Method for determining concentration of Vonoprasone in human plasma by liquid chromatography-tandem mass spectrometry

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for determining the concentration of Vonorrazan in human plasma through liquid chromatography-tandem mass spectrometry. Comprising the following steps: preparing a standard curve solution containing Vonoprazine-d4 and a test solution containing Vonoprazan by taking blank plasma as a matrix, and precipitating the solutions by using acetonitrile; determining the concentration of the test solution containing Vonoprazan after precipitation by using an internal standard method; a liquid chromatography tandem mass spectrometer is used for determination, and a mobile phase of liquid chromatography comprises an acetic acid aqueous solution and acetonitrile; the elution procedure of the liquid chromatography is as follows: the volume content of acetonitrile in a mobile phase is 60-90%, and the flow velocity of the mobile phase is 0.8-1.5 minmL / min. By optimizing chromatographic conditions and mass spectrum parameters, the invention provides an efficient and accurate Vonoprazan drug concentration detection method, the concentration of Vonoprazan in plasma can be accurately determined, and the accuracy and reliability of data are ensured.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for determining the concentration of vonoprazan in human plasma by liquid chromatography-tandem mass spectrometry. Background Art

[0002] Vonoprazan is a new potassium competitive acid blocker (P-CAB) that reversibly inhibits the H + / K + -ATPase, achieving rapid, potent and long-lasting acid suppression. With the popularization of its clinical application, the development of highly sensitive and stable blood drug concentration monitoring methods is crucial for individualized dosing and efficacy evaluation. Liquid chromatography tandem mass spectrometry (LC-MS / MS) has become the preferred method for drug analysis in complex matrices due to its high selectivity, wide linear range and low detection limit. However, the rapid and highly stable quantification of vonoprazan in K2EDTA plasma still lacks systematic method validation. Therefore, this field urgently needs to develop a rapid, highly sensitive and highly stable quantitative analysis method to analyze vonoprazan to meet the needs of clinical pharmacokinetic research.

[0003] Chinese invention patent application CN117607331 A discloses a method for quantitatively analyzing acid-suppressing drugs using a high-performance liquid chromatography-tandem mass spectrometry apparatus. The acid-suppressing drugs include vonoprazan fumarate. The high-performance liquid chromatography employs a mixed mobile phase consisting of mobile phase A and mobile phase B for gradient elution, wherein mobile phase A is an acetonitrile solution containing formic acid or a water-acetonitrile mixture containing ammonia and ammonium acetate (water:acetonitrile volume ratio: 1:99-10:90); and mobile phase B is selected from an aqueous solution containing ammonium acetate, an aqueous solution containing formic acid and ammonium acetate, an aqueous solution containing ammonia and ammonium acetate, and an aqueous solution containing ammonia and ammonium formate. However, the precision and accuracy of the method for detecting vonoprazan fumarate need to be improved, and the detection time needs to be shortened. Summary of the Invention

[0004] The present invention provides a method for determining the concentration of vonoprazan in human plasma by liquid chromatography-tandem mass spectrometry. The method comprises the following steps: using blank plasma as a matrix to prepare a standard curve solution containing vonoprazan-d4 (vonoprazan fumarate-d4) and a test solution containing vonoprazan (vonoprazan fumarate), and precipitating both solutions using acetonitrile; and determining the concentration of the precipitated vonoprazan test solution using an internal standard method.

[0005] This study found that by using acetonitrile for protein precipitation, the peak signal of vonoprazan in liquid chromatography can be increased, thereby improving detection accuracy. Residual endogenous substances will interfere with the retention behavior of the target in the chromatographic column and the mass spectrometry ionization process, resulting in peak tailing or response fluctuations. Acetonitrile, as a highly polar organic solvent, can destroy the hydration layer of plasma proteins and cause them to denature and precipitate, significantly reducing sample complexity. At the same time, it can also effectively remove phospholipids and avoid competitive ionization of the mass spectrometer in the electrospray ionization source (ESI), thereby effectively improving quantitative accuracy.

[0006] The concentration range of vonoprazan in the standard curve solution is 0.5-200 ng / mL.

[0007] The number of vonoprazan concentration gradients in the standard curve solution is 6-10.

[0008] The preparation of the standard curve solution includes diluting the standard curve pre-solution 20 times using blank plasma.

[0009] The standard curve pre-solution includes the following vonoprazan concentration gradient solutions (the solvent is 50 wt% methanol aqueous solution): 0.500 ng / mL, 1.00 ng / mL, 2.50 ng / mL, 10.0 ng / mL, 20.0 ng / mL, 40.0 ng / mL, 80.0 ng / mL and 100 ng / mL.

[0010] The test process also includes preparing quality control solutions, blank samples, zero concentration samples, and samples with no internal standard added to the upper limit of quantitation.

[0011] The concentration range of vonoprazan in the quality control solution is 0.5-100 ng / mL.

[0012] The concentrations of vonoprazan in the quality control solutions were 0.5 ng / mL, 1.5 ng / mL, 8 ng / mL, 30 ng / mL and 75 ng / mL, respectively.

[0013] The determination uses a liquid chromatography tandem mass spectrometer, the mobile phase of the liquid chromatography includes acetic acid aqueous solution and acetonitrile; the elution procedure of the liquid chromatography includes: the volume content of acetonitrile in the mobile phase is 60-90%, and the flow rate of the mobile phase is 0.8-1.5minmL / min.

[0014] The mobile phase of the liquid chromatography includes an aqueous acetic acid solution and acetonitrile; the elution procedure of the liquid chromatography includes: the volume content of acetonitrile in the mobile phase is 60-90%, and the flow rate of the mobile phase is 0.8-1.5minmL / min, which can improve the precision of the detection. The high proportion of acetonitrile significantly reduces the viscosity of the mobile phase, reduces the back pressure of the chromatographic column, and makes vonoprazan elute more quickly, while enhancing hydrophobic interactions and improving the symmetry of the peak shape; the addition of acetic acid inhibits the ionization of the basic group of vonoprazan and improves its retention stability on the C18 column. By optimizing the flow rate, the column efficiency and the running time are balanced to avoid peak broadening caused by too high a flow rate or analysis delays caused by too low a flow rate. The two work together to reduce retention time fluctuations, and by reducing matrix effects and solvent effects, the coefficient of variation of the mass spectrometry response is controlled within 5%, achieving high-precision detection.

[0015] Optionally, the elution procedure of the liquid chromatography includes: the volume content of acetonitrile in the mobile phase is 70-90%, and the flow rate of the mobile phase is 0.8-1.2 minmL / min.

[0016] Optionally, the volume content of acetonitrile in the mobile phase is 70-80%.

[0017] The chromatographic column used in the liquid chromatography is an octadecyl bonded phase; the particle size of the bonded phase is 2.5-8 μm, and the pore size is

[0018] The chromatographic column used in the liquid chromatography is an octadecyl bonded phase (C18); the particle size of the bonded phase is 2.5-8 μm, and the pore size is This can shorten detection times. The pore size of the specific bonded phase likely matches the diffusion rate of vonoprazan within the stationary phase. This, combined with the particle size of the specific bonded phase, shortens the diffusion path of the solute within the stationary phase pores, reduces mass transfer resistance, accelerates separation kinetics, and thus improves column efficiency. This allows for shorter separation times at the same column length while also avoiding the high backpressure associated with small particle size that limits flow rate increases, further shortening detection times.

[0019] Optionally, the particle size of the bonded phase is 2.5-5 μm, and the pore size is

[0020] Optionally, the chromatographic column model is 5μm C18(2) 4.6mm×150mm.

[0021] The elution time of the liquid chromatography is less than 2 minutes.

[0022] Optionally, the elution time of the liquid chromatography is ≤1.5 min.

[0023] The acquisition time of the mass spectrum was 1.5 min.

[0024] The ionization mode of the mass spectrometer is: electrospray ion source, positive ion mode, multiple reaction monitoring.

[0025] The ion source parameters of the mass spectrometer include the following parameters: collision gas 9.00 psi, curtain gas 20.00 psi, first ion source gas 30.00 psi, second ion source gas 60.00 psi, ion source spray voltage 5500.00 V, and ion source temperature 500.00°C.

[0026] The reaction ion parameters of vonoprazan in the mass spectrometer are: monitoring ion pair 346.2 / 315.1, declustering voltage 50.00 V, entrance voltage 10.00 V, exit voltage 10.00 V, collision energy 16.00 eV, and dwell time 200.00 msec.

[0027] The reaction ion parameters of vonoprazan-d4 in the mass spectrometer are: monitoring ion pair 350.1 / 316.0, declustering voltage 50.00 V, entrance voltage 10.00 V, exit voltage 10.00 V, collision energy 17.00 eV, and dwell time 200.00 msec.

[0028] Beneficial effects

[0029] 1. Using acetonitrile for protein precipitation can increase the peak signal of vonoprazan in liquid chromatography, thereby improving detection accuracy.

[0030] 2. The chromatographic column is an octadecyl bonded phase (C18); the particle size of the bonded phase is 2.5-8 μm, and the pore size is 50-200 Can shorten the detection time.

[0031] 3. The particle size of the bonded phase is 2.5-5 μm and the pore size is The elution time can be shortened to 2 minutes.

[0032] 4. The mobile phase of the liquid chromatography comprises aqueous acetic acid and acetonitrile; the elution procedure of the liquid chromatography comprises:

[0033] The volume content of acetonitrile in the mobile phase is 60-90%, and the flow rate of the mobile phase is 0.8-1.5 minmL / min, which can improve the precision of detection.

[0034] 5. By limiting the mass spectrometry parameters, the extraction recovery and selectivity of vonoprazan can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 These are the conditions for liquid chromatography in Example 1.

[0036] Figure 2 These are the mass spectrometry conditions of Example 1 (Q1: quadrupole mass analyzer 1; Q3: quadrupole mass analyzer 3).

[0037] Figure 3 The figures are the precision, accuracy and recovery test results of Example 1.

[0038] Figure 4 This is the selectivity test result of Example 1.

[0039] Figure 5 The durability test results of Example 1 are shown.

[0040] Figure 6 Example 1 Vonolazone daughter ion scanning spectrum.

[0041] Figure 7 Example 1 Vonolazone daughter ion scanning spectrum.

[0042] Figure 8 Example 1 Standard curve The standard curve obtained from the sample.

[0043] Figure 9 This is the liquid chromatogram of the test sample in Example 1.

[0044] Figure 10 This is the liquid chromatogram of the test sample of Comparative Example 1.

[0045] Figure 11 This is the liquid chromatogram of the test sample of Comparative Example 2. DETAILED DESCRIPTION

[0046] Example 1

[0047] A method for determining the concentration of vonoprazan in human plasma by liquid chromatography-tandem mass spectrometry comprises the following steps: using blank plasma as a matrix to prepare a standard curve solution containing vonoprazan-d4 (vonoprazan-d4 fumarate) and a test solution containing vonoprazan (vonoprazan fumarate), and precipitating both solutions using acetonitrile; and determining the concentration of the precipitated vonoprazan test solution using an internal standard method.

[0048] The blank matrix is ​​blank human plasma with K2EDTA as the anticoagulant provided by Zibo Traditional Chinese Medicine Hospital.

[0049] The preparation of the standard curve solution includes diluting the standard curve pre-solution 20 times using blank plasma.

[0050] The standard curve pre-solution includes the following vonoprazan concentration gradient solutions (the solvent is 50 wt% methanol aqueous solution): 0.500 ng / mL, 1.00 ng / mL, 2.50 ng / mL, 10.0 ng / mL, 20.0 ng / mL, 40.0 ng / mL, 80.0 ng / mL and 100 ng / mL.

[0051] The testing process also includes preparing quality control solutions, blank samples (mixing 50 μL of blank matrix with 25 μL of methanol), zero concentration samples (mixing 50 μL of blank matrix with 25 μL of internal standard solution), and quantitative upper limit samples without internal standard (mixing 50 μL of the quality control solution with the highest vonoprazan concentration with 25 μL of methanol).

[0052] Preparation of internal standard solution: Prepare 300 ng / mL internal standard solution of vonoprazan fumarate-d4 standard and methanol.

[0053] The concentrations of vonoprazan in the quality control solution (the solvent is 50 wt % methanol aqueous solution) are 0.5 ng / mL, 1.5 ng / mL, 8 ng / mL, 30 ng / mL and 75 ng / mL, respectively.

[0054] The specific test is as follows: in a 2.2mL 96-well polypropylene plate, at room temperature (25°C) and white light conditions, prepare 75μL of standard curve sample, 75μL of quality control sample, 75μL of test sample, and obtain 75μL of blank sample, zero concentration sample, and quantitative upper limit without internal standard sample; add 300μL of acetonitrile to all the above samples, mix well and precipitate, centrifuge at 4000ppm for 5min after precipitation, take 200μL of supernatant and transfer it to another 96-well polypropylene plate, and detect using liquid chromatography tandem mass spectrometry.

[0055] Chromatogram acquisition and chromatographic peak integration were performed by AB SciexAnalyst software (version 1.7.2). The standard curve was obtained by regression using the Watson LIMS system (version 7.6.1), with the chromatographic response ratio of the analyte to the internal standard as the ordinate and the weighted (W = 1 / x 2 ) The least squares method is used to perform linear regression on the concentration of the analyte in plasma (x) and the response ratio (y). The resulting regression equation (y = ax + b) is the standard curve. The drug concentration of the test sample is calculated using the fitted standard curve equation. Figure 6 As shown, a=0.0376;b=-0.00177;R 2 =0.9993.

[0056] The liquid chromatography conditions are as follows Figure 1 The mass spectrometry conditions are as shown in Figure 2As shown, the ion scanning spectrum of Tivonoplasm-d4 is as shown Figure 7 As shown, the ion scanning spectrum of vonoprazan-d4 is as follows Figure 8 shown.

[0057] Comparative Example 1

[0058] The specific implementation is the same as Example 1; the difference is that the solutions in Comparative Example 1 are all precipitated using methanol.

[0059] Comparative Example 2

[0060] The specific implementation is the same as Example 1; the difference is that the solutions in Comparative Example 2 are all precipitated using acetonitrile with 0.1 wt % formic acid.

[0061] Comparative Example 3

[0062] Specific implementation method is the same as Example 1; except that the mobile phase of the liquid chromatography in Comparative Example 3 includes acetic acid aqueous solution and methanol; the elution procedure of the liquid chromatography includes: the volume content of methanol in the mobile phase is 90%.

[0063] Performance testing methods

[0064] 1. Methodological verification of the test method of Example 1

[0065] 1. Precision and accuracy

[0066] Intra-batch precision and accuracy

[0067] Quality control samples (LLOQ QC, LQC, GMQC, MQC, and HQC) were used to evaluate intra-assay precision and accuracy, with 6 replicates for each concentration of quality control sample.

[0068] The precision was investigated by calculating the coefficient of variation (%CV) of the quality control samples at each concentration level, and the accuracy was investigated by calculating the deviation (Diff%) between the measured concentration mean and the theoretical concentration of the quality control samples at each concentration level.

[0069] Acceptance criteria:

[0070] The deviation of the measured concentration mean of the quality control samples at each concentration level from their theoretical concentration should be within the range of ±15.0% (the deviation of LLOQ QC should be within the range of ±20.0%), and the coefficient of variation should not exceed 15.0% (the coefficient of variation of LLOQ QC should not exceed 20.0%).

[0071] For precision and accuracy analysis batches, the deviation between the measured concentration and the theoretical concentration of at least 2 / 3 of the quality control samples does not exceed ±15.0% (LLOQ QC does not exceed ±20.0%), and at least 1 / 2 of the samples at the same concentration level meet the above standards.

[0072] Inter-assay precision and accuracy

[0073] Inter-assay precision and accuracy were assessed by examining at least three independent validation analytical batches (within-assay precision and accuracy batches completed on at least two days) using freshly prepared quality control samples in blank matrix.

[0074] The quality control samples for calculating inter-batch precision and accuracy were derived from the quality control samples (LLOQ QC, LQC, GMQC, MQC, and HQC) prepared for investigating intra-batch precision and accuracy. Six replicates of each quality control sample were prepared at each concentration level for each validation analysis batch.

[0075] Acceptance criteria:

[0076] The deviation of the overall mean of the measured concentration of the quality control samples at each concentration level from their theoretical concentration should be within the range of ±15.0% (the deviation of the LLOQ QC should be within the range of ±20.0%).

[0077] The overall coefficient of variation of the assay concentrations of quality control samples at each concentration level did not exceed 15.0% (the coefficient of variation of the LLOQ QC did not exceed 20.0%).

[0078] If an analytical batch did not meet the acceptance criteria, the method was validated by repeating the test on three additional accuracy and precision batches.

[0079] like Figure 3 As shown, Example 1 has excellent precision and accuracy.

[0080] 2. Extraction recovery rate

[0081] A blank matrix from the same batch (or source) as the conventional quality control sample (extracted sample, test sample) was used as the blank sample. After extraction, the analyte and internal standard were added to the blank sample extract to prepare low-concentration quality control samples (LQC), medium-concentration quality control samples (MQC), and high-concentration quality control samples (HQC) with 6 replicates at each concentration as reference samples.

[0082] The test samples were routine quality control samples or samples prepared in the same manner as the conventional quality control samples, including low-concentration quality control samples (LQC), medium-concentration quality control samples (MQC), and high-concentration quality control samples (HQC) (six replicates for each concentration).

[0083] Calculation of the extraction recovery rate of the analyte: the peak area of ​​the analyte in the routine quality control sample (test sample) of each concentration is divided by the mean peak area of ​​the analyte in the reference sample of the same concentration.

[0084] Calculation of internal standard extraction recovery: the internal standard peak area in each routine quality control sample (test sample) is divided by the mean internal standard peak area in the reference sample.

[0085] Acceptance criteria: The overall coefficient of variation of the analyte extraction does not exceed 15.0%; the coefficient of variation of the internal standard extraction recovery does not exceed 15.0%.

[0086] If the independent extraction recovery of the analyte or internal standard does not meet the acceptance criteria, the extraction recovery of the analytical method can be evaluated by the internal standard-corrected extraction recovery.

[0087] The internal standard correction extraction recovery rate was calculated by dividing the peak area ratio of the analyte to its internal standard in the conventional quality control sample (test sample) at each concentration by the mean peak area ratio of the analyte to its internal standard in the reference sample at the same concentration.

[0088] Acceptance criteria for internal standard corrected extraction recovery: The overall coefficient of variation of internal standard corrected extraction recovery does not exceed 15.0%.

[0089] like Figure 3 As shown, the extraction recovery rate of Example 1 is excellent.

[0090] Selectivity

[0091] Matrix selectivity (endogenous interference)

[0092] Matrix selectivity was evaluated by examining blank biological matrices from at least six different sources, one high-fat matrix from one source, and one hemolytic matrix from one source, with both blank samples without internal standard addition and samples at the LLOQ level measured.

[0093] High-lipid matrix: commercially available or simulated hyperlipidemic plasma (1±0.1 mg low-density lipoprotein (LDL) and 3±0.3 mg triglycerides added to 1 mL of blank plasma).

[0094] Hemolytic blank matrix: Freeze whole blood at -80°C for at least 30 minutes, thaw, vortex for at least 1 minute, and then mix with conventional blank matrix (1:49, v:v) to prepare hemolytic blank matrix.

[0095] Acceptance criteria: The response value of the interfering component in the blank matrix at the retention time of the analyte shall not exceed 20.0% of the response value of the analyte in the LLOQ sample prepared with the same individual blank matrix; the response value of the internal standard at the retention time shall not exceed 5.0% of the response value of the internal standard in the LLOQ sample prepared with the same individual blank matrix.

[0096] If a blank matrix sample from a particular source (batch) does not meet the acceptance criteria, the same method will be used to evaluate the interference of three additional blank matrices from different sources (batches) on the analytes and internal standards. If a high-fat matrix or hemolytic matrix does not meet the acceptance criteria, the same method will be used to evaluate one additional high-fat matrix or hemolytic matrix from a different source.

[0097] Interference of the analyte on the internal standard

[0098] Three samples containing only the analyte at the upper limit of quantitation without internal standard were prepared, processed and analyzed in parallel.

[0099] Acceptance criterion: The average peak area at the internal standard retention time of samples containing only a single analyte should not be greater than 5.0% of the average peak area of ​​the internal standard in samples with the lower limit of quantitation of the standard curve that meet the acceptance criterion in the same analytical batch.

[0100] Interference of internal standard on analytes

[0101] Three samples containing only a single internal standard without the analyte were prepared, processed, and analyzed in parallel. The internal standard concentration was the actual concentration used.

[0102] Acceptance criterion: The average peak area at the retention time of the analyte should not be greater than 20.0% of the average peak area of ​​the analyte in the samples with the lower limit of quantification of the standard curve that meet the acceptance criterion in the same analytical batch.

[0103] Selectivity of analytical batches

[0104] The selectivity of the analytical batch was evaluated by analyzing the first blank matrix sample and the first blank quality control sample of the analytical batch.

[0105] Acceptance criteria: The peak area of ​​the analyte channel detected in both blank samples does not exceed 20.0% of the average peak area of ​​the analyte in the samples with the lower limit of quantification of the valid standard curve; the peak area of ​​the internal standard channel detected in the first blank matrix sample does not exceed 5.0% of the average peak area of ​​the internal standard in the samples with the lower limit of quantification of the valid standard curve in the same analytical batch.

[0106] like Figure 4 As shown, Example 1 has excellent selectivity.

[0107] Durability

[0108] The samples were operated by different analysts (analyst A and analyst B) or injected on different instruments (LC-MS / MS, two sets of instruments of the same model with the same parameters). The precision was examined by calculating the coefficient of variation (%CV) of the quality control samples at each concentration level. The accuracy was examined by calculating the deviation (Diff%) between the measured mean concentration and the theoretical concentration of the quality control samples at each concentration level.

[0109] Acceptance criteria:

[0110] The deviation of the measured concentration mean of the quality control samples at each concentration level from their theoretical concentration should be within the range of ±15.0% (the deviation of LLOQ QC should be within the range of ±20.0%), and the coefficient of variation should not exceed 15.0% (the coefficient of variation of LLOQ QC should not exceed 20.0%).

[0111] For precision and accuracy analysis batches, the deviation between the measured concentration and the theoretical concentration of at least 2 / 3 of the quality control samples does not exceed ±15.0% (LLOQ QC does not exceed ±20.0%), and at least 1 / 2 of the samples at the same concentration level meet the above standards.

[0112] The mass spectrometer model is TripleQuad 5500+; the chromatography system includes a liquid phase pump: LC-30AD; a controller: CBM-20A; a degasser: DGU-20A5R(C); a column oven: CTO-20A; and a sample injection system: SIL-30ACMP.

[0113] Figure 5 This is the durability test result of Example 1, which meets the requirements.

[0114] 2. Analyze the liquid chromatograms of Example 1 and Comparative Examples 1-2. Figure 9 As shown, the precipitation signal of vonoprazan in the embodiment method is the highest, as shown in FIG. Figure 9-10 As shown, the vornorazan precipitation signals of Comparative Examples 1-2 were significantly reduced.

Claims

1. A method for determining the concentration of vonoprazan in human plasma by liquid chromatography-tandem mass spectrometry, characterized in that: The following steps are involved: A standard curve solution containing vonoprazan-d4 and a test solution containing vonoprazan were prepared using blank plasma as a matrix, and both solutions were precipitated using acetonitrile; and the concentration of the test solution containing vonoprazan after precipitation was determined using an internal standard method. The determination uses a liquid chromatography tandem mass spectrometer, the mobile phase of the liquid chromatography includes acetic acid aqueous solution and acetonitrile; the elution procedure of the liquid chromatography includes: the volume content of acetonitrile in the mobile phase is 60-90%, and the flow rate of the mobile phase is 0.8-1.5minmL / min.

2. The method for determining the concentration of vonoprazan in human plasma by liquid chromatography-tandem mass spectrometry according to claim 1, wherein: The volume content of acetonitrile in the mobile phase is 70-80%.

3. The method for determining the concentration of vonoprazan in human plasma by liquid chromatography-tandem mass spectrometry according to claim 2, wherein: The chromatographic column used in the liquid chromatography is an octadecyl bonded phase; the particle size of the bonded phase is 2.5-8 μm, and the pore size is 4. The method for determining the concentration of vonoprazan in human plasma by liquid chromatography-tandem mass spectrometry according to claim 3, wherein: The particle size of the bonded phase is 2.5-5 μm, and the pore size is 5. The method for determining the concentration of vonoprazan in human plasma by liquid chromatography tandem mass spectrometry according to claim 2 or 3, characterized in that: The elution time of the liquid chromatography is less than 2 minutes.

6. The method for determining the concentration of vonoprazan in human plasma by liquid chromatography tandem mass spectrometry according to claim 5, characterized in that: The acquisition time of the mass spectrum was 1.5 min.

7. The method for determining the concentration of vonoprazan in human plasma by liquid chromatography tandem mass spectrometry according to claim 1 or 6, characterized in that: The ionization mode of the mass spectrometer is: electrospray ion source, positive ion mode, multiple reaction monitoring.

8. The method for determining the concentration of vonoprazan in human plasma by liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The ion source parameters of the mass spectrometer include the following parameters: Collision gas 9.00 psi, curtain gas 20.00 psi, first ion source gas 30.00 psi, second ion source gas 60.00 psi, ion source spray voltage 5500.00 V, ion source temperature 500.00°C.

9. The method for determining the concentration of vonoprazan in human plasma by liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The reaction ion parameters of vonoprazan in the mass spectrometer are: monitoring ion pair 346.2 / 315.1, declustering voltage 50.00 V, entrance voltage 10.00 V, exit voltage 10.00 V, collision energy 16.00 eV, and dwell time 200.00 msec.

10. The method for determining the concentration of vonoprazan in human plasma by liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The reaction ion parameters of vonoprazan-d4 in the mass spectrometer are: monitoring ion pair 350.1 / 316.0, declustering voltage 50.00 V, entrance voltage 10.00 V, exit voltage 10.00 V, collision energy 17.00 eV, and dwell time 200.00 msec.

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