Method for simultaneously detecting compound SYN045 and metabolic compounds thereof in biological sample

Abstract

The invention belongs to the field of drug detection, and relates to an analysis method for simultaneously detecting compounds SYN007 and SYN045 in a biological sample, which comprises the following steps: S1, preparing a mobile phase A, a mobile phase B, an internal standard working solution and SYN045 and SYN007 standard curve sample solutions; s2, adding plasma, SYN045 and SYN007 standard curve sample solutions into the internal standard working solution, performing vortex mixing and centrifugation, and taking supernate to obtain a to-be-detected sample and a loading solution for a standard curve for later use; s3, detecting by adopting a liquid chromatography-mass spectrometry method; and S4, quantifying by adopting an internal standard curve method, and calculating the concentration. According to the analysis method disclosed by the invention, SYN045 and metabolites thereof can be simultaneously detected in a biological sample, and the detection efficiency is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug detection and analysis, and specifically relates to a method for simultaneously detecting a compound SYN045 and its metabolites in a biological sample. Background Art

[0002] Pulmonary hypertension is a rare and difficult-to-cure pulmonary vascular disease that can gradually lead to right heart failure and eventually death. 75% of PAH patients die within 5 years of diagnosis, and the average survival time after the onset of symptoms is 1.9 years. Therefore, it is also called "malignant tumor in the field of cardiopulmonary vascular disease." PGI2 receptor agonists are the dominant drugs for the treatment of pulmonary hypertension, and the representative variety is selexipag.

[0003] SYN045 is a novel long-acting oral PGI2 receptor agonist, with a structural formula as shown in Formula 1. Its chemical name is 6-((5,6-diphenyl-1,2,4-triazine-3-yl)(isopropyl)amino)-N-(methylsulfonyl)hexanamide (referred to as "diphenyltriazine (isopropyl)amino (methylsulfonyl)hexanamide"). The compound has a novel structure and good drugability. Compared with existing drugs, it has strong target selectivity, and the agonist activity of prostacyclin IP receptor is more than 1000 times that of the other 7 prostacyclin receptor targets. SYN045 mainly activates IP receptors to promote the production of cAMP in pulmonary artery smooth muscle cells, thereby inhibiting abnormal contraction of pulmonary arteries, inhibiting the proliferation of pulmonary artery smooth muscle cells, and reducing pulmonary artery pressure to achieve the treatment of pulmonary hypertension. Compared with similar drugs already on the market, it has comparable efficacy, higher safety, and outstanding clinical advantages.

[0004] SYN045 is mainly metabolized in the body to a compound as shown in Formula II, whose chemical name is 6-[N-(5,6-diphenyl-1,2,4-triazine)-2-yl-N-isopropylamino]hexanoic acid (hereinafter referred to as "SYN007").

[0005]

[0006] In order to better study and monitor the metabolic curve of SYN045 in vivo, it is best to simultaneously detect the content of SYN045 and SYN007 in the organism. Currently, there is no literature or patent disclosure on the detection of compound SYN007 and compound SYN045 in biological samples.

[0007] The existing detection technology needs to detect the compound SYN007 and the compound SYN045 in the biological sample separately, which is cumbersome. Therefore, it is necessary to develop a set of analytical detection methods that can simultaneously determine the compound SYN007 and the compound SYN045 in the biological sample to simplify the operation steps. Summary of the invention

[0008] The object of the present invention is to provide a method for simultaneously detecting the compound SYN045 and its metabolites in a biological sample in view of the defects of the prior art.

[0009] The analytical detection method provided by the present invention adopts liquid chromatography-mass spectrometry, uses warfarin as an internal standard, establishes an internal standard standard curve, and optimizes the liquid phase mass spectrometry conditions to achieve simultaneous detection of SYN007 and SYN045 under the same conditions, thereby improving the detection efficiency. At the same time, the biological sample processing method is simple, and there is no need to acidify plasma with hydrochloric acid. The supernatant is directly sampled after vortexing, thereby reducing the sample loss caused by operations such as nitrogen blowing, heating, and re-dissolution.

[0010] To achieve the purpose of the present invention, the following technical solutions are adopted: The present invention provides a method for simultaneously determining the content of compound SYN045 and its metabolites in biological samples, using warfarin as an internal standard, adopting liquid chromatography-mass spectrometry, and the chromatographic conditions are: mobile phase A: 0.5 mM ammonium acetate aqueous solution containing 0.08-0.12 vol% formic acid; mobile phase B: 0.08-0.12 vol% formic acid in acetonitrile solution; mobile phase A and mobile phase B adopt gradient elution mode.

[0011] Furthermore, the formic acid concentration in the mobile phase A and the mobile phase B is preferably 0.1%.

[0012] Furthermore, the gradient elution conditions are shown in the following table.

[0013]

[0014] Furthermore, the specific steps of the determination method include: S1. Preparing a standard curve equation, including: preparing an internal standard working solution and a standard working solution, preparing a sample solution for the standard curve, and detecting the sample solution for the standard curve by liquid chromatography-mass spectrometry to obtain a standard curve equation for the content of SYN045 and its metabolites; S2: Sample processing Collect blood and place it in an anticoagulant tube, centrifuge it, take the plasma, add the internal standard working solution, vortex mix, centrifuge it, and take the supernatant for later use; S3: Liquid quality testing The supernatant was detected and analyzed by liquid chromatography-mass spectrometry, and the concentrations of the compounds SYN045 and SYN007 in the sample solution were calculated according to the standard curve equation.

[0015] Furthermore, in step S3 of the determination method, the chromatographic column used in the liquid chromatography is ACQUITY UPLC® BEH C18, the chromatographic column diameter is 2.1 mm, the chromatographic column length is 50 mm, the filler particle size is 1.7 μm; the column temperature is 35°C to 45°C; and the flow rate is 0.5 mL / min.

[0016] Furthermore, the column temperature is preferably 40°C.

[0017] Furthermore, in step S3 of the determination method, the mass spectrometry conditions are: ionization mode: electrospray ionization, positive ion mode, multiple reaction monitoring; ion spray voltage: 5500V; ion source temperature: 550°C; collision activated dissociation: 10; curtain gas: 35 psi; ion source gas 1: 60 psi; ion source gas 2: 60 psi.

[0018] Furthermore, in step S1 of the assay method, the internal standard working solution is prepared by first preparing a 1 mg / mL warfarin methanol solution as an internal standard stock solution, taking 50 μL of the internal standard stock solution, and using 50 vol% acetonitrile methanol to make the volume to 1000 ml to obtain a 50 ng / mL internal standard working solution.

[0019] Furthermore, in step S1 of the assay method, the standard working solution includes a SYN045 standard curve sample and a SYN007 standard curve sample.

[0020] Furthermore, the SYN045 standard curve samples are as follows: SYN045 methanol solutions with concentrations of 10000 ng / mL, 5000 ng / mL, 2500 ng / mL, 1000 ng / mL, 500 ng / mL, 100 ng / mL, 50 ng / mL, and 10 ng / mL are added to blank matrix plasma respectively to obtain SYN045 standard curve samples with concentrations of 500 ng / mL, 250 ng / mL, 125 ng / mL, 50 ng / mL, 25 ng / mL, 5 ng / mL, 2.5 ng / mL, and 0.5 ng / mL.

[0021] Furthermore, the SYN045 standard curve sample preparation is to first prepare a SYN045 methanol solution with a concentration of 2 mg / mL as a stock solution, further dilute it to SYN045 methanol solutions with concentrations of 10000 ng / mL, 5000 ng / mL, 2500 ng / mL, 1000 ng / mL, 500 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 50 ng / mL, and 10 ng / mL, and then take 20 µL of each of the above concentration solutions and add 380 µL of blank matrix (rat plasma) to obtain SYN045 standard curve samples with concentrations of 500 ng / mL, 250 ng / mL, 125 ng / mL, 50 ng / mL, 25 ng / mL, 5 ng / mL, 2.5 ng / mL, and 0.5 ng / mL.

[0022] Furthermore, the SYN007 standard curve samples are as follows: SYN007 methanol solutions with concentrations of 20000 ng / mL, 10000 ng / mL, 5000 ng / mL, 2000 ng / mL, 1000 ng / mL, 200 ng / mL, 100 ng / mL, and 20 ng / mL are added respectively to blank matrix plasma to obtain SYN007 standard curve samples with concentrations of 1000 ng / mL, 500 ng / mL, 250 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 5 ng / mL, and 1 ng / mL.

[0023] Furthermore, the SYN007 standard curve sample preparation is to first prepare a SYN007 methanol solution with a concentration of 2 mg / mL as a stock solution, further dilute it to SYN007 methanol solutions with concentrations of 20000 ng / mL, 10000 ng / mL, 5000 ng / mL, 2000 ng / mL, 1000 ng / mL, 200 ng / mL, 100 ng / mL, 200 ng / mL, 100 ng / mL, and 20 ng / mL, and then take 20 µL of each of the above concentration solutions and add 380 µL of blank matrix (rat plasma) to obtain SYN007 standard curve samples with concentrations of 1000 ng / mL, 500 ng / mL, 250 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 5 ng / mL, and 1 ng / mL.

[0024] Furthermore, in step S1 of the assay method, the preparation method of the standard curve loading solution is: take a SYN045 standard curve sample and a SYN007 standard curve sample, add the internal standard working solution respectively, vortex mix, centrifuge, and take the supernatant to obtain the SYN045 standard curve loading solution and the SYN007 standard curve loading solution.

[0025] Furthermore, the preparation method of the standard curve loading solution is as follows: take 30 μL of SYN045 standard curve sample and SYN007 standard curve sample, add 200 μL of internal standard working solution respectively, vortex mix, centrifuge at 4°C for 7 minutes, centrifugal force 18000g, and take the supernatant to obtain SYN045 standard curve loading solution and SYN007 standard curve loading solution.

[0026] Furthermore, in step S2, the centrifugation time is 6-7 minutes.

[0027] Furthermore, in step S2, the sample processing is to collect 0.25 mL of blood and place it in an anticoagulant tube containing K2EDTA, centrifuge at 4°C for 6 minutes (centrifugal force 6800g), take 30 μL of plasma, add 200 μL of internal standard working solution, vortex mix, centrifuge at 4°C for 7 minutes (centrifugal force 18000g), and take the supernatant for use.

[0028] Furthermore, in step S3, 180 μL of the supernatant is taken.

[0029] Furthermore, in step S3, the liquid-mass detection is to take 180 μL of supernatant, add it into the corresponding 96-well sample injection plate, use liquid chromatography-mass spectrometry to perform detection and analysis, and record the chromatogram.

[0030] Beneficial effects of the present invention: (1) The analytical detection method provided by the present invention optimizes the liquid phase mass spectrometry conditions to achieve the simultaneous detection of SYN007 and SYN045 in biological samples under the same conditions, thereby improving the detection efficiency.

[0031] (2) The analytical detection method provided by the present invention has good system adaptability, high selectivity and good linearity. The average RE range of the accuracy of SYN045 detected simultaneously is -9.76%~10.03%, the precision RSD≤7.57%, and the average RE range of the accuracy of SYN007 is -7.68%~14.68%, and the precision RSD≤9.64%. The accuracy and precision meet the requirements; the analyte and internal standard recovery rates of SYN045 at all concentration levels are RSD≤6.72%, and the analyte and internal standard recovery rates of SYN007 at all concentration levels are RSD≤4.32%, which meet the acceptance criteria (RSD≤15.00%); the internal standard normalized matrix factor RSDs of SYN045 and SYN007 at 2 concentration levels (LQC and HQC) of 6 batches of matrix are RSD≤12.32% and RSD≤9.00%, respectively, and the results meet the acceptance criteria. The present invention can accurately determine the content of compounds SYN007 and SYN045 in biological samples.

[0032] (3) At the same time, the biological sample processing method is simple. There is no need to acidify the plasma with hydrochloric acid. The supernatant can be directly injected after vortexing, which reduces the sample loss caused by nitrogen blowing, heating, and re-dissolution operations. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a peak area ratio-concentration linear graph of SYN045 and SYN007 in Example 1 of the present invention; Figure 2 The mass spectra of SYN045 and the internal standard in the system suitability test in the verification example of the present invention; Figure 3 The mass spectra of SYN007 and the internal standard in the system suitability test in the verification example of the present invention; Figure 4 This is the mass spectrum of the blank matrix sample in the selectivity test in the verification example of the present invention; Figure 5 The mass spectra of SYN045+LLOQ+IS samples and SYN007+LLOQ+IS samples in the selectivity test in the verification example of the present invention are shown; Figure 6 This is the mass spectrum of blank matrix + IS sample in the selectivity test in the verification example of the present invention; Figure 7 The mass spectra of SYN045+ULOQ samples and SYN007+ULOQ samples in the selectivity test in the verification example of the present invention are shown; Figure 8 This is a linear graph of the peak area ratio-concentration of SYN045 and SYN007 in the standard curve test in the verification example of the present invention. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below through specific implementation modes, but it is only used to help understand the present invention so that professionals in the field can implement or use the present invention, and does not constitute any limitation to the present invention.

[0035] The following is a description with reference to specific embodiments.

[0036] Example 1 The drugs and reagents used in this example are: SYN045 (Shijiazhuang Siyao Co., Ltd., batch number: SYN045-20220503); SYN007 (Shijiazhuang Siyao Co., Ltd., batch number: NEW-INT-20220503); methanol; acetonitrile; isopropanol; ammonium acetate; and formic acid.

[0037] This embodiment provides an analytical method for simultaneously detecting compounds SYN007 and SYN045 in a biological sample, the analytical method comprising the following steps: S1: Solution preparation Mobile phase A: Pipette a certain amount of formic acid into the solvent bottle, add a certain amount of ammonium acetate, add a certain amount of ultrapure water and mix well to prepare a 0.5 mM ammonium acetate aqueous solution containing 0.1% formic acid (v:v=1:1000).

[0038] Mobile phase B: Pipette a certain amount of formic acid into the solvent bottle, add a certain amount of acetonitrile and mix well to prepare an acetonitrile solution containing 0.1% formic acid (v:v=1:1000).

[0039] Internal standard working solution: accurately weigh an appropriate amount of warfarin (internal standard), convert its mass according to the correction factor, and dissolve it in methanol to prepare an internal standard stock solution with a concentration of 1 mg / mL; take 50 μL of the internal standard stock solution and use 50% acetonitrile methanol (v:v=1:1) to make the volume to 1000 ml to obtain a 50 ng / mL internal standard working solution.

[0040] SYN045 standard curve samples: Weigh an appropriate amount of SYN045 and dissolve it in methanol to prepare a 2mg / mL stock solution. Prepare SYN045 solutions with concentrations of 10000ng / mL, 5000ng / mL, 2500ng / mL, 1000ng / mL, 500ng / mL, 100ng / mL, 50ng / mL, and 10ng / mL, respectively. Take 20µL of each of the above concentration solutions and add 380µL of blank matrix (rat plasma) to obtain standard curve samples with concentrations of 500ng / mL, 250ng / mL, 125ng / mL, 50ng / mL, 25ng / mL, 5ng / mL, 2.5ng / mL, and 0.5ng / mL.

[0041] SYN007 standard curve samples: Weigh an appropriate amount of SYN007 and dissolve it in methanol to prepare a 2mg / mL stock solution. Prepare SYN007 solutions of 20000ng / mL, 10000ng / mL, 5000ng / mL, 200ng / mL, 1000ng / mL, 200ng / mL, 100ng / mL, and 20ng / mL respectively; take 20µL of each of the above concentration solutions and add 380µL of blank matrix (rat plasma) to obtain standard curve samples with concentrations of 1000ng / mL, 500ng / mL, 250ng / mL, 100ng / mL, 50ng / mL, 10ng / mL, 5ng / mL, and 1ng / mL.

[0042] S2: Sample processing About 0.25 mL of blood was collected by jugular vein puncture and placed in an anticoagulant tube containing K2EDTA, and centrifuged at 4°C for 6 minutes (centrifugal force 6800g). Take 30μL of plasma, add 200μL of internal standard working solution, vortex mix, centrifuge at 4°C for 7 minutes (centrifugal force 18000g), and take the supernatant to obtain the sample to be tested for later use.

[0043] Take 30 µL of SYN045 standard curve sample and SYN007 standard curve sample, add 200 µL of internal standard working solution respectively, vortex mix, centrifuge at 4°C for 7 minutes (centrifugal force 18000g), and take the supernatant to obtain SYN045 standard curve loading solution and SYN007 standard curve loading solution for use.

[0044] S3: Liquid quality testing Take 180 μL of the sample to be tested, the loading solution for the SYN045 standard curve, and the loading solution for the SYN007 standard curve, add them to the corresponding 96-well injection plate, use liquid chromatography-mass spectrometry (LC-MS / MS) for detection and analysis, and record the chromatogram. The liquid phase conditions and mass spectrometry conditions are shown in Tables 1 and 2.

[0045] Table 1 Liquid phase conditions

[0046] Table 2 Mass spectrometry conditions

[0047] S4: Analysis of quantitative calculation results The compounds SYN045 and SYN007 in the sample solution were determined according to the retention time in the sample chromatogram. The SYN045 linear regression equation was made based on the ratio of the peak area of ​​the SYN045 chromatographic peak measured by the loading solution of the SYN045 standard curve to the peak area of ​​the internal standard and the corresponding SYN045 concentration. The SYN007 linear regression equation was made based on the ratio of the peak area of ​​the SYN007 chromatographic peak measured by the loading solution of the SYN007 standard curve to the peak area of ​​the internal standard and the corresponding SYN007 concentration as shown in Table 3, where y is the peak area ratio and x is the concentration. The resulting linear graph is shown in the attached figure. Figure 1 shown.

[0048] The internal standard curve method was used for quantification. The concentrations of compounds SYN045 and SYN007 in the sample solution were determined by the ratio of the peak area of ​​SYN045 to the peak area of ​​the internal standard and the ratio of the peak area of ​​SYN007 to the peak area of ​​the internal standard. The results are shown in Table 4.

[0049] Table 3 Linear regression equation

[0050] Table 4 Results of plasma concentrations of SYN045 and SYN007 in rats after oral administration of SYN045

[0051] Verification Example 1 System adaptability Test method: Prepare 6 tubes of SYN045 and SYN007 standard solutions with a theoretical concentration of 100 μg / ml in parallel as system adaptability solutions, then insert the needle for detection and record the chromatogram, as shown in the attached figure. Figure 2 , Attachment Figure 3 shown.

[0052] Experimental results: The retention time of SYN045 was 0.678-0.703min, the retention time of SYN007 was 0.698-0.722min, the retention time of the internal standard was 0.458-0.483min, and the separation degree between the analyte peak and the adjacent peak was >1.5.

[0053] Conclusion: In this experiment, the detection method has good system adaptability for detecting SYN045 and SYN007.

[0054] 2 Selectivity Test method: S1: Sample preparation (1) Blank matrix samples: Six different batches of blank matrix were prepared without adding analytes and internal standards.

[0055] (2) Lower limit of quantitation (LLOQ) samples: blank matrices from 6 different batches were spiked with analytes at the LLOQ concentration level (including SYN045 and SYN007) and internal standards at the working concentration level to prepare SYN045 sample solutions with a concentration of 0.5 ng / mL and SYN007 sample solutions with a concentration of 1 ng / mL).

[0056] (3) Blank matrix containing only internal standard: Add only the internal standard at the working concentration level (50 ng / mL) to the mixed blank matrix and prepare three parallel samples.

[0057] (4) Blank matrix containing only analytes at the ULOQ (upper limit of quantitation) concentration level: Add only analytes at the ULOQ concentration level (including SYN045 and SYN007) to the mixed blank matrix to prepare a SYN045 sample solution with a concentration of 500 ng / mL and a SYN007 sample solution with a concentration of 1000 ng / mL), and prepare three parallel samples.

[0058] S2: Detection and Analysis The above sample solution was injected and tested by liquid chromatography-mass spectrometry (LC-MS / MS), and the chromatogram was recorded. Figure 4 , Attachment Figure 5 , Attachment Figure 6 and attached Figure 7 shown.

[0059] Test results: SYN045: The blank matrix had no interference with the analyte SYN045 and the internal standard; the analyte SYN045 had no interference with the internal standard.

[0060] SYN007: The blank matrix had no interference with the analyte SYN007 and the internal standard, and there was no interference between the analyte SYN007 and the internal standard.

[0061] Conclusion: In this study, the assay had good selectivity for detecting SYN045 and SYN007.

[0062] 3 Standard curve Test method: S1: Sample preparation Weigh an appropriate amount of SYN045 and dissolve it in methanol to prepare a 2 mg / ml stock solution. Prepare SYN045 solutions with concentrations of 10000, 5000, 2500, 1000, 500, 100, 50, and 10 ng / ml, respectively. Take 20 µL of each of the above concentration solutions and add 380 µL of blank matrix (rat plasma) to obtain standard curve samples with concentrations of 500, 250, 125, 50, 25, 5, 2.5, and 0.5 ng / mL.

[0063] Weigh an appropriate amount of SYN007 and dissolve it in methanol to prepare a 2 mg / ml stock solution. Prepare SYN007 solutions of 20000, 10000, 5000, 2000, 1000, 200, 100, and 20 ng / ml respectively; take 20 µL of each of the above concentration solutions and add 380 µL of blank matrix (rat plasma) to obtain standard curve samples with concentrations of 1000, 500, 250, 100, 50, 10, 5, and 1 ng / mL.

[0064] S2: Detection and Analysis Take 30 µL of SYN045 standard curve sample and SYN007 standard curve sample, add 200 µL of internal standard working solution, vortex mix, centrifuge at 4°C for 7 min (centrifugal force 18000 g), take 180 µL of supernatant as the loading solution for SYN045 standard curve and SYN007 standard curve, perform LC-MS / MS analysis, and record the chromatogram.

[0065] Test results: The SYN045 linear regression equation was made based on the ratio of the peak area of ​​the SYN045 chromatographic peak measured by the loading solution of the SYN045 standard curve to the peak area of ​​the internal standard and the corresponding SYN045 concentration. The SYN007 linear regression equation was made based on the ratio of the peak area of ​​the SYN007 chromatographic peak measured by the loading solution of the SYN007 standard curve to the peak area of ​​the internal standard and the corresponding SYN007 concentration. The results are shown in Table 5, where y is the peak area, x is the concentration, and the linear graph is shown in the attached figure. Figure 8 shown.

[0066] Table 5 Linear regression equation

[0067] Conclusion: In this study, the detection method had good linearity for SYN045 and SYN007.

[0068] 4 Accuracy and precision Test method: Take appropriate amount of SYN045 and SYN007 standard stock solutions, dilute them with formaldehyde to concentrations of 30, 400, 4000, and 8000 ng / ml of SYN045 and SYN007 standard solutions, take 20 µL of each of the above standard solutions, add 380 µL of blank matrix (rat plasma) to obtain quality control samples with concentrations of 1.5, 20, 200, and 400 ng / mL. Repeat the injection of each quality control sample 6 times. Calculate the accuracy and precision of the SYN045 and SYN007 sample detection, and the results are shown in Tables 6 and 7.

[0069] Test results: The average RE range of accuracy of SYN045 was -9.76%~10.03%, and the precision RSD was ≤7.57%; the average RE range of accuracy of SYN007 was -7.68% ~ 14.68%, and the precision RSD was ≤9.64%.

[0070] Table 6 Accuracy and precision of SYN045

[0071] Table 7 Accuracy and precision of SYN007

[0072] RE (%): Accuracy (%)-100 #RE does not meet the acceptance criteria, but participates in the calculation Conclusion: At least 50% of the quality control samples at each concentration level in the above samples meet the acceptance criteria.

[0073] 5 Recovery rate Test method: Preparation of spiked matrix samples before extraction: Take 1.5 μL of HQC (high concentration quality control sample), MQC (medium concentration quality control sample) and LQC (low concentration quality control sample) working solutions, add them to 28.5 μL blank matrix, vortex mix, add 200 μL internal standard working solution, vortex mix, centrifuge at 4°C for 7 min (18000 g), and prepare 6 copies for each concentration level.

[0074] Preparation of spiked matrix samples after extraction: Take 28.5 μL of mixed blank matrix, add 200 μL of internal standard working solution, vortex mix, add 1.5 μL of HQC, MQC and LQC working solutions respectively after matrix extraction, vortex mix, centrifuge at 4°C for 7 minutes (18000g), and prepare 6 copies for each concentration level.

[0075] The above sample solutions were injected and tested by liquid chromatography-mass spectrometry (LC-MS / MS), and the recoveries of SYN045 and SYN007 samples were calculated. The results are shown in Tables 8 and 9.

[0076] Test results: The analyte recovery rate of SYN045 at each concentration level was between 74.69% and 91.15%, and the internal standard recovery rate was between 95.28% and 101.63%. The RSD of the analyte and internal standard recoveries at each concentration level was ≤3.61%.

[0077] The analyte recovery rate of SYN007 at each concentration level was 77.69%-91.90%, and the internal standard recovery rate was 95.28%-101.63%. The RSD of the analyte and internal standard recovery rates at each concentration level was ≤3.39%.

[0078] Table 8 Recovery rate of SYN045

[0079] Table 9 Recovery rate of SYN007

[0080] Conclusion: The RSDs of the recoveries of the analytes and internal standards at all concentration levels of SYN045 were ≤6.72%, and the RSDs of the recoveries of the analytes and internal standards at all concentration levels of SYN007 were ≤4.32%, which met their acceptance criteria (RSD≤15.00%).

[0081] 6 Matrix Effects Test method: Solution sample preparation: Take 1.5 μL of HQC and LQC working solutions respectively, add them to 28.5 μL ultrapure water, then add 200 μL of internal standard working solution, vortex to mix, and prepare 3 copies for each concentration level.

[0082] Matrix sample preparation: Take 28.5 μL of blank matrix from 6 different donors, add 200 μL of internal standard working solution, vortex mix, add 1.5 μL of HQC and LQC working solution respectively after matrix extraction, vortex mix, and centrifuge at 4°C for 7 minutes (18000g).

[0083] Preparation of quantitative matrix samples: Take 1.5 μL of HQC and LQC working solutions respectively, add 28.5 μL of blank matrix, vortex to mix, and prepare 3 copies for each concentration level.

[0084] The above sample solutions were injected and tested by liquid chromatography-mass spectrometry (LC-MS / MS), and the matrix effect factors of SYN045 and SYN007 samples were calculated. The results are shown in Tables 10 and 11.

[0085] Test results: SYN045: The RSD of the internal standard normalized matrix factor of 6 batches of matrix at 2 concentration levels (LQC and HQC) was ≤12.32%, which met its acceptance criteria.

[0086] SYN007: The RSD of the internal standard normalized matrix factors for 6 batches of matrix at 2 concentration levels (LQC and HQC) was ≤9.00%, which met its acceptance criteria.

[0087] Table 10 SYN045 matrix effect

[0088] Table 11 SYN007 matrix effect

[0089] 7. Durability According to the durability investigation range in Table 12, the influence of slight changes in chromatographic conditions such as column temperature and formic acid concentration in the mobile phase on the separation degree of SYN045 or SYN007 and adjacent peaks was investigated.

[0090] Test method: The solution preparation method and the detection method refer to Example 1, and the chromatographic conditions are fine-tuned according to the durability investigation range of Table 12. The flow rate and gradient elution conditions remain unchanged. The tests are carried out respectively and the chromatograms are recorded. The results are shown in Table 13.

[0091] Test results: The results show that fine-tuning of chromatographic conditions, such as slight changes in column temperature and formic acid concentration in the mobile phase, has no effect on the detection, and the separation between SYN045 or SYN007 and adjacent peaks is not less than 1.5, indicating that the detection method of the present application has good durability.

[0092] Table 12 Durability test range Investigation parameters Standard chromatographic conditions Durability inspection range Column temperature 40℃ 35℃～45℃ Formic acid concentration in mobile phase A 0.1% 0.08%-0.12% Formic acid concentration in mobile phase B 0.1% 0.08%-0.12% Table 13 Durability test results

[0093] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modification, equivalent substitution or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for simultaneously determining the content of compound SYN045 and its metabolites in a biological sample, characterized in that: Liquid chromatography-mass spectrometry was used with warfarin as the internal standard; The chromatographic conditions are: Mobile phase A: 0.08-0.12 vol% formic acid in 0.5 mM ammonium acetate solution; Mobile phase B: 0.08-0.12 vol% formic acid in acetonitrile solution; mobile phase A and mobile phase B were eluted in gradient mode.

2. The method for simultaneously determining the content of compound SYN045 and its metabolites in a biological sample according to claim 1, characterized in that: The gradient elution conditions are shown in the following table: 。 3. The method for simultaneously determining the content of compound SYN045 and its metabolites in a biological sample according to claim 1 or 2, characterized in that: The specific steps include: S1. Preparing a standard curve equation, including: preparing an internal standard working solution and a standard working solution, preparing a sample solution for the standard curve, and detecting the sample solution for the standard curve by liquid chromatography-mass spectrometry to obtain a standard curve equation for the content of SYN045 and its metabolites; S2: Sample processing Collect blood and place it in an anticoagulant tube, centrifuge it, take the plasma, add the internal standard working solution, vortex mix, centrifuge it, and take the supernatant for later use; S3: Liquid quality testing The supernatant was detected and analyzed by liquid chromatography-mass spectrometry, and the concentration of compound SYN045 and its metabolites in the sample solution was calculated according to the standard curve equation.

4. The method according to claim 3, characterized in that In step S3, the chromatographic column used in the liquid chromatography is ACQUITY UPLC® BEH C18, the chromatographic column diameter is 2.1 mm, the chromatographic column length is 50 mm, the filler particle size is 1.7 μm; the column temperature is 35° C. to 45° C.; the flow rate is 0.5 mL / min.

5. The method according to claim 3, characterized in that The analysis method according to claim 1 is characterized in that in step S3, the mass spectrometry conditions are ionization mode: electrospray ionization, positive ion mode, multiple reaction monitoring; ion spray voltage: 5500V; ion source temperature: 550°C; collision activated dissociation: 10; curtain gas: 35 psi; ion source gas 1: 60 psi; ion source gas 2: 60 psi.

6. The analysis method according to claim 3, characterized in that In step S1, the internal standard working solution is prepared: First, prepare a 1 mg / mL warfarin methanol solution as the internal standard stock solution; Take 50 μL of the internal standard stock solution and dilute it to 1000 ml with 50 vol% acetonitrile methanol to obtain an internal standard working solution with a concentration of 50 ng / mL.

7. The analysis method according to claim 3, characterized in that In step S1, the standard working solution includes a SYN045 standard curve sample and a SYN007 standard curve sample; Among them, the SYN045 standard curve samples are: take SYN045 methanol solutions with concentrations of 10000ng / mL, 5000ng / mL, 2500ng / mL, 1000ng / mL, 500ng / mL, 100ng / mL, 50ng / mL, and 10ng / mL, add blank matrix plasma respectively, and obtain SYN045 standard curve samples with concentrations of 500ng / mL, 250ng / mL, 125ng / mL, 50ng / mL, 25ng / mL, 5ng / mL, 2.5ng / mL, and 0.5ng / mL; The SYN007 standard curve samples are as follows: SYN007 methanol solutions with concentrations of 20000 ng / mL, 10000 ng / mL, 5000 ng / mL, 2000 ng / mL, 1000 ng / mL, 200 ng / mL, 100 ng / mL, and 20 ng / mL are added to blank matrix plasma to obtain SYN007 standard curve samples with concentrations of 1000 ng / mL, 500 ng / mL, 250 ng / mL, 100 ng / mL, 50 ng / mL, 10 ng / mL, 5 ng / mL, and 1 ng / mL; The preparation method of the standard curve loading solution is as follows: SYN045 standard curve sample and SYN007 standard curve sample are respectively taken, internal standard working solution is added to each standard curve sample, vortexed and mixed, centrifuged, and supernatant is taken to obtain SYN045 standard curve loading solution and SYN007 standard curve loading solution.

8. The analysis method according to claim 7, characterized in that In step S1, the preparation method of the standard curve loading solution is as follows: 30 µL of SYN045 standard curve sample and SYN007 standard curve sample are taken, 200 µL of internal standard working solution are added respectively, vortexed and mixed, centrifuged at 4°C, and the supernatant is taken to obtain the SYN045 standard curve loading solution and the SYN007 standard curve loading solution.

9. The analysis method according to claim 3, characterized in that In step S2, the sample processing is specifically as follows: 0.25 mL of blood is collected and placed in an anticoagulant tube containing K2EDTA, centrifuged at 4°C for 6 minutes with a centrifugal force of 6800g, 30 μL of plasma is taken, 200 μL of internal standard working solution is added, vortexed to mix, centrifuged at 4°C for 7 minutes with a centrifugal force of 18000g, and the supernatant is taken for later use.

10. The analysis method according to claim 3, characterized in that In step S3, 180 μL of the supernatant is taken.

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