Method for detecting concentration of vonakula in human blood by adopting ultra-high performance liquid chromatography-tandem mass spectrometry

Abstract

The invention discloses a method for detecting the concentration of vinca in human blood by adopting ultra-high performance liquid chromatography-tandem mass spectrometry. The method comprises the following steps: preparing an internal standard stock solution and a standard stock solution, diluting the internal standard stock solution to obtain an internal standard working solution, and carrying out gradient dilution on the standard stock solution to obtain a standard working solution with a series of concentrations; preparing a standard curve blood sample and a clinical blood sample, and pretreating the standard curve blood sample and the clinical blood sample through an organic solvent protein precipitation method to obtain a standard curve sample and a clinical actual sample; respectively detecting the standard curve sample and the clinical actual sample by using an ultra-high performance liquid chromatography and mass spectrometry combined method; and making a standard curve by using the detection result of the standard curve sample, establishing a regression equation, substituting the detection result of the clinical actual sample into the regression equation, and calculating to obtain the concentration of the vinca in the human blood, so that the method has the advantages of wide linear range and high detection speed.

Description

Technical Field

[0001] The present invention relates to the field of medical testing technology, in particular to a method for detecting the concentration of venetoclax in human blood by using ultra-high performance liquid chromatography-tandem mass spectrometry. Background Art

[0002] Venetoclax is an oral B-lymphocytic neoplasm-2 selective inhibitor that can be used to treat chronic lymphocytic leukemia. It can also be used in combination with other anticancer drugs to treat patients with acute myeloid leukemia who are newly diagnosed or not suitable for intensive treatment.

[0003] In the early stage of treatment, a escalating dose is required to avoid tumor lysis syndrome, but venetoclax is poorly water-soluble. Compared with taking the drug on an empty stomach, with a low-fat meal, and with a high-fat meal, the in vivo exposure of venetoclax increases by about 3.4 times and 5.3 times, respectively. At the same time, patients with blood diseases often suffer from multiple complications and need to take many other drugs in combination, which will affect the in vivo exposure of venetoclax, thereby affecting the effect of escalating dose treatment and increasing the risk of medication. Therefore, it is necessary to quickly and accurately quantitatively detect the concentration of venetoclax in human blood. However, the current method for detecting the blood concentration of venetoclax in humans has the following disadvantages: a narrow linear range, a long detection time, and the disadvantage of venetoclax being difficult to dissolve has not been overcome, and it cannot meet the clinical demand for therapeutic drug monitoring of venetoclax. Summary of the invention

[0004] The technical task of the present invention is to address the above shortcomings and provide a method for detecting the concentration of venetoclax in human blood using ultra-high performance liquid chromatography-tandem mass spectrometry. Through this method, the linear concentration range is expanded, the detection speed is improved, and a mixed solvent is used to dissolve venetoclax, thereby solving the above problems.

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

[0006] A method for detecting the concentration of venetoclax in human blood using ultra-high performance liquid chromatography-tandem mass spectrometry, comprising:

[0007] S100, preparation of working solution: dissolving the venetoclax-D8 reference substance and the venetoclax reference substance in a mixed solvent respectively to obtain an internal standard stock solution and a standard stock solution; diluting the internal standard stock solution with the mixed solvent to obtain an internal standard working solution; and performing gradient dilution on the standard stock solution with the mixed solvent to obtain a series of concentrations of standard working solutions;

[0008] S200, sample preparation: preparing standard curve blood samples and clinical blood samples, and pre-treating the standard curve blood samples and clinical blood samples by an organic solvent protein precipitation method to obtain standard curve samples and actual clinical samples;

[0009] S300, ultra-high performance liquid chromatography tandem mass spectrometry detection: using ultra-high performance liquid chromatography coupled with mass spectrometry to detect the obtained standard curve samples and actual clinical samples respectively;

[0010] S400, preparation of standard curve and calculation of venetoclax concentration: using the test results of the standard curve samples obtained in S300 to prepare a standard curve, and establish a regression equation; substituting the test results of the actual clinical samples obtained in S300 into the regression equation, and calculating the venetoclax concentration in human blood.

[0011] Furthermore, the detection range of the method is 20ng / mL-20000ng / mL.

[0012] Furthermore, in S100, the mixed solvent is a mixed solution of ammonium bicarbonate buffer, acetonitrile and dimethyl sulfoxide.

[0013] Furthermore, the concentration of the internal standard working solution is 5000 ng / ml; the concentration of the standard stock solution is 1000000 ng / ml.

[0014] Furthermore, the standard working solutions of the series concentrations are: 50000 ng / mL, 20000 ng / mL, 10000 ng / mL, 5000 ng / mL, 2000 ng / mL, 1000 ng / mL, and 500 ng / mL, respectively.

[0015] Further, in S200, the pretreatment method is to mix the standard curve blood sample and the clinical blood sample with the internal standard working solution and acetonitrile, vortex violently, and centrifuge, add the supernatant after centrifugation to ultrapure water, vortex violently, and centrifuge, and take the supernatant.

[0016] Further, in S300, the chromatographic column for ultra-high performance liquid chromatography detection is BEH-C18, the column temperature is 40°C, the injector temperature is 15°C, the injection volume is 2 μL, the mobile phase A is an acetonitrile solution containing 0.1% formic acid, the mobile phase B is an aqueous solution of 0.1% formic acid, and the flow rate is 0.4 mL / min; the gradient elution conditions are: 0.0 min, mobile phase B: 80%; 1.0 min, mobile phase B: 60%; 3.0 min, mobile phase B: 0%; 3.3 min, mobile phase B: 0%; 3.31 min, mobile phase B: 80%; 3.5 min, mobile phase B: 80%.

[0017] Further, in S300, the mass spectrometer adopts a positive ion electrospray ion source, a capillary voltage of 1.0 kV, a desolvation gas temperature of 450°C, a desolvation gas flow rate of 800 L / Hr, a cone gas flow rate of 60 L / Hr, a multiple reaction monitoring mode, a cone voltage of 65 V, a collision energy of 45 V, and the quantitative analysis ion pairs are venetoclax m / z 868.52→321.18 and venetoclax-D8 m / z 876.32→329.2.

[0018] Furthermore, the method also includes the preparation of quality control working solution, and the use of the quality control working solution for methodological validation, wherein the concentrations of the quality control working solution are 400000 ng / mL, 4000 ng / mL, and 400 ng / mL, respectively.

[0019] Furthermore, the methodological validation includes specificity, linearity validation and lower limit of quantification, accuracy and precision, residual effect, dilution effect, extraction recovery and matrix effect, and stability.

[0020] Compared with the prior art, the advantages and positive effects of the present invention are:

[0021] 1. The method of dissolving venetoclax with a mixed solvent adopted in the present invention overcomes the disadvantage that venetoclax itself is difficult to dissolve. It is in a solid state when stored in a refrigerator at -80°C and in a transparent liquid state when restored to room temperature without precipitation.

[0022] 2. The present invention designs calibration at 7 concentration levels and quality control at 3 concentration levels. Combined with the dilution effect results, the detection range is 20ng / mL-20000ng / mL, which reasonably covers the actual concentration range of venetoclax in clinical samples;

[0023] 3. The sample processing of the present invention is simple and easy to operate, and only requires one step of simple protein precipitation and one step of dilution for analysis. The analysis time after sample loading is only 3.5 minutes, so the present invention is suitable for large-scale clinical sample analysis;

[0024] 4. The quantitative lower limit of the present invention for detecting the blood concentration of venetoclax is 20 ng / mL, and a lower blood concentration of venetoclax can be detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] 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.

[0026] Figure 1 It is a schematic diagram of the process of the present invention;

[0027] Figure 2 The chromatogram of the quantitative lower limit sample of Venetoclax in this embodiment;

[0028] Figure 3 The chromatogram of the quantitative lower limit sample of Venetoclax-D8 in this embodiment;

[0029] Figure 4 is the chromatogram of the blank plasma sample of Venetoclax in this example;

[0030] Figure 5 is the chromatogram of the blank plasma sample of Venetoclax-D8 in this example;

[0031] Figure 6 is a chromatogram of an actual clinical sample of Venetoclax in this embodiment;

[0032] Figure 7 : is a chromatogram of a clinical actual sample of Venetoclax-D8 in this embodiment;

[0033] Figure 8 This is a schematic diagram of the first batch standard curve in this embodiment;

[0034] Fig. 9 This is a schematic diagram of the second batch of standard curves in this embodiment;

[0035] Fig.10 This is a schematic diagram of the third batch of standard curves in this embodiment. DETAILED DESCRIPTION

[0036] 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.

[0037] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0038] This embodiment provides a method for detecting the concentration of venetoclax in human blood using ultra-high performance liquid chromatography-tandem mass spectrometry. Figure 1 .

[0039] Preparation of drugs and reagents: Venetoclax (purity: 99.7%, batch number: 20201103) was purchased from Shanghai Hanxiang Biotechnology Co., Ltd.; Venetoclax-D8 (purity: 99.6%, batch number: 2614-079A4) was purchased from TLC Pharmaceutical Standards, Canada; acetonitrile (mass spectrometry grade, batch number: 217207), dimethyl sulfoxide (mass spectrometry grade, batch number: 121E0313), formic acid (mass spectrometry grade, batch number: A117-50), and acetic acid (mass spectrometry grade, batch number: 165002) were all purchased from Thermo Fisher Scientific (China) Co., Ltd.; ammonium bicarbonate (chromatographic grade, batch number: J2030) was purchased from Honeywell Trading (Shanghai) Co., Ltd.; experimental water was prepared by an ultrapure water system.

[0040] Preparation of 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 vortex mixer (model: LP Vortex Mixer) were all purchased from Thermo Fisher, USA; one-hundred-thousandth balance (model: MS105) was purchased from Mettler, USA; ultrapure water system (model: Milli-Q Direct 8) was purchased from Millipore, USA; pipettes (specifications: 10, 20, 100, 200, 1000 μL) were purchased from Ebende, Germany; volumetric flasks (specifications: 10 mL, 25 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.

[0041] The specific steps are as follows:

[0042] S100, working fluid preparation:

[0043] S101, configuration of mixed solvent:

[0044] Accurately weigh 0.79060 g of ammonium bicarbonate, add it into a 1-L glass container, add 1 L of ultrapure water, shake well until completely dissolved, adjust the pH to 7 with glacial acetic acid, and prepare an ammonium bicarbonate buffer solution.

[0045] Take 6 mL of acetonitrile, 3 mL of dimethyl sulfoxide and 1 mL of ammonium bicarbonate buffer respectively in a 10 mL centrifuge tube, vortex and mix to obtain a mixed solvent.

[0046] S102, preparation of venetoclax-D8 internal standard stock solution:

[0047] Accurately weigh 1 mg of the Venetoclax-D8 reference substance, place it in a 25 mL volumetric flask, make up to volume with a mixed solvent, and shake well to obtain a Venetoclax-D8 internal standard stock solution with a mass concentration of 40,000 ng / mL. The Venetoclax-D8 internal standard stock solution can be stored at -80°C.

[0048] S103, Preparation of Venetoclax Standard Stock Solution:

[0049] Accurately weigh 10 mg of the venetoclax reference substance, place it in a 10 mL volumetric flask, dilute it with a mixed solvent, and shake it well to obtain a venetoclax standard stock solution with a mass concentration of 1,000,000 ng / mL. The venetoclax standard stock solution can be stored at -80°C.

[0050] S104, working fluid configuration:

[0051] Measure 100 μL of the venetoclax-D8 internal standard stock solution into a centrifuge tube, add 700 μL of the mixed solvent to dilute, and vortex to obtain a venetoclax-D8 internal standard working solution with a mass concentration of 5000 ng / mL.

[0052] The standard working solution of venetoclax and the quality control working solution are prepared by stepwise dilution. The standard working solution of venetoclax has 7 concentration levels, S7-S1: 50000ng / mL, 20000ng / mL, 10000ng / mL, 5000ng / mL, 2000ng / mL, 1000ng / mL, 500ng / mL; the quality control working solution has 3 concentration levels, H, M, L: 400000ng / mL, 4000ng / mL, 400ng / mL. The specific preparation method is shown in Table 1. The internal standard working solution of venetoclax-D8, the standard working solution of venetoclax and the quality control working solution can all be stored at -20℃.

[0053] Table 1 Stepwise dilution method for preparing the standard working solution of venetoclax and the quality control working solution

[0054]

[0055] S200, sample preparation: pre-treat the sample by organic solvent protein precipitation method.

[0056] S201. Blood sample preparation:

[0057] Clinical blood samples were obtained by centrifuging clinical EDTA anticoagulated whole blood at 3500 rpm / min for 10 min, and then accurately measuring the upper plasma;

[0058] Preparation method of standard curve blood samples and quality control blood samples: add 180 μL of blank plasma into a 1.5 mL centrifuge tube, then add 20 μL of a certain concentration of venetoclax standard working solution or quality control working solution to obtain standard curve blood samples and quality control blood samples, wherein the theoretical concentrations of the standard curve blood samples are 5000 ng / mL, 2000 ng / mL, 1000 ng / mL, 500 ng / mL, 200 ng / mL, 100 ng / mL, and 50 ng / mL, respectively, and the theoretical concentrations of the quality control blood samples are 4000 ng / mL, 400 ng / mL, and 40 ng / mL, respectively.

[0059] S202, blood sample pretreatment:

[0060] Clinical blood samples, standard curve blood samples and quality control blood samples all need to be pretreated. The pretreatment steps are as follows:

[0061] Take 200 μL of the blood sample that needs to be pretreated and add it to a 1.5 mL centrifuge tube, then add 20 μL of venetoclax-D8 internal standard working solution and 400 μL of acetonitrile in sequence, 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 1.5 mL centrifuge tube, add 200 μL of ultrapure water, vortex vigorously again for 5 minutes, centrifuge at 14000 rpm / min at 10°C for 10 minutes, take the supernatant to obtain the actual clinical samples, standard curve samples and quality control samples.

[0062] S300, ultra-high performance liquid chromatography tandem mass spectrometry detection:

[0063] The ultra-high performance liquid chromatography-mass spectrometry method was used to test actual clinical samples, standard curve samples and quality control samples. The test conditions were as follows:

[0064] (1) Liquid chromatography conditions: the chromatographic column was ACQUITY UPLC BEH-C18 (1.7 μm, 2.1 mm*50 mm), the column temperature was 40°C, the injector temperature was 15°C, the injection volume was 2 μL, the mobile phase A was an acetonitrile solution containing 0.1% formic acid, the mobile phase B was an aqueous solution containing 0.1% formic acid, the flow rate was 0.4 mL / min, and the gradient elution conditions were as follows:

[0065] 0.0min, mobile phase B: 80%;

[0066] 1.0min, mobile phase B: 60%;

[0067] 3.0 min, mobile phase B: 0%;

[0068] 3.3 min, mobile phase B: 0%;

[0069] 3.31min, mobile phase B: 80%;

[0070] 3.5 min, mobile phase B: 80%.

[0071] (2) Mass spectrometry conditions: A positive ion electrospray ion source (ESI+) was used, the capillary voltage was 1.0 kV, the desolvation temperature was 450°C, the desolvation flow rate was 800 L / Hr, the cone gas flow rate was 60 L / Hr, the multiple reaction monitoring mode was used, the cone voltage was 65 V, the collision energy was 45 V, and the quantitative analysis ion pairs were: venetoclax m / z 868.52→321.18, venetoclax-D8 m / z 876.32→329.2.

[0072] S400, standard curve preparation and venetoclax concentration calculation:

[0073] The standard curve samples were analyzed by ultra-high performance liquid chromatography coupled to mass spectrometry. The peak areas of venetoclax and venetoclax-D8 were recorded by Masslynx software. The mass concentration of venetoclax (X) was used as the horizontal axis, and the chromatographic peak area ratio of venetoclax and venetoclax-D8 (Y) was used as the vertical axis. The weighted least square method was used for linear regression. The weighting factor was (1 / X 2 ), make a standard curve, establish a regression equation, and substitute the test results of the actual clinical samples into the regression equation to calculate the concentration of venetoclax in human blood.

[0074] S500, Methodology Validation:

[0075] S501, Linearity verification and determination of lower limit of quantitation:

[0076] The standard curve samples were analyzed by ultra-high performance liquid chromatography coupled to mass spectrometry, and the peak areas of venetoclax and venetoclax-D8 were recorded using Masslynx software.

[0077] The weighted least squares method was used for linear regression with the mass concentration of venetoclax (X) as the horizontal axis and the chromatographic peak area ratio of venetoclax and venetoclax-D8 (Y) as the vertical axis. The weighted factor was (1 / X 2 ), and the operation was continued for 3 days. Venetoclax showed a good linear relationship in the concentration range of 50 ng / mL to 5000 ng / mL. The 3-day calibration curve is shown in Figure 8-10 , the regression equation and determination coefficient (r) are shown in Table 2.

[0078] When the sample concentration is diluted to 20 ng / mL, the instrument response intensity is 10 times the signal-to-noise ratio. Therefore, it can be seen that in this embodiment, the lower limit of quantification is 20 ng / mL.

[0079] Table 2 Regression equations of three batches

[0080]

[0081] S502, Exclusiveness Inspection:

[0082] The test sample with a quantitative lower limit concentration of 20 ng / mL obtained in S501 is the quantitative lower limit sample. The preparation method is the same as S200 and will not be repeated here. Prepare a blank plasma sample. The blank plasma sample does not contain the internal standard working solution. The other operation methods are the same as S200. The chromatograms obtained by injection analysis of the processed quantitative lower limit sample, blank plasma sample and actual clinical sample are shown in Figure 2-7 .

[0083] The results showed that the retention time of venetoclax was between 2.25-2.26 min, the retention time of the internal standard venetoclax-D8 was 2.25 min, the two quantitative detection channels of venetoclax and venetoclax-D8 did not interfere with each other, the endogenous substances in blank plasma did not interfere with the detection of venetoclax and venetoclax-D8, and there was no significant difference in the chromatographic behavior between the simulated quantitative offline samples and the actual clinical samples.

[0084] S503, Accuracy and Precision Verification:

[0085] The samples with the quantitative lower limit of 20 ng / mL obtained by S501 and the three levels of quality control samples of 40, 400, and 4000 ng / mL were configured in parallel, and the operation method was the same as S200. The injection test was carried out for 3 consecutive days. The new standard curve was calculated every day. The relative error (RE%) and relative standard deviation (RSD%) were used to evaluate the accuracy and precision. The intra-day and inter-day accuracy (RE%) were -2.89% to 8.37% and -7.30% to 8.68%, respectively. The intra-day and inter-day precision (RSD%) ranged from 1.41% to 8.46% and 3.43% to 9.94%, respectively. The specific results are shown in Table 3.

[0086] Table 3 Accuracy and precision results

[0087]

[0088] S504, residual effect verification: parallel configuration of 5 samples with a quantitative lower limit of 20ng / mL concentration, the same operation method as S200, sample injection analysis, record the average chromatographic peak area of ​​venetoclax and venetoclax-D8 as a1 and a2. Then parallel configuration of 5 samples with a concentration of 5000ng / mL and 5 blank plasma, the same operation method as S200, in the order of "quantitative upper limit - blank plasma", repeat 5 times, record the average chromatographic peak area of ​​blank plasma venetoclax and venetoclax-D8 as b1 and b2. The residual ratio of venetoclax is b1 / a1, and the residual ratio of venetoclax-D8 is b2 / a2, and the residual ratios are both lower than 20%.

[0089] S505, Dilution effect verification: Add 490μL blank plasma to a 1.5mL centrifuge tube, add 10μL venetoclax standard stock solution, and prepare a high concentration sample of 20000ng / mL. Use blank plasma and dilute it 10 times, 25 times, and 50 times according to the method in Table 4. Prepare 5 copies of each concentration in parallel, operate according to the S200 method, analyze, and calculate using the newly configured standard curve. The results are shown in Table 4, and the RE% is within 5%. The dilution effect verification shows that the detection range of this method can be extended to 20ng / mL-20000ng / mL.

[0090] Table 4 Dilution effect results

[0091]

[0092] S506. Extraction recovery and matrix effect: Prepare 6 samples of 40, 400, and 4000 ng / mL quality control samples in parallel, operate according to the S200 method, analyze the samples, and record the peak areas of venetoclax and venetoclax-D8 as A1 and A2; 6 blank plasmas from different sources were not added with internal standard working solution, and the rest were operated according to the S200 method, with corresponding volumes of quality control working solution and internal standard working solution added to make the injection concentration the same as that of the above-mentioned treated samples, analyze the samples, and record the peak areas of venetoclax and venetoclax-D8. The peak areas of venetoclax and venetoclax-D8 are B1 and B2; ultrapure water, without internal standard working solution, is operated according to the S200 method, and the corresponding volume of quality control and internal standard working solution is added to make the injection concentration the same as the above-mentioned treated sample, and the injection analysis is performed, and the peak areas of venetoclax and venetoclax-D8 are recorded as C1 and C2; extraction recovery rate (%) = A / B × 100%, matrix effect (%) = B / C × 100%, internal standard normalized matrix factor = venetoclax matrix effect / venetoclax-D8 matrix effect. The extraction recovery rate of venetoclax is between 97.62% and 107.65%. The internal standard normalized matrix factor of venetoclax is between 1.0153 and 1.0860, and the RSD% is ≤5.73%. The results are shown in Table 5.

[0093] Table 5 Extraction recovery and matrix effect results

[0094]

[0095] S507, stability verification: Prepare quality control samples of 40, 400, and 4000 ng / mL, and examine the stability under the following five conditions: place in the autosampler (15°C) for 12 hours after treatment, place at room temperature (22°C) for 24 hours, refrigerate at 4°C for 48 hours, freeze and thaw repeatedly at -20°C for 3 times (once a day), refrigerate at 4°C for 72 hours, and freeze at -20°C for 30 days. Examine 6 samples at each concentration under each condition. See Table 6.

[0096] Table 6 Stability results

[0097]

[0098]

[0099] 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 the concentration of venetoclax in human blood using ultra-high performance liquid chromatography-tandem mass spectrometry, characterized in that: include: S100, preparing working solution: dissolving the venetoclax-D8 reference substance and the venetoclax reference substance in a mixed solvent respectively to obtain an internal standard stock solution and a standard stock solution; The internal standard stock solution is diluted using the mixed solvent to obtain an internal standard working solution; the standard stock solution is gradiently diluted using the mixed solvent to obtain a series of concentration standard working solutions; S200, sample preparation: preparing standard curve blood samples and clinical blood samples, and pre-treating the standard curve blood samples and clinical blood samples by an organic solvent protein precipitation method to obtain standard curve samples and actual clinical samples; S300, ultra-high performance liquid chromatography tandem mass spectrometry detection: using ultra-high performance liquid chromatography coupled with mass spectrometry to detect the standard curve samples and actual clinical samples respectively; S400, preparation of standard curve and calculation of venetoclax concentration: using the test results of the standard curve samples obtained in S300 to prepare a standard curve, and establish a regression equation; substituting the test results of the actual clinical samples obtained in S300 into the regression equation, and calculating the venetoclax concentration in human blood.

2. The method for detecting the concentration of venetoclax in human blood by ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The detection range of the method is 20 ng / mL-20000 ng / mL.

3. The method for detecting the concentration of venetoclax in human blood by ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In S100, the mixed solvent is a mixed solution of ammonium bicarbonate buffer, acetonitrile, and dimethyl sulfoxide.

4. The method for detecting the concentration of venetoclax in human blood by ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The concentration of the internal standard working solution is 5000 ng / mL; the concentration of the standard stock solution is 1000000 ng / mL.

5. The method for detecting the concentration of venetoclax in human blood by ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The standard working solutions in the series of concentrations are: 50000 ng / mL, 20000 ng / mL, 10000 ng / mL, 5000 ng / mL, 2000 ng / mL, 1000 ng / mL, and 500 ng / mL, respectively.

6. The method for detecting the concentration of venetoclax in human blood by ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In S200, the pretreatment method is to mix the standard curve blood sample and the clinical blood sample with the internal standard working solution and acetonitrile, vortex violently, and centrifuge, add the supernatant after centrifugation to ultrapure water, vortex violently, and centrifuge, and take the supernatant.

7. The method for detecting the concentration of venetoclax in human blood by ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In S300, the chromatographic column for ultra-high performance liquid chromatography detection is BEH-C18, the column temperature is 40°C, the injector temperature is 15°C, the injection volume is 2 μL, the mobile phase A is an acetonitrile solution containing 0.1% formic acid, the mobile phase B is an aqueous solution of 0.1% formic acid, and the flow rate is 0.4 mL / min; the gradient elution conditions are: 0.0 min, mobile phase B: 80%; 1.0 min, mobile phase B: 60%; 3.0 min, mobile phase B: 0%; 3.3 min, mobile phase B: 0%; 3.31 min, mobile phase B: 80%; 3.5 min, mobile phase B: 80%.

8. The method for detecting the concentration of venetoclax in human blood by ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: In S300, the mass spectrometer uses a positive ion electrospray ion source, a capillary voltage of 1.0 kV, a desolvation gas temperature of 450°C, a desolvation gas flow rate of 800 L / Hr, a cone gas flow rate of 60 L / Hr, a multiple reaction monitoring mode, a cone voltage of 65 V, a collision energy of 45 V, and quantitative analysis ion pairs of venetoclax m / z 868.52→321.18 and venetoclax-D8 m / z 876.32→329.

2.

9. A method for detecting the concentration of venetoclax in human blood using ultra-high performance liquid chromatography-tandem mass spectrometry according to any one of claims 1 to 8, characterized in that: The method also includes the preparation of a quality control working solution, and the use of the quality control working solution for methodological validation, wherein the concentrations of the quality control working solution are 400000 ng / mL, 4000 ng / mL, and 400 ng / mL, respectively.

10. The method for detecting the concentration of venetoclax in human blood by ultra-high performance liquid chromatography-tandem mass spectrometry according to claim 9, characterized in that: The methodological validation includes specificity, linearity validation and lower limit of quantitation, accuracy and precision, carryover effect, dilution effect, extraction recovery and matrix effect, and stability.

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