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

Abstract

The invention relates to the technical field of medicine analysis, in particular to a method for determining the concentration of tigliptin in human plasma through liquid chromatography-tandem mass spectrometry. Wherein a mobile phase of the liquid chromatography comprises an acetic acid aqueous solution and acetonitrile; at 0-0.8 min, the volume content of acetonitrile in the mobile phase is 15-40%; when the time is 0.8-1.2 min, the volume content of acetonitrile in the mobile phase is 80-95%; after 1.2 min, the volume content of acetonitrile in the mobile phase is 15-40%. By optimizing chromatographic conditions, mass spectrum parameters and other steps, the detection process is simplified, the detection efficiency is improved, the operation difficulty and cost are reduced, the efficient and accurate tigliptin drug concentration detection method is provided, the concentration of tigliptin 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 tenegliptin in human plasma by liquid chromatography-tandem mass spectrometry. Background Art

[0002] Tenegliptin is a drug widely used in the treatment of diabetes and belongs to the category of dipeptidyl peptidase-4 (DPP-4) inhibitors. It lowers blood sugar levels by inhibiting the activity of the DPP-4 enzyme, effectively regulates the secretion of insulin and glucagon, improves the utilization efficiency of insulin, and reduces the production of glucose by the liver. Blood drug concentration monitoring is of great significance in clinical drug treatment, and tenegliptin is no exception. By accurately monitoring the drug concentration of tenegliptin in the patient's plasma, doctors can timely understand the absorption, distribution, metabolism and excretion of the drug in the body, thereby evaluating the efficacy and safety of the drug, and adjust the medication regimen accordingly to ensure that the drug is within the effective therapeutic concentration range, while avoiding poor efficacy or adverse reactions caused by excessively high or low concentrations. Although there are some methods for the detection of tenegliptin drug concentration, these methods may have limitations such as insufficient sensitivity, low specificity, complex operation or high cost. These deficiencies may limit the accuracy and timeliness of the test results and fail to meet the needs of clinical and scientific research.

[0003] Chinese invention patent application CN114397380A discloses a method for determining the concentration of sitagliptin in plasma by liquid chromatography-mass spectrometry, wherein sitagliptin-d4 is used as an internal standard, a precipitant is added to precipitate protein, a diluent is added to the supernatant, and after pretreatment, the mixture is separated by a chromatographic column and detected by a mass spectrometer. The detection speed is fast, the accuracy is high, and the sensitivity is excellent. Although sitagliptin and tenegliptin are both dipeptidyl peptidase-4 (DPP-4) inhibitors used to treat type 2 diabetes, there are differences in their chemical structures, physicochemical properties (such as polarity, logP, pKa, etc.) and metabolic characteristics, which directly affect the accuracy and selectivity of liquid chromatography-mass spectrometry. Summary of the invention

[0004] The present invention provides a method for determining the concentration of tenegliptin in human plasma by liquid chromatography-tandem mass spectrometry, comprising the following steps: The tenegliptin standard and methanol were used to prepare standard curve solutions with different tenegliptin concentration gradients; the tenegliptin standard, methanol and blank matrix were used to prepare quality control solutions with different tenegliptin concentration gradients; the tenegliptin-d4 standard and methanol were used to prepare internal standard solutions; the plasma to be tested was used to dilute the quality control solution to obtain the test solution; The standard curve solution, the quality control solution and the test solution were respectively mixed with the internal standard solution to obtain the standard curve sample, the quality control sample and the test sample; the blank matrix was mixed with methanol to obtain the blank sample; the blank matrix was mixed with the internal standard solution to obtain the zero concentration sample; the quality control solution with the highest tenegliptin concentration was mixed with methanol to obtain the quantitative upper limit without internal standard sample; All the above samples were diluted with methanol, mixed evenly and centrifuged, and the supernatants were added with purified water and detected by liquid chromatography-tandem mass spectrometry.

[0005] The mobile phase of the liquid chromatography comprises aqueous acetic acid and acetonitrile; The chromatographic column stationary phase used in the liquid chromatography comprises spherical silica gel; Optionally, the chromatographic column model is InertSustain AQ-C18.

[0006] The present invention has found that the chromatographic column stationary phase used in liquid chromatography includes spherical silica gel; the detection efficiency can be improved and the detection time can be shortened. The spherical silica gel has high purity and strong surface inertness, which can significantly reduce the active interference of silanol groups, cooperate with the bonded octadecyl groups, and is achieved through isometric bonding technology to avoid the "hydrophobic collapse" phenomenon under high water phase conditions.

[0007] Further research found that the selection of the InertSustain AQ-C18 column can further shorten the detection time to within 2 minutes. The end-capping technology is used to further passivate the residual silanol groups. Combined with the specific carbon loading, specific surface area and pore size of the chromatographic column, it effectively matches the polarity of tenegliptin, improves the peak symmetry, and increases the retention time of tenegliptin.

[0008] The mobile phases of liquid chromatography are acetic acid aqueous solution and acetonitrile, which can further improve the detection precision and accuracy on the basis of shortening the detection time. Acetonitrile, as an organic phase, has lower viscosity and higher elution capacity, which can shorten the analysis time and improve the peak shape. Acetic acid can adjust the pH value of the mobile phase, which is helpful for the protonation or deprotonation of tilagliptin, thereby optimizing its retention behavior on the chromatographic column. At the same time, the combination of the two can reduce the matrix effect and reduce the interference of the matrix effect on the ionization of the target, thereby improving the stability of the mass spectrometry signal.

[0009] The volume ratio of the standard curve solution to the internal standard solution in the standard curve sample is (1-3):1.

[0010] The volume ratio of the quality control solution to the internal standard solution in the quality control sample is (1-3):1.

[0011] The volume ratio of the test solution to the internal standard solution in the test sample is (1-3):1.

[0012] The volume ratio of blank matrix to methanol in the blank sample is (1-3):1.

[0013] The volume ratio of the blank matrix to the internal standard solution in the zero-concentration sample is (1-3):1.

[0014] The volume ratio of the quality control solution with the highest concentration of tenegliptin in the quantitative upper limit sample without the addition of the internal standard to methanol is (1-3):1.

[0015] The standard curve sample is diluted with 2-3 volumes of methanol; the quality control sample is diluted with 2-3 volumes of methanol; the test sample is diluted with 2-3 volumes of methanol; the blank sample is diluted with 2-3 volumes of methanol; the zero concentration sample is diluted with 2-3 volumes of methanol; the quantitative upper limit sample without internal standard is diluted with 2-3 volumes of methanol.

[0016] The volume ratio of the supernatant to purified water is 1:(2-6).

[0017] Optionally, the mass concentration of the acetic acid aqueous solution is 0.01-1%.

[0018] The blank matrix includes blank human plasma or whole blood with K2EDTA as anticoagulant.

[0019] The elution procedure of the liquid chromatography includes: at 0-0.8min, the volume content of acetonitrile in the mobile phase is 15-40%; at 0.8-1.2min (excluding 0.8min, the same below), the volume content of acetonitrile in the mobile phase is 80-95%; after 1.2min, the volume content of acetonitrile in the mobile phase is 15-40%.

[0020] Optionally, the elution procedure of the liquid chromatography includes: at 0-0.8 min, the volume content of acetonitrile in the mobile phase is 20-30%; at 0.8-1.2 min, the volume content of acetonitrile in the mobile phase is 85-95%; after 1.2 min, the volume content of acetonitrile in the mobile phase is 20-30%.

[0021] The present invention has found that the detection selectivity can be improved by a specific elution procedure in combination with a specific mobile phase, which may be a specific segmented elution strategy: low acetonitrile content to remove polar interferences - high acetonitrile to quickly elute the target - low acetonitrile content to rebalance, and optimize matrix purification and target recovery in stages; at the same time, increasing the volume content of acetonitrile within 0.8 minutes can shorten the analysis time and reduce the risk of target degradation. At the same time, rapid elution can avoid the co-elution of hydrophobic impurities, and a specific concentration of acetic acid aqueous solution can be used to inhibit secondary interactions, which can effectively improve peak shape and separation.

[0022] The elution time of the liquid chromatography is no more than 2 minutes.

[0023] Optionally, the elution time of the liquid chromatography is no more than 1.5 min.

[0024] The mass spectrometry parameters include: the collision energy for monitoring tenegliptin is 39.5-40.5 eV, and the collision energy for monitoring tenegliptin-d4 is 38.5-39.5 eV.

[0025] The concentration range of tenegliptin in the standard curve solution is 4-400 ng / mL.

[0026] The number of concentration gradients of tenegliptin in the standard curve solution is 6-10.

[0027] The concentrations of tenegliptin in the standard curve solution are 4 ng / mL, 8 ng / mL, 20 ng / mL, 60 ng / mL, 120 ng / mL, 240 ng / mL, 320 ng / mL and 400 ng / mL, respectively.

[0028] The concentration of tenegliptin-d4 in the internal standard solution is 200-400 ng / mL.

[0029] Optionally, the concentration of tenegliptin-d4 in the internal standard solution is 250-350 ng / mL.

[0030] The concentration range of tenegliptin in the quality control solution is 4-300 ng / mL.

[0031] The concentrations of tenegliptin in the quality control solution were 4 ng / mL, 12 ng / mL, 40 ng / mL, 160 ng / mL and 300 ng / mL, respectively.

[0032] The concentration of tenegliptin in the test solution is 200-400 ng / mL.

[0033] Optionally, the concentration of tenegliptin in the test solution is 300-400 ng / mL.

[0034] Beneficial effects: 1. The stationary phase of the chromatographic column used in liquid chromatography includes spherical silica gel, which can improve detection efficiency and shorten detection time.

[0035] 2. Selecting the InertSustain AQ-C18 column can further shorten the detection time to within 2 minutes.

[0036] 3. Selecting acetic acid aqueous solution and acetonitrile as the mobile phase for liquid chromatography can further improve the detection precision and accuracy while shortening the detection time.

[0037] 4. Detection selectivity can be improved by using a specific elution procedure and a specific mobile phase.

[0038] 5. The detection method of the present invention meets the clinical requirements for precision and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 These are the conditions for liquid chromatography in Example 1.

[0040] Figure 2 The mass spectrometry conditions of Example 1 (Q1: quadrupole mass analyzer 1; Q3: quadrupole mass analyzer 3).

[0041] Figure 3 The results of precision, accuracy and recovery test of Example 1 are shown.

[0042] Figure 4 This is the selectivity test result of Example 1.

[0043] Figure 5 The durability test results of Example 1 are shown in FIG.

[0044] Figure 6 The results of precision, accuracy and recovery of comparative example 1 are shown.

[0045] Figure 7 This is the selectivity test result of Comparative Example 1.

[0046] Figure 8 The results of precision, accuracy and recovery of comparative example 2 are shown.

[0047] Fig. 9 This is the selectivity test result of Comparative Example 2.

[0048] Fig.10 Example 1 Scanning spectrum of tenegliptin daughter ions.

[0049] Fig.11 Example 1: Scanning spectrum of tenegliptin-d4 daughter ion.

[0050] Fig.12 Example 1 Standard curve The standard curve obtained from the sample. DETAILED DESCRIPTION

[0051] Example 1 A method for determining the concentration of tenegliptin in human plasma by liquid chromatography-tandem mass spectrometry comprises the following steps: The standard solution of tenegliptin with different concentration gradients of tenegliptin was prepared by using tenegliptin standard (tenegliptin hydrobromide, purchased from TLC, batch number: 3267-055A1) and methanol; the quality control solution of tenegliptin with different concentration gradients of tenegliptin was prepared by using tenegliptin standard, methanol and blank matrix; the internal standard solution of 300 ng / mL was prepared by using tenegliptin-d4 (purchased from TLC, batch number: 1795-061A3) standard and methanol; the quality control solution (quality control solution: 1600 ng / mL, dilution factor: 5) was diluted with the plasma to be tested (provided by Zibo Traditional Chinese Medicine Hospital) to obtain the test solution; In a 2.2 mL 96-well polypropylene plate, at room temperature and under white light conditions, 50 μL of standard curve solution, 50 μL of quality control solution and 50 μL of test solution were mixed with 25 μL of internal standard solution to obtain 75 μL of standard curve sample, 75 μL of quality control sample and 75 μL of test sample; 50 μL of blank matrix was mixed with 25 μL of methanol to obtain 75 μL of blank sample; 50 μL of blank matrix was mixed with 25 μL of internal standard solution to obtain zero concentration sample; 50 μL of quality control solution with the highest concentration of tenegliptin was mixed with 25 μL of methanol to obtain 75 μL of quantitative upper limit sample without internal standard; All the above samples were diluted with 200 μL methanol, mixed evenly and centrifuged at 4000 ppm for 5 min, 100 μL of the supernatant was taken and added to 300 μL purified water, and detected by liquid chromatography tandem mass spectrometry; The chromatographic column stationary phase used in the liquid chromatography is spherical silica gel; The concentrations of tenegliptin in the standard curve solution were 4 ng / mL (lower limit of quantification, LLOQ), 8 ng / mL, 20 ng / mL, 60 ng / mL, 120 ng / mL, 240 ng / mL, 320 ng / mL and 400 ng / mL, respectively.

[0052] The concentrations of tenegliptin in the quality control solutions are 4 ng / mL (lowest quality control concentration, LLOQ QC), 12 ng / mL (low quality control concentration, LQC), 40 ng / mL (medium quality control concentration, GMQC), 160 ng / mL (medium quality control concentration, MQC) and 300 ng / mL (high quality control concentration, HQC).

[0053] The blank matrix is ​​blank human plasma with K2EDTA as the anticoagulant provided by Zibo Traditional Chinese Medicine Hospital.

[0054] The chromatogram collection and chromatographic peak integration of the analyte and internal standard were completed by AB Sciex Analyst software (version 1.7.2). The standard curve was obtained by regression using the Watson LIMS system (version 7.6.1). The chromatographic response ratio of the analyte to the internal standard was used as the ordinate, and the weighted (W=1 / x2) least squares method was used to perform linear regression on the concentration of the analyte in plasma (x) and the response ratio (y). The obtained regression equation (y=ax+b) was the standard curve, and the drug concentration of the sample to be tested was calculated using the standard curve equation obtained by fitting. Fig.12 As shown, a = 0.00588; b = 0.00175; R 2 = 0.9974.

[0055] The conditions of the liquid chromatography are as follows Figure 1 The mass spectrometry conditions are as shown in Figure 2 As shown, the scanning spectrum of the daughter ion of tigliptin is as shown Fig.10 As shown, the ion scanning spectrum of tenegliptin-d4 is as follows Fig.11 shown.

[0056] Comparative Example 1 The specific implementation method is the same as that in Example 1; the difference is that the chromatographic column model in Comparative Example 1 is: Welch Ultimate XB-C18 (stationary phase: spherical silica gel); the mobile phase is 0.1wt% acetic acid aqueous solution and methanol; the elution procedure of the liquid chromatography includes: flow rate 0.5mL / min; at 0-1min, the volume content of methanol in the mobile phase is 30%; at 1-2.5min, the volume content of acetonitrile in the mobile phase is 70%; 2.5-4min, the volume content of acetonitrile in the mobile phase is 30%.

[0057] Comparative Example 2 The specific implementation method is the same as Example 1; the difference is that the chromatographic column model in Comparative Example 2 is: Hypurity C18 (stationary phase: silica gel); the mobile phase is 5mM ammonium formate aqueous solution and acetonitrile; the elution procedure of the liquid chromatography includes: 0-0.5min, acetonitrile in the fluidity is 70%, 0.5-2.0min, the volume content of acetonitrile in the fluidity gradually increases from 70% to 90%, 2.0-3.5min, the volume content of acetonitrile in the fluidity is 90%, 3.5-4.0min, the volume content of acetonitrile in the fluidity gradually decreases from 90% to 70%, 4.0-6.0min, the volume content of acetonitrile in the fluidity is 70%.

[0058] Performance Testing Methods The following tests were carried out in the embodiments and comparative examples: 1. Precision and accuracy Intra-batch precision and accuracy Quality control samples (LLOQ QC, LQC, GMQC, MQC and HQC) were used to evaluate the intra-batch precision and accuracy, and each concentration of quality control sample was repeated 6 times.

[0059] The precision was examined by calculating the coefficient of variation (%CV) of the quality control samples at each concentration level, and the accuracy was examined by calculating the deviation (Diff%) between the measured concentration mean and the theoretical concentration of the quality control samples at each concentration level.

[0060] Acceptance criteria: The deviation between the measured mean concentration of quality control samples at each concentration level and its 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%).

[0061] 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.

[0062] Batch-to-batch precision and accuracy analysis Inter-assay precision and accuracy were assessed by examining at least three independent validation analytical batches (within-assay precision and accuracy analytical batches, completed on at least two days) using freshly prepared quality control samples in blank matrix.

[0063] 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. Each quality control sample at each concentration level was repeated 6 times for each validation analysis batch.

[0064] Acceptance criteria: The deviation of the overall mean of the measured concentration of the quality control samples at each concentration level from its theoretical concentration should be within ±15.0% (the deviation of LLOQ QC should be within ±20.0%).

[0065] The overall coefficient of variation of the measured concentration of quality control samples at each concentration level did not exceed 15.0% (the coefficient of variation of LLOQ QC did not exceed 20.0%).

[0066] If an analytical batch did not meet the acceptance criteria, three additional accuracy and precision analytical batches were repeated for method validation.

[0067] like Figure 3 As shown, the precision and accuracy of Example 1 are relatively high. Figure 6 and Figure 8As shown, the precision and accuracy of Comparative Examples 1 and 2 are significantly reduced.

[0068] 2. Extraction recovery rate A blank matrix from the same batch (or source) as the conventional quality control sample (extracted sample, test sample) was used as a 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.

[0069] The test samples were routine quality control samples or samples with the same preparation process, including low-concentration quality control samples (LQC), medium-concentration quality control samples (MQC) and high-concentration quality control samples (HQC) (6 replicates for each concentration).

[0070] Calculation of extraction recovery rate of the analyte: The peak area of ​​the analyte in the conventional 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.

[0071] Calculation of internal standard extraction recovery: the internal standard peak area in each routine quality control sample (test sample) divided by the mean internal standard peak area in the reference sample.

[0072] 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%.

[0073] If the independent extraction recoveries of the analyte or internal standard do not meet the acceptance criteria, the extraction recovery of the analytical method can be evaluated by the internal standard-corrected extraction recovery.

[0074] Calculation of internal standard correction extraction recovery: the peak area ratio of the analyte to its internal standard in the conventional quality control sample (test sample) at each concentration divided by the mean of the peak area ratio of the analyte to its internal standard in the reference sample of the same concentration.

[0075] Acceptance criteria for internal standard-corrected extraction recovery: The overall coefficient of variation of internal standard-corrected extraction recovery should not exceed 15.0%.

[0076] like Figure 3 As shown, the extraction recovery rate of Example 1 is higher, as shown in Figure 6 and Figure 8 As shown, the extraction recovery rates of Comparative Example 1 and Comparative Example 2 were significantly reduced.

[0077] Selectivity Matrix selectivity (endogenous interference) The matrix selectivity was evaluated by examining at least 6 blank biological matrices from different sources, 1 high-fat matrix from an individual source, and 1 hemolytic matrix from an individual source, and performing blank sample measurements without internal standard and LLOQ level samples, respectively.

[0078] 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).

[0079] Hemolysis matrix: Freeze whole blood at -80℃ for at least 30 minutes, vortex for at least 1 minute after thawing, and mix with conventional blank matrix (1:49, v:v) to prepare hemolysis blank matrix.

[0080] 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 at the retention time of the internal standard shall not exceed 5.0% of the response value of the internal standard in the LLOQ sample prepared with the same individual blank matrix.

[0081] If the blank matrix sample from a certain source (batch) does not meet the acceptance criteria, the same method will be used to evaluate the interference of 3 additional blank matrices from different sources (batches) on the analyte and internal standard. If the high-fat matrix or hemolytic matrix does not meet the acceptance criteria, the same method will be used to evaluate the interference of 1 additional high-fat matrix or hemolytic matrix from different sources.

[0082] Interference of the analyte on the internal standard Three samples containing only a single analyte and no internal standard at the upper limit of quantitation were prepared, processed and analyzed in parallel.

[0083] 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 the quantitative lower limit samples of the standard curve that meet the acceptance criterion in the same analytical batch.

[0084] Interference of internal standard on analytes Three samples containing only a single internal standard without the analyte were prepared, processed and analyzed in parallel, and the internal standard concentration was the actual concentration used.

[0085] Acceptance criteria: 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 quantitative lower limit samples of the standard curve that meet the acceptance criteria in the same analytical batch.

[0086] Selectivity of analytical batches The selectivity of the analytical batch was evaluated by analyzing the first blank matrix sample and the first blank QC sample of the analytical batch.

[0087] Acceptance criteria: The peak areas of the analyte channels of the two blank samples do 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 of 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.

[0088] like Figure 4 As shown, Example 1 has a higher selectivity, as Figure 7 and Fig. 9 As shown, the selectivity of Comparative Example 1 and Comparative Example 2 is significantly reduced.

[0089] Durability The samples were operated by different analysts (analyst A and analyst B) or injected on different instruments (LC-MS / MS, two 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, and 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.

[0090] Acceptance criteria: The deviation between the measured mean concentration of quality control samples at each concentration level and its 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%).

[0091] 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.

[0092] The mass spectrometer model is TripleQuad 5500+; the chromatography system, liquid phase pump is LC-30AD; the controller is CBM-20A; the degasser is DGU-20A5R(C); the column oven is CTO-20A; and the injection system is SIL-30ACMP.

[0093] Figure 5 This is the durability test result of Example 1, which meets the requirements.

Claims

1. A method for determining the concentration of tenegliptin in human plasma by liquid chromatography-tandem mass spectrometry, characterized in that: The following steps are involved: Prepare standard curve samples, quality control samples, test samples, blank samples, zero concentration samples and samples with no internal standard added to the upper limit of quantification; and use liquid chromatography tandem mass spectrometry to detect the standard curve samples, quality control samples, test samples, blank samples, zero concentration samples and samples with no internal standard added to the upper limit of quantification; The mobile phase of the liquid chromatography comprises aqueous acetic acid and acetonitrile; The chromatographic column stationary phase used in the liquid chromatography comprises spherical silica gel; The elution procedure of the liquid chromatography includes: at 0-0.8min, the volume content of acetonitrile in the mobile phase is 15-40%; At 0.8-1.2 min, the volume content of acetonitrile in the mobile phase was 80-95%; after 1.2 min, the volume content of acetonitrile in the mobile phase was 15-40%.

2. The method for determining the concentration of tenegliptin in human plasma by liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The elution time of the liquid chromatography is no more than 2 minutes.

3. The method for determining the concentration of tenegliptin in human plasma by liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The mass spectrometry parameters include: the collision energy for monitoring tenegliptin is 39.5-40.5 eV, and the collision energy for monitoring tenegliptin-d4 is 38.5-39.5 eV.

4. The method for determining the concentration of tenegliptin in human plasma by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The standard curve sample is prepared from a standard curve solution, and the concentration range of tenegliptin in the standard curve solution is 4-400 ng / mL.

5. The method for determining the concentration of tenegliptin in human plasma by liquid chromatography tandem mass spectrometry according to claim 4, characterized in that: The number of concentration gradients of tenegliptin in the standard curve solution is 6-10.

6. The method for determining the concentration of tenegliptin in human plasma by liquid chromatography tandem mass spectrometry according to claim 5, characterized in that: The concentrations of tenegliptin in the standard curve solution are 4 ng / mL, 8 ng / mL, 20 ng / mL, 60 ng / mL, 120 ng / mL, 240 ng / mL, 320 ng / mL and 400 ng / mL, respectively.

7. The method for determining the concentration of tenegliptin in human plasma by liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The zero concentration sample is prepared from an internal standard solution, and the concentration of tenegliptin-d4 in the internal standard solution is 200-400 ng / mL.

8. The method for determining the concentration of tenegliptin in human plasma by liquid chromatography-tandem mass spectrometry according to claim 1, characterized in that: The quality control sample is prepared from a quality control solution, and the concentration range of tenegliptin in the quality control solution is 4-300 ng / mL.

9. The method for determining the concentration of tenegliptin in human plasma by liquid chromatography tandem mass spectrometry according to claim 8, characterized in that: The concentrations of tenegliptin in the quality control solution were 4 ng / mL, 12 ng / mL, 40 ng / mL, 160 ng / mL and 300 ng / mL, respectively.

10. The method for determining the concentration of tenegliptin in human plasma by liquid chromatography tandem mass spectrometry according to claim 1, characterized in that: The test sample is prepared from a test solution, and the concentration of tenegliptin in the test solution is 200-400 ng / mL.

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