Method for determining contents of Lankanzumab and donamazumab in blood sample by using liquid chromatography-tandem mass spectrometry

Abstract

The invention relates to the technical field of pharmaceutical analysis, and provides a method for determining the content of Lankanzumab and donamazumab in a blood sample by using liquid chromatography-tandem mass spectrometry. According to the invention, the LC-MS / MS method capable of simultaneously detecting the Lancanamazumab and the donamazumab is established for the first time, and the defect that multiple antibody drugs cannot be simultaneously detected by an enzyme-linked immunosorbent assay is overcome. The detection method provided by the invention realizes automation of pretreatment, is simple and convenient to operate, and is convenient for high-throughput detection of large-batch samples.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for determining the contents of lencanezumab and donetumab in a blood sample by utilizing liquid chromatography-tandem mass spectrometry. Background Art

[0002] Donanemab and Lecanemab are newly launched drugs for the treatment of Alzheimer's disease. Both are humanized IgG1 monoclonal antibodies that exert their therapeutic effects by clearing β-amyloid (Aβ) plaques and can delay cognitive and functional decline in patients with early Alzheimer's disease.

[0003] At present, the ELISA method is used by pharmaceutical companies in clinical trials to determine the blood concentrations of lencanezumab and donezumab. The ELISA method can only detect lencanezumab and donezumab separately, and cannot achieve rapid detection of the two monoclonal antibodies in the same method. It is urgent to establish a method that can detect lencanezumab and donezumab at the same time. Summary of the invention

[0004] The object of the present invention is to provide a method for simultaneously determining the contents of lencanezumab and donetumab in a blood sample by utilizing liquid chromatography tandem mass spectrometry.

[0005] In order to achieve the purpose of the present invention, the present invention provides a method for determining the content of lencanezumab and donetumab in a blood sample by liquid chromatography tandem mass spectrometry, comprising the following steps: A. Prepare standard solutions of lencanezumab and donezumab respectively: first prepare lencanezumab stock solution and donezumab stock solution respectively with 1%-10% bovine serum albumin (BSA) aqueous solution, and then prepare standard solutions with concentrations of 2μg / mL-200μg / mL by a series of dilutions with human blank serum; B. Simultaneously, the lencanerzumab standard solution and the donezumab standard solution were enriched with immunomagnetic beads and then eluted, and the eluates were respectively digested by enzymes, and the characteristic peptide EGGYYYGR was produced after the digestion of lencanerzumab, and the characteristic peptide YNEKFKGR was produced after the digestion of donezumab, and the digestion products were detected by liquid chromatography tandem mass spectrometry; C. Draw a standard curve based on the concentration of the lencanezumab standard solution and the peak area of ​​the characteristic peptide EGGYYYGR; at the same time, draw a standard curve based on the concentration of the donezumab standard solution and the peak area of ​​the characteristic peptide YNEKFKGR; D. Pre-treating the blood sample to be tested, extracting lencanezumab and donezumab in the blood sample by immunomagnetic bead enrichment, eluting the target substance to be tested from the magnetic beads, performing enzyme digestion, and detecting the enzyme digestion product by liquid chromatography-tandem mass spectrometry; E. According to the test results of step D, the contents of lencanezumab and donetumab in the blood sample to be tested are obtained by comparing with the standard curve.

[0006] Furthermore, in steps B and D, trypsin is used for enzymatic digestion.

[0007] Preferably, the enzyme digestion conditions are: 25°C-45°C (preferably 37°C) overnight.

[0008] Step D is a method for pre-treating the blood sample to be tested, comprising: (1) Mix 20-200 μL of blood sample with 50-500 μL of sample pretreatment solution; (2) Preparation of sample extraction plate: Use a 2 mL 96-well deep-well plate (12 columns and 8 rows in total); place an appropriate amount of magnetic beads in magnetic bead activation solvent in the wells of the 1st and 7th columns, place magnetic bead equilibration solvent in the wells of the 2nd and 8th columns, place sample pretreatment solution in the wells of the 3rd and 9th columns, place magnetic bead washing solution in the wells of the 4th and 10th columns, place magnetic bead washing solution in the wells of the 5th and 11th columns, and place magnetic bead elution solution in the wells of the 6th and 12th columns; The components of the magnetic bead activation solvent, the magnetic bead equilibration solvent, the sample pretreatment solution, and the magnetic bead cleaning solution are all Tris buffer, namely 25mM Tris, 0.15M NaCl, and 0.05% Tween-20.

[0009] The magnetic bead elution solution is 1% formic acid aqueous solution, pH=2.

[0010] (3) Automated extraction: Place the prepared sample extraction plate in the magnetic bead extractor and perform automated extraction according to the following process: 1) Activation of magnetic beads: The magnetic beads in the 1st and 7th columns are shaken for 30 seconds, magnetically attracted for 60 seconds, and then transferred to the 2nd and 8th columns; 2) Magnetic bead balancing: The magnetic beads in the 2nd and 8th columns are shaken for 30 seconds, magnetically attracted for 60 seconds, and then transferred to the 3rd and 9th columns; 3) Sample loading: The magnetic beads in the 3rd and 9th columns are shaken for 3600 seconds, magnetically attracted for 60 seconds, and then transferred to the 3rd and 9th columns. Transfer to the 4th and 10th columns; 4) Magnetic bead washing: shake the magnetic beads in the 4th and 10th columns for 60 seconds, magnetically absorb for 60 seconds, and transfer to the 5th and 11th columns; 5) Magnetic bead washing: shake the magnetic beads in the 5th and 11th columns for 60 seconds, magnetically absorb for 60 seconds, and transfer to the 6th and 12th columns; 6) Magnetic bead elution: shake the magnetic beads in the 6th and 12th columns for 600 seconds, magnetically absorb for 60 seconds, and transfer to the 1st and 7th columns; the eluent contains the target object to be detected.

[0011] Further, add 10-20 μL of eluent pH adjusting reagent to 60-120 μL of eluent, mix well, add 0.5-4 μL of trypsin solution with a concentration of 50-200 μg / mL, and digest overnight at 25°C-45°C (preferably 37°C); ultracentrifuge the digested product, and take the supernatant for detection.

[0012] Furthermore, the liquid chromatography uses a C18 chromatographic column, and uses 2%-95% mobile phase A and 5%-98% mobile phase B for gradient elution; the injection volume is 1-10 μL.

[0013] The mobile phase A contains 0.01%-0.1% formic acid aqueous solution; the mobile phase B contains methanol.

[0014] Gradient elution conditions are shown in Table 1: Table 1 Gradient elution conditions

[0015] The mass spectrometry conditions were as follows: the mass spectrometry selected multiple ion monitoring reaction mode, positive ion mode, electrospray ionization, and ion source temperature of 400~600℃.

[0016] Furthermore, the quantitative ion pairs of the characteristic peptide EGGYYYGR of Lencanerzumab are 482.71 and 395.65, and the qualitative ion pairs are 482.71 and 558.27. The quantitative ion pairs of the characteristic peptide YNEKFKGR of Donenzumab are 521.28 and 434.22, and the qualitative ion pairs are 521.28 and 268.13.

[0017] In the present invention, the blood sample may be serum or plasma.

[0018] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: (I) The present invention is the first to establish an LC-MS / MS method that can simultaneously detect lencanezumab and donezumab, making up for the defect that the enzyme-linked immunosorbent assay cannot simultaneously detect multiple antibody drugs.

[0019] (ii) The detection method provided by the present invention realizes the automation of pre-treatment, is easy to operate, and is convenient for high-throughput detection of large quantities of samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the arrangement of the sample extraction plate and the wells in a preferred embodiment of the present invention.

[0021] Figure 2 The MRM chromatograms of blank serum and blank serum samples added with 20 µg / mL of lencanezumab (lower) and donezumab (upper) in a preferred embodiment of the present invention are shown.

[0022] Figure 3 The linear range of lencanezumab (upper) and donezumab (lower) determined by the LC-MS / MS method in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0023] The present invention establishes a liquid chromatography-tandem mass spectrometry method for simultaneously quantifying lencanezumab and donetumab in blood through a pre-treatment method of trypsin digestion after automated immunomagnetic bead enrichment, and the sample can be serum or plasma.

[0024] The present invention adopts the following technical solution: An automated immunomagnetic bead enrichment method was used to extract lencanezumab and donezumab from the samples. After the analytes were eluted from the magnetic beads, they were enzymatically digested with trypsin and then tested on the machine. The characteristic peptides EGGYYYGR (lencanezumab) and YNEKFKGR (donezumab) produced after enzymatic digestion were separated by liquid phase and then entered the mass spectrometer for electrospray ionization to produce specific mass / charge ratios (mass-to-charge ratios), which were quantified by the chromatographic peaks generated by the scanning mode of multiple reaction detection.

[0025] For the above two determinations, calibration curves were prepared with standard samples of their respective mass concentrations, with the mass concentration of the standard solution as the X-axis and the peak area of ​​the standard sample as the Y-axis, and linear regression analysis was performed. The regression equation was obtained by weighting with "1 / X". The peak area of ​​the component to be tested in the sample was substituted into the standard curve equation to calculate the concentration of each component to be tested in the serum sample. The sample detection signal intensity is proportional to the concentration of the component to be tested in the sample.

[0026] The specific method is as follows: 1. Prepare reagents including magnetic bead activation solvent, magnetic bead equilibration solvent, sample pretreatment solution, magnetic bead cleaning solution, magnetic bead elution solution, elution solution pH adjustment reagent and instrument analysis solution, which are as follows: Magnetic bead activation solvent: Tris buffer solution, i.e. 25mM Tris, 0.15M NaCl, 0.05% Tween-20.

[0027] Magnetic bead equilibration solvent: Tris buffer solution, i.e. 25mM Tris, 0.15M NaCl, 0.05% Tween-20.

[0028] Sample pretreatment solution: Tris buffer solution, i.e. 25mM Tris, 0.15M NaCl, 0.05% Tween-20.

[0029] Magnetic bead washing solution: Tris buffer solution, i.e. 25mM Tris, 0.15M NaCl, 0.05% Tween-20.

[0030] Magnetic bead elution buffer: 1% formic acid aqueous solution, pH=2.

[0031] Eluent pH adjustment reagent: 1M KHCO3 aqueous solution.

[0032] Instrumental analysis liquid: Mobile phase A: Contains 0.01%-0.1% formic acid in water. Mobile phase B: Methanol.

[0033] 2. Calibrator solution: The standards of rocanecuzumab and donetumab were accurately weighed and prepared into stock solutions using 1%-10% BSA aqueous solution, and then a series of dilutions were made using human blank serum to prepare working calibration solutions with concentrations ranging from 2 μg / mL to 200 μg / mL.

[0034] 3. Sample preparation: Sample pretreatment: Add 20-200 μL of blood sample or calibration solution to a 1.5 ml low-adsorption EP tube, add 50-500 μL of sample pretreatment solution, and vortex to mix.

[0035] Preparation of sample extraction plate: Use 2mL 96-well deep well plate ( Figure 1 ), place an appropriate amount of magnetic beads in the magnetic bead activation solvent in the wells of the 1st and 7th columns, place the magnetic bead equilibration solvent in the wells of the 2nd and 8th columns, place the sample pretreatment solution in the wells of the 3rd and 9th columns, place the magnetic bead washing solution in the wells of the 4th and 10th columns, place the magnetic bead washing solution in the wells of the 5th and 11th columns, and place the magnetic bead elution solution in the wells of the 6th and 12th columns; Automated extraction: Place the prepared sample extraction plate in the magnetic bead extractor and perform automated extraction according to the following process. 1) Activation of magnetic beads: Oscillate the magnetic beads in the 1st and 7th columns for 30 seconds, magnetically absorb for 60 seconds, and transfer to the 2nd and 8th columns; 2) Balance of magnetic beads: Oscillate the magnetic beads in the 2nd and 8th columns for 30 seconds, magnetically absorb for 60 seconds, and transfer to the 3rd and 9th columns; 3) Loading: Oscillate the magnetic beads in the 3rd and 9th columns for 3600 seconds, magnetically absorb for 60 seconds, and transfer to the 4th and 10th columns; 4) Washing of magnetic beads: The magnetic beads in the 4th and 10th columns are shaken for 60 seconds, magnetically attracted for 60 seconds, and transferred to the 5th and 11th columns; 5) Magnetic bead washing: the magnetic beads in the 5th and 11th columns are shaken for 60 seconds, magnetically attracted for 60 seconds, and transferred to the 6th and 12th columns; 6) Magnetic bead elution: the magnetic beads in the 6th and 12th columns are shaken for 600 seconds, magnetically attracted for 60 seconds, and transferred to the 1st and 7th columns; the eluent contains the target object to be detected.

[0036] Enzyme digestion: Add 10-20μL of eluent pH adjustment reagent to 60-120μL eluent, mix well, add 0.5-4μL of trypsin solution with a concentration of 50-200μg / mL, and digest overnight at 25℃-45℃ (preferably 37℃). After ultracentrifugation, the digestion product is transferred to a 700μL injection plate and waits for machine testing. The injection volume is 1-10μL.

[0037] 4. Testing: Chromatographic separation was performed on a C18 column using a gradient elution of 2%-95% mobile phase A and 5%-98% mobile phase B.

[0038] The mass spectrometer selected multiple ion monitoring reaction mode, positive ion mode, electrospray ionization, and ion source temperature of 400-600°C. The ion pairs of the characteristic peptides of lencanezumab and donezumab were monitored simultaneously: EGGYYYGR (quantitative): 482.71→395.65; EGGYYYGR (qualitative): 482.71→558.27; YNEKFKGR (quantitative): 521.28→434.22; YNEKFKGR (qualitative): 521.28→268.13.

[0039] In the present invention, the amino acid sequences of the heavy chain and light chain of Lecanemab are shown in SEQ ID NOs: 1-2, respectively, and the amino acid sequences of the heavy chain and light chain of Donanemab are shown in SEQ ID NOs: 3-4, respectively.

[0040] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0041] Example 1 Method for determining the content of lencanezumab and donezumab in blood samples using liquid chromatography tandem mass spectrometry 1. Prepare reagents: including magnetic bead activation solvent, magnetic bead balancing solvent, sample pretreatment solution, magnetic bead cleaning solution, magnetic bead elution solution, elution solution pH adjustment reagent and instrument analysis solution, as follows: Magnetic bead activation solvent: Tris buffer solution, i.e. 25mM Tris, 0.15M NaCl, 0.05% Tween-20.

[0042] Magnetic bead equilibration solvent: Tris buffer solution, i.e. 25mM Tris, 0.15M NaCl, 0.05% Tween-20.

[0043] Sample pretreatment solution: Tris buffer solution, i.e. 25mM Tris, 0.15M NaCl, 0.05% Tween-20.

[0044] Magnetic bead washing solution: Tris buffer solution, i.e. 25mM Tris, 0.15M NaCl, 0.05% Tween-20.

[0045] Magnetic bead elution buffer: 1% formic acid aqueous solution, pH=2.

[0046] Eluent pH adjustment reagent: 1M KHCO3 aqueous solution.

[0047] Instrumental analysis liquid: Mobile phase A: Contains 0.01%-0.1% formic acid in water. Mobile phase B: Methanol.

[0048] 2. Calibration solution: The standards of rocanecuzumab and donetumab were accurately weighed and prepared into stock solutions using 1%-10% BSA aqueous solution, and then a series of dilutions were made using human blank serum to prepare working calibration solutions with concentrations ranging from 2 μg / mL to 200 μg / mL.

[0049] 3. Sample preparation: (1) Sample pretreatment: Add 20-200 μL of blood sample or calibration solution to a 1.5 ml low-adsorption EP tube, add 50-500 μL of sample pretreatment solution, and vortex to mix.

[0050] (2) Preparation of sample extraction plate: Use 2 mL 96-well deep well plate ( Figure 1 ), place an appropriate amount of magnetic beads in the magnetic bead activation solvent in the wells of the 1st and 7th columns, place the magnetic bead equilibration solvent in the wells of the 2nd and 8th columns, place the sample pretreatment solution in the wells of the 3rd and 9th columns, place the magnetic bead washing solution in the wells of the 4th and 10th columns, place the magnetic bead washing solution in the wells of the 5th and 11th columns, and place the magnetic bead elution solution in the wells of the 6th and 12th columns; (3) Automated extraction: Place the prepared sample extraction plate in the magnetic bead extractor and perform automated extraction according to the following process. 1) Activation of magnetic beads: Oscillate the magnetic beads in the 1st and 7th columns for 30 seconds, magnetically absorb for 60 seconds, and transfer to the 2nd and 8th columns; 2) Balance of magnetic beads: Oscillate the magnetic beads in the 2nd and 8th columns for 30 seconds, magnetically absorb for 60 seconds, and transfer to the 3rd and 9th columns; 3) Loading: Oscillate the magnetic beads in the 3rd and 9th columns for 3600 seconds, magnetically absorb for 60 seconds, and transfer to the 4th and 10th columns; 4) Washing of magnetic beads: The magnetic beads in the 4th and 10th columns are shaken for 60 seconds, magnetically attracted for 60 seconds, and transferred to the 5th and 11th columns; 5) Magnetic bead washing: the magnetic beads in the 5th and 11th columns are shaken for 60 seconds, magnetically attracted for 60 seconds, and transferred to the 6th and 12th columns; 6) Magnetic bead elution: the magnetic beads in the 6th and 12th columns are shaken for 600 seconds, magnetically attracted for 60 seconds, and transferred to the 1st and 7th columns; the eluent contains the target object to be detected.

[0051] (4) Enzyme digestion: Add 10 μL of eluent pH adjustment reagent to 100 μL of eluent, mix well, add 2 μL of 100 μg / mL trypsin solution, and digest at 37°C overnight. After ultracentrifugation, transfer to a 700 μL injection plate and wait for machine testing. The injection volume is 10 μL.

[0052] 4. Testing: Chromatographic separation was performed using an ACQUITY UPLC PRIMER HSS C18 column, and the liquid chromatography mobile phase A and mobile phase B were set according to the gradients in Table 1.

[0053] UPLC-TQXS mass spectrometer (Waters, USA) was used in multiple ion monitoring reaction mode, positive ion mode, electrospray ionization, and ion source temperature of 400-600°C. The ion pairs of the characteristic peptides of lencanezumab and donezumab were monitored simultaneously: EGGYYYGR (quantitative): 482.71→395.65; EGGYYYGR (qualitative): 482.71→558.27; YNEKFKGR (quantitative): 521.28→434.22; YNEKFKGR (qualitative): 521.28→268.13.

[0054] 5. Test results: Typical spectra of characteristic peptides of lencanezumab and donezumab in serum determined by chromatography-mass spectrometry are shown in the figure below. Figure 2 shown.

[0055] 6. Performance test of reagents prepared by this method: EGGYYYGR linearity has a correlation of >0.995 in the range of 2-200 μg / mL, and YNEKFKGR linearity has a correlation of >0.995 in the range of 2-200 μg / mL. Figure 3 shown.

[0056] 7. Evaluation of recovery and accuracy of this method: Three concentrations of standard samples, high (100.0 μg / mL), medium (20.0 μg / mL), and low (5.0 μg / mL), were added to the mixed serum for spike recovery tests. The results are shown in Table 2.

[0057] Table 2 Recovery of lencanezumab and donezumab determined by LC-MS / MS method

[0058] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for determining the content of lencanezumab and donezumab in a blood sample using liquid chromatography tandem mass spectrometry, characterized in that: The following steps are involved: A. Prepare standard solutions of lencanezumab and donezumab respectively: first prepare lencanezumab stock solution and donezumab stock solution respectively with 1%-10% bovine serum albumin aqueous solution, and then prepare standard solutions with concentrations of 2μg / mL-200μg / mL by a series of dilutions with human blank serum; B. Simultaneously, the lencanerzumab standard solution and the donezumab standard solution were enriched with immunomagnetic beads and then eluted, and the eluates were respectively digested by enzymes, and the characteristic peptide EGGYYYGR was produced after the digestion of lencanerzumab, and the characteristic peptide YNEKFKGR was produced after the digestion of donezumab, and the digestion products were detected by liquid chromatography tandem mass spectrometry; C. Draw a standard curve based on the concentration of the lencanezumab standard solution and the peak area of ​​the characteristic peptide EGGYYYGR; at the same time, draw a standard curve based on the concentration of the donezumab standard solution and the peak area of ​​the characteristic peptide YNEKFKGR; D. Pre-treating the blood sample to be tested, extracting lencanezumab and donezumab in the blood sample by immunomagnetic bead enrichment, eluting the target substance to be tested from the magnetic beads, performing enzyme digestion, and detecting the enzyme digestion product by liquid chromatography-tandem mass spectrometry; E. According to the test results of step D, the contents of lencanezumab and donetumab in the blood sample to be tested are obtained by comparing with the standard curve.

2. The method according to claim 1, characterized in that Steps B and D were digested with trypsin.

3. The method according to claim 2, characterized in that The enzyme digestion conditions were: 25°C-45°C overnight.

4. The method according to claim 1, characterized in that: The method for pre-treating the blood sample to be tested in step D comprises: (1) Mix 20-200 μL of blood sample with 50-500 μL of sample pretreatment solution; (2) Preparation of sample extraction plate: Use a 2 mL 96-well deep-well plate with 12 columns and 8 rows; Among them, the wells in the first and seventh columns are placed with an appropriate amount of magnetic beads in a magnetic bead activation solvent, the wells in the second and eighth columns are placed with a magnetic bead equilibration solvent, the wells in the third and ninth columns are placed with a sample pretreatment solution, the wells in the fourth and tenth columns are placed with a magnetic bead cleaning solution, the wells in the fifth and eleventh columns are placed with a magnetic bead cleaning solution, and the wells in the sixth and twelfth columns are placed with a magnetic bead elution solution; The components of the magnetic bead activation solvent, the magnetic bead equilibration solvent, the sample pretreatment solution, and the magnetic bead cleaning solution are all Tris buffer, i.e., 25 mM Tris, 0.15 M NaCl, 0.05% Tween-20; The magnetic bead eluent is 1% formic acid aqueous solution, pH=2; (3) Automated extraction: Place the prepared sample extraction plate in the magnetic bead extractor and perform automated extraction according to the following process: 1) Activation of magnetic beads: The magnetic beads in the 1st and 7th columns are shaken for 30 seconds, magnetically attracted for 60 seconds, and then transferred to the 2nd and 8th columns; 2) Magnetic bead balancing: The magnetic beads in the 2nd and 8th columns are shaken for 30 seconds, magnetically attracted for 60 seconds, and then transferred to the 3rd and 9th columns; 3) Sample loading: The magnetic beads in the 3rd and 9th columns are shaken for 3600 seconds, magnetically attracted for 60 seconds, and then transferred to the 3rd and 9th columns. Transfer to the 4th and 10th columns; 4) Magnetic bead washing: shake the magnetic beads in the 4th and 10th columns for 60 seconds, magnetically absorb for 60 seconds, and transfer to the 5th and 11th columns; 5) Magnetic bead washing: shake the magnetic beads in the 5th and 11th columns for 60 seconds, magnetically absorb for 60 seconds, and transfer to the 6th and 12th columns; 6) Magnetic bead elution: shake the magnetic beads in the 6th and 12th columns for 600 seconds, magnetically absorb for 60 seconds, and transfer to the 1st and 7th columns; the eluent contains the target object to be detected.

5. The method according to claim 4, characterized in that Add 10-20 μL of eluent pH adjustment reagent to 60-120 μL of eluent, mix well, add 0.5-4 μL of trypsin solution with a concentration of 50-200 μg / mL, and digest overnight at 25℃-45℃; ultracentrifuge the digested product, and take the supernatant for detection; Wherein, the eluent pH adjusting reagent is a 1M KHCO3 aqueous solution.

6. The method according to claim 1, characterized in that Liquid chromatography used a C18 column, with gradient elution using 2%-95% mobile phase A and 5%-98% mobile phase B; injection volume 1-10 μL; The mobile phase A contains 0.01%-0.1% formic acid aqueous solution; the mobile phase B contains methanol; The gradient elution conditions were as follows: ； The mass spectrometry conditions were as follows: the mass spectrometry selected multiple ion monitoring reaction mode, positive ion mode, electrospray ionization, and ion source temperature of 400~600℃.

7. The method according to claim 6, characterized in that The quantitative ion pairs of the characteristic peptide EGGYYYGR of Lencanermab are 482.71 and 395.65; the qualitative ion pairs are 482.71 and 558.27; The quantitative ion pairs of the characteristic peptide YNEKFKGR of donetumab are 521.28 and 434.22; the qualitative ion pairs are 521.28 and 268.

13.

8. The method according to any one of claims 1 to 7, characterized in that: The blood sample is serum or plasma.

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