Method for simultaneously detecting perfluorinated compounds in mouse tissues and visceral organs

Abstract

The invention provides a method for simultaneously detecting perfluorinated compounds in mouse tissues and organs, and belongs to the technical field of analysis and detection. The method provided by the invention comprises the following steps: mixing a mouse visceral organ tissue sample, an alkaline buffer solution and an internal standard solution, and homogenizing to obtain homogenate; mixing the homogenate with tetrabutylammonium hydrogen sulfate and methyl tert-butyl ether, and carrying out liquid-liquid extraction to obtain extract liquor; and sequentially drying and redissolving the extract liquor, and carrying out ultra-high performance liquid chromatography-tandem mass spectrometry analysis to obtain a result of the perfluorinated compound in the mouse tissue visceral organs. The result of the embodiment shows that the adding standard recovery rate of 14 target objects in the method provided by the invention is 64.9%-120.3%, the RSD of 6 repeated tests is 1.0%-14.0%, the matrix effect of the target objects is 83.7%-134.0%, and the matrix effect is relatively low; and the detection result has excellent accuracy and reproducibility.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, and in particular to a method for simultaneously detecting perfluorinated compounds in mouse tissues and organs. Background Art

[0002] Perfluorinated and polyfluorinated compounds (PFAS) are widely used in industrial products and daily necessities. However, PFAS can exist in the environment for a long time, enter the body through breathing, eating and other ways, and accumulate and amplify in the body, affecting the health of the human reproductive system, immune system, endocrine system and nervous system. It is a persistent organic pollutant. Therefore, the detection and research of PFAS in organisms is imminent.

[0003] For biological tissue samples, taking mouse tissue organs as an example, due to the high content of protein and phospholipids, the matrix effect will seriously affect the quantitative analysis results of UPLC-MS / MS, resulting in the existing PFAS detection methods in mouse tissue organs having problems such as low accuracy and reproducibility.

[0004] Therefore, there is an urgent need to provide a method for simultaneously detecting perfluorinated compounds in mouse tissues and organs with high accuracy and reproducibility. Summary of the invention

[0005] The object of the present invention is to provide a method for simultaneously detecting perfluorinated compounds in mouse tissues and organs with high accuracy and reproducibility.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a method for simultaneously detecting perfluorinated compounds in mouse tissues and organs, comprising the following steps: (1) Mixing a mouse organ tissue sample, an alkaline buffer solution, and an internal standard solution, and then homogenizing the mixture to obtain a homogenate; (2) mixing the homogenate obtained in step (1) with tetrabutylammonium hydrogen sulfate and methyl tert-butyl ether, and performing liquid-liquid extraction to obtain an extract; (3) The extract obtained in step (2) is dried and redissolved in turn, and subjected to ultra-high performance liquid chromatography-tandem mass spectrometry analysis to obtain the results of perfluorinated compounds in mouse tissues and organs.

[0007] Preferably, the mouse organ tissue sample in step (1) includes one or more of mouse liver, kidney, spleen, intestine, heart, lung and brain tissue.

[0008] Preferably, the pH value of the alkaline buffer solution in step (1) is 8-11.

[0009] Preferably, the internal standard substance in the internal standard solution in step (1) comprises13 C 4 -PFBA, 13 C 5 -PFHxA, 13 C 4 -PFHpA, 13 C 8 -PFOA, 13 C 9 -PFNA, 13 C 6 -PFDA, 13 C 2 -PFTeDA, 13 C 3 -PFHxS and 13 C 8 -PFOS.

[0010] Preferably, in step (1), the ratio of the mass of the mouse organ tissue sample to the volume of the alkaline buffer solution is (5-20) mg: (50-200) µL.

[0011] Preferably, in step (2), the ratio of the volume of the homogenate, the amount of tetrabutylammonium hydrogen sulfate and the volume of methyl tert-butyl ether is (1-3) mL: (0.25-0.75) mmol: (6-12) mL.

[0012] Preferably, the solvent for re-dissolving in step (3) is a methanol-water solution, and the volume ratio of methanol to water in the methanol-water solution is 1:(1-3).

[0013] Preferably, the ultra-high performance liquid chromatography conditions for ultra-high performance liquid chromatography tandem mass spectrometry analysis in step (3) include: The chromatographic column is a reversed phase liquid chromatography column with a dimethyl n-octadecylsilane stationary phase chemically bonded to a Poroshell 120 silica gel carrier. The length of the chromatographic column is 50-150 mm, the diameter of the chromatographic column is 2-5 mm, and the filler particle size of the chromatographic column is 1.5-5 µm. The chromatographic column temperature is 30-50 °C. The injection volume is 3-10 µL. A gradient elution program is adopted, and the flow rate of the mobile phase is 300-450 μL / min; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is an ammonium acetate aqueous solution with a concentration of 2-10 mmol / L, and the mobile phase B is an ammonium acetate methanol solution with a concentration of 2-10 mmol / L.

[0014] Preferably, the gradient elution program is: at 0-7.5 min, the mobile phase changes linearly from 55% mobile phase A and 45% mobile phase B to 5% mobile phase A and 95% mobile phase B; at 7.5-8.0 min, the mobile phase remains at 5% mobile phase A and 95% mobile phase B; at 8.0-9.0 min, the mobile phase changes linearly from 5% mobile phase A and 95% mobile phase B to 55% mobile phase A and 45% mobile phase B; at 9.0-12 min, the mobile phase remains at 55% mobile phase A and 45% mobile phase B.

[0015] Preferably, the mass spectrometry conditions of the ultra-high performance liquid chromatography tandem mass spectrometry analysis in step (3) include: ionization in negative ion electrospray mode and negative ion multiple reaction monitoring scanning; the pressure of the collision gas is 6-12 psi; the pressure of the curtain gas is 10-30 psi; the pressure of the nebulizer gas is 40-55 psi; the pressure of the auxiliary gas is 30-60 psi; the ion source voltage is -3500-5500 V; the ionization temperature is 450-550° C.; and the scanning time is 30 ms.

[0016] The invention provides a method for simultaneously detecting perfluorinated compounds in mouse tissues and organs, comprising the following steps: mixing a mouse organ tissue sample, an alkaline buffer solution and an internal standard solution, and then performing a homogenization process to obtain a homogenate; mixing the homogenate with tetrabutylammonium hydrogen sulfate and methyl tert-butyl ether, and performing liquid-liquid extraction to obtain an extract; and sequentially drying and re-dissolving the extract, and performing ultra-high performance liquid chromatography tandem mass spectrometry analysis to obtain the results of perfluorinated compounds in mouse tissues and organs. The present invention uses an alkaline buffer solution as a homogenization medium; adding an internal standard solution during the homogenization process can correct the loss during the sample processing process and improve the accuracy and reliability of quantitative analysis; and the addition of an internal standard solution can also eliminate the influence of the sample matrix on the analysis results and correct the interference caused by the matrix effect; the present invention uses methyl tert-butyl ether as an extractant and tetrabutylammonium hydrogen sulfate as an ion pair reagent to form an ion association complex with the target object, increase the solubility of the target object in the organic phase, thereby realizing the transfer from the aqueous phase to the organic phase, and improving the extraction effect of perfluorinated compounds in mouse organ tissue samples; the present invention uses ultra-high performance liquid chromatography tandem mass spectrometry analysis, the required sample amount is relatively small, and the perfluorinated compounds in the tissue have high sensitivity and specificity. The results of the embodiment show that the spiked recovery rate of 14 kinds of target objects provided by the method of the present invention is between 64.9% and 120.3%, the RSD of 6 repeated tests is between 1.0% and 14.0%, the matrix effect of the target object is between 83.7% and 134.0%, and the matrix effect is low; the detection results have excellent accuracy and reproducibility. DETAILED DESCRIPTION

[0017] The present invention provides a method for simultaneously detecting perfluorinated compounds in mouse tissues and organs, comprising the following steps: (1) Mixing a mouse organ tissue sample, an alkaline buffer solution, and an internal standard solution, and then homogenizing the mixture to obtain a homogenate; (2) mixing the homogenate obtained in step (1) with tetrabutylammonium hydrogen sulfate and methyl tert-butyl ether, and performing liquid-liquid extraction to obtain an extract; (3) The extract obtained in step (2) is dried and redissolved in turn, and subjected to ultra-high performance liquid chromatography-tandem mass spectrometry analysis to obtain the results of perfluorinated compounds in mouse tissues and organs.

[0018] The invention mixes a mouse organ tissue sample, an alkaline buffer solution and an internal standard solution and then performs homogenization treatment to obtain a homogenate.

[0019] In the present invention, the mouse organ tissue sample preferably includes one or more of the liver, kidney, spleen, intestine, heart, lung and brain tissue of the mouse. The present invention has no particular limitation on the source of the mouse organ tissue sample, and any mouse organ tissue sample that can be obtained by a person skilled in the art can be used.

[0020] In the present invention, the pH value of the alkaline buffer solution is preferably 8 to 11, more preferably 10. In the present invention, the alkaline buffer solution is preferably a sodium carbonate sodium bicarbonate buffer solution, and the concentration of the sodium carbonate sodium bicarbonate buffer solution is preferably 0.1 to 0.4 mol / L, more preferably 0.25 mol / L. The present invention utilizes an alkaline buffer solution as a homogenization medium.

[0021] In the present invention, the ratio of the mass of the mouse organ tissue sample to the volume of the alkaline buffer solution during homogenization is preferably (5-20) mg: (50-200) μL, and more preferably 10 mg: 100 μL. The present invention controls the amount of the two within the above range, which is more conducive to the crushing of the mouse organ tissue sample and converting the tissue into a homogenous liquid mixture for subsequent extraction of the target object, thereby increasing the extraction efficiency.

[0022] In the present invention, the internal standard substance in the internal standard solution preferably includes 13 C 4 -PFBA, 13 C 5 -PFHxA, 13 C 4 -PFHpA, 13 C 8 -PFOA, 13 C 9 -PFNA, 13 C 6 -PFDA, 13 C 2 -PFTeDA, 13 C3 -PFHxS and 13 C 8 -PFOS. The present invention has no special limitation on the source of the internal standard, and any commercially available certified standard solution in the art can be used. The present invention can correct the loss during sample processing by adding an internal standard solution, eliminate the influence of the sample matrix on the analysis results, correct the interference caused by the matrix effect, and improve the accuracy and reliability of quantitative analysis.

[0023] The present invention has no particular limitation on the concentration of the internal standard solution, and the concentration of the conventional internal standard solution can be adjusted. In an embodiment of the present invention, the concentration of the internal standard solution can be 50-500 ng / mL, more preferably 100 ng / mL.

[0024] The present invention does not specifically limit the specific addition amount of the mouse organ tissue sample and the internal standard solution, as long as the concentration of the internal standard substance in the standard curve and the sample is consistent. In the present invention, the internal standard is added to accurately quantify the concentration of the target substance, and it is sufficient to ensure that the concentration of the internal standard substance in the standard curve and the sample is consistent.

[0025] In the present invention, the mouse organ tissue sample, alkaline buffer solution and internal standard solution are mixed and then homogenized. The method for obtaining the homogenate is preferably to mix the mouse organ tissue sample, part of the alkaline buffer solution and the internal standard solution, and then homogenize to obtain a homogenate, and then use the remaining part of the alkaline buffer solution to transfer the homogenate to an extraction device.

[0026] In an embodiment of the present invention, when the amount of the alkaline buffer solution is 2 mL, the amount of a portion of the alkaline buffer solution may be 200 μL, and the remaining amount of the alkaline buffer solution may be 1.8 mL.

[0027] In the present invention, the homogenization treatment is preferably carried out in the presence of ceramic beads. The present invention is more conducive to promoting the full fragmentation of mouse organ tissue samples by adding ceramic beads. The present invention has no particular limitation on the size and quantity of the ceramic beads, which can be adjusted according to the amount of the mouse organ tissue sample, as long as the mouse organ tissue sample can be fully fragmented.

[0028] In the present invention, the rotation speed of the homogenization treatment is preferably 4-7 m / s, more preferably 5 m / s; the time of the homogenization treatment is preferably 20-40 s, more preferably 30 s; the homogenization treatment is preferably carried out at room temperature; the number of homogenization treatments is preferably 1-3 times, more preferably 2 times. The present invention controls the parameters of the homogenization treatment within the above range, which is more conducive to converting the mouse organ tissue sample into a homogenous homogenate mixture for subsequent extraction of the target object to increase the extraction efficiency.

[0029] After obtaining the homogenate, the present invention mixes the homogenate with tetrabutylammonium hydrogen sulfate and methyl tert-butyl ether, performs liquid-liquid extraction, and obtains an extract. The present invention uses methyl tert-butyl ether as an extractant, tetrabutylammonium hydrogen sulfate as an ion pair reagent, and an alkaline buffer solution to provide an alkaline environment, forming an ion pair with the target object, thereby increasing the solubility of the target object in the organic phase, thereby achieving transfer from the aqueous phase to the organic phase, and improving the extraction effect of perfluorinated compounds in mouse organ tissue samples.

[0030] In the present invention, the ratio of the volume of the homogenate, the amount of tetrabutylammonium hydrogen sulfate and the volume of methyl tert-butyl ether is preferably (1-3) mL: (0.25-0.75) mmol: (6-12) mL, and more preferably 2 mL: 0.5 mmol: 9 mL. The present invention controls the amount of each component within the above range, which is more conducive to fully extracting the perfluorinated compounds in the homogenate.

[0031] The present invention has no special limitation on the method of mixing the homogenate with tetrabutylammonium hydrogen sulfate and methyl tert-butyl ether. Conventional mixing methods can be used to uniformly mix the components.

[0032] In an embodiment of the present invention, the method for mixing the homogenate with tetrabutylammonium hydrogen sulfate and methyl tert-butyl ether can be: adding tetrabutylammonium hydrogen sulfate to the homogenate in the form of a solution, and then adding methyl tert-butyl ether, the concentration of the tetrabutylammonium hydrogen sulfate is preferably 0.25~0.75mol / L, more preferably 0.5mol / L. The present invention does not specifically limit the volume of the tetrabutylammonium hydrogen sulfate solution, and it is adjusted according to the volume of the homogenate and methyl tert-butyl ether used, so that the ratio of the volume of the homogenate, the amount of tetrabutylammonium hydrogen sulfate and the volume of methyl tert-butyl ether is (1~3)mL: (0.25~0.75)mmol: (6~12)mL. In an embodiment of the present invention, when the volume of the homogenate is 2mL and the volume of methyl tert-butyl ether is 9mL, the concentration of the tetrabutylammonium hydrogen sulfate can be 0.5mol / L, and the volume of the tetrabutylammonium hydrogen sulfate can be 1mL.

[0033] The present invention does not specifically limit the operation method of the liquid-liquid extraction, and a conventional liquid-liquid extraction operation method can be used. In an embodiment of the present invention, the operation method of the liquid-liquid extraction can be: oscillate the homogenate with tetrabutylammonium hydrogen sulfate and methyl tert-butyl ether in a multi-tube oscillator for 20 minutes, centrifuge at 4000 rpm at room temperature for 10 minutes, transfer the organic phase to another 15 mL polypropylene centrifuge tube, repeat the above liquid-liquid extraction process 3 times, combine the extracts, and obtain an extract.

[0034] After obtaining the extract, the present invention sequentially dries and redissolves the extract, performs ultra-high performance liquid chromatography tandem mass spectrometry analysis, and obtains the results of perfluorinated compounds in mouse tissues and organs.

[0035] In the present invention, the drying method is preferably nitrogen drying. The present invention can reduce the loss of perfluorinated compounds and improve the accuracy of the detection results by nitrogen drying. In an embodiment of the present invention, the nitrogen drying operation method can be placing the extract in a nitrogen concentrator and concentrating it to dryness at 40°C.

[0036] In the present invention, the re-dissolving solvent is preferably a methanol-water solution, and the volume ratio of methanol to water in the methanol-water solution is preferably 1:1. In the present invention, the volume of the methanol-water solution is preferably 100-300 μL, more preferably 200 μL. The use of the above solvent in the present invention is more conducive to further dissolving the extracted target analyte.

[0037] In the present invention, the re-dissolution is preferably vortexed in a vortex mixer, and then centrifuged to obtain a supernatant. In the present invention, the rotation speed of the vortex treatment is preferably 1500~2500rpm, more preferably 2000rpm; the time of the vortex treatment is preferably 5~15min, more preferably 10min; the rotation speed of the centrifugation is preferably 10000~15000rpm, more preferably 12000rpm; the time of the centrifugation is preferably 5~15min, more preferably 10min. The present invention can further purify the extraction product and reduce the influence of impurities on the accuracy of the test results by controlling the re-dissolution parameters within the above range.

[0038] In the present invention, the ultra-high performance liquid chromatography conditions for ultra-high performance liquid chromatography tandem mass spectrometry analysis preferably include: the chromatographic column is a reversed phase liquid chromatography column with a dimethyl n-octadecylsilane stationary phase chemically bonded to a Poroshell 120 silica gel carrier, and the length of the chromatographic column is preferably 50-150 mm, more preferably 100 mm; the diameter of the chromatographic column is preferably 2-5 mm, more preferably 3 mm; the filler particle size of the chromatographic column is preferably 1.5-5 µm, more preferably 2.7 µm; the temperature of the chromatographic column is preferably 30-50 ° C, more preferably 40 ° C; the injection volume is preferably 3-10 µL, more preferably 10 µL. In the present invention, the ultra-high performance liquid chromatography preferably adopts a gradient elution program, and the flow rate of the mobile phase is preferably 300-450 μL / min, more preferably 350 μL / min. The mobile phase preferably includes mobile phase A and mobile phase B, wherein the mobile phase A is preferably an aqueous solution of ammonium acetate with a concentration of 2 to 10 mmol / L, and the concentration of the aqueous solution of ammonium acetate is more preferably 5 mmol / L; the mobile phase B is preferably an ammonium acetate methanol solution with a concentration of 2 to 10 mmol / L, and the concentration of the ammonium acetate methanol solution is more preferably 5 mmol / L. The present invention controls the ultra-high performance liquid chromatography conditions within the above range, which is more conducive to improving the efficiency and accuracy of the ultra-high performance liquid chromatography conditions.

[0039] In the present invention, the gradient elution program is preferably: at 0-7.5 min, the mobile phase changes linearly from 55% mobile phase A and 45% mobile phase B to 5% mobile phase A and 95% mobile phase B; at 7.5-8.0 min, the mobile phase maintains 5% mobile phase A and 95% mobile phase B; at 8.0-9.0 min, the mobile phase changes linearly from 5% mobile phase A and 95% mobile phase B to 55% mobile phase A and 45% mobile phase B; at 9.0-12 min, the mobile phase maintains 55% mobile phase A and 45% mobile phase B. The present invention adopts the above-mentioned gradient elution program, which is more conducive to improving the separation degree, so that each component in the sample is separated under suitable conditions, thereby improving the peak shape, improving the column efficiency, reducing the analysis time, and improving the efficiency and accuracy of the ultra-high performance liquid chromatography conditions.

[0040] In the present invention, the mass spectrometry conditions of the ultra-high performance liquid chromatography tandem mass spectrometry analysis preferably include: ionization in negative ion electrospray mode, negative ion multiple reaction monitoring scanning; the pressure of the collision gas is preferably 6-12psi, more preferably 9-12psi; the pressure of the air curtain gas is preferably 10-30psi, more preferably 12-20psi; the pressure of the nebulizer gas is preferably 40-55psi, more preferably 45-50psi; the pressure of the auxiliary gas is preferably 30-60psi, more preferably 35-50psi; the ion source voltage is preferably -3500-5500V, more preferably -4000V; the ionization temperature is preferably 450-550℃, more preferably 550℃; the scanning time is preferably 30ms. The present invention controls the mass spectrometry conditions within the above range, which is more conducive to the sensitivity, selectivity and accuracy of mass spectrometry analysis, thereby obtaining more reliable analysis results.

[0041] The method provided by the present invention is simple to operate, can fully extract perfluorinated compounds in mouse tissues and organs, and the detection results have excellent accuracy and reproducibility.

[0042] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. 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 protection of the present invention.

[0043] Preparation of relevant solutions in the embodiments: (1) Preparation of standard solution Use certified standard solution to accurately pipette appropriate amount of mixed standard solution containing perfluorobutyric acid (PFBA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluorotetradecanoic acid (PFTeDA), perfluorohexane sulfonic acid (PFHxS), perfluoroheptane sulfonic acid (PFHpS), perfluorooctane sulfonic acid (PFOS), perfluorononane sulfonic acid (PFNS), perfluorodecane sulfonic acid (PFDS), 6:2 Cl-PFESA, and 8:2 Cl-PFESA, dilute to volume with methanol to prepare a standard stock solution with a concentration of 100 ng / mL, and store at -20°C in the dark for use.

[0044] (2) Preparation of internal standard solution Use a certified standard solution to accurately pipette an appropriate amount of MPFBA ( 13 C 4 -PFBA)、M5PFHxA( 13 C 5 -PFHxA)、M4PFHpA(13 C 4 -PFHpA), M8PFOA ( 13 C 8 -PFOA)、M9PFNA( 13 C 9 -PFNA)、M6PFDA( 13 C 6 -PFDA), M2PFTeDA ( 13 C 2 -PFTeDA)、M3PFHxS( 13 C 3 -PFHxS), M8PFOS ( 13 C 8 -PFOS) was diluted with methanol to make a standard working solution (internal standard solution) with a concentration of 100 ng / mL; it was stored at -80°C in the dark until use.

[0045] (3) Preparation of standard curve The standard stock solution (stock solution 1) with a mass concentration of 100 ng / mL was diluted 10 times in series with methanol to prepare stock solutions of 10 ng / mL (stock solution 2), 1 ng / mL (stock solution 3), and 0.1 ng / mL (stock solution 4). The stock solutions were shaken and allowed to stand before use or stored at -20°C for later use. The standard stock solutions of the above concentrations were used to prepare a standard series of working solutions of 0 ng / mL, 0.01 ng / mL, 0.02 ng / mL, 0.05 ng / mL, 0.10 ng / mL, 0.20 ng / mL, 0.50 ng / mL, 1.00 ng / mL, 2.00 ng / mL, 5.00 ng / mL, 10.0 ng / mL and 20.0 ng / mL. The concentrations were evenly distributed, with no less than 6 standard concentration points. The final concentration of the internal standard was 2.5 ng / mL. The specific preparation method is shown in Table 1. A calibrated glass injection needle and pipette were used in this step.

[0046] Table 1 Standard curve preparation

[0047] Example 1 A method for simultaneously detecting perfluorinated compounds in mouse tissues and organs, comprising the following steps: (1) Place 20 mg of mouse liver sample in a 2 mL sample tube, add 2 ceramic beads, 200 μL of 0.25 mol / L (pH = 10) sodium carbonate sodium bicarbonate buffer solution and 0.5 ng of internal standard solution in sequence, homogenize with a homogenizer at room temperature, the speed of the homogenizer is 5 m / s, the homogenization time is 30 s, repeat twice, and then transfer the homogenate to a 15 mL polypropylene centrifuge tube with 1.8 mL of sodium carbonate sodium bicarbonate buffer solution to obtain a homogenate; (2) Mix 2 mL of the homogenate obtained in step (1) with 1 mL of 0.5 mol / L tetrabutylammonium hydrogen sulfate and 3 mL of methyl tert-butyl ether for liquid-liquid extraction, shake on a multitube oscillator for 20 min, centrifuge at 4000 rpm at room temperature for 10 min, transfer the organic phase to another 15 mL polypropylene centrifuge tube, repeat the above liquid-liquid extraction process 3 times, combine the extracts, and obtain an extract; (3) The extract obtained in step (2) is placed in a nitrogen concentrator and concentrated to dryness at 40° C., then 200 μL of methanol-water solution (the volume ratio of methanol to water is 1:1) is added to a nitrogen-blown centrifuge tube for re-dissolution, and the extract is placed in a vortex mixer and oscillated at 2000 rpm for 10 min. After centrifugation at 12000 rpm for 10 min, the supernatant layer is transferred to an injection vial for ultra-high performance liquid chromatography tandem mass spectrometry analysis (abbreviated as UHPLC-MS / MS); The ultra-high performance liquid chromatography conditions of the UHPLC-MS / MS are: The chromatographic column is a reversed phase liquid chromatography column with a dimethyl n-octadecylsilane stationary phase chemically bonded to a Poroshell 120 silica gel carrier. The length of the chromatographic column is 100 mm, the diameter of the chromatographic column is 3 mm, and the filler particle size of the chromatographic column is 2.7 µm. The temperature of the chromatographic column is 40°C. The injection volume was 10 µL; A gradient elution program was used, with mobile phase A being an aqueous solution of ammonium acetate at a concentration of 5 mmol / L, and mobile phase B being an ammonium acetate methanol solution at a concentration of 5 mmol / L; the flow rate of the mobile phase was 350 μL / min; The gradient elution program is as follows: at 0-7.5 min, the mobile phase changes linearly from 55% mobile phase A and 45% mobile phase B to 5% mobile phase A and 95% mobile phase B; at 7.5-8.0 min, the mobile phase remains at 5% mobile phase A and 95% mobile phase B; at 8.0-9.0 min, the mobile phase changes linearly from 5% mobile phase A and 95% mobile phase B to 55% mobile phase A and 45% mobile phase B; at 9.0-12 min, the mobile phase remains at 55% mobile phase A and 45% mobile phase B; The mass spectrometry conditions of the UHPLC-MS / MS are as follows: 14 target analytes and internal standard solution were diluted with methanol to form a mixed standard solution and a mixed internal standard solution with a mass concentration of 1000 ng / mL. An electrospray ionization (ESI) source was used; negative ion multiple reaction monitoring (MRM) scanning was performed. The specific process is as follows: 14 target analytes and internal standard solution were diluted with methanol to form a mixed standard solution and a mixed internal standard solution with a mass concentration of 1000 ng / mL, and a syringe pump was used for injection. A full scan was performed in positive and negative ion modes to select the appropriate ionization mode and molecular ion peak. The [MH] of the target compound was used as the - The daughter ion scan was performed for the parent ion, and two daughter ions with higher abundance and less interference were selected, and the collision voltage (CE) and declustering voltage (DP) were optimized. For PFOS, since the ion with the strongest response of m / z=80 was interfered by endogenous substances such as bile acid in animal tissues, the ion with m / z=99 was selected as the quantitative ion to avoid the above interference. For other perfluorinated compounds, the ion with the strongest response was selected as the quantitative ion. Then, a mixed standard solution of 1.00µg / mL was used to optimize the curtain gas (CUR) of 15psi, the collision gas (CAD) of 12psi, the nebulizer gas (GS1) of 50psi, the auxiliary gas (GS2) of 35psi, the ion source voltage (IS) of -4000V, the ionization temperature (TEM) of 550℃ and the scanning time of 30ms in the negative ionization mode with multiple reaction monitoring (MRM).

[0048] Table 2 Mass spectrometry parameters of 14 perfluorinated compounds and internal standard substances

[0049] Note: * is the quantitative ion Example 2 The difference from Example 1 is that the mouse organ tissue sample is mouse kidney tissue, and the remaining steps are the same as Example 1.

[0050] Example 3 The difference from Example 1 is that the mouse organ tissue sample is mouse spleen tissue, and the remaining steps are the same as Example 1.

[0051] Example 4 The difference from Example 1 is that the mouse organ tissue sample is mouse intestinal tissue, and the remaining steps are the same as Example 1.

[0052] Example 5 The difference from Example 1 is that the mouse organ tissue sample is mouse heart tissue, and the remaining steps are the same as Example 1.

[0053] Example 6 The difference from Example 1 is that the mouse organ tissue sample is mouse lung tissue, and the remaining steps are the same as Example 1.

[0054] Example 7 The difference from Example 1 is that the mouse organ tissue sample is mouse brain tissue, and the remaining steps are the same as Example 1.

[0055] Test Example 1 (1) Accuracy test The accuracy of the method was evaluated by a spike recovery test. According to the operating methods of Examples 1 to 7, the treatment and determination were repeated 6 times, and the results were shown in Tables 3 to 6.

[0056] Table 3 Recovery of 14 target compounds in low-concentration matrix spikes (1 ng / mL, %)

[0057] Table 4 Recovery of 14 target compounds in high concentration matrix (10 ng / mL, %)

[0058] Table 5 Precision of the results of the spiked determination of 14 target compounds at low concentrations (1 ng / mL, %)

[0059] Table 6 Precision of the results of spiked determination of 14 target substances at high concentrations (10 ng / mL, %)

[0060] As can be seen from Tables 3 to 6, the recoveries of the 14 target substances ranged from 64.9% to 120.3%, and the RSDs of the six repeated tests were in the range of 1.0% to 14.0%.

[0061] (2) Method detection limit and quantification limit The detection limit of the method provided in the embodiment is shown in Table 7, and the quantification limit is shown in Table 8.

[0062] Table 7 Detection limits of 14 target substances in different matrices (ng / g, based on 0.02g sample)

[0063] Table 8 Quantitative limits of the determination methods for 14 target substances in different matrices (ng / g, based on 0.02g sample)

[0064] Example 8 The difference from Example 1 is that kidneys of 6 mice were selected to represent liver, spleen, heart and kidney matrix and used as mouse organ tissue samples, and the remaining steps were the same as Example 1.

[0065] Example 9 The difference from Example 1 is that the lungs of 6 mice were selected to represent the intestine and lung matrix and used as mouse organ tissue samples, and the remaining steps were the same as Example 1.

[0066] Example 10 The difference from Example 1 is that brain tissues of 6 mice were selected as mouse organ tissue samples, and the remaining steps were the same as in Example 1.

[0067] Test Example 2 The eluent was obtained according to the method of Examples 8 to 10, and then 0.500 ng of internal standard was added to 200 μL of the eluent. The matrix effect was evaluated by comparing the stability of the internal standard peak area, and the results are shown in Table 9.

[0068] Table 9 Representative matrix effect evaluation (%)

[0069] It can be seen from Table 9 that the matrix effects of all target substances are between 83.7% and 134.0%, indicating that the matrix effect of the method is relatively weak and meets the requirements of quantitative analysis.

[0070] Embodiment 11 Liver, kidney, spleen, intestine, heart, lung and brain tissue samples of mice orally exposed to representative perfluorinated compounds PFOA, PFOS and 6:2 Cl-PFESA for 3 months were collected and used as mouse tissue samples. The samples were tested according to the method of Example 1. The results are shown in Table 10.

[0071] Table 10 Detection results of PFOA, PFOS and 6:2 Cl-PFESA in tissue samples of mice exposed orally (μg / g)

[0072] It can be seen from Table 10 that the method provided by the present invention can accurately detect PFOA, PFOS and 6:2 Cl-PFESA in orally exposed mouse tissue samples, which shows that the detection method provided by the present invention is feasible.

[0073] It can be seen from the above results that the method results provided by the present invention have excellent accuracy and reproducibility. This is because the addition of an internal standard solution during the homogenization process can correct the loss during the sample processing process and improve the accuracy and reliability of quantitative analysis; and the addition of an internal standard solution can also eliminate the influence of the sample matrix on the analysis results and correct the interference caused by the matrix effect; methyl tert-butyl ether is used as an extractant and tetrabutylammonium hydrogen sulfate is used as an ion pair reagent to form an ion pair with the target, thereby increasing the solubility of the target in the organic phase, thereby achieving transfer from the aqueous phase to the organic phase, and improving the extraction effect of perfluorinated compounds in mouse organ tissue samples; the present invention uses ultra-high performance liquid chromatography tandem mass spectrometry analysis, which requires a relatively small amount of sample and has a highly sensitive and specific detection result for perfluorinated compounds in tissues.

[0074] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for simultaneously detecting perfluorinated compounds in mouse tissues and organs, comprising the following steps: (1) Mixing a mouse organ tissue sample, an alkaline buffer solution, and an internal standard solution, and then homogenizing the mixture to obtain a homogenate; (2) mixing the homogenate obtained in step (1) with tetrabutylammonium hydrogen sulfate and methyl tert-butyl ether, and performing liquid-liquid extraction to obtain an extract; (3) The extract obtained in step (2) is dried and redissolved in turn, and subjected to ultra-high performance liquid chromatography-tandem mass spectrometry analysis to obtain the results of perfluorinated compounds in mouse tissues and organs.

2. The method according to claim 1, characterized in that The mouse organ tissue sample in step (1) includes one or more of the mouse liver, kidney, spleen, intestine, heart, lung and brain tissue.

3. The method according to claim 1, characterized in that The pH value of the alkaline buffer solution in step (1) is 8-11.

4. The method according to claim 1, characterized in that: The internal standard substance in the internal standard solution in step (1) includes 13 C4-PFBA, 13 C5-PFHxA, 13 C4-PFHpA, 13 C8-PFOA, 13 C9-PFNA, 13 C6-PFDA, 13 C2-PFTeDA, 13 C3-PFHxS and 13 C8-PFOS.

5. The method according to claim 1, characterized in that In step (1), the ratio of the mass of the mouse organ tissue sample to the volume of the alkaline buffer solution is (5-20) mg: (50-200) µL.

6. The method according to claim 1, characterized in that In the step (2), the ratio of the volume of the homogenate, the amount of tetrabutylammonium hydrogen sulfate and the volume of methyl tert-butyl ether is (1-3) mL: (0.25-0.75) mmol: (6-12) mL.

7. The method according to claim 1, characterized in that The solvent for re-dissolving in step (3) is a methanol-water solution, and the volume ratio of methanol to water in the methanol-water solution is 1:(1-3).

8. The method according to claim 1, characterized in that The ultra-high performance liquid chromatography conditions for ultra-high performance liquid chromatography tandem mass spectrometry analysis in step (3) include: The chromatographic column is a reversed phase liquid chromatography column with a dimethyl n-octadecylsilane stationary phase chemically bonded to a Poroshell 120 silica gel carrier. The length of the chromatographic column is 50-150 mm, the diameter of the chromatographic column is 2-5 mm, and the filler particle size of the chromatographic column is 1.5-5 µm. The chromatographic column temperature is 30-50 °C. The injection volume is 3-10 µL. A gradient elution program is adopted, and the flow rate of the mobile phase is 300-450 μL / min; the mobile phase includes mobile phase A and mobile phase B, the mobile phase A is an ammonium acetate aqueous solution with a concentration of 2-10 mmol / L, and the mobile phase B is an ammonium acetate methanol solution with a concentration of 2-10 mmol / L.

9. The method according to claim 8, characterized in that The gradient elution program is as follows: at 0-7.5 min, the mobile phase changes linearly from 55% mobile phase A and 45% mobile phase B to 5% mobile phase A and 95% mobile phase B; at 7.5-8.0 min, the mobile phase remains at 5% mobile phase A and 95% mobile phase B; at 8.0-9.0 min, the mobile phase changes linearly from 5% mobile phase A and 95% mobile phase B to 55% mobile phase A and 45% mobile phase B; at 9.0-12 min, the mobile phase remains at 55% mobile phase A and 45% mobile phase B.

10. The method according to claim 1, characterized in that The mass spectrometry conditions of the ultra-high performance liquid chromatography tandem mass spectrometry analysis in the step (3) include: ionization in negative ion electrospray mode and negative ion multiple reaction monitoring scanning; the pressure of the collision gas is 6-12 psi; the pressure of the curtain gas is 10-30 psi; the pressure of the nebulizer gas is 40-55 psi; the pressure of the auxiliary gas is 30-60 psi; the ion source voltage is -3500-5500 V; the ionization temperature is 450-550° C.; and the scanning time is 30 ms.

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