A pretreatment method for serum liquid chromatography-tandem mass spectrometry analysis

Abstract

The present invention relates to a pretreatment method for serum liquid chromatography-tandem mass spectrometry analysis, comprising the following steps: (S1) mixing a serum sample with an acetonitrile solution containing formic acid, ultrasonicating, vortexing, and centrifuging, taking the supernatant and diluting it with an aqueous solution containing formic acid to obtain a liquid to be purified; (S2) mixing the liquid to be purified with PCN-777, ultrasonicating, vortexing, and centrifuging, taking the supernatant and obtaining a liquid to be tested; the PCN-777 is C 24 H 23 N3O 16 Zr3 phospholipid removal material, the PCN-777 is C 24 H 23 N3O 16 Zr3. The present invention uses a known MOFs material PCN-777 to develop a pretreatment method for serum ultra-high performance liquid chromatography-tandem mass spectrometry analysis, which can specifically, quickly and effectively remove different types of phospholipids in serum, and achieve efficient recovery and detection of pesticides and veterinary drugs, avoiding interference from phospholipids.

Description

Technical Field

[0001] The invention belongs to the technical field of liquid chromatography-mass spectrometry detection pretreatment, and particularly relates to a pretreatment method for serum liquid chromatography-tandem mass spectrometry analysis. Background Art

[0002] Detecting pollutants in human blood not only assesses an individual's exposure to harmful substances and provides a scientific basis for health risks, but also allows for the early detection of pollutant accumulation and preventive measures to reduce the risk of disease. Furthermore, test results help trace the source of pollutants, providing a basis for pollution control and environmental protection, and providing scientific support for the formulation of relevant policies and legal accountability, thus playing a vital role in protecting public health and improving environmental quality. Recent advances in high-resolution mass spectrometry have enabled researchers to use nontargeted methods to simultaneously and reproducibly analyze thousands of compounds in biological samples. However, the concentrations of exogenous chemicals (such as pesticides and plasticizers) in the blood of the general population are typically hundreds to thousands of times lower than those of endogenous compounds in the sample. Ion suppression caused by these highly abundant endogenous chemicals often prevents the detection of trace and ultratrace levels of biologically significant exogenous exposure chemicals. Therefore, ensuring that sample analysis can unbiasedly reflect human chemical exposure is a critical issue.

[0003] Before performing LC / MS / MS testing, removing interference from endogenous compounds in blood samples during sample pretreatment is an effective solution. In non-targeted monitoring of plasma or serum samples, the most commonly used sample preparation method is protein precipitation with icy methanol or acetonitrile. While this method can extract the most compounds, due to interference from endogenous compounds such as phospholipids, it not only requires large amounts of organic solvents, but also requires long processing times, resulting in suboptimal purification results.

[0004] Phospholipids are lipids containing phosphoric acid and are essential components of cell membranes. Serum contains a wide variety of phospholipids, including phosphatidylcholine, lysophosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, and sphingomyelin. These phospholipids are complex and subject to significant interference. Phospholipids have been a major cause of matrix effects in blood samples analyzed by liquid chromatography-mass spectrometry (LC-MS). Phospholipids and lysophosphatidylcholine are particularly prominent endogenous compounds responsible for matrix effects, affecting the mass spectrometry response of many compounds and severely impacting the ionization efficiency of electrospray ionization sources. Furthermore, conventional methods are difficult to remove phospholipids.

[0005] CN114755355A reports a phospholipid removal agent and its use in dephospholipid removal from biological samples. The phospholipid removal agent comprises component A and component B; component A is a dextran salt, component B is a zirconium source, and the molar ratio of component A to component B is 0.1-1.7:2.5-5; the dextran salt is dextran sulfate, and the zirconium source is zirconium oxychloride; and the phospholipid contains at least lysophosphatidylcholine. However, this polyanion-metal ion system has difficulty in efficiently removing phospholipids while maintaining good recovery of certain analytes.

[0006] Shanghai Jiao Tong University reported on the selective adsorption of phospholipids by a zirconium dioxide-coated silica core-shell filler. Taking advantage of its large specific surface area and selective adsorption of phospholipids, serum samples were pretreated using a ZrO2 / SiO2 SPE cartridge. However, the article only explored the adsorption efficiency of the zirconium dioxide-coated silica core-shell filler for phospholipids and did not investigate the recovery of other substances.

[0007] Therefore, it is necessary to develop a method that can effectively remove phospholipids in serum and provide new technical support and support for sample pretreatment during pollutant analysis. Summary of the Invention

[0008] To address the shortcomings of existing technologies in removing phospholipids from blood samples, including insufficient efficiency, limited recovery of certain analytes, and complex operation, the present invention proposes a method for removing phospholipids from blood by adsorbing phospholipids using MOFs. This method is simple to operate, boasts high phospholipid removal efficiency, and excellent analyte recovery, making it widely applicable for residual analysis of chemical contaminants in serum. Specifically, the present invention provides the following technical solutions to address the aforementioned issues:

[0009] A pretreatment method for serum liquid chromatography-tandem mass spectrometry analysis comprises the following steps:

[0010] (S1) mixing a serum sample with an acetonitrile solution containing formic acid, sonicating, vortexing, and centrifuging, and diluting the supernatant with an aqueous solution containing formic acid to obtain a solution to be purified;

[0011] (S2) The liquid to be purified is mixed with PCN-777, ultrasonicated, vortexed, and centrifuged, and the supernatant is taken to obtain the liquid to be tested; the PCN-777 is C 24 H 23 N3O 16 Zr3 is a complex formed by 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine and Zr in a molar ratio of 1:3.

[0012] Furthermore, after step (S2), there is a step (S3): the test solution is separated and measured by UPLC-MS / MS to obtain the removal efficiency of phospholipids;

[0013] Furthermore, after step (S2), there is a step (S4): the test liquid is separated and measured by UPLC-MS / MS, and quantified by an external standard method to obtain the content of chemical pollutants.

[0014] Furthermore, in step (S1), the formic acid concentration in the acetonitrile containing formic acid and the aqueous solution containing formic acid is 0.1-0.2 wt %. The volume ratio of the serum sample to the formic acid acetonitrile solution is 1:3-5, for example, 1:4.

[0015] Furthermore, in step (S1) and step (S2), the ultrasonic frequency is 60-120 kHz; the vortex oscillation time is 30-60 s; the ultrasonic time is 5-30 min; the centrifugal speed is 8000-15000 rpm, the centrifugal temperature is 0-10°C, the centrifugation time is 5-20 min, and the supernatant is diluted with an aqueous solution (0.1% formic acid) in a 1:1 ratio.

[0016] Furthermore, in step (S2), the ratio of the liquid to be purified to PCN-777 is 1 mL: 10-50 mg, preferably 1 mL: 10-30 mg.

[0017] Furthermore, in step (S3), the representative phospholipids selected are 2 lysophosphatidylethanolamines (LPE): LPE 16:0, LPE 18:0; 4 lysophosphatidylcholines (LPC): LPC 15:0, LPC 16:0, LPC 18:0, LPC 22:4; 4 phosphatidylcholines (PC): PC p- 16:0 / 18:0, PC p -16:0 / 20:1, PC p -18:0 / 14:0, PC p- 18:0 / 18:0; and 4 sphingomyelins (SM): SM d18:1 / 12:0, SM d18:1 / 14:0, SM d18:1 / 14:1, SM d18:1 / 16:0;

[0018] Furthermore, in step (S3), the chromatographic column used in the ultra-high performance liquid chromatography method is ACQUITY CSH C18 (2.1 mm×100 mm, 1.7 μm). Mobile phase: 10-20 mM ammonium formate + 0.1-0.2% formic acid in acetonitrile / water (6:4 to 4:6, v / v) (A) and 10-20 mM ammonium formate + 0.1-0.2% formic acid in isopropanol / acetonitrile (9:1 to 2:2, v / v) (B); gradient elution program: 40%-43% B (0-2.0 min), 43%-50% B (2.0-2.1 min), 50%-54% B (2.1-12.0 min), 54%-70% B (12.0-12.1 min), 70%-99% B (12.1-18.0 min), 99%-40% B (18.0-18.1 min), 40% B (18.1-20.0 min); flow rate: 0.35-0.40 mL / min; column temperature 40-45°C; injection volume 5-10 μL. In gradient elution programs, the mobile phase and time are expressed as is well known in the art. For example, 40%-43% B (0-2.0 min) means that from 0 to 2.0 minutes, the mobile phase gradually changes from 40% mobile phase B (the remainder is mobile phase A) to 43% mobile phase B (the remainder is mobile phase A). Another example is 99%-40% B (18.0-18.1 min) means that from 18.0 to 18.1 minutes, the mobile phase changes from 99% mobile phase B (the remainder is mobile phase A) to 40% mobile phase B (the remainder is mobile phase A). Percentages are expressed by volume.

[0019] Furthermore, in step (S4), for pesticides, the chromatographic column used in the ultra-high performance liquid chromatography method is ACQUITY UPLC HSS T3 (2.1 mm × 100 mm, 1.8 μm); for veterinary drugs, the chromatographic column used in the ultra-high performance liquid chromatography method is ACQUITY UPLC BEH C18 (2.1 mm × 100 mm, 1.7 μm).

[0020] Furthermore, in step (S4), for pesticides, the mobile phases used in the ultra-high performance liquid chromatography method are: 2-5 mM ammonium formate + 0.01-0.05% (v / v) formic acid in water (A) and 2-5 mM ammonium formate + 0.01-0.05% (v / v) formic acid in methanol (B); the gradient elution program is 3% B (0-1.0 min), 3%-15% B (1.0-1.5 min), 15%-50% B (1.5-2.5 min), 50%-70% B (2.5-18.0 min), 70%-98% B (18.0-23.0 min), 98% B (23.0-27.0 min), 98%-3% B (27.0-27.1 min), 3% B (27.1-30.0 min); the flow rate is 0.3-0.5 mL / min; the column temperature is 40-45°C, and the injection volume is 2-5 μL; for veterinary drugs, the mobile phases used in the ultra-high performance liquid chromatography method are: 0.5-1.0 mM ammonium fluoride + 0.1-0.2% (v / v) formic acid in water (A) and acetonitrile / methanol (v / v = 1 / 1 to 1 / 1.5) (B); the flow rate is 0.3-0.5 mL / min. The gradient elution program was 3-10% B (0-2.0 min), 10%-15% B (2.0-5.0 min), 10-15% B (5.0-10.0 min), 15%-30% B (10.0-15.0 min), 30%-50% B (15.0-20.0 min), 50%-100% B (20.0-24.0 min), 100% B (24.0-28.0 min), 100%-3% B (28.0-28.5 min), 3-5% B (28.5-29.0 min); the flow rate was 0.3-0.5 mL / min; the column temperature was 40-45°C, and the injection volume was 3-5 μL.

[0021] The present invention also provides a use of a metal organic framework PCN-777 in clearing phospholipids from a blood sample, wherein the PCN-777 is C 24 H 23 N3O 16 Zr3: The structural formula is as follows:

[0022] .

[0023] PCN-777 is a known MOF material, as described in the literature "A Highly Stable Zeotype Mesoporous Zirconium Metal–Organic Framework with Ultralarge Pores, Angew Chem Int Ed Engl. 2015 Jan 2;54(1):149-54.doi:10.1002 / anie.201409334." This study is the first to use this MOF as a phospholipid adsorbent in serum samples, demonstrating its ability to specifically adsorb and remove phospholipids without interfering with other components to be detected. This specific and efficient phospholipid adsorption property has not been observed in other MOFs.

[0024] Furthermore, the metal organic framework PCN-777 is prepared by a method comprising the following steps: dissolving a zirconium source and a ligand 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine (TATB) in an organic solvent in the presence of a catalyst, heating the mixture, cooling it, washing it, collecting a white powder precipitate, and drying it. The catalyst is selected from at least one of trifluoroacetic acid, acetic acid, and formic acid.

[0025] Furthermore, the zirconium source is selected from ZrOCl2, ZrCl4, and hydrates thereof; the organic solvent is selected from at least one of N,N-diethylformamide, N,N-dimethylformamide, and tetrahydrofuran; and the heating temperature is 100-140°C for 5-15 hours.

[0026] Furthermore, the ratio of the zirconium source, 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine, catalyst and solvent is 200-360 mg: 60-90 mg: 0.4-0.6 mL: 10-20 mL.

[0027] The inventors unexpectedly discovered that among many organic metal frameworks, PCN-777 showed excellent adsorption and removal of phospholipids in serum. The possible reasons are that PCN-777 has a large specific surface area and a pore size that matches that of phospholipids. In addition, the Zr 4+ The phosphate groups in phospholipids can bind through electrostatic interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 is a SEM image of PCN-777 obtained in the preparation example;

[0029] Figure 2 is the N2 adsorption-desorption curve and pore size distribution diagram of PCN-777 obtained in the preparation example;

[0030] Figure 3 is the liquid chromatogram before and after adsorption of PCN-777 on phospholipid SM d18:1 / 16:0;

[0031] Figure 4 The adsorption performance of PCN-777 on 14 phospholipids. DETAILED DESCRIPTION

[0032] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0033] Preparation Example

[0034] Place 360 ​​mg of ZrOCl2·8H2O, 90 mg of 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine (TATB), and 0.6 mL of trifluoroacetic acid in a Pyrex glass bottle. Add 12 mL of N,N-diethylformamide (DEF) and sonicate to dissolve the mixture. Heat the mixture to 120°C for 12 hours. After cooling to room temperature, wash the mixture five times with N,N-dimethylformamide (DMF) and acetone. Centrifuge the mixture to collect the white powder product, PCN-777, and vacuum dry it for later use.

[0035] Figure 1 : is a SEM image of PCN-777 obtained in Preparation Example 1. It can be seen that the prepared PCN-777 has an octahedral structure.

[0036] Figure 2 The following is the N2 adsorption-desorption curve and pore size distribution diagram of PCN-777 obtained in the preparation example. The nitrogen adsorption experiment was carried out at 77K. Figure 2 The results show that the nitrogen adsorption capacity of PCN-777 at 1 bar is about 103 cm 3 ·g -1 The Brunauer–Emmett–Teller specific surface area is 300.14 m 2 ·g -1 , with a mesoporous cage of 3.5 nm.

[0037] Example 1

[0038] The representative phospholipids selected are: 2 lysophosphatidylethanolamines (LPEs): LPE 16:0, LPE 18:0; 4 lysophosphatidylcholines (LPCs): LPC 15:0, LPC 16:0, LPC 18:0, LPC 22:4; 4 phosphatidylcholines (PCs): PC p-16:0 / 18:0, PC p-16:0 / 20:1, PC p-18:0 / 14:0, PC p-18:0 / 18:0; and 4 sphingomyelins (SMs): SMd18:1 / 12:0, SM d18:1 / 14:0, SMd18:1 / 14:1, SM d18:1 / 16:0, as target representative phospholipids.

[0039] Phospholipid analytes were chromatographically separated using an ACQUITY CSH C18 column (2.1 mm × 100 mm, 1.7 μm). The mobile phases were 10 mM ammonium formate + 0.1% formic acid in acetonitrile / water (6:4, v / v) (A) and 10 mM ammonium formate + 0.1% formic acid in isopropanol / acetonitrile (9:1, v / v) (B); the gradient elution program was 40%-43% B (0-2.0 min), 43%-50% B (2.0-2.1 min), 50%-54% B (2.1-12.0 min), 54%-70% B (12.0-12.1 min), 70%-99% B (12.1-18.0 min), 99%-40% B (18.0-18.1 min), and 40% B (18.1-20.0 min); the flow rate was 0.35 mL / min; the column temperature was 45°C, the injection volume was 5 μL, and the total cycle time for each sample was 30 min.

[0040] Mass spectrometric analysis was performed using a Waters Xevo TQ-S triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode. Key parameters include: electrospray ionization (ESI) source, positive ionization mode, and source temperature of 350°C. Parameters such as retention time (RT), quantitative ion pairs, qualitative ion pairs, collision energy, and declustering potential are shown in Table 1.

[0041] Table 1 Mass spectrometry parameters of 14 phospholipids

[0042] .

[0043] Take 500µL of serum, add 5mg of PCN-777 powder, vortex for 5 minutes, centrifuge at 10000 rpm for 5 minutes, take 100 µL of supernatant and 400 µL of acetonitrile solution containing 0.1% formic acid, mix and sonicate for 15 minutes, centrifuge at 12000 rpm at 4℃ for 10 minutes, take 250 µL of supernatant, dilute with an equal amount of water containing 0.1% formic acid, and take 200 µL for detection. The results showed that there was no effect on phospholipid removal, indicating that adding the material before acetonitrile protein precipitation cannot remove phospholipids.

[0044] 100 μL of supernatant and 400 μL of acetonitrile solution containing 0.1% formic acid were mixed and sonicated for 15 min. The mixture was then centrifuged at 12,000 rpm at 4°C for 10 min. 250 μL of supernatant was taken and diluted with an equal volume of water containing 0.1 wt% formic acid. 5 mg of PCN-777 powder was added to 500 μL of the dilution solution and vortexed for 5 min. The mixture was centrifuged at 10,000 rpm for 5 min, and 200 μL of the supernatant was taken for detection. Figure 3 The adsorption performance of PCN-777 on 14 phospholipids is shown in Table 2. After protein precipitation with acetonitrile, PCN-777 showed excellent adsorption performance on all 14 phospholipids. This indicates that protein precipitation with acetonitrile is required before using PCN-777 to adsorb phospholipids. The adsorption rate is calculated as follows:

[0045] ;

[0046] Peak area refers to the peak area of ​​the characteristic peak in the liquid chromatogram. The retention time corresponding to each phospholipid is different, as shown in Table 1. The results showed that after protein precipitation, the adsorption rate of PCN-777 on phospholipids was as follows: the adsorption rate for two LPEs was greater than 95%, the adsorption rate for four LPCs was 80%-95%, the adsorption rate for three PCs was greater than 95%, and the adsorption rate for four SMs was 80%-99%. These results indicate that PCN-777 has excellent phospholipid adsorption properties for serum after acetonitrile protein precipitation.

[0047] Figure 3 The liquid chromatograms of PCN-777 before and after adsorption on phospholipid SM d18:1 / 16:0. Figure 4 The authors also tested other MOFs, such as ZIF-8 and UIO-66, and found that their adsorption performance for these 14 phospholipids was far inferior to that of PCN-777, with adsorption rates of only 40-60%.

[0048] Example 2

[0049] In the spike recovery experiment, 1 mL of serum was transferred to a 15 mL polypropylene centrifuge tube, and then 4 mL of acetonitrile solution containing 0.1% formic acid was added and vortexed for 30 seconds, followed by sonication for 15 minutes and centrifugation at 12000 rpm at 4°C for 10 minutes. 4 mL of the supernatant was taken and diluted with an equal amount of water containing 0.1% formic acid and eluted at 50 μg kg-1. 1 Chemical hazard spikes were performed at the same concentration level. 5 mg of PCN-777 was dispersed in 500 µL of the supernatant, vortexed for 5 minutes, and centrifuged at 10,000 rpm for 5 minutes to separate the PCN-777. 200 µL of the supernatant was then analyzed for chemical hazard concentrations using HPLC-MS / MS.

[0050] Pesticide standards were analyzed by liquid chromatography using a Waters Acquity Ultra Performance LC. Chromatographic separation of the analytes was performed using an ACQUITY UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm). The column temperature was 40°C, and the injection volume was 2 μL. The mobile phases consisted of 2 mM ammonium formate with 0.01% (v / v) formic acid in water (A) and 2 mM ammonium formate with 0.01% (w / v) formic acid in methanol (B); the flow rate was 0.3 mL / min. The gradient elution program was 3% B (0-1.0 min), 3%-15% B (1.0-1.5 min), 15%-50% B (1.5-2.5 min), 50%-70% B (2.5-18.0 min), 70%-98% B (18.0-23.0 min), 98% B (23.0-27.0 min), 98%-3% B (27.0-27.1 min), and 3% B (27.1-30.0 min). The total cycle time for each sample was 30.0 min.

[0051] Mass spectrometric analysis was performed using a Waters Xevo TQ-S triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode. Key parameters included: electrospray ionization (ESI) source, positive ionization mode, and source temperature of 350°C.

[0052] Veterinary drug standards were analyzed by liquid chromatography using a Waters Acquity Ultra Performance LC. Analytes were separated using an ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm). The column temperature was 40°C, and the injection volume was 3 μL. The mobile phases consisted of 0.5 mM ammonium fluoride in 0.1 wt% formic acid in water (A) and acetonitrile / methanol (v / v = 1 / 1) (B); the flow rate was 0.3 mL / min. The gradient elution program was 3% B (0-2.0 min), 3%-15% B (2.0-5.0 min), 15% B (5.0-10.0 min), 15%-30% B (10.0-15.0 min), 30%-50% B (15.0-20.0 min), 50%-100% B (20.0-24.0 min), 100% B (24.0-28.0 min), 100%-3% B (28.0-28.5 min), and 3% B (28.5-29.0 min). The total cycle time for each sample was 29.0 min.

[0053] Mass spectrometric analysis was performed using a Waters Xevo TQ-XS triple quadrupole mass spectrometer operated in multiple reaction monitoring (MRM) mode. Key parameters included: electrospray ionization (ESI) source, positive ionization mode, and source temperature of 400°C.

[0054] 553 pesticides and veterinary drugs were tested, and the results are shown in Table 2 below.

[0055] To evaluate the material's full recovery performance, the accuracy and precision of the method were assessed through recovery experiments at a spike level of 50 ppb (n=3). The recovery rate and relative standard deviation (RSD) represent the accuracy and precision of the method, respectively. Tables 2 and 3 show that PCN-777 achieved excellent recoveries (R) (35%-130%) for 553 chemical hazards (pesticides and veterinary drugs) at a 50 ppb concentration in serum samples, with RSDs ranging from 0.01%-20%. Although the recoveries for some pesticides were lower, all compounds were detected, meeting the qualitative requirements for non-targeted screening. Furthermore, matrix effect factors (MFs) for 507 (88.4%) of the 553 chemical hazards in human serum ranged from 0.7 to 1.3, indicating a slight inhibitory or enhancing effect of the matrix. Without PCN-777 treatment of serum, significant matrix effects can occur, resulting in the failure to detect trace amounts of these chemical hazards. For example, for acephate, after treatment with PCN-777, the matrix effect ME was 1.12; if PCN-777 was not used, the ME reached 2.36; if UIO-66 was used, the ME was 1.74.

[0056] Table 2 Information and spiked recoveries of 337 pesticides (50 μg kg -1 )

[0057] .

[0058] Table 3 Information and spiked recoveries of 216 veterinary drugs (50 μg kg -1 )

[0059] 。

Claims

1. A pretreatment method for serum liquid chromatography-tandem mass spectrometry analysis, characterized in that: The pretreatment method utilizes the organic metal framework PCN-777 to adsorb phospholipids to remove phospholipids from the blood, and the method comprises the following steps: (S1) mixing a serum sample and an acetonitrile solution containing formic acid, sonicating, vortexing, and centrifuging, taking the supernatant and diluting it with an aqueous solution containing formic acid to obtain a solution to be purified; the formic acid concentration in the acetonitrile containing formic acid and the aqueous solution containing formic acid is 0.1-0.2 wt %, and the volume ratio of the serum sample to the formic acid acetonitrile solution is 1:3-5; (S2) The liquid to be purified is mixed with PCN-777, ultrasonicated, vortexed, and centrifuged, and the supernatant is taken to obtain the liquid to be tested; the PCN-777 is C 24 H 23 N3O 16 Zr3, a complex formed by 2,4,6-tris(4-carboxyphenyl)-1,3,5-triazine and Zr in a molar ratio of 1:3; (S3) separating and measuring the test solution by UPLC-MS / MS to obtain the phospholipid removal efficiency; (S4) The test solution is separated and measured by UPLC-MS / MS, and quantified by an external standard method to obtain the content of the chemical pollutant; the chemical pollutant is a pesticide or a veterinary drug.

2. The pre-treatment method according to claim 1, characterized in that In step (S1) and step (S2), the ultrasonic frequency is 60-120 kHz; the vortex oscillation time is 30-60 s; the ultrasonic time is 5-30 min; the centrifugal speed is 8000-15000 rpm, the centrifugal temperature is 0-10°C, the centrifugation time is 5-20 min, and the supernatant is diluted with an aqueous solution containing 0.1-0.2 wt% formic acid.

3. The pre-treatment method according to claim 1, characterized in that In step (S2), the ratio of the liquid to be purified to PCN-777 is 1 mL: 10-50 mg.

4. The pre-treatment method according to claim 1, characterized in that: In step (S2), the ratio of the liquid to be purified to PCN-777 is 1 mL: 10-30 mg.

5. The pre-treatment method according to claim 1, characterized in that: In step (S3), the chromatographic column used in the ultra-performance liquid chromatography method is ACQUITY CSH C18; mobile phase A: 10-20 mM ammonium formate + 0.1-0.2 wt% formic acid in acetonitrile / water, the volume ratio of acetonitrile to water is 6:4 to 4:6, mobile phase B: 10-20 mM ammonium formate + 0.1-0.2 wt% formic acid in isopropanol / acetonitrile, the volume ratio of propanol to acetonitrile is 9:1 to 2:2; the gradient elution program is 40%-43% B, 0-2.0 min; 43%-50% B, 2.0-2.1 min; 50%-54% B, 2.1-12.0 min; 54%-70% B, 12.0-12.1 min; 70%-99% B, 12.1-18.0 min; 99%-40% B, 18.0-18.1 min; 40% B, 18.1-20.0 min; flow rate: 0.35-0.40 mL / min; column temperature: 40-45°C, injection volume: 5-10 μL.

6. The pre-treatment method according to claim 1, characterized in that: In step (S4), for pesticides, the chromatographic column used in the ultra-high performance liquid chromatography method is ACQUITY UPLC HSS T3; for veterinary drugs, the chromatographic column used in the ultra-high performance liquid chromatography method is ACQUITY UPLC BEH C18; For pesticides, the mobile phases used in ultra-high performance liquid chromatography are: mobile phase A: 2-5 mM ammonium formate + 0.01-0.05 wt% formic acid in water, mobile phase B: 2-5 mM ammonium formate + 0.01-0.05 wt% formic acid in methanol; the gradient elution program is 3% B, 0-1.0 min; 3%-15% B, 1.0-1.5 min; 15%-50% B, 1.5-2.5 min; 50%-70% B, 2.5-18.0 min; 70%-98% B, 18.0-23.0 min; 98% B, 23.0-27.0 min; 98%-3% B, 27.0-27.1 min; 3% B, 27.1-30.0 min; flow rate: 0.3-0.5 mL / min; column temperature: 40-45°C, injection volume: 2-5 μL; for veterinary drugs, the mobile phase used in ultra-high performance liquid chromatography is: mobile phase A: 0.5-1.0 mM ammonium fluoride + 0.1-0.2wt% formic acid aqueous solution, mobile phase B: acetonitrile / methanol volume ratio of 1:1-1.5; flow rate: 0.3-0.5 mL / min; gradient elution program: 3-10% B, 0-2.0 min; 10%-15% B, 2.0-5.0 min; 10-15% B, 5.0-10.0 min; 15%-30% B, 10.0-15.0 min; 30%-50% B, 15.0-20.0 min; 50%-100% B, 20.0-24.0 min; 100% B, 24.0-28.0 min; 100%-3% B, 28.0-28.5 min; 3-5% B, 28.5-29.0 min; flow rate: 0.3-0.5 mL / min; column temperature: 40-45°C; injection volume: 3-5 μL.

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