and then re-dissolve to obtain the sample to be tested. When the number of extractions was 1 and 2, the average recovery rates of PFNA and PFHxS of traditional PFASs were high or low, the average recovery rates of N-EtFOSAA, PAPs and PFPAs in new PFASs were lower than 70%, and the average recovery rates of 6:8PFPi, 8:8PFPi and diPAPs were higher than 120%.

[0046] According to one embodiment of the present invention, step S3 is: repeating step S2 2 to 3 times, and obtaining the sample to be tested after re-dissolving. Preferably, step S3 is: repeating step S2 3 times, and obtaining the sample to be tested after re-dissolving.

[0047] For the most preferred method, step S2 is repeated three times, and the sample to be tested is obtained after redissolution. The average recovery rates of new and traditional PFASs in urine can be between 77.25% and 117.93% and 73.33% and 119.00%, respectively. In one embodiment of the present invention, when the most preferred extraction step is repeated three times, the optimal recovery rate can be obtained.

[0048] According to one embodiment of the present invention, the reagent used for reconstitution includes methanol.

[0049] According to one embodiment of the present invention, the amount of methanol used is 0.2 to 1.5 mL.

[0050] According to one embodiment of the present invention, the amount of methanol used is 0.2 to 0.5 mL.

[0051] According to one embodiment of the present invention, the amount of methanol used is 0.5 to 1.5 mL.

[0052] According to one embodiment of the present invention, step S3 further includes the step of filtering the sample to be tested through a filter after re-dissolution.

[0053] According to one embodiment of the present invention, filtering includes the step of filtering using a filter. The filter includes at least one of a filter membrane and a syringe.

[0054] According to one embodiment of the present invention, the filter is made of at least one of polyethersulfone (PES), polyamide, mixed cellulose, polypropylene, and glass fiber.

[0055] According to one embodiment of the present invention, the filter is made of polyamide. Preferably, the polyamide is nylon 66.

[0056] According to one embodiment of the present invention, the filter is a needle filter.

[0057] According to one embodiment of the present invention, the diameter of the needle filter is 10 to 15 mm.

[0058] According to one embodiment of the present invention, the diameter of the needle filter is 10-13 mm.

[0059] According to one embodiment of the present invention, the diameter of the needle filter is 13-15 mm.

[0060] According to one embodiment of the present invention, the pore size of the filter is 0.1-0.6 μm, preferably one of 0.2, 0.22, 0.45 or 0.6 μm, and preferably, the pore size of the filter is 0.22 μm.

[0061] According to one embodiment of the present invention, in the ultra-high performance liquid chromatography-tandem mass spectrometry, the mobile phases used include mobile phase A and mobile phase B, the mobile phase A includes a first acidic solution, and the mobile phase B includes a second acidic solution and an alcohol solution.

[0062] According to one embodiment of the present invention, the first acidic solution and the second acidic solution include formic acid solutions.

[0063] According to one embodiment of the present invention, the alcohol solution includes a methanol solution.

[0064] According to one embodiment of the present invention, the volume fraction of the mobile phase A is 0.01-0.5%, and the volume fraction of the mobile phase B is 0.01-0.5%.

[0065] According to one embodiment of the present invention, the volume fraction of the mobile phase A is 0.1-0.2%, preferably 0.1%.

[0066] According to one embodiment of the present invention, the volume fraction of the mobile phase B is 0.1-0.2%, preferably 0.1%.

[0067] According to one embodiment of the present invention, in the ultra high performance liquid chromatography-tandem mass spectrometry, the ultra high performance liquid chromatography adopts the American Agilent 1290 ultra high performance liquid chromatograph.

[0068] According to one embodiment of the present invention, in the ultra-high performance liquid chromatography-tandem mass spectrometry, the ultra-high performance liquid chromatography uses an Agilent ZOBRAX SB-C18 chromatographic column with a length of 100 mm, an inner diameter of 2.1 mm, and a filler particle diameter of 3.5 μm.

[0069] According to one embodiment of the present invention, in the ultra-high performance liquid chromatography-tandem mass spectrometry, the column temperature of the ultra-high performance liquid chromatography is 20-25°C.

[0070] According to one embodiment of the present invention, in the ultra-high performance liquid chromatography-tandem mass spectrometry, the column temperature of the ultra-high performance liquid chromatography is 20-23°C.

[0071] According to one embodiment of the present invention, in the ultra-high performance liquid chromatography-tandem mass spectrometry, the injection volume is 1 to 3 μL, preferably 3 μL.

[0072] According to one embodiment of the present invention, in the ultra-high performance liquid chromatography-tandem mass spectrometry, the flow rate is 0.1 to 0.5 mL / min.

[0073] According to one embodiment of the present invention, in the ultra-high performance liquid chromatography-tandem mass spectrometry, the flow rate is 0.1 to 0.3 mL / min.

[0074] According to one embodiment of the present invention, in the ultra-high performance liquid chromatography-tandem mass spectrometry, the flow rate is 0.3 to 0.5 mL / min.

[0075] According to one embodiment of the present invention, in the ultra-high performance liquid chromatography-tandem mass spectrometry, the elution method used is gradient elution, and the elution program of the gradient elution is as shown in Table 1:

[0076] Table 1

[0077] Time (min) Mobile phase A (%) Mobile phase B (%) Flow rate (mL / min) 0～7 80～99 1～20 0.3～0.5 7～8.5 15～35 65～85 0.3～0.5 8.5～10.5 0～10 90～100 0.3～0.5 10.5～12 0～10 90～100 0.3～0.5 12～18 80～99 1-20 0.3～0.5

[0078] According to one embodiment of the present invention, in the ultra-high performance liquid chromatography-tandem mass spectrometry, the triple quadrupole mass spectrometry adopts an American AB SCIEX 5500 mass spectrometer.

[0079] According to one embodiment of the present invention, in the ultra-high performance liquid chromatography-tandem mass spectrometry, the triple quadrupole mass spectrometry adopts an electrospray ion source and a negative ion mode.

[0080] According to one embodiment of the present invention, in the ultra-high performance liquid chromatography-tandem mass spectrometry, the acquisition mode of the heavy quadrupole mass spectrometry is a multiple reaction monitoring mode.

[0081] According to one embodiment of the present invention, the ion source temperature of the triple quadrupole mass spectrometry is 250-450°C, and the ionization voltage is 250-450V.

[0082] According to one embodiment of the present invention, the data processing system of the triple quadrupole mass spectrometry is Analystsoftware.

[0083] According to one embodiment of the present invention, the perfluoro and polyfluoroalkyl substances (PFASs) include at least one of perfluoroalkyl sulfonic acid, perfluoroalkyl carboxylic acid, fluoropolymer sulfonic acid, fluoropolymer unsaturated carboxylic acid, perfluorooctane sulfonamide, chlorinated polyfluoro ether sulfonic acid, hexafluoropropylene oxide dimer acid, 4,8-dioxy-3-hydrogen-polyfluorononanoic acid ammonium, perfluoroethane cyclohexane sulfonate, perfluoroalkyl phosphinic acid, polyfluoroalkyl phosphate, polyfluoroalkyl phosphoric diester and perfluorophosphoric acid.

[0084] According to one embodiment of the present invention, the perfluoro and polyfluoroalkyl substances include perfluorobutyl carboxylic acid (PFBA), perfluoropentyl carboxylic acid (PFPeA), perfluorohexyl carboxylic acid (PFHxA), perfluoroheptyl carboxylic acid (PFHpA), perfluorononyl carboxylic acid (PFNA), perfluorodecyl carboxylic acid (PFDA), perfluoroundecyl carboxylic acid (PFUnDA), perfluorododecyl carboxylic acid (PFDoDA), perfluorotridecyl carboxylic acid (PFTrDA), perfluorotetradecyl carboxylic acid (PFTeDA), perfluorobutyl sulfonic acid (PFBS), perfluorohexyl sulfonic acid (PFBS), Acid (PFHxS), perfluorooctane sulfonic acid (PFOS), perfluorononyl sulfonic acid (PFNS), perfluorodecane sulfonic acid (PFDS), 4:2 fluorotelomer sulfonic acid (4:2FTSA), 6:2 fluorotelomer sulfonic acid (6:2FTSA), 8:2 fluorotelomer sulfonic acid (8:2FTSA), 6:2 fluorotelomer unsaturated carboxylic acid (6:2FTUCA), 8:2 fluorotelomer unsaturated carboxylic acid (8:2FTUCA), 10:2 fluorotelomer unsaturated carboxylic acid (10:2FTUCA), perfluorooctane sulfonamide (FOSA), 2-(N-methyl perfluorooctane sulfonamide)acetic acid (N-MeFOSAA), 2-(N-ethyl perfluorooctane sulfonamide)acetic acid (N-EtFOSAA), 6:2 chlorinated polyfluoroether sulfonic acid (6:2Cl-PFESA), 8:2 chlorinated polyfluoroether sulfonic acid (8:2Cl-PFESA), hexafluoropropylene oxide dimer acid (HFPO-DA), 4,8-dioxy-3-hydro-polyfluorononanoic acid ammonium (ADONA), perfluoroethane cyclohexane sulfonate (PFECHS), 6:6 perfluoroalkyl phosphinic acid (6:6PFPi), 6:8 perfluoroalkyl phosphinic acid At least one of phosphonic acid (6:8PFPi), 8:8 perfluoroalkyl phosphinic acid (8:8PFPi), 6:2 polyfluoroalkyl phosphate (6:2PAP), 6:2 polyfluoroalkyl phosphate diester (6:2diPAP), 8:2 polyfluoroalkyl phosphate (8:2PAP), 8:2 polyfluoroalkyl phosphate diester (8:2diPAP), 6:2 / 8:2 polyfluoroalkyl phosphate diester (6:2 / 8:2diPAP), perfluorohexylphosphonic acid (PFHxPA), perfluorooctylphosphonic acid (PFOPA), and perfluorodecylphosphonic acid (PFDPA).

[0085] According to one embodiment of the present invention, during the detection of test samples by ultra-high performance liquid chromatography-tandem mass spectrometry, a standard curve is established to calculate the average recovery rate, intra-day and inter-day relative standard deviation of at least 40 PFASs in urine.

[0086] Other features and advantages of the present invention will be set forth in the description which follows, and in part will be apparent from the description, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0088] Figure 1 This is a flow chart of the steps of the method for simultaneous detection of perfluoro and polyfluoroalkyl substances in urine in Example 1.

[0089] Figure 2 This is the extraction chromatogram of perfluorobutylcarboxylic acid to perfluorodecylcarboxylic acid in the target analytes of Example 1.

[0090] Figure 3 This is the extraction chromatogram of perfluoroundecyl carboxylic acid to perfluorohexane sulfonic acid in the target analytes in Example 1.

[0091] Figure 4 This is the extraction chromatogram of perfluorooctane sulfonic acid to 8:2 fluorotelomer sulfonic acid in the target analyte of Example 1.

[0092] Figure 5 This is the extraction chromatogram of the target analyte in Example 1, 6:2 fluorotelomerization of unsaturated carboxylic acid to 2-(N-ethylperfluorooctanesulfonamide)acetic acid.

[0093] Figure 6 This is the extraction chromatogram of 6:2 chlorinated polyfluoroether sulfonic acid to 6:6 perfluoroalkyl hypophosphorous acid in the target analyte of Example 1.

[0094] Figure 7 This is the extraction chromatogram of 6:8 perfluoroalkyl hypophosphite to 8:2 polyfluoroalkyl phosphoric acid diester in the target analyte of Example 1.

[0095] Figure 8 This is the extraction chromatogram of 6:2 / 8:2 polyfluoroalkyl phosphoric acid diester to perfluorodecyl phosphoric acid in the target analyte of Example 1.

[0096] Fig. 9 This is a test chart of the recovery rate test of 15 target analytes of different concentrations using the method of Example 1.

[0097] Fig.10This is a test chart of the spiked recovery test of 25 target analytes of different concentrations using the method of Example 1.

[0098] Fig.11 The figure is a test chart for comparing the average recovery rates of the methods of Example 1 and the comparative example.

[0099] Fig.12 This is a test chart for comparing the average recovery rates of the methods of Examples 1-3. DETAILED DESCRIPTION

[0100] In the description of the present invention, if there is a description of first, second, etc., it is only for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0101] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of the present invention.

[0102] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0103] Example 1

[0104] A method for simultaneous detection of perfluorinated and polyfluoroalkyl substances in urine, the detection method steps are as follows Figure 1 Specifically, the following steps are included:

[0105] S1 sample pretreatment:

[0106] (1) Add 2.5 ng of the stable isotope-labeled mixed internal standard compound to 0.5 mL of the urine sample to be tested, place in a 15 mL polyethylene centrifuge tube, cover tightly, and vortex for 0.5 min to mix the sample evenly;

[0107] (2) Then, 0.5 mL of acetonitrile was added as an extraction solvent to extract the target analytes in the sample. After vortexing for 30 seconds and shaking for 30 minutes, 0.1 g of NaCl was added and vortexed for 30 seconds. The sample was centrifuged at 8000 rpm for 15 minutes and the supernatant was extracted.

[0108] (3) Repeat the above step (2) three times;

[0109] (4) Collect and combine the supernatants of the three extractions in another 15 mL centrifuge tube, blow nitrogen to dryness, and then reconstitute with 0.5 mL of methanol; install an organic phase syringe filter with a diameter of 13 mm and a pore size of 0.22 μm between the syringe and the needle of a sterile syringe with a needle, inject the reconstituted solution into the syringe, filter it through the syringe filter, and collect it in a 9 mm threaded brown glass injection bottle;

[0110] S2 Sample detection: UPLC-MS / MS was used to analyze the pretreated samples, and a standard curve was established to calculate the average recovery rate, intra-day and inter-day relative standard deviation of 40 PFASs in urine. When the recovery rate was in the range of 70% to 120%, the method had good accuracy.

[0111] The target substances in urine include: perfluorobutyl carboxylic acid (PFBA), perfluoropentyl carboxylic acid (PFPeA), perfluorohexyl carboxylic acid (PFHxA), perfluoroheptyl carboxylic acid (PFHpA), perfluorononyl carboxylic acid (PFNA), perfluorodecyl carboxylic acid (PFDA), perfluoroundecyl carboxylic acid (PFUnDA), perfluorododecyl carboxylic acid (PFDoDA), perfluorotridecyl carboxylic acid (PFTrDA), perfluorotetradecyl carboxylic acid (PFTeDA), perfluorobutane sulfonic acid (PFBS), perfluorohexane sulfonic acid (PFH xS), perfluorooctane sulfonic acid (PFOS), perfluorononane sulfonic acid (PFNS), perfluorodecane sulfonic acid (PFDS), 4:2 fluorotelomer sulfonic acid (4:2FTSA), 6:2 fluorotelomer sulfonic acid (6:2FTSA), 8:2 fluorotelomer sulfonic acid (8:2FTSA), 6:2 fluorotelomer unsaturated carboxylic acid (6:2FTUCA), 8:2 fluorotelomer unsaturated carboxylic acid (8:2FTUCA), 10:2 fluorotelomer unsaturated carboxylic acid (10:2FTUCA), perfluorooctane sulfonamide (FOSA), 2-(N-methylperfluorooctanesulfonamide) 2-(N-ethylperfluorooctanesulfonamide)acetic acid (N-MeFOSAA), 2-(N-ethylperfluorooctanesulfonamide)acetic acid (N-EtFOSAA), 6:2 chlorinated polyfluoroethersulfonic acid (6:2Cl-PFESA), 8:2 chlorinated polyfluoroethersulfonic acid (8:2Cl-PFESA), hexafluoropropylene oxide dimer acid (HFPO-DA), 4,8-dioxy-3-hydro-polyfluorononanoic acid ammonium (ADONA), perfluoroethane cyclohexane sulfonate (PFECHS), 6:6 perfluoroalkyl hypophosphorous acid (6:6PFPi), 6:8 perfluoroalkyl Hypophosphorous acid (6:8PFPi), 8:8 perfluoroalkyl hypophosphorous acid (8:8PFPi), 6:2 polyfluoroalkyl phosphate (6:2PAP), 6:2 polyfluoroalkyl phosphate diester (6:2diPAP), 8:2 polyfluoroalkyl phosphate (8:2PAP), 8:2 polyfluoroalkyl phosphate diester (8:2diPAP), 6:2 / 8:2 polyfluoroalkyl phosphate diester (6:2 / 8:2diPAP), perfluorohexaalkyl phosphoric acid (PFHxPA), perfluorooctane phosphoric acid (PFOPA), and perfluorodecane phosphoric acid (PFDPA).

[0112] The extraction chromatograms of the above target analytes are shown in Figure 2-Figure 8 shown.

[0113] Specifically, Figure 2 The extracted chromatograms are perfluorobutylcarboxylic acid (PFBA), perfluoropentylcarboxylic acid (PFPeA), perfluorohexylcarboxylic acid (PFHxA), perfluoroheptylcarboxylic acid (PFHpA), perfluorononylcarboxylic acid (PFNA), and perfluorodecylcarboxylic acid (PFDA).

[0114] Specifically, Figure 3These are the extraction chromatograms of perfluoroundecylcarboxylic acid (PFUnDA), perfluorododecylcarboxylic acid (PFDoDA), perfluorotridecylcarboxylic acid (PFTrDA), perfluorotetradecylcarboxylic acid (PFTeDA), perfluorobutane sulfonic acid (PFBS), and perfluorohexane sulfonic acid (PFHxS).

[0115] Specifically, Figure 4 These are the extraction chromatograms of perfluorooctane sulfonic acid (PFOS), perfluorononane sulfonic acid (PFNS), perfluorodecane sulfonic acid (PFDS), 4:2 fluorotelomer sulfonic acid (4:2FTSA), 6:2 fluorotelomer sulfonic acid (6:2FTSA), and 8:2 fluorotelomer sulfonic acid (8:2FTSA).

[0116] Specifically, Figure 5 These are the extraction chromatograms of 6:2 fluoropolymeric unsaturated carboxylic acid (6:2FTUCA), 8:2 fluoropolymeric unsaturated carboxylic acid (8:2FTUCA), 10:2 fluoropolymeric unsaturated carboxylic acid (10:2FTUCA), perfluorooctane sulfonamide (FOSA), 2-(N-methylperfluorooctane sulfonamide)acetic acid (N-MeFOSAA), and 2-(N-ethylperfluorooctane sulfonamide)acetic acid (N-EtFOSAA).

[0117] Specifically, Figure 6 The extracted chromatograms are 6:2 chlorinated polyfluoroether sulfonic acid (6:2Cl-PFESA), 8:2 chlorinated polyfluoroether sulfonic acid (8:2Cl-PFESA), hexafluoropropylene oxide dimer acid (HFPO-DA), 4,8-dioxy-3-hydro-polyfluorononanoic acid ammonium (ADONA), perfluoroethane cyclohexane sulfonate (PFECHS), and 6:6 perfluoroalkyl hypophosphorous acid (6:6PFPi).

[0118] Specifically, Figure 7 These are the extraction chromatograms of 6:8 perfluoroalkyl hypophosphorous acid (6:8PFPi), 8:8 perfluoroalkyl hypophosphorous acid (8:8PFPi), 6:2 polyfluoroalkyl phosphate (6:2PAP), 6:2 polyfluoroalkyl phosphate diester (6:2diPAP), 8:2 polyfluoroalkyl phosphate (8:2PAP), and 8:2 polyfluoroalkyl phosphate diester (8:2diPAP).

[0119] Specifically, Figure 8 The extracted chromatograms are of 6:2 / 8:2 polyfluoroalkyl phosphoric acid diester (6:2 / 8:2diPAP), perfluorohexaalkyl phosphoric acid (PFHxPA), perfluorooctyl phosphoric acid (PFOPA), and perfluorodecyl phosphoric acid (PFDPA).

[0120] In the above-mentioned method for simultaneous detection of perfluoroalkyl and polyfluoroalkyl substances in urine, the detection conditions of ultra-high performance liquid chromatography are shown in Table 2.

[0121] Table 2

[0122]

[0123] In the above-mentioned simultaneous detection method for perfluorinated and polyfluoroalkyl substances in urine, the detection conditions of mass spectrometry are as follows:

[0124] AB SCIEX 5500 triple quadrupole mass spectrometer (AB SCIEX, Framingham. MA);

[0125] Ion source: ESI;

[0126] Source temperature: 600°C;

[0127] The ion spray voltage was -4500 V;

[0128] The air curtain gas is 35Psi;

[0129] Atomizing gas is 60Psi;

[0130] Auxiliary gas is 70Psi;

[0131] The collision gas is 9Psi;

[0132] Monitoring mode: Multiple reaction monitoring (MRM).

[0133] In the above-mentioned simultaneous detection method for perfluoroalkyl and polyfluoroalkyl substances in urine, the mass spectrometry parameters are shown in Table 3.

[0134] Table 3

[0135]

[0136]

[0137] In the above-mentioned simultaneous detection method for perfluorinated and polyfluoroalkyl substances in urine, the basic information of the target analytes is shown in Table 4.

[0138] Table 4

[0139]

[0140]

[0141] Example 2

[0142] The difference between Example 2 and Example 1 is that:

[0143] In the S1 sample pretreatment of Example 2, step (3) is: repeating the above step (2) once.

[0144] In the pretreatment of the S1 sample in Example 1, step (3) is: repeating the above step (2) three times.

[0145] Example 3

[0146] The difference between Example 3 and Example 1 is that:

[0147] In the S1 sample pretreatment of Example 3, step (3) is: repeating the above step (2) twice.

[0148] In the pretreatment of the S1 sample in Example 1, step (3) is: repeating the above step (2) three times.

[0149] Comparative Example

[0150] A method for pre-treatment and detection of 40 new and traditional PFASs using traditional solid phase extraction, comprising the following steps:

[0151] Nine human urine samples were mixed evenly, and the target analyte was added at 10 ng / mL. Three parallels were set up, and a urine sample with only the internal standard solution added was set up as a blank control. The sample spike recovery rate was corrected using the isotope internal standard method, and the internal standards corresponding to the target analytes are shown in Table 3.

[0152] The recoveries of 40 PFASs in urine were compared using a WAX solid phase extraction column with a filler of 60 mg and a specification of 3 mL.

[0153] The specific steps are as follows:

[0154] S1 sample pretreatment:

[0155] (1) Take 2 mL of urine sample into a 15 mL PP tube, add 10 ng / mL of the original standard solution, set up 3 parallel tubes, add 5 ng / mL of the internal standard solution, and set up a urine sample with only the internal standard added as a blank control. Vortex and shake for 30 minutes. Dilute the urine sample to 4 mL with 0.1 M formic acid.

[0156] (2) Activation: The solid phase extraction column was eluted with 4 mL of 1% ammonia methanol solution, 4 mL of methanol, and 4 mL of 0.1 M formic acid in sequence;

[0157] (3) Sample loading: Load the urine sample diluted to 4 mL;

[0158] (4) Wash the column with 4 mL of 0.1 M formic acid and 4 mL of 0.1 M formic acid / methanol (v:v=1:1) to remove impurities, and then vacuum dry for 5 minutes;

[0159] (5) Elution: After eluting the analyte with 4 mL of 1% ammonia methanol solution, nitrogen blowing was performed;

[0160] (6) Concentration: Blow the eluate to near dryness with liquid nitrogen, and reconstitute it with a mixture of 100 μL HPLC grade methanol and 100 μL Milli-Q water. Install an organic phase syringe filter with a diameter of 13 mm and a pore size of 0.22 μm between the syringe and the needle of a sterile needle syringe, inject the reconstituted solution into the syringe, filter it through the syringe filter, and collect it in a 9 mm threaded brown glass injection bottle;

[0161] S2 Sample detection: UPLC-MS / MS was used to analyze the pretreated samples, and a standard curve was established to calculate the average recovery rate and intra-day relative standard deviation of 40 PFASs in urine. When the recovery rate was in the range of 70% to 120%, the method had good accuracy.

[0162] Performance Test:

[0163] Based on the simultaneous detection method for perfluoro and polyfluoroalkyl substances in urine in Example 1, sample spike recovery rate detection was performed to evaluate the accuracy of the method.

[0164] Nine human urine samples were mixed evenly and three concentration levels (0.1, 1 and 10 ng / mL) of target analytes were added. Five parallels were run for each concentration, and a urine sample with only the internal standard solution added was set as a blank control. The sample spike recovery was corrected using the isotope internal standard method, and the internal standards corresponding to the target analytes are shown in Table 3. Within one day, urine samples with three concentration levels (0.1, 1 and 10 ng / mL, n = 5) of the original were added for intraday evaluation; for three consecutive days, urine samples with three concentration levels (0.1, 1 and 10 ng / mL, n = 15) of the original were added for interday evaluation.

[0165] The relative standard deviations (RSDs) of the PFASs spike recoveries were calculated to evaluate the intra-day and inter-day precision, repeatability, and reproducibility of the method. The test results are shown in Table 5.

[0166] A bar graph of the test results, such as Fig. 9 and Fig.10 shown.

[0167] in, Fig. 9The following targets are recorded in the test: perfluorobutylcarboxylic acid (PFBA), perfluoropentylcarboxylic acid (PFPeA), perfluorohexylcarboxylic acid (PFHxA), perfluoroheptylcarboxylic acid (PFHpA), perfluorononylcarboxylic acid (PFNA), perfluorodecylcarboxylic acid (PFDA), perfluoroundecylcarboxylic acid (PFUnDA), perfluorododecylcarboxylic acid (PFDoDA), perfluorotridecylcarboxylic acid (PFTrDA), perfluorotetradecylcarboxylic acid (PFTeDA), perfluorobutane sulfonic acid (PFBS), perfluorohexane sulfonic acid (PFHxS), perfluorooctane sulfonic acid (PFOS), perfluorononane sulfonic acid (PFNS), and perfluorodecane sulfonic acid (PFDS).

[0168] in, Fig.10 The following targets were recorded: 4:2 fluorotelomer sulfonic acid (4:2FTSA), 6:2 fluorotelomer sulfonic acid (6:2FTSA), 8:2 fluorotelomer sulfonic acid (8:2FTSA), 6:2 fluorotelomer unsaturated carboxylic acid (6:2FTUCA), 8:2 fluorotelomer unsaturated carboxylic acid (8:2FTUCA), 10:2 fluorotelomer unsaturated carboxylic acid (10:2FTUCA), perfluorooctane sulfonamide (FOSA), 2-(N-methylperfluorooctane sulfonamide)acetic acid (N-MeFOSAA), 2-(N-ethylperfluorooctane sulfonamide)acetic acid (N-EtFOSAA), 6:2 chlorinated polyfluoroether sulfonic acid (6:2Cl-PFESA), 8:2 chlorinated polyfluoroether sulfonic acid (8:2Cl-PFESA), hexafluoropropylene oxide dimer acid (HFPO -DA), 4,8-dioxy-3-hydrogen-polyfluorononanoic acid ammonium (ADONA), perfluoroethane cyclohexane sulfonate (PFECHS), 6:6 perfluoroalkyl hypophosphorous acid (6:6PFPi), 6:8 perfluoroalkyl hypophosphorous acid (6:8PFPi), 8:8 perfluoroalkyl hypophosphorous acid (8:8PFPi), 6:2 polyfluoroalkyl phosphate (6:2PAP), 6:2 polyfluoroalkyl phosphate diester (6:2diPAP), 8:2 polyfluoroalkyl phosphate (8:2PAP), 8:2 polyfluoroalkyl phosphate diester (8:2diPAP), 6:2 / 8:2 polyfluoroalkyl phosphate diester (6:2 / 8:2diPAP), perfluorohexaalkyl phosphoric acid (PFHxPA), perfluorooctyl phosphoric acid (PFOPA), and perfluorodecyl phosphoric acid (PFDPA).

[0169] Table 5

[0170]

[0171]

[0172] Test correlation coefficient (R 2 ), LOD and LOQ, the test results are shown in Table 6.

[0173] Table 6

[0174]

[0175]

[0176] It can be seen from Table 5 that when the PFASs spike concentration is 10 ng / mL, the average recovery rate of the method ranges from 70% to 123%, the intra-day RSD is 2% to 19% (n=5), and the inter-day RSD is 4% to 26% (n=15). Therefore, the detection method of the present invention has good accuracy, repeatability and reproducibility, and meets the analysis requirements of new and traditional PFASs in urine.

[0177] The average recovery rate of the solid phase extraction method of Example 1 and the comparative example was tested:

[0178] During the test, the urine matrix was 10 ng / mL. The test method refers to the above spike recovery test. The test results are as follows Fig.11 shown.

[0179] The solvent consumption of the solid phase extraction method of Example 1 and the comparative example was compared and tested:

[0180] The neonicotinoid insecticides and their metabolites in surface water were detected by the detection method of Example 1 and the solid phase extraction method of the comparative example, respectively. The consumption of solvents required for pretreatment of the two detection methods was compared and summarized in Table 7.

[0181] The detection method is as follows:

[0182] (1) Take 2 mL of urine sample into a 15 mL PP tube, add 10 ng / mL of target analyte, set up 3 parallels, add 5 ng / mL of internal standard solution, and set up a urine sample with only internal standard added as a blank control. Vortex and shake for 30 minutes. Dilute the urine sample to 4 mL with 0.1 M formic acid and calculate the required solvent;

[0183] (2) Activation: Elute the solid phase extraction column with 4 mL of 1% methanolic ammonia solution, 4 mL of methanol, and 4 mL of 0.1 M formic acid in sequence, and calculate the required solvent;

[0184] (3) Sample loading: Load the urine sample diluted to 4 mL and calculate the required solvent;

[0185] (4) Wash the column with 4 mL of 0.1 M formic acid and 4 mL of 0.1 M formic acid / methanol (v:v = 1:1) at a rate of 4 mL / min to remove impurities, and then vacuum dry for 5 minutes to calculate the required solvent;

[0186] (5) Elution: After eluting the analyte with 4 mL of 1% methanolic ammonia solution, perform nitrogen blowdown and calculate the required solvent;

[0187] (6) Concentration: Blow the eluate to near dryness with liquid nitrogen, and reconstitute it with a mixture of 100 μL HPLC-grade methanol and 100 μL Milli-Q water. Install an organic phase syringe filter with a diameter of 13 mm and a pore size of 0.22 μm between the syringe and the needle of a sterile syringe with a needle, inject the reconstituted solution into the syringe, filter it through the syringe filter, and collect it in a 9 mm threaded brown glass injection bottle. Calculate the required solvent.

[0188] Table 7

[0189]

[0190] It can be seen from Table 7 that the total consumption of solvent required for pretreatment in Example 1 is 2 mL, and the total consumption of solvent required for pretreatment by solid phase extraction method in Comparative Example 1 is 16.72 mL. The results show that the detection method of Example 1 can save more solvent, is simpler and more convenient, does not require solid phase extraction column enrichment and purification, and is more economical and environmentally friendly.

[0191] The average recovery rates of the methods of Examples 1-3 were compared as follows:

[0192] Take 9 human urine samples and mix them evenly, and add 10 ng / mL of the target analyte. According to the method of Examples 1-3, set up three parallels, and set up a urine sample with only the internal standard solution added as a blank control. The sample spike recovery rate was corrected using the isotope internal standard method, and the internal standard corresponding to the target analyte is shown in Table 3.

[0193] The test results are as follows Fig.12 As shown. It can be seen that the average recovery rates of new and traditional PFASs in urine in Example 1 are between 70% and 121% and 81% and 123%, respectively. When the number of extractions is 1 and 2, the average recovery rates of PFNA and PFHxS of traditional PFASs are higher or lower, the average recovery rates of N-EtFOSAA, PAPs and PFPAs in new PFASs are lower than 70%, and the average recovery rates of 6:8PFPi, 8:8PFPi and diPAPs are higher than 120%. The results show that the use of 3 extractions in the present invention is a preferred step and a key step.

[0194] The method of Example 1 is put into practical application:

[0195] The specific steps are as follows:

[0196] This embodiment provides a practical application of a method for simultaneous detection of 40 new and traditional PFASs in urine. Urine samples of 9 volunteers (aged between 22 and 28) are first collected and three parallel sets are set up to perform separate tests in sequence. 40 PFASs in the same urine are selected as target analytes for simultaneous extraction and detection.

[0197] In the process of testing real volunteer urine, after every 20 samples were tested, one spiked quality control sample, one 10 ng / mL repeat sample in the standard curve, two instrument (methanol) blank samples and one procedural blank were analyzed for quality control and assurance. The test results are shown in Table 8.

[0198] Table 8

[0199]

[0200]

[0201] As shown in Table 8, the median concentrations of new and traditional PFASs in urine samples ranged from 0.001 ng / mL to 2.426 ng / mL, and the geometric means were 0.001 ng / mL to 2.687 ng / mL.

[0202] In general, the method for simultaneous detection of perfluoroalkyl and polyfluoroalkyl substances in urine provided by the present invention overcomes many shortcomings of existing detection technologies, especially the problem that most existing detection methods focus on traditional PFASs (perfluoroalkyl sulfonic acids (PFSAs) and perfluoroalkyl carboxylic acids (PFCAs)) and a few substitutes. By improving sample pretreatment and instrumental analysis methods, 40 new and traditional PFASs in urine are simultaneously extracted and analyzed, which is convenient and fast, and saves solvents. At the same time, the average recovery rate of this method ranges from 70% to 123%, and the intra-day and inter-day relative standard deviations are within 19% and 26%, respectively; the linearity is good, R 2 Both were greater than 0.99; the sensitivity was high, and the LOQs and LODs of the method were 0.001ng / mL~3ng / mL and 0.0003ng / mL~0.9ng / mL.

[0203] The simultaneous detection method for perfluorinated and polyfluoroalkyl substances in urine provided by the present invention overcomes many shortcomings of existing detection technologies, analyzes 40 new and traditional PFASs, simplifies the detection steps, reduces the detection cost, provides the necessary conditions for further exploring the impact of exposure to new PFASs substitutes on human health issues, and expands the application scope of health exposure risk assessment.

[0204] The above are only embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent transformations made using the contents of the present invention specification, or directly or indirectly applied in related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for simultaneous detection of perfluoroalkyl and polyfluoroalkyl substances in urine, characterized in that: The following steps are involved: Sample pretreatment: S1 adds a stable isotope-labeled internal standard compound to the urine sample to be tested; S2 adds an extractant to the sample of step S1 to extract the target analyte in the sample; S3 repeats step S2 three times to obtain the sample to be tested after re-dissolving; The extractant comprises acetonitrile; Testing of samples to be tested: Ultra-high performance liquid chromatography-tandem mass spectrometry was used to detect the samples to be tested; In the ultra-high performance liquid chromatography-tandem mass spectrometry, the mobile phases used include mobile phase A and mobile phase B, the mobile phase A includes a first acidic solution, and the mobile phase B includes a second acidic solution and an alcohol solution; the first acidic solution and the second acidic solution include formic acid solutions; In the ultra-high performance liquid chromatography-tandem mass spectrometry, the elution method used is gradient elution, and the elution procedure of the gradient elution is as follows: ； The perfluoro and polyfluoroalkyl substances include perfluorobutyl carboxylic acid, perfluoropentyl carboxylic acid, perfluorohexyl carboxylic acid, perfluoroheptyl carboxylic acid, perfluorononyl carboxylic acid, perfluorodecyl carboxylic acid, perfluoroundecyl carboxylic acid, perfluorododecyl carboxylic acid, perfluorotridecyl carboxylic acid, perfluorotetradecyl carboxylic acid, perfluorobutyl sulfonic acid, perfluorohexyl sulfonic acid, perfluorooctane sulfonic acid, perfluorononyl sulfonic acid, perfluorodecane sulfonic acid, 4:2 fluorotelomer sulfonic acid, 6:2 fluorotelomer sulfonic acid, 8:2 fluorotelomer sulfonic acid, 6:2 fluorotelomer unsaturated carboxylic acid, 8:2 fluorotelomer unsaturated carboxylic acid, 10:2 fluorotelomer unsaturated carboxylic acid, perfluorooctane sulfonamide, 2-(N-methylperfluorooctane sulfonamide) and 2-(N-methylperfluorooctane sulfonamide). Perfluorooctane sulfonamide) acetic acid, 2-(N-ethyl perfluorooctane sulfonamide) acetic acid, 6:2 chlorinated polyfluoroether sulfonic acid, 8:2 chlorinated polyfluoroether sulfonic acid, hexafluoropropylene oxide dimer acid, 4,8-dioxy-3-hydro-polyfluorononanoic acid ammonium, perfluoroethane cyclohexane sulfonate, 6:6 perfluoroalkyl phosphinic acid, 6:8 perfluoroalkyl phosphinic acid, 8:8 perfluoroalkyl phosphinic acid, 6:2 polyfluoroalkyl phosphate, 6:2 polyfluoroalkyl phosphate diester, 8:2 polyfluoroalkyl phosphate, 8:2 polyfluoroalkyl phosphate diester, 6:2 / 8:2 polyfluoroalkyl phosphate diester, perfluorohexylphosphonic acid, perfluorooctylphosphonic acid, perfluorodecylphosphonic acid.

2. The method according to claim 1, characterized in that: The concentration of the matrix in the urine to be tested is greater than or equal to 0.001 ng / mL.

3. The method according to claim 1, characterized in that: The ratio of the volume of the urine sample to be tested in step S1 to the volume of the extractant in step S2 is 1-1.5:1-1.

5.

4. The method according to claim 1, characterized in that: The ratio of the volume of the urine sample to be tested in step S1 to the volume of the solvent required for reconstitution in step S3 is 1-1.5:1-1.

5.

5. The method according to claim 1, characterized in that: The volume fraction of the mobile phase A is 0.01-0.5%, and the volume fraction of the mobile phase B is 0.01-0.5%.

6. The method according to claim 1, characterized in that: In the ultra-high performance liquid chromatography-tandem mass spectrometry, the flow rate is 0.1 to 0.5 mL / min.

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