Serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction

Abstract

The invention relates to the technical field of biological analytical chemistry, in particular to a serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction, which comprises the steps of acidified acetonitrile protein precipitation, nitrogen blowing concentration, composite extractant liquid-liquid extraction and redissolution filtration. According to the method, a methyl tert-butyl ether and ethyl acetate compound extraction system is adopted, the process of first concentration and then extraction is combined, the pretreatment effects of high recovery rate, low matrix effect and high enrichment multiple are achieved in a single centrifugal tube, and the method is suitable for high-flux, low-cost and green detection of C4-C14 perfluorinated compounds in trace serum.

Description

Technical Field

[0001] This invention belongs to the field of environmental and bioanalytical chemistry technology, specifically a serum pretreatment method for detecting perfluorinated compounds based on acidified protein precipitation and liquid-liquid extraction. Background Technology

[0002] In the field of pretreatment for the detection of perfluorinated and polyfluoroalkyl substances (PFASs) in serum, existing technologies mainly rely on methods such as protein precipitation or solid-phase extraction (SPE). While protein precipitation offers advantages such as ease of operation and low cost, its purification capacity is limited. It typically only removes protein components and is less effective at removing coexisting interfering substances such as phospholipids, salts, and endogenous small molecules. This can easily lead to significant matrix effects during liquid chromatography-mass spectrometry (LC-MS) analysis, thus affecting the accuracy and precision of quantitative results. Furthermore, when processing low-concentration PFASs samples, the method may reduce detection sensitivity due to sample dilution, making it unsuitable for trace analysis.

[0003] On the other hand, while solid-phase extraction (SPE) can achieve good purification results, its process is relatively cumbersome, involving multiple steps such as activation, sample loading, rinsing, and elution. This requires highly skilled operators and necessitates the use of disposable SPE columns, increasing both detection costs and the burden of significant plastic waste, which conflicts with the current trend towards green analytical chemistry. Furthermore, lipids and particulate matter in serum samples can easily clog SPE columns, affecting the stability of recovery rates and even leading to column failure.

[0004] Among the relevant patents retrieved, CN214310339U discloses a rapid sample pretreatment device based on liquid chromatography-mass spectrometry (LC-MS), employing a combination of filter tubes and ultrafiltration tubes for the mixing and separation purification of solid samples. However, this device is primarily designed for solid samples and is not optimized for the characteristics of perfluorinated compounds in complex biological liquid samples such as serum. It also lacks an effective removal mechanism for lipids and small molecule interfering substances, making it difficult to meet the pretreatment requirements for highly sensitive and selective detection of PFASs.

[0005] Another related patent, CN209946091U, discloses a dedicated pretreatment device for pesticide residue detection based on the QuEChERS method. This device improves pretreatment efficiency by replacing manual operation with mechanical vibration. Although the QuEChERS method has wide applications in pesticide residue detection, its core still relies on salting out and adsorbent purification. For highly polar and stable perfluorinated compounds in the serum matrix, conventional QuEChERS adsorbents (such as PSA and C18) have limited retention and purification effects on PFASs. Furthermore, this device does not integrate an acidification step, failing to effectively promote protein denaturation and precipitation and target analyte release, nor does it incorporate a liquid-liquid extraction mechanism to further remove lipophilic interfering substances.

[0006] In summary, existing technologies for serum perfluorinated compound detection pretreatment either suffer from insufficient purification capabilities leading to severe matrix interference, or are limited in large-scale screening due to complex procedures, high costs, and significant environmental impact. Therefore, there is an urgent need for a pretreatment method that is efficient in removing proteins, effectively removes lipids and small molecule interference, is easy to operate, and is environmentally friendly, in order to improve the accuracy, sensitivity, and applicability of perfluorinated compound detection. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by proposing a serum pretreatment method for the detection of perfluorinated compounds based on acidified protein precipitation and liquid-liquid extraction.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: A serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction includes the following steps: S10: Take 500 μL of serum sample and place it in a 1.5 mL centrifuge tube. Add 1000~1500 μL of acetonitrile solution containing 0.1%~1.0% formic acid. Vortex for 30 seconds to 2 min. Centrifuge at 12000~15000 r / min at 4℃ for 5~10 min. Discard the precipitate and keep the supernatant. S20: Transfer the supernatant obtained in step S10 to a new 1.5ml centrifuge tube and concentrate it to 200~300μL under nitrogen blowing at 37℃. S30: Add 500 μL of a composite extractant made of methyl tert-butyl ether and ethyl acetate in a volume ratio of 3:1 to 1:1 to the supernatant after concentration in step S20, vortex for 0.5 to 3 min, and centrifuge at 12000 to 15000 r / min at 4℃ for 5 to 8 min to separate the upper organic phase. S40: The organic phase obtained in step S30 is purged to near dryness under a nitrogen flow at 37°C, and then redissolved with 65 μL of methanol aqueous solution, wherein the volume ratio of methanol to water in the methanol aqueous solution is 3:2. After vortexing and mixing, the mixture is centrifuged at 12000~15000 r / min for 5~8 min at 4°C. The resulting supernatant is used for liquid chromatography-tandem mass spectrometry analysis.

[0009] Preferably, in step S10, the concentration of formic acid in the acetonitrile solution is 0.1% to 0.5%, and the volume ratio of acetonitrile to serum is 1:1 to 3:1.

[0010] Preferably, in step S10, the vortex mixing time is 1 min, and the centrifugation conditions are 4℃, 14000 r / min, and 8 min.

[0011] Preferably, in step S20, the final volume of nitrogen blowing concentration is 250 μL, the nitrogen flow rate is 1.5~2.0 L per minute, and the temperature is 37°C.

[0012] Preferably, in step S30, the volume ratio of methyl tert-butyl ether to ethyl acetate is 1:1.

[0013] Preferably, in step S30, the vortex oscillation time is 2 min, and the centrifugation conditions are 4℃, 14000 r / min, and 6 min.

[0014] Preferably, in step S40, the volume ratio of methanol to water in the methanol-water solution used for resolution is 3:2, and the resolution volume is 65 μL.

[0015] Preferably, in step S40, the nitrogen blowing drying endpoint is when no obvious droplets remain visible to the naked eye, but a trace amount of moisture is retained.

[0016] Preferably, the composite extractant is composed of methyl tert-butyl ether and ethyl acetate in a volume ratio of 3:1 to 1:1, and is used to remove phospholipids, triglycerides and cholesterol, and to enrich perfluorocarboxylic acids, perfluorosulfonic acids and their precursor compounds.

[0017] Preferably, the perfluorinated compounds include, but are not limited to, perfluorobutyric acid, perfluorovalerate, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanic acid, perfluorobutyric acid, perfluorohexanesulfonic acid, perfluorooctanesulfonic acid and their chlorinated or hydrogenated precursors.

[0018] The present invention has the following beneficial effects: In summary, this invention achieves high recovery rate, low matrix effect, and high enrichment factor in trace serum samples through a two-step synergistic purification process of "acidified acetonitrile protein precipitation" and "liquid-liquid extraction with composite extractant," combined with the optimized sequence of "concentration followed by extraction." By utilizing a mixed solvent system tailored to the molecular characteristics of PFASs, this invention solves the core problems of incomplete purification, insufficient sensitivity, high cost, and environmental unfriendliness in existing technologies. Attached Figure Description

[0019] Figure 1 This is the chromatogram of perfluorobutyric acid in this invention; Figure 2 This is the chromatogram of perfluorobutane sulfonic acid in this invention; Figure 3 This is the chromatogram of perfluoroheptanoic acid in this invention; Figure 4 This is the chromatogram of perfluorohexylsulfonic acid in this invention; Figure 5 This is the chromatogram of perfluorohexanoic acid in this invention; Figure 6 This is the chromatogram of perfluorononanoic acid in this invention; Figure 7 This is the chromatogram of perfluorododecanoic acid in this invention; Figure 8 This is the chromatogram of perfluoroundecanoic acid in this invention; Figure 9 This is the chromatogram of perfluorovalerate in this invention; Figure 10 This is the chromatogram of perfluorooctylsulfonamide in this invention; Figure 11 This is the chromatogram of perfluorooctanoic acid in this invention. Detailed Implementation

[0020] The various embodiments or implementation schemes in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments.

[0021] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0022] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0023] As described in the background section above, current pretreatment methods for detecting perfluorinated compounds (PFASs) in serum mainly rely on solid-phase extraction (SPE) or single-protein precipitation. While SPE offers good purification, it suffers from high consumable costs, cumbersome procedures, and the potential for plastic contamination. Conventional protein precipitation, though simple and rapid, is incomplete in removing lipophilic interfering substances such as phospholipids and triglycerides, leading to significant matrix effects in LC-MS / MS analysis and impacting quantitative accuracy. Therefore, a pretreatment method that balances purification efficiency, ease of operation, environmental friendliness, and cost control is urgently needed.

[0024] Based on this, this application provides a serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction. Through a two-step synergistic purification strategy, an optimized concentration-extraction sequence, a specific composite extractant system, and a micro-volume high-throughput operation process, it achieves efficient enrichment of trace PFASs in serum and effective removal of matrix interference.

[0025] In a first aspect, this application provides a serum pretreatment method for detecting perfluorinated compounds based on acidified protein precipitation and liquid-liquid extraction, comprising the following steps: S10: Take 500 μL of serum sample and place it in a 1.5 ml centrifuge tube. Add 500~1500 μL of acetonitrile solution containing 0.1% to 1.0% formic acid. Vortex mix for 30 seconds to 2 minutes. Centrifuge at 12000~15000 r / min at 4℃ for 5 to 10 minutes. Discard the precipitate and keep the supernatant. S20: Transfer the supernatant obtained in step S10 to a new 1.5 ml centrifuge tube and concentrate it to about 200~300 μL under nitrogen blowing at 37°C. S30: Add 500 μL of a composite extractant made of methyl tert-butyl ether and ethyl acetate in a volume ratio of 3:1 to 1:1 to the supernatant after concentration in step S20, vortex for 1 to 3 min, and centrifuge at 12000 to 15000 r / min at 4℃ for 5 to 8 min to separate the upper organic phase. S40: The organic phase obtained in step S30 is purged to near dryness under a nitrogen flow at 37°C, and then reconstituted with 65 μL of a methanol-water solution containing 0.1% formic acid (methanol to water volume ratio of 3:1 to 1:1). After vortexing and mixing, the solution is centrifuged at 12000 to 15000 r / min for 5 to 8 min at 4°C. The supernatant obtained is used for liquid chromatography-tandem mass spectrometry analysis.

[0026] According to this application, in step S10, acetonitrile containing formic acid is used for protein precipitation. The addition of formic acid lowers the pH of the system to 2.5-3.5, effectively promoting the denaturation and aggregation of serum proteins, while simultaneously disrupting the non-covalent binding between PFASs and serum albumin, releasing the target analyte. Acetonitrile, as a highly polar organic solvent, can efficiently precipitate more than 95% of proteins under acidic conditions and reduce the loss of the target analyte during precipitation. This step is completed in a single centrifuge tube, avoiding losses caused by sample transfer.

[0027] In some embodiments, in step S10, the concentration of formic acid in the acetonitrile solution is 0.1% to 0.5%, and the volume ratio of acetonitrile to serum is 1:1 to 3:1. This ratio range ensures sufficient protein precipitation while maintaining the solubility and stability of PFASs in the supernatant.

[0028] In some embodiments, in step S10, the vortex mixing time is 1 min, and the centrifugation conditions are 4°C, 14000 r / min, and 8 min. This combination of parameters shortens the processing time and reduces the risk of degradation of heat-sensitive substances while ensuring complete protein sedimentation.

[0029] In some embodiments, in step S20, the nitrogen blowing concentration endpoint is controlled at 250 ml, the nitrogen flow rate is maintained at 1.5 to 2.0 L / min, and the temperature is strictly controlled at 37°C. This temperature is lower than the volatilization temperature of most PFASs and avoids excessive acetonitrile residue affecting the efficiency of subsequent liquid-liquid extraction.

[0030] In some embodiments, in step S30, the volume ratio of methyl tert-butyl ether to ethyl acetate is 1:1. Methyl tert-butyl ether has low polarity and good lipophilicity, which can effectively extract long-chain PFASs and neutral lipids; ethyl acetate has a certain polarity, which is beneficial to the partitioning of short-chain PFASs (such as PFBA and PFPeA). The combination of the two forms a composite extraction system with both polarity gradient and solubility selectivity, so that the recovery rate of PFASs with chain lengths from C4 to C14 is maintained between 85% and 105%.

[0031] In some embodiments, in step S30, the vortex oscillation time is 2 minutes, and the centrifugation conditions are 4°C, 14000 r / min, and 6 minutes. These conditions ensure sufficient contact between the two phases and achieve rapid stratification.

[0032] In some embodiments, in step S40, the reconstitution solvent is a mixture of methanol and water at a volume ratio of 3:2, and the reconstitution volume is precisely controlled to be 65 μL to achieve a 7.7-fold enrichment from 500 ml of raw serum to 65 μL of final injection solution. This volume matches the capacity of the LC-MS / MS injection loop, avoiding secondary dilution.

[0033] In some embodiments, in step S40, the nitrogen blowing drying endpoint is defined as when no obvious droplets remain visible to the naked eye, but a slight degree of moisture is retained to avoid complete drying and subsequent PFAS adsorption loss. This operation can be judged empirically or observed using graduated centrifuge tubes.

[0034] In some embodiments, the method is applicable to serum samples from humans, rats, mice, or pigs. Samples are stored at -80°C after collection and thawed at room temperature and thoroughly mixed before pretreatment. Pretreatment of thawed samples must be completed within 2 hours to prevent PFAS degradation or adsorption.

[0035] In some implementations, the method can process 1 to 96 samples simultaneously, with a total processing time of no more than 90 minutes per sample. This throughput is suitable for large-scale epidemiological screening.

[0036] Secondly, this application provides the application of the composite extractant in the pretreatment of serum perfluorinated compounds. The composite extractant is composed of methyl tert-butyl ether and ethyl acetate in a volume ratio of 3:1 to 1:1, and is used in the liquid-liquid extraction step to remove lipophilic matrix interferences such as phospholipids, triglycerides, and cholesterol, while simultaneously enriching perfluorocarboxylic acids, perfluorosulfonic acids, and their precursor compounds.

[0037] According to this application, the selection of the composite extractant is based on the amphiphilic nature of the PFAS molecular structure: its perfluoroalkyl chain is strongly hydrophobic, while the carboxylic acid or sulfonic acid head group is hydrophilic. Methyl tert-butyl ether has good solubility for the hydrophobic portion, while ethyl acetate interacts with the polar head group through dipole-dipole interactions, and the two synergistically improve the partition coefficient. Experiments show that when methyl tert-butyl ether is used alone, the recovery rate of short-chain PFASs (C4-C6) is less than 70%; when ethyl acetate is used alone, the recovery rate of long-chain PFASs (C10 and above) is less than 80%; while when a 1:1 mixing ratio is used, the recovery rates of all target compounds are stable between 85% and 105%.

[0038] In some embodiments, the amount of the composite extractant used is 500 μL, which reduces the generation of organic waste liquid by 50% to 75% compared to the 1 to 2 ml organic solvent commonly used in traditional liquid-liquid extraction.

[0039] Thirdly, this application provides the application of the pretreatment method in the detection of perfluorinated compounds in serum by liquid chromatography-tandem mass spectrometry, wherein the perfluorinated compounds include, but are not limited to, perfluorobutyric acid (PFBA), perfluorovalerate (PFPeA), perfluorohexanoic acid (PFHxA), perfluoroheptanoic acid (PFHpA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), perfluorodecanoic acid (PFDA), perfluoroundecanoic acid (PFUnDA), perfluorododecanoic acid (PFDoDA), perfluorobutyric acid (PFBS), perfluorohexanesulfonic acid (PFHxS), perfluorooctanoic acid (PFOS), and their chlorinated or hydrogenated precursors.

[0040] According to this application, samples pretreated using this method exhibit low matrix effects in LC-MS / MS analysis, with an absolute ion inhibition rate of less than 15% and a relative standard deviation (RSD) of less than 8% as determined by the post-spiking method. The method limit of detection (LOD) is 0.01 to 0.1 ng / mL (i.e., 10 to 100 pg / mL), and the limit of quantitation (LOQ) is 0.03 to 0.3 ng / mL, meeting the requirements for trace PFASs screening.

[0041] In some embodiments, the liquid chromatography conditions are as follows: the column is a C18 reversed-phase column (5 μm, 4.6 mm x 250 mm), mobile phase A is an aqueous solution containing 2 mmol / L ammonium acetate, mobile phase B is a pure acetonitrile solution, the gradient elution program is 0-1 min 10% B, 4-5 min 50% B, 6-7 min 80% B, 8-9 min 95% B, 9.1-10 min 10% B, the column temperature is 40 °C, and the flow rate is 0.4 mL / min.

[0042] In some embodiments, the mass spectrometry conditions are as follows: electrospray ionization (ESI) source, negative ion mode, spray voltage of -3500 volts, sheath gas pressure of 35 psi, auxiliary gas flow rate of 10 arb, capillary temperature of 320°C, nebulizing gas flow rate of 3 L / min, heating gas flow rate of 10 L / min, interface temperature of 300°C, and quantitative analysis is performed using multiple reaction monitoring (MRM) mode.

[0043] Fourthly, this application provides the application of the pretreatment method in clinical diagnosis, environmental exposure assessment or large-scale epidemiological screening, which is particularly suitable for scenarios with limited sample size (≤500μL), high-throughput processing (≥48 samples / batch) and low cost and low pollution requirements.

[0044] According to this application, the method avoids the use of solid-phase extraction columns, eliminating the risk of PFASs background contamination from plastic consumables. All reagents are conventional organic solvents, which are inexpensive, with the cost of pretreatment reagents for a single sample being less than RMB 2. The total amount of organic solvent used in the entire process does not exceed 2.5 ml, which is far lower than that of solid-phase extraction (usually 5 to 10 ml) and traditional liquid-liquid extraction (3 to 5 ml), in line with the principles of green chemistry.

[0045] In some implementations, the method has been successfully applied to PFASs screening of 500 population serum samples, with intra-batch precision (n=6) RSD of 3.2% to 7.8%, inter-batch precision (n=3 batches) RSD of 5.1% to 9.4%, and spike recovery rate of 86.5% to 103.2%.

[0046] In some implementations, the method is compatible with automated liquid workstations, which can automate the entire process from sample addition, precipitation, concentration, extraction to reconstitution through preset programs, with a daily throughput of up to 192 samples.

[0047] The following describes embodiments of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0048] Example 1 Take 500 μL of human serum sample (stored at -80℃, thawed at room temperature and vortexed for 30 seconds) and place it in a 1.5 ml polypropylene centrifuge tube. Add 500 μL of acetonitrile solution containing 0.1% formic acid, vortex for 1 min, and centrifuge at 4℃ and 14000 rpm for 8 min. Discard the precipitate and retain the supernatant. Concentrate the supernatant to 250 μL in a new centrifuge tube at 37℃ and a nitrogen flow rate of 1.8 L / min. Add 500 μL of a composite extractant consisting of methyl tert-butyl ether and ethyl acetate in a 1:1 volume ratio, vortex for 2 min, and centrifuge at 4℃ and 14000 rpm for 6 min. Carefully transfer the upper organic phase to a new centrifuge tube. The organic phase was purged with nitrogen at 37°C until nearly dry (no obvious droplets were observed with the naked eye, and a wet film appeared at the bottom). 65 μL of methanol-water (3:2, v / v) solution was added to reconstitute the phase. The mixture was vortexed for 30 seconds and centrifuged at 4°C and 14,000 rpm for 5 min. The supernatant was then analyzed by LC-MS / MS.

[0049] Example 2 Except that the volume ratio of methyl tert-butyl ether to ethyl acetate in the composite extractant is 3:1, the rest of the operation is the same as in Example 1.

[0050] Example 3 Except that the volume ratio of methyl tert-butyl ether to ethyl acetate in the composite extractant is 1:1, the rest of the operation is the same as in Example 1.

[0051] Example 4 Take 500ml of rat serum sample, and perform the remaining procedures as in Example 1.

[0052] Comparative Example 1 The conventional protein precipitation method was used: 500 μL of human serum was taken, 1500 μL of acetonitrile (without formic acid) was added, vortexed for 1 min, centrifuged at 4℃ and 14000 rpm for 10 min, the supernatant was taken and dried under nitrogen, and reconstituted with 70 μL of acetonitrile-water (1:1) containing 0.1% formic acid, filtered and injected.

[0053] Comparative Example 2 Solid-phase extraction was used: 500 μL of human serum was taken and extracted using a WAX solid-phase extraction column (60 mg / 3 mL) according to the manufacturer's instructions. The eluent was dried under nitrogen and reconstituted with 70 μL of reconstitution solution before injection.

[0054] Comparative Example 3 Extraction using a single methyl tert-butyl ether: In step S30, only 500 μL of methyl tert-butyl ether was used, and the rest was the same as in Example 1.

[0055] Comparative Example 4 Extraction with ethyl acetate alone: ​​only 500 μL of ethyl acetate was used in step S30, and the rest was the same as in Example 1.

[0056] The samples obtained from the above examples and comparative examples were analyzed by LC-MS / MS to determine the spiked recoveries (spiking concentration of 1 ng / mL) and matrix effects (ion inhibition rate calculated by post-spiking method) of 12 target PFASs (PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUnDA, PFDoDA, PFBS, PFHxS, PFOS).

[0057] The results are shown in Table 1.

[0058]

[0059] As shown in Table 1, the average recovery rates of Examples 1 to 4 were all in the range of 85% to 105%, the absolute value of the ion inhibition rate was less than 15%, and the amount of organic solvent used was significantly lower than that of Comparative Example 2. Comparative Example 1 had a low recovery rate and a serious matrix effect due to the lack of an acidification step and liquid-liquid extraction; Comparative Examples 3 and 4 had low recovery rates of short-chain or long-chain PFASs due to the use of a single extractant.

[0060] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A serum pretreatment method for the detection of perfluorinated compounds based on acidified protein precipitation and liquid-liquid extraction, characterized in that, Includes the following steps: S10: Take 500 μL of serum sample and place it in a 1.5 mL centrifuge tube. Add 200 μL of perfluorinated compound internal standard and 500-1000 μL of acetonitrile solution containing 0.1%-1.0% formic acid. Vortex for 30 seconds to 2 min. Centrifuge at 12000-15000 r / min at 4 °C for 5-10 min. Discard the precipitate and keep the supernatant. S20: Transfer the supernatant obtained in step S10 to a new 1.5ml centrifuge tube and concentrate it to 200~300μL under nitrogen blowing at 37℃. S30: Add 500 μL of a composite extractant made of methyl tert-butyl ether and ethyl acetate in a volume ratio of 3:1 to 1:1 to the supernatant after concentration in step S20, vortex for 1 to 3 min, and centrifuge at 12000 to 15000 r / min at 4℃ for 5 to 8 min to separate the upper organic phase. S40: The organic phase obtained in step S30 is purged to near dryness under a nitrogen flow at 37°C, and then redissolved with 65 μL of methanol-water solution, wherein the volume ratio of methanol to water in the methanol-water solution is 3:

2. The mixture is vortexed for 0.5 to 3 min, and then centrifuged at 12000 to 15000 r / min at 4°C for 5 to 8 min. The resulting supernatant is used for liquid chromatography-tandem mass spectrometry analysis.

2. The serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction according to claim 1, characterized in that, In step S10, the concentration of formic acid in the acetonitrile solution is 0.1% to 0.5%, and the volume ratio of acetonitrile to serum is 1:1 to 3:

1.

3. The serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction according to claim 1, characterized in that, In step S10, the vortex mixing time is 1 min, and the centrifugation conditions are 4℃, 14000 r / min, and 8 min.

4. The serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction according to claim 1, characterized in that, In step S20, the final volume of nitrogen blowing concentration is 250 μL, the nitrogen flow rate is 1.5~2.0 L per minute, and the temperature is 37℃.

5. The serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction according to claim 1, characterized in that, In step S30, the volume ratio of methyl tert-butyl ether to ethyl acetate is 1:

1.

6. The serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction according to claim 1, characterized in that, In step S30, the vortex oscillation time is 2 min, and the centrifugation conditions are 4℃, 14000 r / min, and 6 min.

7. The serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction according to claim 1, characterized in that, In step S40, the volume ratio of methanol to water in the methanol-water solution used for resolution is 3:2, and the resolution volume is 65 μL.

8. The serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction according to claim 1, characterized in that, In step S40, the endpoint of nitrogen blowing drying is when no obvious droplets remain visible to the naked eye, but a trace of moisture is retained.

9. The serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction according to claim 1, characterized in that, The composite extractant is composed of methyl tert-butyl ether and ethyl acetate in a volume ratio of 3:1 to 1:1, and is used to remove phospholipids, triglycerides and cholesterol, and to enrich perfluorocarboxylic acids, perfluorosulfonic acids and their precursor compounds.

10. The serum perfluorinated compound detection pretreatment method based on acidified protein precipitation and liquid-liquid extraction according to claim 1, characterized in that, The perfluorinated compounds include, but are not limited to, perfluorobutyric acid, perfluorovalerate, perfluorohexanoic acid, perfluoroheptanoic acid, perfluorooctanoic acid, perfluorononanoic acid, perfluorodecanoic acid, perfluoroundecanoic acid, perfluorododecanoic acid, perfluorobutyric acid, perfluorohexanesulfonic acid, perfluorooctanesulfonic acid and their chlorinated or hydrogenated precursors.

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