Method for detecting perfluorinated and polyfluoroalkyl substances in skeleton

Abstract

The invention provides a method for detecting perfluorinated and polyfluoroalkyl substances in bones, and belongs to the field of analytical chemistry. The detection method comprises the following steps: homogenizing a skeleton sample to obtain a sample 1; adding a recovery rate indicator and ammonia water into the sample 1 to obtain a sample 2; adding a solvent into the sample 2 for extraction, and taking supernate; concentrating and purifying the supernate, and adding an internal standard substance to obtain a sample solution; and detecting by using a high performance liquid chromatography-tandem mass spectrometry method to obtain the concentration of each perfluoroalkyl substance monomer and the concentration of each polyfluoroalkyl substance monomer. The detection method disclosed by the invention is high in recovery rate and sensitivity, and 24 PFASs substances can be detected. The method is simple to operate, low in cost, fast, efficient and beneficial to large-scale popularization.

Description

Technical Field

[0001] The present invention provides a method for detecting perfluoroalkyl and polyfluoroalkyl substances in bones, and belongs to the field of analytical chemistry. Background Art

[0002] Per- and polyfluoroalkyl substances (PFASs) are organic compounds in which hydrogen atoms on hydrocarbon chains are fully or partially replaced by fluorine atoms. PFAS exposure is widespread and occurs through diverse pathways. Toxicological studies have demonstrated cytotoxic, neurotoxic, reproductive, and endocrine-toxic effects in humans. Several epidemiological studies have found associations between PFAS exposure and bone health, such as decreased bone mineral density and an increased risk of osteoporosis.

[0003] Several liquid chromatography-mass spectrometry detection methods are described in the "Research Progress on Detection and Screening Technologies for Perfluoroalkyl and Polyfluoroalkyl Substances" (DOI:10.15898 / j.ykcs.202408120172). These methods detected 15-23 types of PFASs, and the number of types detected needs to be improved. Furthermore, the recovery rates varied significantly between different sample types. In the direct determination of 23 perfluorinated compounds in crude plant extracts using online solid-phase extraction cleanup-liquid chromatography-tandem mass spectrometry (DOI:10.19756 / j.issn.0253-3820.231167), the recovery rates in leaves were 55.3-118.7%, and the recovery rates in plants were 0.06-8.8%. Currently, no research has been conducted on the detection of PFASs in bones. For example, the test samples in the above-mentioned literature are geological soil, pollutants, plants, etc. Chinese patent CN117783362A discloses a method for detecting the content of perfluorinated and polyfluorinated compounds, and the test sample is food (milk).

[0004] At present, the main research on humans focuses on measuring the concentration of PFASs in plasma and serum. For example: A study on the association between perfluorinated compound exposure and the risk and severity of acute coronary syndrome (Li Haoran, Hebei Medical University, doctoral dissertation) used ultra-high performance liquid chromatography-quadrupole-electrostatic field orbital trap high-resolution mass spectrometry to detect the content of PFASs in plasma samples. However, it only detected 12 PFASs substances, which is a relatively small number, and its sensitivity was not investigated. Moreover, compared with plasma and serum, bones are target organs, and their PFASs concentration is more closely related to bone health. Existing technologies have not conducted research on this.

[0005] Therefore, further research is needed to develop methods for detecting PFAS in bones and to improve their sensitivity and recovery. Summary of the Invention

[0006] To address the deficiencies in the prior art, the present invention provides a method for detecting perfluoro and polyfluoroalkyl substances in bones and its application, which has high recovery rate and sensitivity and can detect 24 PFASs substances.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a method for detecting perfluoro and polyfluoroalkyl substances in bones, comprising the following steps:

[0009] (1) Homogenize the bone sample to obtain Sample 1;

[0010] (2) Add a recovery rate indicator and ammonia water to Sample 1 obtained in step (1) to obtain Sample 2;

[0011] (3) Add a solvent for extraction to Sample 2 obtained in step (2) and take the supernatant;

[0012] (4) Concentrate and purify the supernatant obtained in step (3), and add an internal standard to obtain a sample solution;

[0013] (5) Detect the sample solution by high performance liquid chromatography tandem mass spectrometry to obtain the concentrations of each perfluoro and polyfluoroalkyl substance monomer;

[0014] The perfluoro and polyfluoroalkyl substances include at least one of perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnDA), perfluorotridecanoic acid (PFTrDA), perfluorotetradecanoic acid (PFTeDA), perfluorododecanoic acid (PFDoDA), perfluoropentane sulfonic acid (PFPeS), perfluorohexane sulfonic acid (PFHxS), perfluoroheptane sulfonic acid (PFHpS), perfluorooctane sulfonic acid (PFOS), perfluorodecane sulfonic acid (PFDS), hexafluoropropylene oxide trimeric acid (HFPO-TA), hexafluoropropylene oxide dimeric acid (HFPO-DA), perfluorooctane sulfonamide (FOSA), ammonium 4,8-dioxy-3H-perfluorononanoate (ADONA), 6:2 chlorinated polyfluorinated ether sulfonic acid (6:2Cl-PFESA), 8:2 chlorinated polyfluorinated ether sulfonic acid (8:2Cl-PFESA), 4:2 fluorotelomer sulfonic acid (4:2FTSA), 8:2 fluorotelomer sulfonic acid (8:2FTSA), 6:2 fluorotelomer carboxylic acid (6:2FTCA), 8:2 fluorotelomer carboxylic acid (8:2FTCA), 8:2 fluorotelomer unsaturated carboxylic acid (8:2FTUCA);

[0015] The purification is carried out by solid phase extraction using an ENVI-18 column;

[0016] The recovery rate indicator is at least one of MPFOA, MPFUnDA, M2PFTeDA, MPFHxS, MPFOS, d3MeFOSA, M2-8:2FTSA, and M2-8:2FTCA;

[0017] The internal standard is at least one of M8PFOA, M7PFUnDA, M3PFHxS, and M8PFOS.

[0018] Further, the bone is a bone of a mammal.

[0019] Further, the mammals include, but are not limited to, cattle, sheep, pigs, horses, and humans.

[0020] Further, the homogenization in step (1) is to freeze-dry and grind the bone sample.

[0021] Further, the temperature of the freeze-drying is -30°C to -40°C, the vacuum degree of the drying is 0.4 - 0.8 MPa, and the drying time is 36 - 60 h.

[0022] Preferably, the recovery rate indicator is MPFOA, MPFUnDA, M2PFTeDA, MPFHxS, MPFOS, d3MeFOSA, M2-8:2FTSA, and M2-8:2FTCA with equal concentrations.

[0023] Preferably, the internal standard is M8PFOA, M7PFUnDA, M3PFHxS, and M8PFOS with equal concentrations.

[0024] Further, the concentrations of the recovery rate indicator and the internal standard are 1 - 1.5 ppm, and the solvent is methanol.

[0025] Preferably, the concentrations of the recovery rate indicator and the internal standard are 1 ppm.

[0026] Further, the addition amount of the ammonia water in step (2) is 0.5 - 1.5 mL.

[0027] ]>Preferably, the addition amount of the ammonia water in step (2) is 1 mL.

[0028] Preferably, the mass concentration of the ammonia water is 25 - 28%.

[0029] Preferably, after obtaining sample 2 in step (2), it further includes standing for 6 - 12 h.

[0030] Further, the solvent in step (3) is: an organic solvent capable of dissolving perfluoro and polyfluoroalkyl substances.

[0031] Preferably, the organic solvent is acetonitrile.

[0032] Further, the extraction in step (3) is: ultrasonic extraction, repeated 2 - 4 times.

[0033] Preferably, the conditions for the ultrasonic extraction are: vortex oscillation for 0.5 - 2 min, ultrasonic treatment at 80 - 150 Hz for 15 - 30 min, centrifugation to obtain the supernatant; the bone tissue sample matrix is mixed with the extraction solvent again, and ultrasonic extraction is continued.

[0034] More preferably, the conditions for the ultrasonic extraction are: ultrasonic treatment at 100 Hz for 20 min, centrifugation at 5000 rpm for 10 min.

[0035] Further, the concentration in step (4) is: at 30 - 40 °C, the supernatant obtained in step (3) is concentrated to 5 - 15% of the supernatant using an inert gas stream.

[0036] Preferably, the concentration is: at 35 °C, the supernatant obtained in step (3) is concentrated to 10% of the supernatant using a nitrogen gas stream.

[0037] Further, the solid-phase extraction is: pretreat the ENVI-18 column with methanol and water respectively, then load the supernatant obtained in step (3) onto the ENVI-18 column, vacuum dry, and elute with methanol.

[0038] Further, the elution gradient of the high-performance liquid chromatography in step (5) is:

[0039] Time (min) Mobile phase A (%) Mobile phase B (%) 0 90 10 2 55 45 8 5 95 14.9 5 95 15 90 10 18 90 10

[0040] The mobile phase A of the high-performance liquid chromatography is an aqueous solution of ammonium acetate at 1 - 2 mmol / L, and the mobile phase B is a mixture of methanol and acetonitrile with a volume ratio of 1:(1 - 2).

[0041] Preferably, the mobile phase A of the high-performance liquid chromatography is an aqueous solution of ammonium acetate at 2 mmol / L, and the mobile phase B is a mixture of methanol and acetonitrile with a volume ratio of 1:1.

[0042] The beneficial effects of the present invention are:

[0043] The detection method of the present invention can simultaneously enrich and detect 24 traditional and emerging PFASs in bone samples, use HPLC-MS / MS for quantitative analysis of PFASs, and improve the sensitivity and recovery rate of the detection method. At the same time, the detection method of the present invention is simple to operate, low in cost, fast and efficient, and is conducive to large-scale promotion. Description of the Drawings

[0044] Figure 1It is the chromatogram result map of detecting 24 kinds of PFASs spiked by using a high performance liquid chromatography-mass spectrometry instrument in Example 1 of the present invention.

[0045] Figure 2 It is the result map of the spiked recovery rate of each PFAS in Example 1 of the present invention. Specific implementation manners

[0046] In order to make the technical means, creative features, achieved purposes and functions of the present invention easy to understand, the present invention will be further illustrated below in conjunction with specific embodiments. However, the following embodiments are only the preferred embodiments of the present invention, not all of them. Based on the embodiments in the implementation manners, other embodiments obtained by those skilled in the art without creative efforts all belong to the protection scope of the present invention. It is worth noting that the raw materials used in the present invention are all ordinary commercially available products, and no specific limitation is made on their sources. The technical and scientific terms used in the embodiments have the meanings commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0047] Unless otherwise specified, the concentrations described in the present invention are all mass concentrations.

[0048] The present invention does not limit the sources of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all ordinary commercially available products in the technical field.

[0049] The bone samples used in the present invention are from the Third Hospital of Peking University and belong to the lower limb bones of the human body.

[0050] Table 1 shows the basic information of the target analytes of the present invention.

[0051]

[0052]

[0053] 1. Example 1 A method for detecting perfluoro- and polyfluoroalkyl substances in bones

[0054] It includes the following steps:

[0055] S1 Sample pretreatment:

[0056] (1) Add 10 ng of recovery rate indicator to 0.5 g of the bone sample to be detected, add 1 mL of ammonia water, place it in a 15 mL polyethylene centrifuge tube, tighten the lid, and vortex to mix the sample evenly, and leave it overnight for 12 h;

[0057] (2) Add 5 mL of acetonitrile as the extraction solvent, extract three times repeatedly, ultrasonically treat each extraction step for 20 minutes, and then centrifuge at 5000 rpm for 10 minutes. Mix the supernatant and blow it to dryness with nitrogen to 1 mL, and then add 50 mL of ultrapure water for dilution and mix evenly;

[0058] (3) Further enrichment and purification were carried out using ENVI-18 cartridges (6 mL, 500 mg; Supelco). The cartridges were pretreated with 10 mL of methanol and ultrapure water respectively, then the samples were loaded onto the cartridges. After vacuum drying for 30 minutes, the PFASs analytes were eluted with 10 mL of methanol;

[0059] (4) The eluate was concentrated under a gentle nitrogen stream in a 35 °C water bath and made up to a volume of 100 μL, then the internal standard was added to obtain the sample to be measured, waiting for on-machine detection.

[0060] The recovery rate indicator is: MPFOA, MPFUnDA, M2PFTeDA, MPFHxS, MPFOS, d3MeFOSA, M2-8:2FTSA and M2-8:2FTCA with equal concentrations were dissolved in methanol to prepare a 1 ppm solution.

[0061] The internal standard is M8PFOA, M7PFUnDA, M3PFHxS, M8PFOS with equal concentrations dissolved in methanol to prepare a 1 ppm solution.

[0062] S2 Sample Detection:

[0063] PFASs were detected using a Thermo Scientific Vanquish high performance liquid chromatograph in tandem with a TSQ Quantis triple quadrupole mass spectrometer.

[0064] (1) The high performance liquid chromatography conditions were as follows: The chromatographic column used was a reversed-phase C18 column: Acclaim RSLC 120C18 (150×2.1 mm, 2.2 μm); The mobile phase included mobile phase A and mobile phase B; Mobile phase A was a 2 mmol / L ammonium acetate solution; Mobile phase B was methanol:acetonitrile (1:1); The flow rate was 0.3 mL / min, the temperature of the injector was 4 °C, the injection volume was 5 μL, and gradient elution was used. The gradient elution program was carried out according to the following procedure: The sum of mobile phase A and mobile phase B was 100%; The initial proportion of the mobile phase was 10% of mobile phase B; From 0 to 2 min, the volume percentage of mobile phase B increased from 10% to 45%; From 2 to 8 min, the volume percentage of mobile phase B increased from 45% to 95%, maintaining an equilibrium for 6.9 min; From 14.9 to 15 min, the volume percentage of mobile phase B returned to the initial conditions and was maintained for 3 min;

[0065] (2) The mass spectrometry detection conditions were as follows: The ESI source negative ion mode and MRM mode were used, the spray voltage was 3000 V, the sheath gas and auxiliary gas pressures were respectively 40 and 5 Arb, the ion transfer tube temperature was set at 320 °C, and the ion source temperature was 300 °C;

[0066] (3) Data collection: TraceFinder 4.1 General Quan software is used for data collection and peak collection. Qualitative analysis is performed based on ion pair information and retention time, and quantitative analysis is carried out using the external standard method and multi-point calibration standard curve.

[0067] The method of the present invention can quantitatively detect and analyze 24 PFASs in bones, and the ion pair information in the mass spectrometry is shown in Table 2.

[0068] Table 2

[0069]

[0070]

[0071] 2. Investigation of MDL

[0072] The smaller the MDL value, the higher the detection sensitivity. The calculation method of MDL is: the mean value of the compound concentration in the blank sample plus 3 times the standard deviation. The experimental results are shown in Table 3.

[0073] Table 3

[0074]

[0075]

[0076] The experimental results show that when the method of the present invention is used to detect the above 24 PFASs, the MDL values are in the range of 0.0003 - 0.0648 ng / g dw, which can meet the detection of 24 PFASs in actual bone samples in clinical practice.

[0077] 3. Investigation of linearity and spiked recovery

[0078] In the present invention, the standard curve is directly prepared in solution. Using methanol as the solution, standard solutions of 24 PFASs with concentrations of 0.1 - 100 ng / mL (0.1, 0.2, 0.5, 1, 2, 5, 10, 20, 50, 100 ng / mL) are prepared, and 6 batches are continuously made to investigate the linear correlation and spiked recovery of 24 PFASs.

[0079] Calculation method of recovery rate: The recovery rate of this method in bone tissue samples is tested through matrix spiking experiments. Recovery rate (%) = (C2 - C1) / C0 × 100, where C2 is the measured value of the spiked sample; C1 is the measured value of the unspiked sample; C0 is the concentration of the spiked sample.

[0080] The spiked sample is: 10 ng of the standard solution is added to the sample to be tested in Example 1. The unspiked sample is the sample to be tested in Example 1, and the concentration of the spiked sample is the concentration of the standard solution.

[0081] The results are shown in Table 4.

[0082] Table 4

[0083]

[0084]

[0085]

[0086] The experimental results show that: each compound has a good linearity in the range of 0.1 - 100 ng / mL, and the linear correlation coefficient R 2 > 0.995, and the average recovery rate is 66.17 ± 1.28% - 114.08 ± 3.54%, indicating that the method of the present invention has a good effect in detecting PFASs in bone samples.

[0087] 4. Comparative Examples

[0088] Comparative Example 1

[0089] Different from Example 1, in step S1(1), ammonia water was not added.

[0090] The remaining steps are the same as those in Example 1, and the average recovery rate results of 24 PFASs are shown in Table 5.

[0091] Comparative Example 2

[0092] Different from Example 1, in step S1(1), ammonia water was not added, and in step S1(2), the extraction solvent acetonitrile was replaced with an equal amount of methanol.

[0093] The remaining steps are the same as those in Example 1, and the average recovery rate results of 24 PFASs are shown in Table 5.

[0094] Comparative Example 3

[0095] Different from Example 1, in step S1(2), the extraction solvent acetonitrile was replaced with methanol. The remaining steps are the same as those in Example 1, and the average recovery rate results of 24 PFASs are shown in Table 5.

[0096] Comparative Example 4

[0097] Different from Example 1, in step S1(3), the solid phase extraction column ENVI-18 (6 mL, 500 mg; Supelco) was replaced with WAX (6 mL, 500 mg; Waters). The column was activated with 1% ammonia water methanol, methanol and water in sequence, and finally eluted with 5 mL of methanol and 5 mL of 1% ammonia water methanol in sequence.

[0098] The remaining steps are the same as those in Example 1, and the average recovery rate results of 24 PFASs are shown in Table 5.

[0099] Comparative Example 5

[0100] Different from Example 1, in step S1(3), the solid-phase extraction column ENVI-18 (6 mL, 500 mg; Supelco) was replaced with PWAX (3 mL, 60 mg; CNW). The column was activated successively with 5% triethylamine methanol and 2% formic acid water, and finally eluted with 12 mL of 5% triethylamine methanol.

[0101] The remaining steps were the same as those in Example 1, and the average recovery results of 24 PFASs are shown in Table 5.

[0102] Table 5 (unit: %)

[0103]

[0104]

[0105]

[0106] Analysis of the experimental results of this example and comparative examples:

[0107] Generally, a recovery rate of 60-120% is considered qualified. The recovery rate of Example 1 of the present invention was 66.17±1.28%-115.14±2.27%, within the range of 60-120%; the average recovery rates of the target PFASs in Comparative Examples 1-5 were 34.88±0.38%-139.14±4.31%, 40.14±3.07%-80.14±0.93%, 56.52±2.55%-89.08±3.74%, 19.47±1.36%-55.87±1.67% and 25.48±2.40%-175.06±2.09% respectively. There were problems with unqualified recovery rates for some PFASs substances.

[0108] The recovery rates of Comparative Examples 1-5 were not as good as those of Example

[0109] In summary, the present invention provides a method for detecting PFASs in bones. The method of ultrasonic extraction, solid-phase extraction and HPLC-MS / MS detection for specific bones has the characteristics of being simple and fast, having a low detection limit, strong versatility, high sensitivity, good stability and reproducibility, and can determine 24 PFASs in bones.

[0110] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the protection scope of the present invention. Any simple modification or equivalent replacement of the technical solution of the present invention by those of ordinary skill in the art shall not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for detecting per- and polyfluoroalkyl substances in bones, characterized in that, It includes the following steps: (1) Homogenize the bone sample to obtain Sample 1; (2) Add a recovery indicator and ammonia water to Sample 1 obtained in step (1) to obtain Sample 2; (3) Add solvent extraction to Sample 2 obtained in step (2), and take the supernatant; (4) Concentrate and purify the supernatant obtained in step (3), add an internal standard to obtain a sample solution; (5) Use high performance liquid chromatography-tandem mass spectrometry to detect the sample solution to obtain the concentrations of each perfluoro and polyfluoroalkyl substance monomer; The perfluoro and polyfluoroalkyl substances include at least one of perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluorododecanoic acid, perfluoropentanesulfonic acid, perfluorohexanesulfonic acid, perfluoroheptanesulfonic acid, perfluorooctanesulfonic acid, perfluorodecanesulfonic acid, hexafluoropropylene trimer acid, hexafluoropropylene dimer acid, perfluorooctanesulfonamide, 4,8-dioxy-3H-polyfluorononanoic acid ammonium, 6:2 chlorinated polyfluoroether sulfonic acid, 8:2 chlorinated polyfluoroether sulfonic acid, 4:2 fluorotelomer sulfonic acid, 8:2 fluorotelomer sulfonic acid, 6:2 fluorotelomer carboxylic acid, 8:2 fluorotelomer carboxylic acid, 8:2 fluorotelomer unsaturated carboxylic acid; The purification is solid phase extraction using an ENVI-18 column; The recovery indicator is at least one of MPFOA, MPFUnDA, M2PFTeDA, MPFHxS, MPFOS, d3MeFOSA, M2-8:2FTSA and M2-8:2FTCA; The internal standard is at least one of M8PFOA, M7PFUnDA, M3PFHxS and M8PFOS.

2. The detection method according to claim 1, wherein The homogenization in step (1) is freeze-drying and grinding of the bone sample.

3. The detection method according to claim 2, characterized in that The temperature of the freeze-drying is -30°C to -40°C, the vacuum degree of the drying is 0.4 - 0.8 MPa, and the drying time is 36 - 60 h.

4. The detection method according to claim 1, characterized in that, The addition amount of the ammonia water in step (2) is 0.5 - 1.5 mL.

5. The detection method according to claim 1, characterized in that, The solvent in step (3) is: an organic solvent capable of dissolving perfluoro and polyfluoroalkyl substances.

6. The detection method according to claim 1, wherein The organic solvent is acetonitrile.

7. The detection method according to claim 1, characterized in that The extraction in step (3) is: ultrasonic extraction, repeated 2 - 4 times.

8. The detection method according to claim 1, characterized in that, The concentration in step (4) is: at 30 - 40°C, use an inert gas stream to concentrate the supernatant obtained in step (3) to 5 - 15% of the supernatant.

9. The detection method according to claim 1, wherein The solid phase extraction is: pretreat the ENVI-18 column with methanol and water respectively, then load the supernatant obtained in step (3) onto the ENVI-18 column, vacuum dry, and elute with methanol.

10. The detection method according to claim 1, characterized in that, The elution gradient of the high performance liquid chromatography in step (5) is: The mobile phase A is an aqueous solution of ammonium acetate at 1 - 2 mmol / L, and the mobile phase B is methanol and acetonitrile with a volume ratio of 1:(1 - 2).

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