Method for Determining the Concentration of Teneligliptin in Human Plasma by Liquid Chromatography-Tandem Mass Spectrometry

Abstract

The present invention relates to the technical field of pharmaceutical analysis. Specifically, it relates to a method for determining the concentration of teneligliptin in human plasma by liquid chromatography-tandem mass spectrometry. The mobile phase of the liquid chromatography includes an aqueous acetic acid solution and acetonitrile; at 0-0.8 min, 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 is 80-95%; after 1.2 min, the volume content of acetonitrile in the mobile phase is 15-40%. By optimizing steps such as chromatographic conditions and mass spectrometry parameters, the detection process is simplified, the detection efficiency is improved, the operation difficulty and cost are reduced, and an efficient and accurate method for detecting the concentration of teneligliptin drug is provided, which can accurately determine the concentration of teneligliptin in plasma and ensure the accuracy and reliability of the data.

Description

Technical Field

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

[0002] Teneligliptin is a drug widely used in the field of diabetes treatment and belongs to the class of dipeptidyl peptidase-4 (DPP-4) inhibitors. It reduces blood glucose 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. Monitoring of blood drug concentration is of great significance in clinical drug treatment, and teneligliptin is no exception. By accurately monitoring the drug concentration of teneligliptin 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 adjusting the medication plan accordingly to ensure that the drug is within the effective therapeutic concentration range, while avoiding poor efficacy or adverse reactions caused by too high or too low concentrations. Although there are currently some methods for detecting the drug concentration of teneligliptin, 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 detection results and cannot meet the needs of clinical and scientific research.

[0003] Chinese Patent Application CN114397380A discloses a method for determining the concentration of sitagliptin in plasma by liquid chromatography-mass spectrometry. Using sitagliptin-d4 as the internal standard, a precipitant is added for protein precipitation. After taking the supernatant and adding a diluent, and after pretreatment, it is separated by a chromatographic column and detected with a mass spectrometer detector. The detection speed is fast, the accuracy is high, and the sensitivity is excellent. Although both sitagliptin and teneligliptin belong to the class of dipeptidyl peptidase-4 (DPP-4) inhibitor drugs for the treatment of type 2 diabetes, there are differences in their chemical structures, physical and chemical properties (such as polarity, logP, pKa, etc.), and metabolic characteristics. These differences 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 teneligliptin in human plasma by liquid chromatography-tandem mass spectrometry, comprising the following steps:

[0005] Take teneligliptin standard and methanol to prepare standard curve solutions with different teneligliptin concentration gradients; take teneligliptin standard, methanol, and blank matrix to prepare quality control solutions with different teneligliptin concentration gradients; take teneligliptin-d4 standard and methanol to prepare internal standard solutions; take the plasma to be tested to dilute the quality control solutions to obtain test solutions;

[0006] Mix the standard curve solution, quality control solution, and test solution with the internal standard solution respectively to obtain standard curve samples, quality control samples, and test samples; mix blank matrix with methanol to obtain blank samples; mix blank matrix with the internal standard solution to obtain zero-concentration samples; mix the quality control solution with the highest concentration of trelagliptin with methanol to obtain upper limit of quantification samples without internal standard.

[0007] Dilute all the above samples with methanol respectively, mix evenly and centrifuge, take the supernatant and add purified water, and detect by liquid chromatography-tandem mass spectrometry.

[0008] The mobile phase of the liquid chromatography includes aqueous acetic acid solution and acetonitrile.

[0009] The stationary phase of the chromatographic column used in the liquid chromatography includes spherical silica gel.

[0010] Optionally, the model of the chromatographic column is InertSustain AQ-C18.

[0011] The research of the present invention finds that the stationary phase of the chromatographic column used in liquid chromatography includes spherical silica gel, which can improve the detection efficiency and shorten the detection time. The spherical silica gel has high purity and strong surface inertness, significantly reducing the active interference of silanol groups, and cooperating with the bonded octadecyl groups achieved by the equidistant bonding technology to avoid the "hydrophobic collapse" phenomenon under high aqueous phase conditions.

[0012] Further research finds that choosing a chromatographic column with the model of InertSustain AQ-C18 can further shorten the detection time to within 2 minutes. It adopts end-capping technology to further passivate the residual silanols, and cooperates with the specific carbon loading, specific surface area, and pore size of this chromatographic column to effectively match the polarity of trelagliptin, improve the peak shape symmetry, and increase the retention time of trelagliptin.

[0013] Selecting aqueous acetic acid solution and acetonitrile as the mobile phase of liquid chromatography can further improve the detection precision and accuracy on the basis of shortening the detection time. Acetonitrile, as the organic phase, has low viscosity and high elution ability, which can shorten the analysis time and improve the peak shape. Acetic acid can adjust the pH value of the mobile phase, which helps the protonation or deprotonation of trelagliptin, thereby optimizing its retention behavior on the chromatographic column. At the same time, the combination of the two can reduce the matrix effect and the interference of the matrix effect on the ionization of the target substance, thus improving the stability of the mass spectrometry signal.

[0014] The volume ratio of the standard curve solution to the internal standard solution in the standard curve sample is (1 - 3):1.

[0015] The volume ratio of the quality control solution to the internal standard solution in the quality control sample is (1 - 3):1.

[0016] The volume ratio of the test solution to the internal standard solution in the test sample is (1 - 3):1.

[0017] The volume ratio of the blank matrix to methanol in the blank sample is (1 - 3):1.

[0018] The volume ratio of the blank matrix to the internal standard solution in the zero - concentration sample is (1 - 3):1.

[0019] The volume ratio of the quality control solution with the highest concentration of trelagliptin in the sample without internal standard at the upper limit of quantification to methanol is (1 - 3):1.

[0020] The standard curve samples are diluted with 2 - 3 volumes of methanol; the quality control samples are diluted with 2 - 3 volumes of methanol; the test samples are diluted with 2 - 3 volumes of methanol; the blank samples are diluted with 2 - 3 volumes of methanol; the zero - concentration samples are diluted with 2 - 3 volumes of methanol; the samples without internal standard at the upper limit of quantification are diluted with 2 - 3 volumes of methanol.

[0021] The volume ratio of the supernatant to purified water is 1:(2 - 6).

[0022] Optionally, the mass concentration of the aqueous acetic acid solution is 0.01 - 1%.

[0023] The blank matrix includes blank human plasma or whole blood with K2EDTA as an anticoagulant.

[0024] The elution program of the liquid chromatography includes: from 0 - 0.8 min, the volume content of acetonitrile in the mobile phase is 15 - 40%; from 0.8 - 1.2 min (excluding 0.8 min, the same below), 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%.

[0025] Optionally, the elution program of the liquid chromatography includes: from 0 - 0.8 min, the volume content of acetonitrile in the mobile phase is 20 - 30%; from 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%.

[0026] The present invention has found through research that by means of a specific elution procedure in combination with a specific mobile phase, the detection selectivity can be improved. It may be a specific segmented elution strategy: a low acetonitrile content to remove polar interfering substances - a high acetonitrile content to rapidly elute the target substances - a low acetonitrile content for rebalancing, optimizing matrix purification and target recovery in stages; at the same time, increasing the volume content of acetonitrile within 0.8 min can shorten the analysis time, reduce the risk of target degradation, and avoid the co-elution of hydrophobic impurities through rapid elution. In combination with an acetic acid aqueous solution at a specific concentration to inhibit secondary interactions, the peak shape and resolution can be effectively improved.

[0027] The elution time of the liquid chromatography is not more than 2 min.

[0028] Optionally, the elution time of the liquid chromatography is not more than 1.5 min.

[0029] The parameters of the mass spectrometry include: the collision energy for monitoring trelagliptin is 39.5 - 40.5 eV, and the collision energy for monitoring trelagliptin-d4 is 38.5 - 39.5 eV.

[0030] The concentration range of trelagliptin in the standard curve solution is 4 - 400 ng / mL.

[0031] The number of concentration gradients of trelagliptin in the standard curve solution is 6 - 10.

[0032] The concentrations of trelagliptin 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.

[0033] The concentration of trelagliptin-d4 in the internal standard solution is 200 - 400 ng / mL.

[0034] Optionally, the concentration of trelagliptin-d4 in the internal standard solution is 250 - 350 ng / mL.

[0035] The concentration range of trelagliptin in the quality control solution is 4 - 300 ng / mL.

[0036] The concentrations of trelagliptin in the quality control solution are 4 ng / mL, 12 ng / mL, 40 ng / mL, 160 ng / mL, and 300 ng / mL respectively.

[0037] The concentration of trelagliptin in the test solution is 200 - 400 ng / mL.

[0038] Optionally, the concentration of trelagliptin in the test solution is 300 - 400 ng / mL.

[0039] Beneficial effects:

[0040] 1. The stationary phase of the chromatographic column used in liquid chromatography includes spherical silica gel, which can improve the detection efficiency and shorten the detection time.

[0041] 2. Selecting a chromatographic column with the model InertSustain AQ-C18 can further shorten the detection time to within 2 minutes.

[0042] 3. Selecting acetic acid aqueous solution and acetonitrile as the mobile phase of liquid chromatography can further improve the detection precision and accuracy on the basis of shortening the detection time.

[0043] 4. By a specific elution program, cooperating with a specific mobile phase can improve the detection selectivity.

[0044] 5. The detection method of the present invention meets the clinical requirements for precision and accuracy. Description of the Drawings

[0045] Figure 1 It is the conditions of liquid chromatography for Example 1.

[0046] Figure 2 It is the conditions of mass spectrometry for Example 1 (Q1: quadrupole mass analyzer 1; Q3: quadrupole mass analyzer 3).

[0047] Figure 3 It is the test results of precision, accuracy and recovery rate for Example 1.

[0048] Figure 4 It is the test results of selectivity for Example 1.

[0049] Figure 5 It is the test results of durability for Example 1.

[0050] Figure 6 It is the test results of precision, accuracy and recovery rate for Comparative Example 1.

[0051] Figure 7 It is the test results of selectivity for Comparative Example 1.

[0052] Figure 8 It is the test results of precision, accuracy and recovery rate for Comparative Example 2.

[0053] Figure 9 It is the test results of selectivity for Comparative Example 2.

[0054] Figure 10 Product ion scan spectrum of tigliptin in Example 1.

[0055] Figure 11 Product ion scan spectrum of tigliptin-d4 in Example 1.

[0056] Figure 12Standard curve obtained from the standard curve samples in Example 1. Detailed implementation

[0057] Example 1

[0058] A method for determining the concentration of teneligliptin in human plasma by liquid chromatography - tandem mass spectrometry, which comprises the following steps:

[0059] Take teneligliptin standard (teneligliptin hydrobromide, purchased from TLC, batch number: 3267 - 055A1) and methanol to prepare standard curve solutions with different teneligliptin concentration gradients; take teneligliptin standard, methanol and blank matrix to prepare quality control solutions with different teneligliptin concentration gradients; take teneligliptin - d4 (purchased from TLC, batch number: 1795 - 061A3) standard and methanol to prepare an internal standard solution of 300 ng / mL; take the plasma to be tested (provided by Zibo Traditional Chinese Medicine Hospital) and dilute the quality control solution (quality control solution: 1600 ng / mL, dilution factor: 5) to obtain the test solution;

[0060] In a 2.2 mL 96 - well polypropylene plate, at room temperature and under white light conditions, mix 50 μL of the standard curve solution, 50 μL of the quality control solution and 50 μL of the test solution with 25 μL of the internal standard solution respectively to obtain 75 μL of standard curve samples, 75 μL of quality control samples and 75 μL of test samples; mix 50 μL of the blank matrix with 25 μL of methanol to obtain 75 μL of blank samples; mix 50 μL of the blank matrix with 25 μL of the internal standard solution to obtain zero - concentration samples; mix 50 μL of the quality control solution with the highest teneligliptin concentration with 25 μL of methanol to obtain 75 μL of upper - limit - of - quantification samples without internal standard;

[0061] Dilute all the above samples with 200 μL of methanol respectively, mix evenly and centrifuge at 4000 ppm for 5 min, then take 100 μL of the supernatant and add 300 μL of purified water, and perform detection by liquid chromatography - tandem mass spectrometry;

[0062] The stationary phase of the chromatographic column used in the liquid chromatography is spherical silica gel;

[0063] The concentrations of teneligliptin in the standard curve solutions are 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.

[0064] The concentrations of teneligliptin in the quality control solutions are 4 ng / mL (lower limit of 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) respectively.

[0065] The blank matrix is blank human plasma with K2EDTA provided by Zibo Hospital of Traditional Chinese Medicine as the anticoagulant.

[0066] The collection of chromatograms of the analyte and internal standard and the integration of chromatographic peaks 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). With the chromatographic response ratio of the analyte to the internal standard as the ordinate, weighted (W = 1 / x2) least squares method was used to perform linear regression on the concentration (x) of the analyte in plasma and the response ratio (y). The resulting regression equation (y = ax + b) was the standard curve, and the drug concentration of the sample to be tested was calculated from the fitted standard curve equation. As Figure 12 shown, a = 0.00588; b = 0.00175; R 2 = 0.9974.

[0067] The conditions of the liquid chromatography are as Figure 1 shown, and the mass spectrometry conditions are as Figure 2 shown, among which the product ion scan spectrum of teneligliptin is as Figure 10 shown, and the product ion scan spectrum of teneligliptin-d4 is as Figure 11 shown.

[0068] Comparative Example 1

[0069] The specific implementation method is the same as that of Example 1; the difference is that in Comparative Example 1, the chromatographic column model is: Welch Ultimate XB-C18 (stationary phase: spherical silica gel); the mobile phase is 0.1 wt% acetic acid aqueous solution and methanol; the elution program of the liquid chromatography includes: flow rate 0.5 mL / min; from 0 to 1 min, the volume content of methanol in the mobile phase is 30%; from 1 to 2.5 min, the volume content of acetonitrile in the mobile phase is 70%; from 2.5 to 4 min, the volume content of acetonitrile in the mobile phase is 30%.

[0070] Comparative Example 2

[0071] The specific implementation method is the same as that of Example 1; the difference is that in Comparative Example 2, the chromatographic column model is: Hypurity C18 (stationary phase: silica gel); the mobile phase is 5 mM ammonium formate aqueous solution and acetonitrile; the elution program of the liquid chromatography includes: from 0 to 0.5 min, acetonitrile in the mobile phase is 70%, from 0.5 to 2.0 min, the volume content of acetonitrile in the mobile phase gradually increases from 70% to 90%, from 2.0 to 3.5 min, the volume content of acetonitrile in the mobile phase is 90%, from 3.5 to 4.0 min, the volume content of acetonitrile in the mobile phase gradually decreases from 90% to 70%, from 4.0 to 6.0 min, the volume content of acetonitrile in the mobile phase is 70%.

[0072] Performance test method

[0073] The following tests were carried out in the examples and comparative examples:

[0074] 1. Precision and accuracy

[0075] Intra-batch precision and accuracy

[0076] Quality control samples (LLOQ QC, LQC, GMQC, MQC and HQC) were used to evaluate intra-batch precision and accuracy, with 6 replicates for each concentration level of the quality control samples.

[0077] Precision was investigated by calculating the coefficient of variation (%CV) of the quality control samples at each concentration level, and accuracy was investigated by calculating the deviation (Diff%) between the mean measured concentration and the theoretical concentration of the quality control samples at each concentration level.

[0078] Acceptance criteria:

[0079] The deviation between the mean measured concentration and the theoretical concentration of the quality control samples at each concentration level should be within ±15.0% (the deviation of LLOQ QC is within ±20.0%), and the coefficient of variation does not exceed 15.0% (the coefficient of variation of LLOQ QC does not exceed 20.0%).

[0080] For the precision and accuracy analysis batches, the deviation of at least 2 / 3 of the quality control samples from their theoretical concentrations 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 criteria.

[0081] Inter-batch precision and accuracy analysis batches

[0082] Inter-batch precision and accuracy were evaluated by examining at least 3 independent validation analysis batches (intra-batch precision and accuracy analysis batches, completed in at least two days) within at least two days, using quality control samples freshly prepared in blank matrix.

[0083] The quality control samples for calculating inter-batch precision and accuracy were from the quality control samples prepared for investigating intra-batch precision and accuracy (LLOQ QC, LQC, GMQC, MQC and HQC), with 6 replicates for each concentration level of the quality control samples in each validation analysis batch.

[0084] Acceptance criteria:

[0085] The deviation between the overall mean measured concentration and the theoretical concentration of the quality control samples at each concentration level should be within ±15.0% (the deviation of LLOQ QC is within ±20.0%).

[0086] The overall coefficient of variation of the measured concentrations of the quality control samples at each concentration level does not exceed 15.0% (the coefficient of variation of the LLOQ QC does not exceed 20.0%).

[0087] If an analytical run does not meet the acceptance criteria, repeat the testing of an additional 3 analytical runs for accuracy and precision for method validation.

[0088] As Figure 3 shown, Example 1 has higher precision and accuracy. As Figure 6 and Figure 8 shown, the precision and accuracy of Comparative Example 1 and Comparative Example 2 are significantly reduced.

[0089] 2. Extraction recovery

[0090] Use the blank matrix of the same batch (or source) as the conventional quality control samples (extracted samples, test samples) as the blank sample. After extraction, add the analyte and internal standard to the extract of the blank sample to prepare low-concentration quality control samples (LQC), medium-concentration quality control samples (MQC), and high-concentration quality control samples (HQC), with 6 replicates for each concentration, as reference samples.

[0091] The test samples are conventional quality control samples or samples prepared in the same process as them, 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).

[0092] Calculation of analyte extraction recovery: The peak area of the analyte in the conventional quality control samples (test samples) at each concentration is divided by the average peak area of the analyte in the reference samples at the same concentration.

[0093] Calculation of internal standard extraction recovery: The peak area of the internal standard in each conventional quality control sample (test sample) is divided by the average peak area of the internal standard in the reference samples.

[0094] Acceptance criteria: The overall coefficient of variation of analyte extraction does not exceed 15.0%; the coefficient of variation of internal standard extraction recovery does not exceed 15.0%.

[0095] If the independent extraction recovery of the analyte or internal standard does not meet this acceptance criteria, the extraction recovery of this analytical method can be evaluated by the extraction recovery corrected by the internal standard.

[0096] Calculation of extraction recovery corrected by internal standard: The ratio of the peak area of the analyte to its internal standard in the conventional quality control samples (test samples) at each concentration is divided by the average ratio of the peak area of the analyte to its internal standard in the reference samples at the same concentration.

[0097] Acceptance criteria for extraction recovery corrected by internal standard: The overall coefficient of variation of extraction recovery corrected by internal standard does not exceed 15.0%.

[0098] AsFigure 3 As shown, the extraction recovery rate of Example 1 is relatively high, such as Figure 6 and Figure 8 As shown, the extraction recovery rates of Comparative Example 1 and Comparative Example 2 are significantly reduced.

[0099] Selectivity

[0100] Matrix selectivity (endogenous interference)

[0101] Matrix selectivity was evaluated by examining blank biological matrices from at least 6 different individual sources, a high-fat matrix from 1 individual source, and a hemolyzed matrix from 1 individual source, and performing determinations on blank samples without internal standard and samples at the LLOQ level, respectively.

[0102] High-fat matrix: Prepared by adding commercially available or simulated hyperlipidemic plasma (1 ± 0.1 mg of low-density lipoprotein (LDL) and 3 ± 0.3 mg of triglyceride to 1 mL of blank plasma).

[0103] Hemolyzed matrix: Whole blood was frozen at -80 °C for at least 30 minutes, thawed and vortexed for at least 1 minute, and then mixed with a conventional blank matrix (1:49, v:v) to prepare a hemolyzed blank matrix.

[0104] Acceptance criteria: The response value of interfering components 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 from the same individual's 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 from the same individual's blank matrix.

[0105] If the blank matrix samples from a certain source (batch) do not meet this acceptance criteria, the interference of the analyte and the internal standard by an additional 3 different sources (batches) of blank matrices will be evaluated using the same method. If the high-fat matrix or the hemolyzed matrix does not meet this acceptance criteria, the interference of an additional 1 different source of high-fat matrix or hemolyzed matrix will be evaluated using the same method.

[0106] Interference of the analyte on the internal standard

[0107] Three samples at the upper limit of quantification containing only a single analyte and without internal standard were prepared, processed, and analyzed in parallel.

[0108] Acceptance criteria: The average peak area at the retention time of the internal standard in the sample containing only a single analyte should not be greater than 5.0% of the average peak area of the internal standard in the lower limit of quantification sample of the standard curve that meets the acceptance criteria in the same analytical batch.

[0109] Interference of the internal standard on the analyte

[0110] Three samples containing only a single internal standard and without analyte were prepared, processed, and analyzed in parallel, with the internal standard concentration being the actual concentration used.

[0111] 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 sample at the lower limit of quantitation of the calibration curve that meets the acceptance criteria in the same analytical batch.

[0112] Selectivity of the analytical batch

[0113] The selectivity of the analytical batch is evaluated by analyzing the first blank matrix sample and the first blank quality control sample of the analytical batch.

[0114] Acceptance criteria: The peak areas of the analytes detected in the analyte channels of the two blank samples should not exceed 20.0% of the average peak area of the analyte in the sample at the lower limit of quantitation of the valid calibration curve; the peak area of the analyte detected in the internal standard channel of the first blank matrix sample should not exceed 5.0% of the average peak area of the internal standard in the sample at the lower limit of quantitation of the valid calibration curve in the same analytical batch.

[0115] As Figure 4 shown, Example 1 has higher selectivity. As Figure 7 and Figure 9 shown, the selectivities of Comparative Example 1 and Comparative Example 2 are significantly reduced.

[0116] Robustness

[0117] Performed by different analysts (Analyst A and Analyst B) or injected on different instruments (LC-MS / MS, two sets of the same model instruments with the same parameters), the precision is investigated by calculating the coefficient of variation (%CV) of the quality control samples at each concentration level, and the accuracy is investigated by calculating the deviation (Diff%) between the mean measured concentration and the theoretical concentration of the quality control samples at each concentration level.

[0118] Acceptance criteria:

[0119] The deviation between the mean measured concentration and the theoretical concentration of the quality control samples at each concentration level should be within the range of ±15.0% (the deviation of LLOQ QC is within the range of ±20.0%), and the coefficient of variation does not exceed 15.0% (the coefficient of variation of LLOQ QC does not exceed 20.0%).

[0120] For the precision and accuracy analytical batches, the deviation of at least 2 / 3 of the quality control samples from their theoretical concentrations 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 criteria.

[0121] Among them, the mass spectrometry model: TripleQuad 5500+; chromatographic system, liquid phase pump: LC-30AD; controller: CBM-20A; degasser: DGU-20A5R(C); column oven: CTO-20A; injection system: SIL-30ACMP.

[0122] Figure 5 For the durability test results of Example 1, they meet the requirements.

Claims

1. A method for determining the concentration of teneligliptin in human plasma by liquid chromatography-tandem mass spectrometry, characterized in that, It includes the following steps: Prepare standard curve samples, quality control samples, test samples, blank samples, zero-concentration samples, and upper limit of quantification samples without internal standard; detect the standard curve samples, quality control samples, test samples, blank samples, zero-concentration samples, and upper limit of quantification samples without internal standard by liquid chromatography-tandem mass spectrometry; The mobile phase of the liquid chromatography: 0.1 wt% acetic acid aqueous solution and acetonitrile; The stationary phase of the chromatographic column used in the liquid chromatography includes spherical silica gel; the model of the chromatographic column is InertSustain AQ-C18; The elution program of the liquid chromatography: at 0 min, the volume content of acetonitrile in the mobile phase is 25%; at 0.3 min, the volume content of acetonitrile in the mobile phase is 25%; at 0.8 min, the volume content of acetonitrile in the mobile phase is 90%; at 1.2 min, the volume content of acetonitrile in the mobile phase is 90%; at 1.21 min, the volume content of acetonitrile in the mobile phase is 25%; at 1.5 min, the volume content of acetonitrile in the mobile phase is 25%; The flow rate of the mobile phase from 0 to 1.5 min is 0.5 mL / min; The mass spectrometry parameters are as follows: Ion source parameters, collision gas: 7.00 psi; Curtain gas: 25.00 psi; Ion source gas 1: 50.00 psi; Ion source gas 2: 50.00 psi; Ion source spray voltage: 5000.00 V; Ion source temperature: 550.00 °C; Resolution Q1 / Q3: Unit / Unit; Pause time: 5.0070 msec; Mass spectrometry acquisition time: 1.50 min; Ionization mode: electrospray ionization source, positive ion mode, multiple reaction monitoring; Teneligliptin: monitor the ion pair 427.2 / 243.2; the declustering voltage is 100.00 V; the entrance voltage is 10.00 V; the exit voltage is 10.00 V; the collision energy is 40.00 eV; the dwell time is 200.00 msec; Teneligliptin-d4: monitor the ion pair 431.4 / 247.3; the declustering voltage is 100.00 V; the entrance voltage is 10.00 V; the exit voltage is 10.00 V; the collision energy is 39.00 eV; the dwell time is 200.00 msec.

2. The method for determining the concentration of teneligliptin in human plasma by liquid chromatography-tandem mass spectrometry according to claim 1, wherein The standard curve samples are prepared from the standard curve solution, and the concentration range of teneligliptin in the standard curve solution is 4 - 400 ng / mL.

3. The method for determining the concentration of teneligliptin in human plasma by liquid chromatography-tandem mass spectrometry according to claim 2, wherein The number of concentration gradients of teneligliptin in the standard curve solution is 6 - 10.

4. The method for determining the concentration of teneligliptin in human plasma by liquid chromatography-tandem mass spectrometry according to claim 3, characterized in that, The concentrations of teneligliptin 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.

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

6. The method for determining the concentration of teneligliptin in human plasma by liquid chromatography-tandem mass spectrometry according to claim 1, wherein The quality control samples are prepared from the quality control solution, and the concentration range of teneligliptin in the quality control solution is 4 - 300 ng / mL.

7. The method for determining the concentration of teneligliptin in human plasma by liquid chromatography-tandem mass spectrometry according to claim 6, wherein The concentrations of trelagliptin in the quality control solution are 4 ng / mL, 12 ng / mL, 40 ng / mL, 160 ng / mL, and 300 ng / mL, respectively.

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

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