A method for rapid determination of tigecycline and GD-MET-1 in human liver microsomal incubation system by LC-MS / MS

Abstract

This invention belongs to the field of drug metabolism and analytical chemistry, specifically relating to a method for the rapid determination of tigustastat and its active metabolite GD-MET-1 concentrations in a human liver microsome incubation system using liquid chromatography-tandem mass spectrometry (LC-MS / MS). The method first prepares stock solutions of tigustastat, GD-MET-1, and an internal standard, and prepares a series of standard curve solutions, quality control solutions, and precipitant solutions containing the internal standard. Next, the sample with the added precipitant solution undergoes vortexing and centrifugation pretreatment. Finally, chromatographic and mass spectrometric conditions are set, and the analysis is performed by LC-MS / MS. This method has been validated for specificity, accuracy, precision, matrix effects, recovery, residues, and stability, and can be reliably used for metabolic stability studies of tigustastat and GD-MET-1 in a human liver microsome incubation system, GD-MET-1 formation studies, metabolic enzyme identification, enzyme kinetic studies, and drug interaction studies.

Description

Technical Field

[0001] This invention belongs to the field of drug metabolism and analytical chemistry technology, specifically relating to a method for rapidly determining the concentration of tigoustastat and its active metabolite GD-MET-1 in a human liver microsomal incubation system using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Background Technology

[0002] Commonly used medications in clinical practice include those that inhibit uric acid synthesis, such as allopurinol and febuxostat, and those that promote uric acid excretion, such as benzbromarone. Allopurinol is the first-line drug for treating hyperuricemia and gout, but it carries a risk of allergic reactions, and HLA-B*5801 gene testing is required before use. Febuxostat is suitable for patients with mild to moderate renal insufficiency, but it may increase the risk of cardiovascular disease. Benzbromarone effectively increases uric acid excretion, but may cause liver damage. Tigustastat (also known as tigustostat), a novel xanthine oxidase inhibitor, is currently in an international multicenter phase III clinical trial. In previous phase II clinical studies, tigustastat has demonstrated good uric acid-lowering effects and safety. GD-MET-1 is its main active metabolite.

[0003] Establishing an analytical method for the simultaneous detection of tigustastat and GD-MET-1 in a human liver microsomal incubation system is of great significance for evaluating drug metabolic stability, studying metabolite formation, identifying metabolic enzymes, and investigating drug interactions. Detecting tigustastat and GD-MET-1 in the human liver microsomal incubation system allows for accurate assessment of drug metabolic stability, optimization of drug chemical structures, and improvement of in vivo stability and bioavailability. Identification of metabolic enzymes helps clarify the metabolic pathways of drugs and the specific enzymes involved in metabolism, playing a crucial role in predicting drug interaction risks, guiding personalized medication, and optimizing drug dosage and dosing regimens. However, due to differences in physicochemical properties and ionic responses between tigustastat and GD-MET-1, and the presence of complex endogenous impurities and phospholipid interference in the human liver microsomal incubation system, traditional analytical methods struggle to simultaneously achieve high resolution, sensitivity, and matrix adaptability. Currently, there is a lack of a highly sensitive LC-MS / MS analytical method suitable for complex liver microsomal incubation systems that can simultaneously determine the concentrations of tegustastat and GD-MET-1. This is especially true in in vitro drug metabolism studies, where a standardized detection system that can be directly applied is lacking. Therefore, establishing a highly sensitive, specific, and easy-to-operate simultaneous detection method suitable for complex liver microsomal incubation systems is of great significance. Summary of the Invention

[0004] Based on this, the purpose of this invention is to provide an LC-MS / MS analytical method for the simultaneous determination of tigustastat and GD-MET-1. This method has been validated in terms of specificity, accuracy, precision, matrix effect, recovery, residue and stability, and can be reliably used for the study of the metabolic stability of tigustastat and GD-MET-1 in liver microsome incubation system, the study of GD-MET-1 generation, the identification of metabolic enzymes, the study of enzyme kinetics and drug interaction.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for rapid determination of tigustastat and GD-MET-1 in a human liver microsomal incubation system using LC-MS / MS, comprising the following steps: S1, Preparation of stock solutions: Take tigustastat standard and GD-MET-1 standard respectively, dissolve and dilute to volume to prepare tigustastat stock solution and GD-MET-1 stock solution; S2, Preparation of standard curve solutions: Take the tigustastat stock solution and GD-MET-1 stock solution mentioned in S1, mix them, and then dilute them stepwise to prepare a series of mixed standard working solutions of different concentrations; add the mixed standard working solutions of different concentrations to blank human liver microsome incubation matrix and mix them to prepare a series of standard curve solutions of different concentrations. S3, Preparation of quality control solutions: Take the tigustastat stock solution and GD-MET-1 stock solution mentioned in S1, mix them thoroughly, and then dilute them stepwise to prepare a series of quality control mixed working solutions of different concentrations; add the series of quality control mixed working solutions of different concentrations to blank human liver microsome incubation matrix and mix them to prepare a series of quality control solutions of different concentrations. S4, Preparation of precipitant solution containing internal standard: Take the internal standard, dissolve and dilute to obtain internal standard stock solution; take the internal standard stock solution, dilute to obtain organic precipitant solution containing internal standard; S5, Sample pretreatment: Take the standard curve solution, quality control solution or human liver microsome incubation system sample to be tested, add the precipitant solution described in S4, and vortex and centrifuge to obtain the sample solution to be tested; S6, LC-MS / MS determination: Inject the sample solution described in S5, set the high performance liquid chromatography test conditions and mass spectrometry test conditions, and perform LC-MS / MS analysis and determination.

[0006] Furthermore, the solution used for dissolution in S1 includes dimethyl sulfoxide, the concentration of the tigustastat stock solution is 10±0.5 mM, and it is stored at -20±0.5 ℃; the concentration of the GD-MET-1 stock solution is 10±0.5 mM, and it is stored at -20±0.5 ℃.

[0007] Furthermore, the solvent used for the stepwise dilution in S2 includes acetonitrile; the concentrations of tegustastat and GD-MET-1 in the standard curve solutions of the series of concentrations are 5, 10, 20, 50, 100, 500, 1000 and 2500 nM, respectively.

[0008] Furthermore, the solvent used for the stepwise dilution in S3 includes acetonitrile; the concentrations of tegustastat and GD-MET-1 in the series of quality control solutions are 10, 250, and 2000 nM, respectively, and they are stored at -80±0.5 °C.

[0009] Further, the internal standard mentioned in S4 includes diazepam; the solvent used for dissolution and dilution includes acetonitrile; the concentration of the internal standard stock solution is 1 ± 0.05 mg / mL; and the concentration of the precipitant solution is 40 ± 0.05 μg / mL, stored at 4 ± 0.5 ℃. Furthermore, in S5, the volume ratio of the standard curve solution, quality control solution, or human liver microsome incubation system sample to the precipitant solution is 1:1, the vortexing time is 1~3 min, the centrifugation speed is 13000~15000 rpm, and the time is 9~11 min.

[0010] Furthermore, the test conditions for high performance liquid chromatography described in S6 are as follows: the chromatographic column is a Waters Symmetry C18 column; the autosampler temperature is 6~10 ℃, the column temperature is 25~40 ℃, the injection volume is 3~10 μL, and the flow rate is 0.3~0.5 mL / min; the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is acetonitrile, and gradient elution is used.

[0011] Furthermore, the gradient elution conditions are as follows: initially, the mobile phase A:B (volume ratio) is 90:10 and is maintained for 1.0 min; then, it linearly changes to A:B at 10:90 within 1.0-5.0 min and is maintained at this ratio for 7.0 min; then, it rapidly returns to the initial ratio of A:B at 90:10 within 7.0-7.1 min and is maintained for 9.0 min to complete column equilibration.

[0012] Furthermore, the mass spectrometry test conditions are as follows: the ion source is an electrospray ionization source in positive ion mode; the spray voltage is 5500 V; the ion source temperature is 400~600 ℃; the auxiliary gas 1 is 55 psi; the auxiliary gas 2 is 50 psi; the curtain gas is 35 psi; and the scanning mode is multiple reaction monitoring (MRM) scanning mode.

[0013] Furthermore, the mass spectrometry testing conditions are as follows: the ion pair of tegustastat and GD-MET-1 is... m / z295.1→235.0 and 252.8→235.2, the ion pairs of the internal standard are m / z 285.3→154.1; the declustering voltage of tigusta is 103.0V, the declustering voltage of GD-MET-1 is 85.0V, and the declustering voltage of the internal standard is 118.0V; the collision voltage of tigusta is 31V, the collision voltage of GD-MET-1 is 24V, and the collision voltage of the internal standard is 32V.

[0014] The beneficial effects of this invention are: 1. This invention provides an LC-MS / MS analytical method for the simultaneous determination of tigustastat and GD-MET-1. Due to differences in physicochemical properties, ion responses, and retention behaviors between tigustastat and GD-MET-1, and the presence of complex endogenous impurities and phospholipid interference in the human liver microsome incubation system, traditional analytical methods struggle to simultaneously achieve optimal resolution, sensitivity, and matrix adaptability. This invention achieves simultaneous high-sensitivity detection of tigustastat and GD-MET-1 through systematic optimization of sample pretreatment conditions, chromatographic separation conditions, and mass spectrometry detection parameters. Currently, there are no publicly reported methods for the simultaneous quantitative analysis of tigustastat and GD-MET-1.

[0015] 2. This invention optimizes the complex liver microsomal matrix, which can effectively reduce matrix interference caused by lipids and endogenous impurities, and improve the stability and accuracy of analysis.

[0016] 3. The sample pretreatment method is simple, requiring only protein precipitation, and does not rely on expensive reagents or complex extraction equipment; it requires a small sample volume, has high processing efficiency, and low cost, making it suitable for routine metabolic kinetics and drug screening experiments.

[0017] 4. The method of this invention has high sensitivity, wide linear range, strong matrix adaptability, and simple experimental operation. It has good scalability and application prospects and can be widely used in the study of the metabolic stability of tigustastat and GD-MET-1, the study of GD-MET-1 generation, the identification of metabolic enzymes, the study of enzyme kinetics, and the study of drug interactions. Attached Figure Description

[0018] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is the mass spectrum of tegustabolite in Example 1 of the present invention; Figure 2This is the mass spectrum of GD-MET-1 in Example 1 of the present invention; Figure 3 This is the mass spectrum of the internal standard in Example 1 of the present invention; Figure 4 The chromatogram of tigousstat in the human liver microsomal incubation system in Example 1 of this invention; Figure 5 This is a chromatogram of GD-MET-1 in the human liver microsomal incubation system of Example 1 of the present invention; Figure 6 This is a chromatogram of the internal standard in the human liver microsome incubation system of Example 1 of the present invention; Figure 7 This is a standard curve diagram of tegustastat and GD-MET-1 in Embodiment 1 of the present invention, where a is tegustastat and b is GD-MET-1. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. All mentioned embodiments are implemented based on the technical solutions of the present invention, and detailed implementation processes are given. However, it should be stated that the scope of protection of the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials, reagents, or equipment used, unless otherwise specified, are commercially available.

[0021] Method optimization (1) Optimization of sample pretreatment conditions The responses, peak shapes, and matrix interferences of tegustamol and GD-MET-1 were investigated using methanol, acetonitrile, and acetonitrile containing formic acid as protein precipitants. The results showed that when methanol was used as the protein precipitant, GD-MET-1 exhibited a low response and some matrix interference. While acetonitrile containing formic acid improved the analyte response, its peak shape stability was poor. In contrast, when acetonitrile was used as the protein precipitant, both tegustamol and GD-MET-1 showed high responses, good peak shapes, and low matrix interference. Therefore, acetonitrile was ultimately chosen as the protein precipitant.

[0022] (2) Optimization of chromatographic conditions The effects of different chromatographic columns and mobile phase systems on the separation of tegustamol and GD-MET-1 were investigated. The results showed that under certain column conditions, tegustamol and GD-MET-1 exhibited insufficient retention or peak tailing. However, when using a Waters Symmetry C18 column, both analytes showed good retention behavior, peak shape, and resolution. Further comparison of different mobile phase systems revealed that a 0.1% formic acid aqueous solution-acetonitrile system yielded higher responses and more stable peak shapes for both tegustamol and GD-MET-1; therefore, this mobile phase system was selected.

[0023] (3) Optimization of mass spectrometry conditions Mass spectrometry parameters, including precursor ion, daughter ion, declustering voltage, and collision voltage, for tigustastat, GD-MET-1, and the internal standard were optimized. Results showed that both tigustastat and GD-MET-1 exhibited high ion response and stable signal intensity in electrospray ionization positive ion mode; therefore, multiple reaction monitoring (MRM) was employed for detection.

[0024] Example 1

[0025] A method for the simultaneous and rapid determination of tigustastat and GD-MET-1 concentrations in a human liver microsomal incubation system using ultra-high performance liquid chromatography-tandem mass spectrometry (LC-MS / MS) includes the following steps: (1) Preparation of stock solutions: Accurately weigh appropriate amounts of tegustastat and GD-MET-1 standard, place them in 1 mL volumetric flasks, add dimethyl sulfoxide to dissolve and dilute to the mark to obtain stock solutions of tegustastat and GD-MET-1 with a concentration of approximately 10 mM, and store them at -20 ℃ for later use.

[0026] (2) Preparation of standard curve solutions: Accurately measure appropriate amounts of tegustastat and GD-MET-1 stock solutions, mix them well, and then dilute them stepwise with acetonitrile to prepare a series of mixed standard working solutions. For example, mixed standard working solutions containing tegustastat and GD-MET-1 at concentrations of 50, 100, 200, 500, 1000, 5000, 10000 and 25000 nM were obtained respectively.

[0027] Take 90 μL each of blank human liver microsome incubation matrix (containing only buffer and liver microsomes, excluding tigustastat and GD-MET-1) and add 10 μL of the above mixed standard working solution (8 concentrations). Vortex mix to obtain the standard curve solutions in the liver microsome matrix. The final concentrations of tigustastat and GD-MET-1 are 5, 10, 20, 50, 100, 500, 1000, and 2500 nM, respectively. All standard curve solutions should be prepared fresh and used immediately.

[0028] Preparation of quality control solutions: Accurately measure appropriate amounts of tigustastat and GD-MET-1 stock solutions, mix well, and dilute stepwise with acetonitrile to prepare low, medium, and high quality control mixed working solutions containing tigustastat and GD-MET-1 at concentrations of 100, 2500, and 20000 nM, respectively. Add 10 μL of each of the above quality control working solutions to 90 μL of blank human liver microsome incubation matrix, vortex to mix, and obtain low, medium, and high quality control solutions with final concentrations of tigustastat and GD-MET-1 of 10, 250, and 2000 nM, respectively. All quality control solutions were stored at -80 ℃ for later use.

[0029] Preparation of the precipitant solution containing internal standard: Accurately weigh an appropriate amount of diazepam (internal standard), place it in a 1 mL volumetric flask, add acetonitrile to dissolve and dilute to the mark to obtain an internal standard stock solution with a concentration of 1 mg / mL. Take the above diazepam internal standard stock solution and further dilute it with acetonitrile as the solvent to prepare a protein precipitant solution containing a diazepam internal standard concentration of 40 μg / mL. Store the above solutions at 4 °C for later use.

[0030] (5) Sample pretreatment: Take 100 μL of human liver microsome incubation system sample, add 100 μL of precipitant solution, vortex for 2 min to precipitate protein, centrifuge at 14000 rpm for 10 min, take 150 μL of supernatant into the injection bottle, and inject 5 μL of sample.

[0031] (6) High performance liquid chromatography column: Waters Symmetry C18 column (5 μm, 2.1 mm × 150 mm).

[0032] (7) High performance liquid chromatography conditions: The mobile phase was acetonitrile (B)-water (0.1% formic acid) (A), using gradient elution. Initially, the mobile phase A:B (volume ratio) was 90:10 and held for 1.0 min; then linearly changed to A:B 10:90 within 1.0-5.0 min and held at this ratio for 7.0 min; then rapidly restored to the initial ratio of A:B 90:10 within 7.0-7.1 min and held for 9.0 min to complete column equilibration; flow rate 0.3 mL / min; autosampler temperature 8 ℃; column temperature 40 ℃; injection volume 5 mL.

[0033] (8) Mass spectrometry conditions: Electrospray ionization (ESI) source, positive ion mode. Ion source parameters were as follows: spray voltage (IS) = 5500 V; ion source temperature (TEM) = 550 ℃; auxiliary gas 1 (GS1) = 55 psi; auxiliary gas 2 (GS2) = 50 psi; curtain gas (CUR) = 35 psi. Multiple reaction monitoring was used. The ion pair of tegustastat and GD-MET-1 was...m / z 295.1→235.0 and 252.8→235.2, the ion pairs of the internal standard are m / z 285.3→154.1; the declustering voltages of tegustastat and GD-MET-1 are 103.0 and 85.0 V, respectively, and the declustering voltage of the internal standard is 118.0 V; the collision voltages of tegustastat and GD-MET-1 are 31 and 24 V, respectively, and the collision voltage of the internal standard is 32 V.

[0034] Implementation effect analysis

[0035] The above-mentioned determination methods underwent methodological validation for specificity, accuracy, precision, matrix effect, recovery, residue, and stability. Details are as follows: 1. The instruments and reagents used for verification are: Instrument: AB SCIEX ExionLC (USA) TM The Triple Quad 4500MD mass spectrometer with tandem liquid chromatography system is equipped with a chromatography workstation: Analyst Software (version 1.6.2).

[0036] HPLC column: Waters Symmetry C18 column (5 μm, 2.1 mm × 150 mm).

[0037] SB25-12 DTD Ultrasonic Cleaner (Ningbo Xinzhi Biotechnology Co., Ltd.); Centrifuge 5810R high-speed centrifuge (Eppendorf Life Sciences); XS205 model 1 / 100,000 balance (METTLER TOLEDO); Vortex Genie2 vortex analyzer (Scientific Industries, USA); Reagents: Mass spectrometry grade pure water, mass spectrometry grade acetonitrile, and mass spectrometry grade methanol were purchased from Thermo Fisher Scientific, Inc., USA; mass spectrometry grade formic acid was purchased from Sigma-Aldrich, Inc., USA. Standards: Tigustastat (batch number: 461061, purity: 99.18%) was purchased from MedChemExpress LLC, USA; GD-MET-1 (purity >99%, synthesized in our laboratory); and diazepam injection (internal standard) was purchased from Jiangsu Enhua Pharmaceutical Co., Ltd.

[0038] 2. Methodological Examination Specificity: Take blank human liver microsome incubation matrix samples (excluding tigustastat and GD-MET-1), process them according to the sample pretreatment method described in section (5) above, wherein no internal standard solution is added to the precipitant; separately take tigustastat and GD-MET-1 standard curve samples at the lower limit of quantitation (LLOQ) concentration, process them in the same way, and then inject them for analysis. The mass spectra of tigustastat, GD-MET-1, and internal standard are shown below. Figures 1-3 As shown. Under the described chromatographic and mass spectrometric conditions, the presence of endogenous interference peaks near the retention times of tigustastat, GD-MET-1, and diazepam internal standards was investigated. The test results are shown below. Figures 4-6 As shown. Figures 4-6 The results showed that no obvious interference peaks were observed at the retention times of tigustastat, GD-MET-1, and the internal standard in the blank liver microsome incubation matrix. The response of the interference peaks was less than 20% of the response value of the analyte quantification limit and less than 5% of the response value of the internal standard, indicating that the method has good specificity and selectivity and is suitable for the quantitative analysis of tigustastat and GD-MET-1 in the liver microsome incubation system.

[0039] Standard curve: Prepare 100 μL of standard curve solution according to the operating steps in section (2), and process the rest according to the method described in section (5) to obtain liver microsome incubation samples of different concentrations. Inject and analyze the samples separately, performing three consecutive analytical batches. Plot the analyte concentration on the x-axis and the ratio of the analyte peak area to the internal standard peak area on the y-axis, using the weighted least squares method (W = 1 / x). 2 Perform linear regression to obtain the standard curve as shown below. Figure 7 As shown. Figure 7 The results showed that tegustabolone (Tegustabolone) Figure 7 a) and GD-MET-1 ( Figure 7 b) The correlation coefficient r in the linear range of 5 nM to 2500 nM 2 The linear range is >0.99, indicating good linearity and meeting the needs of analysis and testing.

[0040] Limit of Quantification (LLOQ): Prepare 100 mL of the LLOQ solution according to the operating procedure in section (2), and process the rest according to the method described in section (5). Prepare 6 parallel solutions and use them with the standard curve of the day. Calculate their mass concentration, RE, and RSD values. The results are shown in Table 1 below. The results in Table 1 show that the RE of tegustastat and GD-MET-1 is 1.20%, and the RSD is 7.72%; the RE of GD-MET-1 is 4.23%, and the RSD is 1.26%, which meet the test requirements.

[0041] Table 1. Precision and accuracy of the lower limit of quantification for tegustastat and GD-MET-1 in the human liver microsomal incubation system.

[0042] Precision and accuracy: Standard solutions of four concentrations (limit of quantitation, low quality control, medium quality control, and high quality control) were prepared according to the operating steps in (2) and (3). The remaining solutions were processed according to the method described in (5). Six replicates were prepared for each concentration, and the intra-batch precision and accuracy were calculated. One analytical batch was measured each day for three consecutive days, for a total of three analytical batches. The standard curve of the day was used as a reference, and the measured concentration of the sample at that concentration was calculated based on the standard curve of the day. The inter-batch accuracy and precision were calculated, and the results are shown in Table 2 below. The results in Table 2 show that the intra-batch and inter-batch accuracy and precision of the analyte were good at the four concentrations (limit of quantitation, low quality control, medium quality control, and high quality control), with RSDs all <13.57%, indicating that the method has good precision and high accuracy.

[0043] Table 2. Intra-assay and inter-assay precision and accuracy of tegustastat and GD-MET-1 in the human liver microsomal incubation system.

[0044] Matrix effect and recovery rate: Prepare standard solutions of four concentrations (limit of quantitation, low quality control, medium quality control, and high quality control) according to the operating steps in (2) and (3). Process the rest according to the method described in (5). Prepare six parallel samples of each concentration and analyze them separately. The peak area obtained is A1. Replace the blank human liver microsome incubation system with acetonitrile. Prepare four matrix-free standard solutions of four concentrations (limit of quantitation, low quality control, medium quality control, and high quality control) according to the operating steps in (2) and (3). Process the rest according to the method described in (5). Prepare six parallel samples of each concentration and analyze them separately. The peak area obtained is A2. Take 100 mL of the blank human liver microsome incubation system, add 200 mL of acetonitrile solution containing internal standard, vortex for 1 min to precipitate protein, centrifuge at 14000 rpm for 10 min, take all supernatant, and add 100 mL of matrix-free standard solutions of four concentrations (limit of quantitation, low quality control, medium quality control, and high quality control). Centrifuge the sample again as described in section (5), prepare six parallel samples for each concentration, and analyze the peak area obtained by each sample. The matrix effect factor is calculated by internal standard normalization, i.e., (A3 / IS3) / (A2 / IS2)×100%. Calculate the A1 / A3×100% value for the same concentration, which is the extraction recovery rate of the analyte. The results are shown in Table 3 below. The results in Table 3 show that, at the four concentrations (lower limit of quantitation, low quality control, medium quality control, and high quality control), the normalized matrix effect factor of tigustastat (internal standard) ranged from 0.97 to 1.06, with an RSD of <9.77%, and the extraction recovery rates were all above 97.49%, with an RSD of <5.34%. Similarly, the normalized matrix effect factor of GD-MET-1 (internal standard) ranged from 0.89 to 0.96, with an RSD of <10.36%, and the extraction recovery rates were all above 89.58%, with an RSD of <11.00%. These results indicate that the matrix effect of this method is not significant, the recovery rate is good, and it meets the requirements for testing biological samples.

[0045] Table 3. Matrix effects and recoveries of tigustastat and GD-MET-1 in the human liver microsomal incubation system.

[0046] Sample residue: Prepare 100 mL of the upper limit of quantitation solution according to the operating steps in section (2), and process the rest according to the method described in section (5). Prepare 3 parallel samples. Test the upper limit of quantitation sample 3 times consecutively, and then test the blank treatment sample. The test results are shown in Table 4 below. The results in Table 4 show that, under the set chromatographic conditions, the sample residue is less than 20% of the lower limit of quantitation response value of the analyte and 5% of the internal standard, that is, the analyte and internal standard are not affected by the sample residue.

[0047] Table 4. Residues of tegustastat, GD-MET-1, and internal standard in the human liver microsomal incubation system.

[0048] Stability: Stability test at room temperature: Prepare low, medium and high concentration quality control samples according to the operation steps in (3), 6 in parallel, and place at room temperature for 6 hours. Process according to the method in (5), and take the supernatant for analysis. Freeze-thaw stability test: Prepare low, medium and high concentration quality control samples according to the operation steps in (3), 6 in parallel, freeze at -80 ℃ (≥12h), take out and thaw, repeat the freeze-thaw cycle 3 times, process according to the method in (5), and take the supernatant for analysis. Stability test after sample processing: Prepare low, medium and high concentration quality control samples according to the operation steps in (3), 6 in parallel, process according to the method in (5), place the obtained samples in the sample injector for 21 h, take the supernatant for analysis, and the test results are shown in Table 5 below. The results in Table 5 show that, under the above conditions, the accuracy of tegustastat was 82.82-109.46%, with RSDs all less than 12.79%, and the accuracy of GD-MET-1 was 93.62-109.52%, with RSDs all less than 11.79%, indicating good stability.

[0049] Table 5. Stability of tegustastat and GD-MET-1 human liver microsomal incubation system solutions under different conditions.

[0050] In summary, this invention provides a method for the simultaneous detection of tigustastat and GD-MET-1 in a liver microsomal incubation system. Currently, there is no existing technology capable of simultaneously and accurately quantifying tigustastat and GD-MET-1 with high sensitivity, and a standardized detection system readily applicable to in vitro drug metabolism studies is particularly lacking. This method, by optimizing sample pretreatment conditions, chromatographic separation conditions, and mass spectrometry detection parameters, achieves the simultaneous and stable detection of tigustastat and GD-MET-1. It can be used for studies on the metabolic stability of tigustastat and GD-MET-1 in a liver microsomal incubation system, studies on GD-MET-1 formation, identification of metabolic enzymes, enzyme kinetics, and drug interaction studies.

[0051] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for rapid determination of tigustastat and GD-MET-1 in a human liver microsomal incubation system using LC-MS / MS, characterized in that, Includes the following steps: S1, Preparation of stock solutions: Take tigustastat standard and GD-MET-1 standard respectively, dissolve and dilute to volume to prepare tigustastat stock solution and GD-MET-1 stock solution; S2, Preparation of standard curve solutions: Take the tigustastat stock solution and GD-MET-1 stock solution mentioned in S1, mix them, and then dilute them stepwise to prepare a series of mixed standard working solutions of different concentrations; add the mixed standard working solutions of different concentrations to blank human liver microsome incubation matrix and mix them to prepare a series of standard curve solutions of different concentrations. S3, Preparation of quality control solutions: Take the tigustastat stock solution and GD-MET-1 stock solution mentioned in S1, mix them thoroughly, and then dilute them stepwise to prepare a series of quality control mixed working solutions of different concentrations; add the series of quality control mixed working solutions of different concentrations to blank human liver microsome incubation matrix and mix them to prepare a series of quality control solutions of different concentrations. S4, Preparation of precipitant solution containing internal standard: Take the internal standard, dissolve and dilute to obtain internal standard stock solution; take the internal standard stock solution, dilute to obtain precipitant solution; S5, Sample pretreatment: Take the standard curve solution, quality control solution or human liver microsome incubation system sample to be tested, add the precipitant solution described in S4, and vortex and centrifuge to obtain the sample solution to be tested; S6, LC-MS / MS determination: Inject the sample solution described in S5, set the high performance liquid chromatography test conditions and mass spectrometry test conditions, and perform LC-MS / MS analysis and determination.

2. The method according to claim 1, characterized in that, The solution used for dissolution in S1 includes dimethyl sulfoxide, the concentration of the tigustastat stock solution is 10±0.5 mM, and it is stored at -20±0.5 ℃; the concentration of the GD-MET-1 stock solution is 10±0.5 mM, and it is stored at -20±0.5 ℃.

3. The method according to claim 1, characterized in that, The solvent used for the stepwise dilutions described in S2 includes acetonitrile; the concentrations of tegustastat and GD-MET-1 in the standard curve solutions of the series of concentrations are 5, 10, 20, 50, 100, 500, 1000 and 2500 nM, respectively.

4. The method according to claim 1, characterized in that, The solvent used for the stepwise dilutions described in S3 includes acetonitrile; the concentrations of tegustastat and GD-MET-1 in the series of quality control solutions are 10, 250, and 2000 nM, respectively, and they are stored at -80±0.5℃.

5. The method according to claim 1, characterized in that, The internal standard mentioned in S4 includes diazepam; the solvent used for dissolution and dilution includes acetonitrile; the concentration of the internal standard stock solution is 1 ± 0.05 mg / mL; the concentration of the precipitant solution is 40 ± 0.05 μg / mL, and it is stored at 4 ± 0.5℃.

6. The method according to claim 1, characterized in that, The volume ratio of the standard curve solution, quality control solution, or human liver microsome incubation system sample to the precipitant solution in S5 is 1:

1. The vortexing time is 1-3 min, the centrifugation speed is 13000-15000 rpm, and the time is 9-11 min.

7. The method according to claim 1, characterized in that, The test conditions for high performance liquid chromatography described in S6 are as follows: the chromatographic column is a Waters Symmetry C18 column; the autosampler temperature is 6~10 ℃, the column temperature is 25~40 ℃, the injection volume is 3~10 μL, and the flow rate is 0.3~0.5 mL / min; the mobile phase A is 0.1% formic acid aqueous solution, the mobile phase B is acetonitrile, and gradient elution is used.

8. The method according to claim 7, characterized in that, The gradient elution conditions are as follows: initially, the mobile phase A:B (volume ratio) is 90:10 and held for 1.0 min; then, it linearly changes to A:B at 10:90 within 1.0-5.0 min and is held at this ratio for 7.0 min; then, it rapidly returns to the initial ratio of A:B at 90:10 within 7.0-7.1 min and is held for 9.0 min to complete column equilibration.

9. The method according to claim 1, characterized in that, The mass spectrometry test conditions were as follows: the ion source was an electrospray ionization source in positive ion mode; the spray voltage was 5500 V; the ion source temperature was 400~600℃; and the auxiliary gas was 55 psi. The auxiliary gas was 50 psi; the curtain gas was 35 psi; and the scanning mode was multiple reaction monitoring.

10. The method according to claim 1, characterized in that, The mass spectrometry test conditions were as follows: the ion pairs of tigustastat and GD-MET-1 were m / z 295.1→235.0 and 252.8→235.2, respectively, and the ion pair of the internal standard was m / z 285.3→154.1; the declustering voltage of tigustastat was 103.0 V, the declustering voltage of GD-MET-1 was 85.0 V, and the declustering voltage of the internal standard was 118.0 V; the collision voltage of tigustastat was 31 V, the collision voltage of GD-MET-1 was 24 V, and the collision voltage of the internal standard was 32 V.

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