One-step determination method for perfluoro / polyfluoroalkyl substances in serum

Abstract

The present invention discloses a one-step determination method for perfluoro / polyfluoroalkyl compounds in serum, belonging to the field of biotechnology. By using a stepped split-type HMR purification tube, the sample to be loaded is purified through a through-type process, and the pretreatment of the sample can be completed in one step in the purification tube, improving the sample recovery rate, saving the pre-treatment time of the sample, and improving the efficiency. In the above method, the extraction and purification are completed in one step in the HMR degreasing column without the need for back-and-forth transfer operations, simplifying the experimental process and ensuring good method stability. At the same time, due to the small size of the stepped split-type HMR purification tube, only 50 μL of sample is required to complete the detection, solving the pain point of serum sample waste; the entire process is carried out in one tube, reducing the reagent usage and being more environmentally friendly; the HMR filler used specifically adsorbs phospholipid groups in the HMR-Lipid column, which can eliminate the matrix effect problem caused by phospholipids.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a one-step determination method for perfluoro / polyfluoroalkyl compounds in serum. Background Art

[0002] Perfluoro / polyfluoroalkyl compounds (PFASs) are a class of widely used synthetic compounds. Due to their strong hydrophobicity and unique physicochemical properties, they are widely used in fields such as non-stick coatings for kitchen utensils, textile fabrics, and leather products. However, due to their stable chemical properties, they are difficult to degrade in the natural environment. A large number of domestic wastes containing PFASs easily enter the food chain and accumulate and amplify after entering the environment, and are ingested by the human body through food and drinking water. Currently known studies show that PFASs can be widely distributed in multiple organs of the human body, causing harm to various important organs such as the nervous system, reproductive system, and immune system.

[0003] For blood samples, traditional extraction methods require at least 200 μL of sample volume. Add the internal standard solution, then vortex and mix well, sonicate for 15 min, centrifuge (12,000 rpm / min) for 10 min, take the supernatant to a new centrifuge tube, and add methanol to the original centrifuge tube to repeat the extraction once. Then, a dispersive solid-liquid extraction and purification step is adopted: combine the above extraction solutions, add PSA filler, vortex and mix well, transfer to a new centrifuge tube, blow dry with nitrogen, re-dissolve with methanol aqueous solution, vortex and mix well, centrifuge, and take the supernatant for on-machine detection. The above extraction and purification method not only requires a large amount of sample volume, but also requires a large number of centrifugation, extraction, drying, re-dissolution and other processes, which is very cumbersome and complex.

[0004] The internationally popular traditional liquid-liquid extraction method requires at least 200 μL of sample volume. Add a buffer solution composed of sodium carbonate and sodium bicarbonate, add TBAHs solution, internal standard solution, add methyl tert-butyl ether, shake for 20 min, centrifuge and take the supernatant, repeat the above extraction step twice, combine the supernatants, blow dry with nitrogen, re-dissolve with methanol aqueous solution, vortex and mix well, centrifuge for 15 min, and take the supernatant for on-machine detection. Although this liquid-liquid extraction method can remove most impurities, lecithin and phosphatidylglycerol in the blood cannot be effectively removed. The unremoved phospholipids will accumulate in the ion source of the mass spectrometer and ionize competitively with the target analyte, resulting in ion suppression and inhibiting the response of the target. On the one hand, this affects the lifespan of the mass spectrometer, and on the other hand, it affects the accuracy of the detection results, and the experimental operation process is cumbersome.

[0005] In summary, the technical defects of the existing technology for the detection method of perfluoro compounds in serum are as follows:

[0006] 1) The serum sample loading volume is large; at least 200 μL of sample volume is required, wasting precious serum samples;

[0007] 2) The pre-treatment extraction steps are cumbersome: Repeated extraction and transfer are required, which affects the recovery rate.

[0008] 3) The pre-treatment takes a long time, reducing the experimental efficiency.

[0009] 4) The reagent consumption is large and it is not environmentally friendly.

[0010] 5) The purification effect is poor: In traditional liquid-liquid extraction, the purification packing cannot completely remove phospholipid and protein impurities, which affects the purification effect. Summary of the Invention

[0011] The purpose of the present invention is to provide a one-step determination method for perfluoro / polyfluoroalkyl compounds in serum. By using a stepped split-type HMR purification tube and adopting a through-type purification process for the loaded sample, the pre-treatment of the sample can be completed in one step inside the purification tube, improving the recovery rate of the sample, saving the pre-treatment time of the sample, and improving the efficiency. At the same time, due to the small size of the stepped split-type HMR purification tube, only 50 μL of the sample volume is required to complete the detection, solving the pain point of serum sample waste. The entire process is carried out in one tube body, reducing the reagent usage and being more environmentally friendly. The used HMR packing is an HMR-Lipid small column that specifically adsorbs phospholipid groups, which can eliminate the matrix effect problem caused by phospholipids.

[0012] The present invention is realized through the following technical solutions:

[0013] A one-step determination method for perfluoro / polyfluoroalkyl compounds in serum, comprising the following steps:

[0014] S1. Pretreatment of the sample:

[0015] Select an in vitro serum sample and add it to a stepped split-type HMR purification tube, add an isotope internal standard, vortex and mix evenly, slowly add acetonitrile along the tube wall, and let it stand for protein precipitation.

[0016] S2. Sample extraction and elution: Set a positive pressure device at the top of the stepped split-type HMR purification tube. After pressurizing the positive pressure device until the liquid in the stepped split-type HMR purification tube slowly drops, increase the pressure until no liquid is discharged from the stepped split-type HMR purification tube and collect it for standby;

[0017] Then add acetonitrile again, let it stand, and perform positive pressure treatment to discharge the liquid in the stepped split-type HMR purification tube, and collect it for standby;

[0018] S3. Nitrogen blowing treatment:

[0019] Place all the filtrates collected in S2 in a nitrogen blowing device for nitrogen blowing treatment;

[0020] S4. Instrument detection:

[0021] After nitrogen blowing treatment, add methanol-aqueous solution, mix well, and then conduct on-machine detection;

[0022] The stepped split-type HMR purification tube includes a sample loading area, a purification area, and a liquid discharge area. A protein precipitation sieve plate is arranged between the sample loading area and the purification area; HMR packing is provided in the purification area; the liquid in S2 is discharged from the purification tube through the liquid discharge area.

[0023] Preferably, the usage amount of the in vitro serum sample is 50 μL.

[0024] Preferably, the perfluoro / polyfluoroalkyl compounds include any one or more of perfluorobutyric acid, perfluoropentanoic acid, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanoic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluorobutanesulfonic acid, perfluorohexanesulfonic acid, perfluoroheptanesulfonic acid, perfluorooctanesulfonic acid, 6:2 chlorinated polyfluoroalkyl ether sulfonic acid, and 8:2 chlorinated polyfluoroalkyl ether sulfonic acid.

[0025] Preferably, the isotope internal standard is a PFASs internal standard use solution with a concentration of 100 ng / mL.

[0026] Preferably, the volume ratio of the in vitro serum sample to the isotope internal standard is 1:1;

[0027] The volume ratio of the in vitro serum sample to acetonitrile is 1:4.

[0028] Preferably, in S1, the standing time is 3 - 5 min.

[0029] Preferably, in S2, the positive pressure device pressurizes to a positive pressure of 0 - 0.1 PSI.

[0030] Preferably, the sample elution program is: 0 - 6.0 min, 40% mobile phase A, 60% mobile phase B; 6.0 - 8.0 min, 95% mobile phase A, 5% mobile phase B; 8.0 - 8.1 min, 40% mobile phase A, 60% mobile phase B; 8.1 - 10.0 min, 40% mobile phase A, 60% mobile phase B;

[0031] The mobile phase A is a 5 mM ammonium acetate methanol solution; the mobile phase B is a 5 mM ammonium acetate aqueous solution.

[0032] Preferably, in S4, the volume ratio of methanol to water is 1:1, and the total volume is 50 μL;

[0033] The mixing time is 1 - 2 min.

[0034] Preferably, the HMR packing is modified inorganic zirconium packing.

[0035] Compared with the prior art, the present invention has at least the following technical effects:

[0036] The object of the present invention is to provide a one-step determination method for perfluoro / polyfluoroalkyl compounds in serum. By using a stepped split-type HMR purification tube, the sample to be loaded is purified by a through-flow purification process, and the pretreatment of the sample can be completed in one step in the purification tube, improving the sample recovery rate, saving the pretreatment time of the sample, and improving work efficiency.

[0037] In the above method, extraction and purification are completed in one step in the HMR defatting column without the need for back-and-forth transfer operations, simplifying the experimental process and ensuring method stability.

[0038] At the same time, due to the small size of the stepped split-type HMR purification tube, only 50 μL of sample is required to complete the detection, solving the pain point of serum sample waste; the entire process is carried out in one tube, reducing the reagent usage and being more environmentally friendly; the HMR filler used is the HMR-Lipid column that specifically adsorbs phospholipid groups, which can eliminate the matrix effect problem caused by phospholipids.

[0039] The above determination method has the following advantages:

[0040] (1) Better purification effect: A cleaner purification effect. The HMR-Lipid specifically adsorbs phospholipid groups, which can eliminate the matrix effect problem caused by phospholipids. In addition, the column efficiency far exceeds that of the through-type SPE, providing a higher theoretical plate number; the recovery rate is stable at about 90%.

[0041] (2) Simpler and more efficient: Through-flow purification greatly simplifies the pretreatment steps, and protein precipitation and sample purification are completed in one step. Traditional methods require cumbersome steps such as repeated liquid-liquid extraction, ultrasonic centrifugation, and changing centrifuge tubes.

[0042] (3) Smaller sample loading volume: Traditional and international methods require a 200-μL sample loading, while the HMR S-micro 96-well plate only requires 50 μL of sample, greatly saving precious samples.

[0043] (4) Shorter pretreatment time: Taking 30 samples as an example, the HMR ultra-efficient split 96-well plate only requires 45 minutes, while traditional and international methods require 2-3 hours. Description of the Drawings

[0044] Figure 1 Schematic diagram of the one-step determination method for perfluoro / polyfluoroalkyl compounds in serum in Example 1;

[0045] Figure 2 Flow chart of the one-step determination method for perfluoro / polyfluoroalkyl compounds in serum in Example 1;

[0046] Figure 3 Total ion current comparison chart of liquid-liquid extraction and purification method in Example 1

[0047] Figure 4 Lecithin chromatogram comparison chart of liquid-liquid extraction and purification method in Example 1

[0048] Figure 5 Phosphatidylglycerol chromatogram comparison chart of liquid-liquid extraction and purification method in Example 1 Detailed implementation mode

[0049] The following will describe the implementation scheme of the present invention in detail in combination with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0050] Example 1:

[0051] 1. Experimental process: The specific process is as shown in Figure 1 and 2 shown

[0052] 1.1 Take 50 μL of serum into an HMR purification tube;

[0053] 1.2 Add 50 μL of isotope internal standard;

[0054] 1.3 Slowly add 200 μL of acetonitrile and let it stand for 5 min;

[0055] 1.4 Place it on a positive pressure device, apply a pressure of 0.1 PSI, and wait for the liquid droplets to drip slowly (one by one). Gradually increase the pressure to ensure that there is no liquid in the purification tube;

[0056] 1.5 Place the plate for collecting the filtrate in a nitrogen evaporator and blow it to nearly dry with filtered liquid nitrogen;

[0057] 1.6 Add 50 μL of methanol-water (1:1), mix for 1 min, and then perform on-machine detection.

[0058] 2. Instrument conditions:

[0059] The chromatographic column uses Poroshell 120 EC-C18 (100 mm × 3 mm, 2.7 μm). The mobile phases are methanol solution (phase A) and 5 mM ammonium acetate aqueous solution (phase B). The column temperature is 40 °C, the injection volume is 2 μL, the flow rate is 0.4 mL / min, and the mobile phase gradient conditions are shown in Table 1.

[0060] Table 1 Liquid chromatography analysis conditions

[0061] Time (min) Flow rate (mL / min) ％A ％B Gradient curve 0.0 0.4 40 60 6 6.0 0.4 95 5 6 8.0 0.4 95 5 6 8.1 0.4 40 60 6 10.0 0.4 40 60 6

[0062] 3. Mass Spectrometry Conditions

[0063] The multiple reaction monitoring (MRM) mode and the ESI negative ion mode were adopted. The mass spectrometry parameters were as follows: ion source temperature, 400 °C; electrospray voltage: -4500 V; curtain gas: 30 psi; collision gas: medium; Ion Source Gas 1: 60 psi; Ion Source Gas 2: 60 psi. The parameters such as ion pairs, declustering voltage, and collision energy of the target PFASs are shown in Table 2.

[0064] Perfluorobutyric acid (PFBA), perfluoropentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnDA), perfluorododecanoic acid (PFDoDA), perfluorotridecanoic acid (PFTriDA), perfluorotetradecanoic acid (PFTeDA), perfluorobutane sulfonic acid (PFBS), perfluorohexane sulfonic acid (PFHxS), perfluoroheptane sulfonic acid (PFHpS), perfluorooctane sulfonic acid (PFOS), 6:2 chlorinated polyfluoroalkyl ether sulfonic acid (6:2Cl-PFESA), 8:2 chlorinated polyfluoroalkyl ether sulfonic acid (8:2Cl-PFESA).

[0065] Table 2 Mass Spectrometry Parameters of PFASs

[0066]

[0067]

[0068] 4. Method Quantification Limit

[0069] The maximum value of the following two methods was taken as the method quantification limit (LOQ) of this project: (1) the lowest concentration in the actual serum matrix that satisfied a signal-to-noise ratio greater than 10, and the signal-to-noise ratio was calculated using the RMS (Root Mean Square) method; (2) the lowest concentration within the working range of the standard curve that satisfied the measured concentration within ±20% of the theoretical value. Since the samples in this study were not concentrated or diluted and there was no dilution factor, the method quantification limit was the LOQ, as shown in Tables 3 and 4.

[0070] Table 3 Quantification Limits of 16 PFASs

[0071]

[0072]

[0073] Table 4 Spike recoveries and precisions of 16 perfluorinated compounds in serum at different levels (n = 6)

[0074]

[0075]

[0076] Comparison between Example 1 and the prior art:

[0077] Conclusion of the comparison with the liquid-liquid extraction method for serum samples

[0078] As Figure 3 shown, it is the total ion current comparison chart of liquid-liquid extraction and the purification method of Example 1. Among them, A is the total ion current chart of liquid-liquid extraction; B is the total ion current chart of the purification method of Example 1

[0079] As Figure 4 shown, it is the lecithin chromatogram comparison chart of liquid-liquid extraction and the purification method of Example 1. Among them, A is the lecithin chromatogram of liquid-liquid extraction; B is the lecithin chromatogram of the purification method of Example 1

[0080] As Figure 5 shown, it is the phosphatidylglycerol chromatogram comparison chart of liquid-liquid extraction and the purification method of Example 1. Among them, A is the phosphatidylglycerol chromatogram of liquid-liquid extraction; B is the phosphatidylglycerol chromatogram of the purification method of Example 1

[0081] Summary of experimental results

[0082] Lecithin Phosphatidylglycerol Elution time 25.24 25.25 Peak height of liquid-liquid extraction <![CDATA[3.43*10 7 > <![CDATA[1.110*10 6 > Peak height of HMR purification <![CDATA[2.27*10 5 > 0 Removal efficiency 99.3％ 100％

[0083] From the above experimental schematic diagrams and tables, it can be seen that only liquid-liquid extraction cannot effectively remove the phospholipid interference in blood samples. After purification by HMR-Lipid, most of the interfering substances in the samples can be removed. Among them, the removal rate of lecithin is 99.3%, and the removal rate of phosphatidylglycerol is 100%. It can effectively protect the mass spectrometry instrument, reduce matrix interference, and improve the accuracy of detection data

[0084] 6. Taking 30 actual samples as an example

[0085] 6.1 Implementation method of the above Example 1: Total: 47 min

[0086] ① Add 50 μL of serum sample and internal standard solution into the HMR purification tube respectively, slowly add 200 μL of acetonitrile along the tube wall, and let it stand for 5 min for protein precipitation; (about 7 min)

[0087] ② Apply positive pressure until the liquid drops slowly drip. Gradually increase the pressure to ensure that there is no liquid in the purification tube; (about 5 min) Repeat the extraction with 200 μL of acetonitrile under positive pressure once; (about 5 min)

[0088] ③ Place the receiving plate for collecting the filtrate in a nitrogen evaporator and evaporate to nearly dryness with nitrogen; redissolve with 50 μL of methanol-water (50:50) solution and measure on the machine (about 30 min).

[0089] 6.2 Traditional method: a total of 1 h 40 min

[0090] ① Take 200 μL of serum sample and internal standard solution into a 1.5 mL centrifuge tube; add 1 mL of methanol solution, vortex and mix well, and sonicate for 15 min; (about 18 min);

[0091] ② Transfer the supernatant to a new centrifuge tube and centrifuge for 10 min; (about 18 min)

[0092] ③ Repeat the extraction once with 1 mL of methanol solution; (about 30 min)

[0093] ④ Combine the supernatants, add 50 mg of PSA, mix well, and shake for 10 min,

[0094] ⑤ Evaporate the supernatant to dryness with nitrogen; redissolve with 200 μL of methanol-water (50:50) solution and measure on the machine. (about 30 min).

[0095] 6.3 International method: a total of 3 h 10 min

[0096] ① Take 200 μL of serum sample and internal standard solution into a 1.5 mL centrifuge tube;

[0097] ② 2 mL of buffer solution (Na 2 CO 3 5.30 g, NaHCO 3 4.20 g dissolved in 200 mL of water, concentration 0.25 mol / L pH = 10),

[0098] ③ 1 mL of TBAH S (17.00 g of tetrabutylammonium hydrogen sulfate in 100 mL of water, 0.5 mol / L) solution;

[0099] ④ 4 mL of methyl tert-butyl ether (complicated solution preparation, about 60 min);

[0100] ⑤ Shake for 20 min and centrifuge, take the supernatant; (about 30 min)

[0101] ⑥ Repeat the extraction twice with the above extraction solution; (about 60 min)

[0102] ⑦ Combine the supernatants and evaporate to dryness with nitrogen (about 40 min)

[0103] ⑧ Redissolve with 200 μL of methanol-water (50:50) solution and measure on the machine.

[0104] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A one-step method for the determination of perfluoro / polyfluoroalkyl compounds in serum, characterized in that: The steps include: S1. Sample pretreatment: Select an in vitro serum sample and add it to a step-split HMR purification tube, add an isotope internal standard, vortex mix, slowly add acetonitrile along the tube wall, and let it stand for protein precipitation; S2. Sample extraction and elution: a positive pressure device is set on the top of the stepped split HMR purification tube. After the liquid in the stepped split HMR purification tube slowly drips, the pressure is increased until no liquid is discharged from the stepped split HMR purification tube and collected for later use; Then, acetonitrile is added again, the mixture is allowed to stand, and the liquid in the stepped split HMR purification tube is discharged by positive pressure treatment and collected for later use; S3, nitrogen blowing treatment: All the filtrate collected in S2 is placed in a nitrogen blowing device for nitrogen blowing treatment; S4. On-machine testing: After nitrogen blowing, add methanol-water solution and mix well, then test on the machine; The stepped split HMR purification tube comprises a sample loading area, a purification area and a liquid discharge area, a protein precipitation screen plate is arranged between the sample loading area and the purification area; and an HMR filler is arranged in the purification area; The liquid in S2 is discharged from the purification pipe through the liquid discharge area; The HMR filler used is the HMR-Lipid column that specifically adsorbs phospholipid groups; The HMR filler is a modified inorganic zirconium filler.

2. The one-step determination method for perfluoro / polyfluoroalkyl compounds in serum according to claim 1, characterized in that: The usage volume of the in vitro serum sample is 50 μL.

3. The one-step determination method for perfluoro / polyfluoroalkyl compounds in serum according to claim 1, characterized in that: The perfluoro / polyfluoroalkyl compounds include any one or more of perfluorobutyric acid, perfluoropentanoic acid, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanoic acid, perfluorotridecanoic acid, perfluorotetradecanoic acid, perfluorobutane sulfonic acid, perfluorohexane sulfonic acid, perfluoroheptane sulfonic acid, perfluorooctane sulfonic acid, 6:2 chloropolyfluoroalkyl ether sulfonic acid and 8:2 chloropolyfluoroalkyl ether sulfonic acid.

4. The one-step determination method of perfluoro / polyfluoroalkyl compounds in serum according to claim 1, characterized in that: The isotope internal standard is a 100 ng / mL PFASs internal standard solution.

5. The one-step determination method of perfluoro / polyfluoroalkyl compounds in serum according to claim 1, characterized in that: The volume ratio of the in vitro serum sample to the isotope internal standard is 1:1; The volume ratio of the in vitro serum sample to acetonitrile is 1:

4.

6. The one-step determination method for perfluoro / polyfluoroalkyl compounds in serum according to claim 1, characterized in that: In S1, the standing time is 3 to 5 minutes.

7. The one-step determination method for perfluoro / polyfluoroalkyl compounds in serum according to claim 1, characterized in that: In S2, the positive pressure device pressurizes the device to a positive pressure of 0 to 0.1 PSI.

8. The one-step determination method for perfluoro / polyfluoroalkyl compounds in serum according to claim 1, characterized in that: The sample elution program was: 0-6.0 min, 40% mobile phase A, 60% mobile phase B; 6.0-8.0 min, 95% mobile phase A, 5% mobile phase B; 8.0-8.1 min, 40% mobile phase A, 60% mobile phase B; 8.1-10.0 min, 40% mobile phase A, 60% mobile phase B; the mobile phase A is 5 mM ammonium acetate methanol solution; the mobile phase B is 5 mM ammonium acetate aqueous solution.

9. The one-step determination method for perfluoro / polyfluoroalkyl compounds in serum according to claim 1, characterized in that: In S4, the volume ratio of methanol and water is 1:1, and the total volume is 50 μL; The mixing time is 1 to 2 minutes.

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