Method for detecting Zebrutinib and Ibrutinib in human blood

Abstract

The invention discloses a method for detecting zerbutinib and ibrutinib in human blood. The method comprises the following steps: preparing a mixed stock solution by using zerbutinib and ibrutinib reference substances, diluting step by step to prepare a standard curve working solution, and preparing a standard curve sample by using the standard curve working solution; preparing a mixed internal standard working solution by using a Zebrutini-D5 reference substance and an ibrutini-D5 reference substance, and pretreating the standard curve sample and the sample to be detected by using the mixed internal standard working solution; performing mass spectrum acquisition on the preprocessed to-be-detected sample and the preprocessed standard curve sample by using an electrospray ionization source and a multi-reaction monitoring mode to respectively obtain the signal intensity of the to-be-detected sample and the signal intensity of the standard curve sample; performing linear regression according to the signal intensity of the standard curve sample and the theoretical concentration of the standard curve sample to obtain a regression equation; and according to the regression equation and the signal intensity of the to-be-detected sample, calculating to obtain the concentration of the zebrutinib and the ibrutinib in the to-be-detected sample. The method has the advantages of wide detection linear range and high detection speed.

Description

Technical Field

[0001] The present invention relates to the field of medical testing technology, and in particular to a method for detecting zanubrutinib and ibrutinib in human blood. Background Art

[0002] Zebutinib and ibrutinib are a class of Bruton's tyrosine kinase inhibitors with extensive clinical use. The oral absorption of this type of drug varies greatly and can be affected by food. It is mainly metabolized by the liver CYP3A enzyme. When taken simultaneously with strong CYP3A inhibitors, its in vivo exposure can be greatly increased, leading to dose-related adverse reactions in patients, such as bleeding and hypertension. In addition, poor patient compliance can also lead to abnormal in vivo exposure. All of these require therapeutic drug monitoring to assess the actual drug exposure in patients, so as to provide individualized treatment for patients, improve clinical efficacy, and reduce adverse reactions.

[0003] The drug detection methods currently reported are complex to operate, and multiple drugs use the same internal standard, which cannot avoid interference caused by matrix effects. In addition, there are defects such as inappropriate linear range and obvious residual effects of the method, which makes it impossible to quickly and accurately determine the drug exposure in the patient's body. Summary of the invention

[0004] The technical task of the present invention is to address the above deficiencies and provide a method for detecting zebutinib and ibrutinib in human blood, which simplifies the sample processing operation in the method, and the addition of internal standard working solution can simultaneously complete the internal standard addition and protein precipitation operations. The on-machine detection time is only 3 minutes, which is suitable for large-scale clinical sample analysis; the use of 2 isotope internal standards can effectively reduce the interference of matrix effects; 6 concentration levels of standard curve samples and 3 concentration levels of quality control samples are set to reasonably cover the actual concentration range of zebutinib and ibrutinib in clinical samples.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for detecting zanubrutinib and ibrutinib in human blood, comprising:

[0007] Use zanubrutinib and ibrutinib reference substances to prepare mixed stock solutions, dilute them step by step to prepare standard curve working solutions, and use the standard curve working solutions to prepare standard curve samples;

[0008] Use zanubrutinib-D5 and ibrutinib-D5 reference substances to prepare a mixed internal standard working solution, and use the mixed internal standard working solution to pretreat the standard curve sample and the sample to be tested;

[0009] The pre-treated samples to be tested and the standard curve samples were mass spectrometrically acquired using an electrospray ion source and a multiple reaction monitoring mode to obtain the signal intensity of the samples to be tested and the signal intensity of the standard curve samples respectively;

[0010] Perform linear regression based on the signal intensity of the calibration curve sample and the theoretical concentration of the calibration curve sample to obtain a regression equation;

[0011] The concentrations of zanubrutinib and ibrutinib in the sample to be tested are calculated according to the regression equation and the signal intensity of the sample to be tested.

[0012] In some embodiments, zanubrutinib and ibrutinib reference substances are used to prepare a mixed stock solution, which is diluted stepwise to prepare a standard curve working solution, and the standard curve sample is prepared using the standard curve working solution, comprising:

[0013] The zanubrutinib reference substance and the ibrutinib reference substance are mixed with a Biaoqu solvent to obtain a mixed Biaoqu stock solution, wherein the Biaoqu solvent is a methanol aqueous solution or an acetonitrile aqueous solution, preferably a 90% methanol aqueous solution; the mixed Biaoqu stock solution is gradiently diluted to obtain a Biaoqu working solution, and blank plasma is added to the Biaoqu working solution to obtain a Biaoqu sample, wherein the volume ratio of the Biaoqu working solution to the blank sample plasma is 1:5-20, preferably 1:9.

[0014] In some embodiments, the method uses zanubrutinib-D5 and ibrutinib-D5 reference substances to prepare a mixed internal standard working solution, and uses the mixed internal standard working solution to pretreat the sample to be tested, including: mixing zanubrutinib-D5 and ibrutinib-D5 reference substances with an internal standard stock solvent to obtain zanubrutinib-D5 stock solution and ibrutinib-D5 stock solution, wherein the internal standard stock solvent is a methanol aqueous solution or an acetonitrile aqueous solution, preferably a 90% methanol aqueous solution; mixing the zanubrutinib-D5 stock solution and the ibrutinib-D5 stock solution with the internal standard working solvent Mixing to obtain a mixed internal standard working solution, wherein the internal standard working solvent is one or more of methanol, acetonitrile, and formic acid, preferably acetonitrile with 0.05% formic acid, and the concentration of the mixed internal standard working solution is 10-100 ng / mL, preferably 20 ng / mL; mixing the mixed internal standard working solution with the sample to be tested, centrifuging to obtain the supernatant, and using a diluent to dilute the supernatant to obtain the pretreated sample to be tested, wherein the diluent is a formic acid aqueous solution, an acetonitrile aqueous solution or a mixture thereof, preferably an aqueous solution containing 0.1% formic acid and 38% acetonitrile.

[0015] In some embodiments, in the mass spectrometry acquisition, the chromatographic column filler is C18, the particle size is 1.7-5μm, preferably 1.7μm; the injection volume is 2-10μL, preferably 2μL; the column temperature is 35-45°C, preferably 40°C; the capillary voltage is 0.5-2.0kV, preferably 1.0kV; the desolvation temperature is 400-500°C, preferably 450°C; the desolvation gas flow rate is 600-800L / Hr, preferably 800L / Hr; the cone gas flow rate is 40-60L / Hr, preferably 60L / Hr.

[0016] In some embodiments, in the mass spectrometry acquisition, the organic mobile phase is an acetonitrile solution containing formic acid, and the aqueous mobile phase is an aqueous solution of formic acid and ammonium formate, wherein the volume fraction of formic acid is 0.01% to 1%, preferably 0.1%, and the molar concentration of ammonium formate is 5 to 20 mM, preferably 10 mM; the elution flow rate is 0.3 to 0.5 ml / min, preferably 0.4 ml / min.

[0017] In some embodiments, in the mass spectrometry acquisition, the gradient elution conditions are as follows: 0.0 min, aqueous mobile phase: 62%; 0.5 min, aqueous mobile phase: 62%; 1.0 min, aqueous mobile phase: 40%; 2.0 min, aqueous mobile phase: 28%; 2.5 min, aqueous mobile phase: 0%; 3.0 min, aqueous mobile phase: 62%.

[0018] In some embodiments, the method further comprises cleaning the injection needle with a needle wash solution after mass spectrometry acquisition and injection, wherein the needle wash solution is a methanol or acetonitrile aqueous solution containing ammonia, preferably an aqueous solution containing 0.5% volume ammonia and 49.5% volume acetonitrile.

[0019] In some embodiments, the theoretical concentrations of zanubrutinib and ibrutinib in the standard curve sample are 500, 250, 50, 25, 5, and 2.5 ng / mL, respectively.

[0020] In some embodiments, the method further comprises a quality control test, wherein in the quality control samples of the quality control test, the theoretical concentrations of zanubrutinib and ibrutinib are 400, 40, and 4 ng / mL, respectively.

[0021] In some embodiments, the method detects zanubrutinib and ibrutinib in a concentration range of 2.5-500 ng / mL.

[0022] Compared with the prior art, the advantages and positive effects of the present invention are:

[0023] 1. By setting standard curve samples at 6 concentration levels and quality control samples at 3 concentration levels, the actual concentration range of zanubrutinib and ibrutinib in clinical samples was reasonably covered;

[0024] 2. Use two isotope internal standards, and the quantitative method can choose single internal standard or double internal standard scheme, which can effectively reduce the interference of matrix effect and ensure the accuracy of the test results;

[0025] 3. Use special needle washing solution to reduce the residue caused by high-concentration samples;

[0026] 4. Simplify sample processing operations. The mixed internal standard working solution can complete the internal standard addition and protein precipitation operations in one step, and the machine detection time only takes 3 minutes, which is suitable for large-scale clinical sample analysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0028] Figure 1 Schematic diagram of the application method flow chart.

[0029] Figure 2 This is a chromatogram of the quantitative lower limit concentration sample in the embodiment of the present application, wherein a is zanubrutinib-D5, b is zanubrutinib, c is ibrutinib-D5, and d is ibrutinib.

[0030] Figure 3 It is the chromatogram of the blank sample in this embodiment, wherein a is zanubrutinib-D5, b is zanubrutinib, c is ibrutinib-D5, and d is ibrutinib.

[0031] Figure 4 : is the chromatogram of the clinical sample in this embodiment, wherein a is zanubrutinib-D5, b is zanubrutinib, c is ibrutinib-D5, and d is ibrutinib. DETAILED DESCRIPTION

[0032] In order to more clearly understand the above-mentioned purpose, features and advantages of the present invention, the present invention is further described below in conjunction with the accompanying drawings and embodiments. It should be noted that the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

[0033] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0034] Embodiment 1:

[0035] Drugs and reagents: Zebutinib (purity: 98.5%, batch number: 20200328), Zebutinib-D5 (purity: 98.6%, batch number: 4298-043A3), and Ibrutinib-D5 (purity: 99.3%, batch number: 3070-078A4) were purchased from TLC Pharmaceutical Standards, Canada; Ibrutinib (purity: 99%, batch number: 20200507) was purchased from Shanghai Hanxiang Biotechnology Co., Ltd.; Acetonitrile (MS grade, batch number: 217207), methanol (MS grade, batch number: 224617), formic acid (MS grade, batch number: 220614), and ammonia (MS grade, batch number: 7115080) were all purchased from Thermo Fisher Scientific (China) Co., Ltd. The experimental water was prepared by an ultrapure water system.

[0036] Instruments and consumables: LC-MS / MS instrument (model: Xevo TQD IVD) was purchased from Waters, USA; low-temperature high-speed centrifuge (model: SCIENTIFIC Legend Micro 21R), low-speed centrifuge (model: SL8), and vortexer (model: LPVortex Mixer) were all purchased from Thermo Fisher, USA; one-hundred-thousandth balance (model: MS105) was purchased from Mettler, USA; ultrapure water system (model: Milli-QDirect 8) was purchased from Millipore, USA; pipettes (specifications: 10, 20, 100, 200, 1000 μL) were purchased from Eppendorf, Germany; volumetric flasks (specifications: 10 mL, 50 mL) were purchased from Beijing Xinweier Glass Instrument Co., Ltd.; centrifuge tubes (specifications: 0.2 mL, 1.5 mL, 10 mL) were purchased from Aisin Biotechnology (Hangzhou) Co., Ltd.

[0037] See also Figure 1 This embodiment provides a method for detecting zanubrutinib and ibrutinib in human blood, comprising the following steps, and the flowchart is shown in Figure 1 :

[0038] (1) Prepare the stock solution:

[0039] Accurately weigh 10 mg of each of the zanubrutinib and ibrutinib reference substances, place them in 10 mL volumetric flasks, dilute to volume with 90% methanol aqueous solution, and shake well to obtain zanubrutinib standard stock solutions and ibrutinib standard stock solutions with a concentration of 1,000,000 ng / mL.

[0040] Take 1 mL of each of the zanubrutinib and ibrutinib standard stock solutions, place them in the same 10 mL volumetric flask, and make up to volume with 90% methanol aqueous solution to obtain a mixed standard stock solution with a concentration of 100,000 ng / mL.

[0041] Weigh 1 mg of zanubrutinib-D5 reference substance, place it in a 50 mL volumetric flask, and dilute to volume with 90% methanol aqueous solution to obtain 20,000 ng / mL zanubrutinib-D5 stock solution. The ibrutinib-D5 stock solution can be prepared in the same way.

[0042] It should be noted that the above stock solutions can be stored at -80°C.

[0043] (2) Prepare standard curve working solution and quality control working solution:

[0044] The mixed standard stock solution was gradiently diluted using 90% methanol in water. The gradient dilution method is detailed in Table 1. The concentrations of the six standard working solutions were 5000, 2500, 500, 250, 50, and 25 ng / mL, and the concentrations of the three quality control working solutions were 4000, 400, and 40 ng / mL, respectively.

[0045] The above-mentioned standard curve working solution and quality control working solution can be stored at -20℃.

[0046] Table 1 Gradient dilution method of standard curve working solution and quality control working solution

[0047]

[0048]

[0049] (3) Preparation of standard curve samples and quality control samples:

[0050] Add 20 μL of a certain concentration of standard curve working solution or quality control working solution to 180 μL of blank plasma to prepare standard curve samples and quality control samples.

[0051] (4) Prepare the mixed internal standard working solution:

[0052] Take 100 μL each of the zanubrutinib-D5 stock solution and the ibrutinib-D5 stock solution and add them to a 100 mL volumetric flask, and make up to volume with 0.05% formic acid in acetonitrile to prepare a mixed internal standard working solution with a concentration of 20 ng / mL.

[0053] (5) Sample pretreatment:

[0054] Accurately measure 200 μL of standard curve sample or quality control sample or clinical sample, add 400 μL of mixed internal standard working solution, vortex vigorously for 5 minutes, centrifuge at 14000 rpm / min at 10°C for 5 minutes, take 200 μL of the supernatant after centrifugation and place it in a new centrifuge tube, add 200 μL of an aqueous solution containing 0.1% formic acid and 38% acetonitrile, vortex vigorously again for 5 minutes, mix well and centrifuge again, centrifuge at 14000 rpm / min at 10°C for 10 minutes, and take the supernatant.

[0055] Among them, the theoretical concentrations of 6 standard curve samples of Zebutinib and Ibrutinib are 500, 250, 50, 25, 5, and 2.5 ng / mL, respectively; the theoretical concentrations of 3 quality control samples of Zebutinib and Ibrutinib are 400, 40, and 4 ng / mL, respectively.

[0056] (6) On-machine testing:

[0057] The supernatant of the pretreated samples was tested using liquid chromatography-mass spectrometry.

[0058] Liquid chromatography detection conditions: the chromatographic column is ACQUITY UPLC BEH-C18 (1.7 μm, 2.1 mm*50 mm), the column temperature is 40°C, the injector temperature is 15°C, the injection volume is 2 μL, the needle wash solution is an aqueous solution containing 0.5% ammonia and 49.5% acetonitrile, the organic mobile phase (mobile phase A) is an acetonitrile solution containing 0.1% formic acid, the aqueous mobile phase (mobile phase B) is an aqueous solution containing 0.1% formic acid and 10 mM ammonium formate, the flow rate is 0.4 mL / min, and the gradient elution conditions are as follows:

[0059] 0.0min, mobile phase B: 62%;

[0060] 0.5min, mobile phase B: 62%;

[0061] 1.0min, mobile phase B: 40%;

[0062] 2.0 min, mobile phase B: 28%;

[0063] 2.5 min, mobile phase B: 0%;

[0064] 3.0 min, mobile phase B: 62%.

[0065] It should be noted that the use of needle wash solution can reduce the residue of high-concentration samples in the injection system.

[0066] Mass spectrometry detection conditions: positive ion electrospray ion source (ESI+), capillary voltage (Capilary) of 1.0 kV, desolvation temperature (Desolvation Temprature) of 450 ° C, desolvation gas flow (Desolvbation) of 800 L / Hr, cone gas (Cone) flow rate of 60 L / Hr, multiple reaction monitoring mode, ion information, collision energy and cone voltage are shown in Table 2.

[0067] Table 2 Ion information, collision energy and cone voltage

[0068]

[0069] Embodiment 2:

[0070] This example refers to the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Rules, 9012 Biological Sample Quantitative Analysis Method Validation Guidance, and the method of Example 1 was methodologically validated as follows:

[0071] (1) Linear parameters:

[0072] The standard curve samples at 6 concentration levels were processed according to Example 1 and then tested on a computer. The Masslynx software recorded the chromatographic peak areas of zanubrutinib, ibrutinib, zanubrutinib-D5, and ibrutinib-D5.

[0073] The mass concentration of zanubrutinib or ibrutinib (X) was used as the horizontal axis, and the peak area ratio of zanubrutinib to zanubrutinib-D5 or ibrutinib to ibrutinib-D5 (Y) was used as the vertical axis. The weighted least square method was used for linear regression, and the weighting factor was (1 / X 2 ), and obtain the linear regression equation, as shown in Table 3. Repeat the operation and measure 3 batches, and the determination coefficient (r) is greater than 0.99. Substitute the Y value into the regression equation of the day to back-calculate its concentration, and the deviation from the theoretical value is less than 15%.

[0074] There is a good linear relationship between zanubrutinib and ibrutinib in the concentration range of 2.5 to 500 ng / mL. When the sample concentration is 2.5 ng / mL, the instrument response intensity is about 10 times the signal-to-noise ratio. Therefore, in this embodiment, the lower limit of quantification is 2.5 ng / mL.

[0075] Table 3 Regression equations of three batches

[0076]

[0077] (2) Specificity:

[0078] Prepare the quantitative lower limit concentration sample, treat it according to Example 1, and then test it on the machine.

[0079] A blank plasma sample was prepared, and the internal standard working solution was replaced with acetonitrile containing 0.05% formic acid. Other samples were processed as in Example 1 and then tested on the machine.

[0080] The clinical samples were processed according to Example 1 and then tested on the machine.

[0081] The above three samples were tested on the machine, and the chromatograms of the three samples were obtained. See Figure 2-4 ,in, Figure 2 , 3 , 4 are the chromatograms of the quantitative lower limit concentration sample, blank plasma sample, and clinical sample in this embodiment, respectively, wherein a is zanubrutinib-D5, b is zanubrutinib, c is ibrutinib-D5, and d is ibrutinib.

[0082] As can be seen from the figure, the retention time of zanubrutinib is 1.17min, the retention time of ibrutinib is 1.30, the retention time of the internal standard zanubrutinib-D5 is between 1.16 and 1.17min, and the retention time of the internal standard ibrutinib-D5 is 1.29min. The above four detection channels do not interfere with each other, the endogenous substances in blank plasma do not interfere with the detection of the target and the corresponding isotope internal standard, and there is no significant difference in the chromatographic behavior of the simulated quantitative lower limit concentration sample and the clinical sample.

[0083] (3) Accuracy and precision:

[0084] Five quality control samples of the lower limit of quantitation and three concentration levels were prepared in parallel, and after being processed according to Example 1, they were tested on the machine and operated continuously for 3 days, and their concentrations were calculated using a newly prepared standard curve every day. The relative standard deviation (RSD) and relative error (RE) were used to evaluate the precision and accuracy. The intra-day and inter-day RSD% and RE% of the two target drugs were within 15% (the limit of the lower limit of quantitation was 20%), which met the requirements of the guidelines. The results are shown in Table 4.

[0085] Table 4 Accuracy and precision

[0086]

[0087]

[0088] (4) Residual effect:

[0089] Five samples with a lower limit of quantitation were prepared in parallel, and after being processed according to Example 1, they were tested on the machine, and the average chromatographic peak areas of the target drug and the corresponding isotope internal standard were recorded as a1 and a2. Five samples with a high concentration of 500 ng / mL were then prepared in parallel and processed according to Example 1; five blank samples, the internal standard working solution was replaced with acetonitrile containing 0.05% formic acid, and the others were processed according to Example 1; the samples were tested on the machine in the injection order of "high concentration sample-blank sample", and repeated 5 times, and the average chromatographic peak areas of the blank sample target drug and the corresponding isotope internal standard were recorded as b1 and b2. The target drug residual ratio was b1 / a1, both lower than 20%, and the isotope internal standard residual ratio was b2 / a2, both lower than 5%.

[0090] (5) Extraction recovery and matrix effect:

[0091] Six quality control samples of three concentration levels were prepared in parallel. After being processed according to Example 1, they were tested on the machine, and the peak areas of the target drug and the corresponding isotope internal standard were recorded as A1 and A2. Six blank plasmas from different sources were treated according to Example 1, except that the internal standard working solution was replaced with acetonitrile containing 0.05% formic acid. The other six blank plasmas were added with corresponding volumes of quality control and internal standard working solutions so that their theoretical injection concentrations were the same as those of the above quality control samples. The peak areas of the target drug and the corresponding isotope internal standard were recorded as B1 and B2. B2; Ultrapure water, except for the internal standard working solution replaced with acetonitrile containing 0.05% formic acid, was treated according to Example 1, and the corresponding volume of quality control and internal standard working solution was added to make its theoretical injection concentration the same as the above quality control sample, and the peak area of ​​the target drug and the corresponding isotope internal standard was recorded as C1 and C2; Extraction recovery (%) = A / B × 100%, matrix effect (%) = B / C × 100%, internal standard normalized matrix factor = target drug matrix effect / internal standard matrix effect. The extraction recovery of zabutinib was between 88.08% and 94.63%, the extraction recovery of ibrutinib was between 95.96% and 99.83%, the normalized matrix factor of zabutinib-D5 was between 1.03-1.07, and the normalized matrix factor of ibrutinib was between 1.03-1.06. The results are shown in Table 5.

[0092] Table 5 Extraction recovery and matrix effect

[0093]

[0094] (6) Stability:

[0095] Quality control samples at three concentration levels were prepared to examine the stability under the following five conditions: placed in an automatic sampler (15°C) for 12 hours after treatment, placed at room temperature (22°C) for 8 hours, refrigerated at 4°C for 48 hours, repeatedly frozen and thawed at -20°C for 3 times (once a day), and frozen at -20°C for 15 days. Six samples were examined at each concentration under each condition. The stability was good under various conditions. The results are shown in Table 6.

[0096] Table 6 Stability under five conditions

[0097]

[0098]

[0099] In some embodiments, the quantitative method can be performed using an external standard method or an internal standard method, preferably an internal standard method. The internal standard method can use a monoisotope and a dual-isotope internal standard standard curve method, wherein the monoisotope zanubrutinib uses zanubrutinib-D5 as the internal standard, the ibrutinib uses ibrutinib-D5 as the internal standard, and the dual-isotope zanubrutinib and ibrutinib use the average of the response values ​​of zanubrutinib-D5 and ibrutinib-D5 as the internal standard.

[0100] Through the above specific implementations, those skilled in the art can easily implement the present invention. However, it should be understood that the present invention is not limited to the above specific implementations. On the basis of the disclosed implementations, those skilled in the art can arbitrarily combine different technical features to achieve different technical solutions.

Claims

1. A method for detecting zanubrutinib and ibrutinib in human blood, characterized in that: include: Use zanubrutinib and ibrutinib reference substances to prepare mixed stock solutions, dilute them step by step to prepare standard curve working solutions, and use the standard curve working solutions to prepare standard curve samples; Use zanubrutinib-D5 and ibrutinib-D5 reference substances to prepare a mixed internal standard working solution, and use the mixed internal standard working solution to pretreat the standard curve sample and the sample to be tested; The pre-treated samples to be tested and the standard curve samples were mass spectrometrically acquired using an electrospray ion source and a multiple reaction monitoring mode to obtain the signal intensity of the samples to be tested and the signal intensity of the standard curve samples respectively; Perform linear regression based on the signal intensity of the calibration curve sample and the theoretical concentration of the calibration curve sample to obtain a regression equation; The concentrations of zanubrutinib and ibrutinib in the sample to be tested are calculated according to the regression equation and the signal intensity of the sample to be tested.

2. The method for detecting zanubrutinib and ibrutinib in human blood according to claim 1, characterized in that: Use zanubrutinib and ibrutinib reference substances to prepare mixed stock solutions, dilute them step by step to prepare standard curve working solutions, and use the standard curve working solutions to prepare standard curve samples, including: Mixing the zanubrutinib reference substance and the ibrutinib reference substance with a standard curve solvent to obtain a mixed standard curve stock solution, wherein the standard curve solvent is a methanol aqueous solution or an acetonitrile aqueous solution, preferably a 90% methanol aqueous solution; The mixed Biaoqu stock solution is gradiently diluted to obtain Biaoqu working solution, and blank plasma is added to the Biaoqu working solution to obtain a Biaoqu sample, wherein the volume ratio of the Biaoqu working solution to the blank plasma is 1:5-20, preferably 1:

9.

3. The method for detecting zanubrutinib and ibrutinib in human blood according to claim 1, characterized in that: Use zanubrutinib-D5 and ibrutinib-D5 reference substances to prepare a mixed internal standard working solution, and use the mixed internal standard working solution to pretreat the standard curve sample and the sample to be tested, including: Respectively mixing zanubrutinib-D5 and ibrutinib-D5 reference substances with an internal standard stock solvent to obtain zanubrutinib-D5 stock solution and ibrutinib-D5 stock solution, wherein the internal standard stock solvent is a methanol aqueous solution or an acetonitrile aqueous solution, preferably a 90% methanol aqueous solution; Mixing the zanubrutinib-D5 stock solution and the ibrutinib-D5 stock solution with an internal standard working solvent to obtain a mixed internal standard working solution, wherein the internal standard working solvent is one or more of methanol, acetonitrile, and formic acid, preferably acetonitrile with 0.05% formic acid, and the concentration of the mixed internal standard working solution is 10-100 ng / mL, preferably 20 ng / mL; The mixed internal standard working solution is mixed with the sample to be tested, and the supernatant is taken out by centrifugation, and the supernatant is diluted with a diluent to obtain the pretreated sample to be tested, wherein the diluent is a formic acid aqueous solution, an acetonitrile aqueous solution or a mixture thereof, preferably an aqueous solution containing 0.1% formic acid and 38% acetonitrile.

4. The method for detecting zanubrutinib and ibrutinib in human blood according to claim 1, characterized in that: In the mass spectrometry acquisition, the chromatographic column filler is C18, the particle size is 1.7-5μm, preferably 1.7μm; the injection volume is 2-10μL, preferably 2μL; the column temperature is 35-45°C, preferably 40°C; the capillary voltage is 0.5-2.0kV, preferably 1.0kV; the desolvation temperature is 400-500°C, preferably 450°C; the desolvation gas flow rate is 600-800L / Hr, preferably 800L / Hr; the cone gas flow rate is 40-60L / Hr, preferably 60L / Hr.

5. The method for detecting zanubrutinib and ibrutinib in human blood according to claim 1, characterized in that: In the mass spectrometry acquisition, the organic mobile phase is an acetonitrile solution containing formic acid, and the aqueous mobile phase is an aqueous solution of formic acid and ammonium formate, wherein the volume fraction of formic acid is 0.01% to 1%, preferably 0.1%, and the molar concentration of ammonium formate is 5 to 20 mM, preferably 10 mM; the elution flow rate is 0.3 to 0.5 ml / min, preferably 0.4 ml / min.

6. The method for detecting zanubrutinib and ibrutinib in human blood according to claim 5, characterized in that: In the mass spectrometry acquisition, the gradient elution conditions are as follows: 0.0min, water mobile phase: 62%; 0.5min, water mobile phase: 62%; 1.0min, water mobile phase: 40%; 2.0min, aqueous mobile phase: 28%; 2.5min, water mobile phase: 0%; 3.0min, aqueous mobile phase: 62%.

7. The method for detecting zanubrutinib and ibrutinib in human blood according to claim 3, characterized in that: The method further comprises cleaning the injection needle with a needle washing solution after mass spectrometry sampling, wherein the needle washing solution is a methanol or acetonitrile aqueous solution containing ammonia water, preferably an aqueous solution containing 0.5% volume ammonia water and 49.5% volume acetonitrile.

8. The method for detecting zanubrutinib and ibrutinib in human blood according to claim 1, characterized in that: The theoretical concentrations of zanubrutinib and ibrutinib in the standard curve samples are 500, 250, 50, 25, 5, and 2.5 ng / mL, respectively.

9. The method for detecting zanubrutinib and ibrutinib in human blood according to claim 1, characterized in that: The method also includes a quality control test, wherein in the quality control samples of the quality control test, the theoretical concentrations of zanubrutinib and ibrutinib are 400, 40, and 4 ng / mL, respectively.

10. A method for detecting zanubrutinib and ibrutinib in human blood according to any one of claims 1 to 9, characterized in that: The method detects the concentration range of zanubrutinib and ibrutinib in the range of 2.5-500 ng / mL.

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