Quantitative detection method for related substances of remegapam intermediate

Abstract

The invention provides a rapid, sensitive and accurate quantitative detection method for (R)-9-triisopropylsiloxy-6, 7, 8, 9-tetrahydro-5H-cycloheptyl [b] pyridine-5-ketone and related substances of (R)-9-triisopropylsiloxy-6, 7, 8, 9-tetrahydro-5H-cycloheptyl [b] pyridine-5-ketone. According to the method, the content of (R)-9-triisopropylsiloxy-6, 7, 8, 9-tetrahydro-5H-cycloheptyl [b] pyridine-5-ketone and related substances thereof can be quantitatively detected in the same chromatographic system, and effective separation is realized.

Description

Technical Field

[0001] The present invention belongs to the field of chemical pharmacy and drug analysis technology, and relates to a quantitative detection method for a remigipam intermediate. More specifically, the present invention relates to a quantitative detection method for (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-5-one and its related substances. Background Art

[0002] Rimegepant is a small molecule calcitonin gene-related peptide (CGRP) antagonist developed by Biohaven. It exerts its clinical effect by blocking the binding of CGRP ligand to receptors. It has been approved by the US FDA for the acute treatment and preventive treatment of migraine in adults. Rimegepant has the characteristics of high safety, good efficacy, and mild side effects, providing a new treatment option for migraine patients. The structure of Rimegepant is shown in the following formula:

[0003]

[0004] Remigipan structure

[0005] At present, the original research company has disclosed in detail the process synthesis route of Remigipam API in patents WO2012050764 and WO2013130402. Among them, (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-5-one (Formula I compound) is a key chiral intermediate for the synthesis of Remigipam, and its purity greatly affects the quality of Remigipam API. (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-5-one is usually synthesized from (R)-9-hydroxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-5-one (Formula II compound) containing chiral hydroxyl groups, and the specific route is shown in Scheme 1.

[0006]

[0007] Solution 1

[0008] However, in the synthesis process of (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-5-one, the introduced triisopropylsilyl group as a hydroxyl protecting group often acts on other sites at the same time to generate corresponding impurities, as shown in Scheme 2. The introduced impurities may not only react with reactants or intermediates to generate new impurities, but may also remain in the subsequent process, resulting in a decrease in the yield of the target product and having an important impact on the quality and safety of the final Remigipam. In order to ensure the safety and effectiveness of the drug, high performance liquid chromatography (HPLC) technology is usually used to qualitatively and quantitatively analyze the impurities in the drug. In the analysis of the compound of formula I and related substances, the compounds of formula III and formula IV exhibit lower polarity due to the presence of two triisopropylsilyl groups, and are difficult to be eluted by the mobile phase in a conventional reversed phase liquid chromatography system; and the structural differences between the two are small, and it is difficult to achieve effective separation in the same chromatographic system. At the same time, some of the unreacted compound of formula II remains in the intermediate (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-5-one, which also needs to be controlled and detected. The compound of formula II contains a hydroxyl group and has a relatively large polarity. It is easier to elute in a reversed phase liquid chromatography system and has an earlier peak time.

[0009]

[0010] Solution 2

[0011] Therefore, how to achieve effective separation of (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-5-one and its related substances in the same chromatographic system has become the focus of research, which is also of great significance for the subsequent quality control of Remigipan API. After investigation, no existing technology can be used to detect (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-5-one and its related substances.

[0012] Based on this, the present invention provides a rapid, sensitive and accurate high performance liquid chromatography analysis method, which greatly improves the quality detection efficiency of (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-5-one by detecting (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-5-one and its related substances in the same chromatography system, thereby achieving effective control of the quality of the target product (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-5-one. Summary of the invention

[0013] The present invention provides a quantitative detection method for (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-5-one and related substances, characterized in that:

[0014] A quantitative detection method for related substances of (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-5-one as shown in formula I, characterized in that: a liquid chromatograph equipped with an ultraviolet detector is used in the quantitative detection method; a bonded silica gel material is used as the stationary phase; a ternary elution system is used as the mobile phase, wherein mobile phase A is an aqueous solution system, and mobile phase B and mobile phase C are both organic solvents or organic solvent-aqueous solution mixed systems;

[0015] I.

[0016] The quantitative detection method described in the present invention is characterized in that: the method is used for quantitative analysis and detection of a mixture of any one or any combination of the compound represented by Formula I and the compounds represented by Formula II, Formula III, and Formula IV, II, III, IV.

[0017] The quantitative detection method described in the present invention is characterized in that: in the liquid chromatography method, the stationary phase adopts a bonded silica gel material selected from a carbon octadecyl bonded silica gel material and a carbon octadecyl bonded silica gel material, the particle size of the stationary phase bonded silica gel material is selected from 3μm or 5μm, and the size of the chromatographic column filled with the stationary phase bonded silica gel material is selected from 4.6mm×150mm and 4.6mm×250mm.

[0018] The quantitative detection method described in the present invention is characterized in that: in the liquid chromatography method, mobile phase A is an aqueous solution selected from purified water or a salt solution of purified water; mobile phase B and mobile phase C are both organic solvents or organic solvent-aqueous solution mixed systems selected from acetonitrile, tetrahydrofuran or a mixture of the two in any ratio, an acetonitrile-aqueous solution mixture, and a tetrahydrofuran-water homogeneous mixture.

[0019] The quantitative detection method described in the present invention is characterized in that: in the liquid chromatography method, the elution gradient of the mobile phase is 0→3 minutes, mobile phase A: mobile phase B: mobile phase C=90:8:2; 3→15 minutes, mobile phase A: mobile phase B: mobile phase C=0:85:15; 15→40 minutes, mobile phase A: mobile phase B: mobile phase C=0:85:15; 40→42 minutes, mobile phase A: mobile phase B: mobile phase C=90:8:2; 42→50 minutes, mobile phase A: mobile phase B: mobile phase C=90:8:2.

[0020] The quantitative detection method described in the present invention is characterized in that: in the liquid chromatography method, the column temperature of the chromatographic column is maintained at 45±5°C.

[0021] The quantitative detection method described in the present invention is characterized in that: in the liquid chromatography method, the detection wavelength is 262±5nm.

[0022] The quantitative detection method described in the present invention is characterized in that: in the liquid chromatography method, the injection volume is 10±5μL.

[0023] The quantitative detection method described in the present invention is characterized in that: in the liquid chromatography method, the flow rate of the mobile phase is 1.1±0.2 ml / min.

[0024] The quantitative detection method described in the present invention is characterized in that: in the liquid chromatography method, the sample diluent is an acetonitrile-water mixture.

[0025] The quantitative detection method described in the present invention realizes the quantitative detection of the content of (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridine-5-one and its related substances in the same chromatographic system, and the detection is accurate, sensitive and efficient.

[0026] Terminology: The specification appendix in this invention Figure 1-Figure 3 , Figure 8-Figure 12 "Detector" refers to the detector. "Peak" refers to the peak. "Ret. Time" refers to the retention time. "Height" refers to the peak height. "Area" refers to the peak area. "Separation Factor" refers to the separation factor. "Area%" refers to the peak area percentage. "RG5T" represents the compound of formula I, "RG5" represents the compound of formula II, "RG5E" represents the compound of formula III, and "RG5F" represents the compound of formula IV. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Attached Figure 1 Typical spectra of the system applicability of the method described in the present invention.

[0028] Attached Figure 2 Typical spectra of the quantitative limit of the compound of formula I in the method of the present invention.

[0029] Attached Figure 3 Typical spectra of the detection limit of the compound of formula I in the method of the present invention.

[0030] Attached Figure 4 The method of the present invention provides a linear equation standard curve for the compound of formula I.

[0031] Attached Figure 5 The method of the present invention provides a linear equation standard curve for the compound of formula II.

[0032] Attached Figure 6 The method of the present invention provides a linear equation standard curve for the compound of formula III.

[0033] Attached Figure 7 The method of the present invention provides a linear equation standard curve for the compound of formula IV.

[0034] Attached Figure 8 The method of the present invention detects a typical spectrum of a test solution.

[0035] Attached Fig. 9 Liquid phase spectrum of method comparative example 1.

[0036] Attached Fig.10 Liquid phase spectrum of method comparative example 2.

[0037] Attached Fig.11 Liquid phase spectrum of method comparative example 3.

[0038] Attached Fig.12 Liquid phase spectrum of method comparative example 4. DETAILED DESCRIPTION

[0039] The following specific embodiments are provided to enable those skilled in the art to fully understand the present invention, but are not intended to limit the present invention in any way.

[0040] In the following examples, unless otherwise specified, all temperatures are Celsius; unless otherwise specified, the room temperature is 20-30°C; unless otherwise specified, all starting materials and reagents are commercially available and are used directly without further purification; unless otherwise specified, all solvents used for chromatographic detection are chromatographic grade reagents and are used directly without further treatment; unless otherwise specified, commercial manufacturers include but are not limited to Hangzhou Chemical Reagent, Sinopharm Reagent, etc. In the following examples, (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cycloheptyl[b]pyridine-5-one reference substance and its test samples are self-made products of Zhejiang Hongyuan Pharmaceutical Co., Ltd. In the following examples, the liquid chromatography instrument is: Agilent 1260 liquid chromatograph.

[0041] Example 1: System Applicability and Specificity Investigation of the Quantitative Detection Method of the Present Invention

[0042] (1) Brief description: The system suitability was evaluated by repeated injection of 6 times, with the relative standard deviation (RSD) of the main component no greater than 2.0%, the RSD of the programmed control solution no greater than 3.0%, and the separation degree between the main peak and related impurities >1.5; the method specificity was evaluated by comparison with the blank solution.

[0043] (2) Solution preparation:

[0044] Impurity stock solution: Weigh 12.55 mg of the compound of formula II, 12.49 mg of the compound of formula III, and 12.43 mg of the compound of formula IV respectively, place them in a 50 mL volumetric flask, dissolve them in ethanol and make up to the mark, and mix well.

[0045] Sample stock solution: Weigh 500.00 mg of the compound of formula I into a 50 mL volumetric flask, dilute to volume with 90% acetonitrile water, and mix well.

[0046] Sample standard solution: Pipette 5 mL of sample stock solution into a 25 mL volumetric flask, dilute with 60% acetonitrile water to volume, mix well. Take 1 mL each and dispense into 3 different injection vials, and place at room temperature.

[0047] System suitability solution: Pipette 2 mL of impurity stock solution and 5 mL of sample stock solution into a 25 mL volumetric flask, dilute with 60% acetonitrile water to volume, mix well. Take 1 mL of each into 3 different injection vials and place at room temperature.

[0048] Correction factor solution: Pipette 0.5 mL of sample standard solution and 4 mL of impurity stock solution into a 50 mL volumetric flask, dilute to volume with 60% acetonitrile water, and mix well.

[0049] (3) Experimental results

[0050] Table 1: System suitability solution test results

[0051]

[0052] Table 2: Separation test results

[0053]

[0054] Table 2 System suitability The minimum separation between the sample and related impurities in the solution is 2.8>1.5, which meets the specified requirements for separation.

[0055] Table 3: Blank solution test results

[0056]

[0057] The blank solution experimental results showed that the blank solution had no interference with the detection of samples and related impurities and had good specificity.

[0058] Table 4: Correction factor experimental results

[0059]

[0060] As shown in Table 4, the correction factor of the compound of formula II is 0.73, the correction factor of the compound of formula IV is 0.87, and the correction factor of the compound of formula III is 1.89.

[0061] The typical spectra of the relevant detection in this embodiment are shown in the appendix of the specification. Figure 1 .

[0062] Example 2: Investigation of the quantitative limit, detection limit and linear range of the quantitative detection method of the present invention

[0063] (1) Brief description: The limit of quantification (LOQ) is the lowest amount of the substance in the sample that can be quantitatively determined, and the limit of detection (LOD) is the lowest amount of the substance in the sample that can be detected. The signal-to-noise ratio method is used to calculate the lowest concentration or amount of the substance that can be reliably detected. The LOD is determined when the signal-to-noise ratio is 3, and the LOQ is determined when the signal-to-noise ratio is 10.

[0064] Take 5 relatively even concentration points in the concentration range of 80% to 120% of the compound of formula I, and take 6 relatively even concentration points in the concentration range of the impurity LOQ to 300%, draw a curve with concentration as the abscissa and peak area as the ordinate, and calculate the linear regression coefficient R of the curve 2 The numerical value of .

[0065] (2) Solution preparation

[0066] Linear stock solution of the compound of formula I: weigh 500.00 mg of the compound of formula I and place it in a 50 mL volumetric flask, dilute to the mark with 90% acetonitrile water, and mix well.

[0067] Linear stock solution of compound of formula II: Weigh 14.36 mg of compound of formula II into a 25 mL volumetric flask, dilute to scale with 60% acetonitrile water, and mix well.

[0068] Linear stock solution of compound III: Weigh 20.08 mg of compound III into a 10 mL volumetric flask, dilute to scale with acetonitrile, and mix well.

[0069] Linear stock solution of compound IV: Weigh 21.54 mg of compound IV into a 10 mL volumetric flask, dilute to scale with ethanol, and mix well.

[0070] Impurity linear stock solution: Pipette 5 mL of the linear stock solution of the compound of formula II, 5 mL of the linear stock solution of the compound of formula III, and 5 mL of the linear stock solution of the compound of formula IV into a 25 mL volumetric flask, dilute to the scale with 60% acetonitrile water, and mix well.

[0071] LOQ solution: Pipette 1 mL of impurity linear stock solution into a 100 mL volumetric flask, dilute to scale with 60% acetonitrile water, and mix well.

[0072] LOD solution: Pipette 3 mL of LOQ solution into a 10 mL volumetric flask, dilute to the mark with 60% acetonitrile water, and mix well.

[0073] Standard curve solution: Pipette the linear stock solution of the compound of formula I into a 25 mL volumetric flask, dilute to the mark with acetonitrile, and mix well.

[0074] Impurity standard curve solution: Transfer the impurity linear stock solution into a 20 mL volumetric flask, dilute to the mark with acetonitrile, and mix well.

[0075] (3) Experimental results

[0076] Table 5: Results of limit of quantitation experiments

[0077]

[0078] The LOQ shown in Table 5 is equivalent to a sample concentration of 0.05%. Among the three-injection LOQs, the minimum S / N value of the compound of formula II is 11.64, the minimum S / N value of the compound of formula IV is 56.11, and the minimum S / N value of the compound of formula III is 22.34, all of which are greater than 10 and meet the specified requirements.

[0079] Table 6: Detection limit experimental results

[0080]

[0081] The LOD shown in Table 6 is equivalent to a sample concentration of 0.015%. The S / N value of the compound of formula II is 4.15, the S / N value of the compound of formula IV is 22.52, and the S / N value of the compound of formula III is 8.89, all of which are greater than 3 and meet the specified requirements.

[0082] The typical spectra of the relevant detection in this embodiment are shown in the appendix of the specification. Figure 2-Figure 3 .

[0083] Table 7: Linear range experimental results of compounds of formula I

[0084]

[0085] The concentration and peak area of ​​the compound of formula I shown in Table 7 are in the range of 1.6 mg / mL to 2.4 mg / mL and the linear regression coefficient R 2 is 0.9998.

[0086] Table 8: Linear range experimental results of compounds of formula II

[0087]

[0088] The concentration and peak area of ​​the compound of formula II shown in Table 8 are in the range of 0.0011 mg / mL to 0.0345 mg / mL, and the linear regression coefficient R 2 is 0.9998.

[0089] Table 9: Linear range experimental results of compounds of formula III

[0090]

[0091] The concentration and peak area of ​​the compound of formula III shown in Table 9 are in the range of 0.0040 mg / mL to 0.1205 mg / mL, and the linear regression coefficient R 2 is 0.9984.

[0092] Table 10: Linear range experimental results of compounds of formula IV

[0093]

[0094] The concentration and peak area of ​​the compound of formula IV shown in Table 10 are in the range of 0.0043 mg / mL to 0.1292 mg / mL, and the linear regression coefficient R 2 is 0.9987.

[0095] The standard curve of the linear equation of the method in this embodiment is shown in the appendix of the specification. Figure 4-Figure 7 .

[0096] Example 3: Investigation of solution stability of the quantitative detection method of the present invention

[0097] (1) Brief description: The sample standard solution and system suitability solution were evaluated for their changes over time. The sample standard solution and system suitability solution in the injection vial of Example 1 were placed for 2 days and compared with the contents on day 0. The relative deviation of purity was ≤2.0%, and the stability of the solution was investigated.

[0098] (2) Solution preparation

[0099] For the preparation of system suitability solutions and sample standard solutions, refer to the method for system suitability & specificity.

[0100] (3) Experimental results

[0101] Table 11: Results of stability test of sample standard solutions

[0102]

[0103] Table 12: System suitability solution stability test results

[0104]

[0105] The relative deviations of the purity of the compound of formula I after the sample standard solution and the system suitability solution were placed at room temperature for 2 days and 0 day were 0.33% and 0.31%, respectively, which were within the range of 2.0%, that is, the sample solution and the system suitability solution were stable after being placed at room temperature for 2 days.

[0106] Example 4: Study on the Durability of the Quantitative Detection Method 5 of the Present Invention

[0107] (1) Brief description: By changing the flow rate, column temperature, chromatographic column and other conditions, the effect of parameter changes on the analytical method is evaluated. The separation degree between the main peak and related impurities>1.5 is used to examine the robustness of the method.

[0108] (2) Solution preparation

[0109] For the preparation of system suitability solutions, refer to the System Suitability & Specificity Method.

[0110] (3) Experimental results

[0111] Table 13: Method durability test results

[0112]

[0113] The method durability test results shown in Table 13 show that under normal conditions, the minimum separation of the components in the system suitability solution is 2.8, which is greater than 1.5 and meets the specified requirements. After the condition parameters are changed, the minimum separation of the components in the system suitability solution is 2.6, which is greater than 1.5 and meets the specified requirements.

[0114] Example 5: Repeatability study of the quantitative detection method of the present invention

[0115] (1) Brief description: Repeated injection of 6 injections of sample standard solution, RSD is no more than 2.0% to examine the repeatability of the method.

[0116] (2) Solution preparation

[0117] Blank solution: 60% acetonitrile water.

[0118] Sample Stock Solution: See System Suitability & Specificity for preparation.

[0119] Sample standard solution: Weigh 20.02 mg, 20.06 mg, 19.99 mg, 20.04 mg, 20.06 mg and 20.05 mg of the compound of formula I into 6 different 10 mL volumetric flasks, dilute to the mark with 60% acetonitrile water, and mix well.

[0120] (3) Experimental results

[0121] Table 14: Repeatability test results

[0122]

[0123] The above repeatability experimental results show that the RSD value of the 6-injection sample standard solution is 0.55%, which is less than 2%, meeting the specified requirements. The detection method has good repeatability.

[0124] Example 6: Accuracy of the quantitative detection method of the present invention

[0125] (1) Brief description: The accuracy of the method was evaluated by measuring the recovery rate between the measured results and the theoretical value of sample solutions with different concentrations (80%, 100%, 120%).

[0126] (2) Solution preparation

[0127] Blank solution: 60% acetonitrile water.

[0128] Sample Stock Solution: See System Suitability & Specificity for preparation.

[0129] Sample standard solution: weigh 10.00 mg of the compound of formula I into a 5 mL volumetric flask, dilute to the mark with 60% acetonitrile water, and mix well.

[0130] 80% sample standard solution: Weigh 16.04 mg, 16.04 mg, and 16.02 mg of the compound of formula I into three different 10 mL volumetric flasks, dilute to the mark with 60% acetonitrile water, and mix well.

[0131] 100% sample standard solution: Weigh 19.98 mg, 20.03 mg, and 20.04 mg of the compound of formula I into three different 10 mL volumetric flasks, dilute to the mark with 60% acetonitrile water, and mix well.

[0132] 120% sample standard solution: Weigh 23.99 mg, 24.02 mg, and 24.00 mg of the compound of formula I into three different 10 mL volumetric flasks, dilute to the mark with 60% acetonitrile water, and mix well.

[0133] (3) Experimental results

[0134] Table 15: Accuracy test results

[0135]

[0136] For each sample in the sample standard solution shown in Table 15, the minimum recovery rate was 99.8% and the maximum recovery rate was 100.8%, both within the range of 98% to 102%. The recovery rate was good and met the specified requirements.

[0137] Example 7: Detection of (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-5-one by the quantitative detection method of the present invention

[0138] Chromatographic conditions: column: Inertsustain AQ-C18 4.6*150mm, 3μm or equivalent column; flow rate: 1.1mL / min; column temperature: 45℃; wavelength: 262nm; injection volume: 10μL; running time: 50min; mobile phase A: purified water; mobile phase B: acetonitrile; mobile phase C: tetrahydrofuran; diluent: 60% acetonitrile water.

[0139] Blank solution preparation: 60% acetonitrile water.

[0140] Preparation of test solution: Weigh 100 mg of (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-5-one test sample, dissolve it in a 50 mL volumetric flask with 60% acetonitrile water and dilute to the mark, and mix well.

[0141] Preparation of impurity stock solution: Weigh 12.5 mg of compound of formula II, dissolve it in 60% acetonitrile water in a 50 mL volumetric flask and make up to the scale; weigh 12.5 mg of compound of formula III, dissolve it in acetonitrile in a 50 mL volumetric flask and make up to the scale; weigh 12.5 mg of compound of formula IV, dissolve it in ethanol in a 50 mL volumetric flask and make up to the scale, and mix well.

[0142] System adaptability solution: Weigh 50 mg of the compound of formula I into a 25 mL volumetric flask, add an appropriate amount of diluent to dissolve it, then transfer 2.00 mL of each impurity stock solution, dilute to the mark with diluent, and mix well.

[0143] Injection procedure: blank solution (2 injections), system suitability solution (1 injection), sample solution (1 injection)

[0144] Experimental results: The content of each component in the test solution was calculated according to the area normalization result. The test results were: 96.27% for the compound of formula I, 1.77% for the compound of formula II, 0.63% for the compound of formula III, and 1.33% for the compound of formula IV. The typical spectra of the relevant tests in this example are shown in the appendix of the specification. Figure 8 .

[0145] Example 8: Detection of (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-5-one by the quantitative detection method of the present invention

[0146] Chromatographic conditions: chromatographic column: Inertsustain AQ-C18 4.6*150mm, 3μm or equivalent chromatographic column; flow rate: 1.1mL / min; column temperature: 45℃; wavelength: 267nm; injection volume: 15μL; running time: 50min; mobile phase A: purified water; mobile phase B: acetonitrile; mobile phase C: tetrahydrofuran; diluent: 60% acetonitrile water.

[0147] Blank solution preparation: 60% acetonitrile water.

[0148] Preparation of test solution: Weigh 100 mg of (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-5-one test sample, dissolve it in a 50 mL volumetric flask with 60% acetonitrile water and dilute to the mark, and mix well.

[0149] Preparation of impurity stock solution: Weigh 12.5 mg of compound of formula II, dissolve it in 60% acetonitrile water in a 50 mL volumetric flask and make up to the scale; weigh 12.5 mg of compound of formula III, dissolve it in acetonitrile in a 50 mL volumetric flask and make up to the scale; weigh 12.5 mg of compound of formula IV, dissolve it in ethanol in a 50 mL volumetric flask and make up to the scale, and mix well.

[0150] System adaptability solution: Weigh 50 mg of the compound of formula I into a 25 mL volumetric flask, add an appropriate amount of diluent to dissolve it, then transfer 2.00 mL of each impurity stock solution, dilute to the mark with diluent, and mix well.

[0151] Injection procedure: blank solution (2 injections), system suitability solution (1 injection), sample solution (1 injection)

[0152] Experimental results: The content of each component in the test solution was calculated according to the area normalization results. The detection results were: 95.62% for the compound of formula I, 2.16% for the compound of formula II, 0.59% for the compound of formula III, and 1.63% for the compound of formula IV.

[0153] Example 9: Detection of (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-5-one by the quantitative detection method of the present invention

[0154] Chromatographic conditions: column: Inertsustain AQ-C18 4.6*150mm, 3μm or equivalent column; flow rate: 1.1mL / min; column temperature: 45℃; wavelength: 257nm; injection volume: 10μL; running time: 50min; mobile phase A: purified water; mobile phase B: acetonitrile; mobile phase C: tetrahydrofuran; diluent: 60% acetonitrile water.

[0155] Blank solution preparation: 60% acetonitrile water.

[0156] Preparation of test solution: Weigh 100 mg of (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-5-one test sample, dissolve it in a 50 mL volumetric flask with 60% acetonitrile water and dilute to the mark, and mix well.

[0157] Preparation of impurity stock solution: Weigh 12.5 mg of compound of formula II, dissolve it in 60% acetonitrile water in a 50 mL volumetric flask and make up to the scale; weigh 12.5 mg of compound of formula III, dissolve it in acetonitrile in a 50 mL volumetric flask and make up to the scale; weigh 12.5 mg of compound of formula IV, dissolve it in ethanol in a 50 mL volumetric flask and make up to the scale, and mix well.

[0158] System adaptability solution: Weigh 50 mg of the compound of formula I into a 25 mL volumetric flask, add an appropriate amount of diluent to dissolve it, then transfer 2.00 mL of each impurity stock solution, dilute to the mark with diluent, and mix well.

[0159] Injection procedure: blank solution (2 injections), system suitability solution (1 injection), sample solution (1 injection)

[0160] Experimental results: The content of each component in the test solution was calculated according to the area normalization results. The detection results were: 96.16% for the compound of formula I, 1.81% for the compound of formula II, 0.64% for the compound of formula III, and 1.39% for the compound of formula IV.

[0161] Example 10: Detection of (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-5-one by the quantitative detection method of the present invention

[0162] Chromatographic conditions: column: Inertsustain AQ-C18 4.6*150mm, 3μm or equivalent column; flow rate: 1.1mL / min; column temperature: 45℃; wavelength: 262nm; injection volume: 8μL; running time: 50min; mobile phase A: purified water; mobile phase B: acetonitrile; mobile phase C: tetrahydrofuran; diluent: 60% acetonitrile water.

[0163] Blank solution preparation: 60% acetonitrile water.

[0164] Preparation of test solution: Weigh 100 mg of (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-5-one test sample, dissolve it in a 50 mL volumetric flask with 60% acetonitrile water and dilute to the mark, and mix well.

[0165] Preparation of impurity stock solution: Weigh 12.5 mg of compound of formula II, dissolve it in 60% acetonitrile water in a 50 mL volumetric flask and make up to the scale; weigh 12.5 mg of compound of formula III, dissolve it in acetonitrile in a 50 mL volumetric flask and make up to the scale; weigh 12.5 mg of compound of formula IV, dissolve it in ethanol in a 50 mL volumetric flask and make up to the scale, and mix well.

[0166] System adaptability solution: Weigh 50 mg of the compound of formula I into a 25 mL volumetric flask, add an appropriate amount of diluent to dissolve it, then transfer 2.00 mL of each impurity stock solution, dilute to the mark with diluent, and mix well.

[0167] Injection procedure: blank solution (2 injections), system suitability solution (1 injection), sample solution (1 injection)

[0168] Experimental results: The content of each component in the test solution was calculated according to the area normalization results. The test results were: 95.89% for the compound of formula I, 1.97% for the compound of formula II, 0.65% for the compound of formula III, and 1.49% for the compound of formula IV.

[0169] Example 11: Quantitative Detection Method Comparative Example 1

[0170] Chromatographic conditions: chromatographic column ACE C18-AR 4.6*150mm, 3μm; column temperature: 40℃; wavelength: 235nm; flow rate: 1.0mL / min; injection volume: 20uL; running time: 45min; mobile phase A: purified water, mobile phase B: acetonitrile; diluent: 60% acetonitrile water.

[0171] Blank solution preparation: 60% acetonitrile water.

[0172] Preparation of reference solution: Accurately weigh 25 mg of compound I, compound II, and compound III into a 25 mL volumetric flask, dissolve in 60% acetonitrile water and dilute to the mark, and shake well. Weigh 25 mg of compound IV into a 25 mL volumetric flask, dissolve in 80% acetonitrile water and dilute to the mark, and shake well.

[0173] Preparation of system adaptability solution: Accurately pipette 1 mL of each of the reference solution of the compound of formula I, the compound of formula II, the compound of formula III and the compound of formula IV into the same 100 mL volumetric flask, dissolve it with 80% acetonitrile water and make up to the mark, and mix well.

[0174] Experimental results: The positions of the compounds of formula II and formula I can be determined by running the system adaptability solution, but the peak time and peak order of the compounds of formula III and formula IV cannot be known; the positions of the main components and impurities of the compounds of formula I can be known by running the reference solution for positioning, but the peak shapes of the two impurity peaks of the compounds of formula III and formula IV are poor, and the separation degree does not meet the standard. Fig. 9 .

[0175] Example 12: Quantitative Detection Method Comparative Example 2

[0176] Chromatographic conditions: chromatographic column: Inertsustain AQ-C18 4.6*150mm, 3μm; flow rate: 0.6mL / min; injection volume: 20μL; column temperature: 40℃; wavelength: full wavelength; running time: 50min; mobile phase A: purified water, mobile phase B: acetonitrile.

[0177] Preparation of reference solution: see the preparation method in comparative example 1 of the quantitative detection method.

[0178] Preparation of system suitability solution: Take 2 drops of 1 mL of the reference solution of the compound of formula I, the compound of formula II, the compound of formula III and the compound of formula IV into the injection vial.

[0179] Experimental results: The peaks of the compounds of formula III and IV are asymmetric, with severe tailing, and the peak time of the compound of formula IV is too late. Fig.10 .

[0180] Example 13: Quantitative Detection Method Comparative Example 3

[0181] Chromatographic column: Waters Xbridge C18 4.6*100mm, 3.5μm; flow rate: 1.0mL / min; injection volume: 20μL; column temperature: 25℃; wavelength: full wavelength; running time: 40min; mobile phase A: 0.1% trifluoroacetic acid water, mobile phase B: acetonitrile, mobile phase D: tetrahydrofuran; diluent: 60% acetonitrile water.

[0182] Preparation of reference solution: see the preparation method in comparative example 1 of the quantitative detection method.

[0183] Preparation of system suitability solution: Take 4 drops of 1 mL of the reference solution of the compound of formula I, the compound of formula II, the compound of formula III and the compound of formula IV into a sample injection vial.

[0184] Experimental results: The separation degree between the main component and the impurity meets the standard. The peak shape of the compound of formula II is asymmetric, with tailing after the peak, and the retention time is early. Due to the low proportion of organic phase, the peak width of the compound of formula III and the compound of formula IV is wide, and the peak shape is not good enough. Fig.11 .

[0185] Example 14: Quantitative Detection Method Comparative Example 4

[0186] Chromatographic conditions: Inertsustain AQ-C18 4.6*150mm, 3μm; flow rate: 1.0mL / min; injection volume: 20μL; column temperature: 40℃; wavelength: full wavelength; running time: 60min; mobile phase A: purified water, mobile phase B: acetonitrile, mobile phase D: tetrahydrofuran.

[0187] Preparation of reference solution: Weigh 25.04 mg of compound I, 25.95 mg of compound II, and 25.03 mg of compound III respectively, dissolve them in 60% acetonitrile water in a 25 mL volumetric flask, dilute to the mark, and shake well. Weigh 25.06 mg of compound IV, dissolve them in 80% acetonitrile water in a 25 mL volumetric flask, dilute to the mark, and shake well.

[0188] Preparation of system adaptability solution: Accurately pipette 1 mL of each of the reference solutions of the compound of formula I, the compound of formula II, the compound of formula III and the compound of formula IV into the same 100 mL volumetric flask.

[0189] Experimental results: The peak shape of the compound of formula II is asymmetric, the peak shape of the compound of formula I is good, and the compounds of formula III and formula IV have tailing phenomenon. Fig.12 .

[0190] The method of the present invention is described by preferred embodiments. It is obvious that persons skilled in the relevant art can make appropriate common sense adjustments, changes and combinations to the methods and applications described in the present invention where necessary within the content and scope of the present invention to implement and apply the technology of the present invention. Personnel in the art can also refer to the content of the present invention and implement it by appropriately improving the process parameters. It is particularly important to point out that all similar improvements and adjustments are obvious to those skilled in the art and should be deemed to be included in the present invention.

Claims

1. A method for quantitatively detecting related substances of (R)-9-triisopropylsilyloxy-6,7,8,9-tetrahydro-5H-cyclohepta[b]pyridin-5-one as shown in Formula I, characterized in that: The quantitative detection method uses a liquid chromatograph equipped with an ultraviolet detector; the stationary phase uses a bonded silica gel material; the mobile phase uses a ternary elution system, wherein the mobile phase A is an aqueous solution system, and the mobile phases B and C are both organic solvents or organic solvent-aqueous solution mixed systems. 。 2. The quantitative detection method as claimed in claim 1, characterized in that: The method is used for quantitative analysis and detection of a mixture of any one or any combination of the compound represented by Formula I and the compounds represented by Formula II, Formula III, and Formula IV. 。 3. The quantitative detection method as claimed in claim 1, characterized in that: In the liquid chromatography method, the stationary phase adopts a bonded silica gel material selected from a carbon octadecyl bonded silica gel material and a carbon octadecyl bonded silica gel material, the particle size of the stationary phase bonded silica gel material is selected from 3 μm or 5 μm, and the size of the chromatographic column filled with the stationary phase bonded silica gel material is selected from 4.6 mm×150 mm or 4.6 mm×250 mm.

4. The quantitative detection method as claimed in claim 1, characterized in that: In the liquid chromatography method, mobile phase A is an aqueous solution selected from purified water or a salt solution of purified water; mobile phase B and mobile phase C are both organic solvents or organic solvent-aqueous solution mixed systems selected from acetonitrile, tetrahydrofuran or a mixture of the two in any ratio, an acetonitrile-aqueous solution mixture, and a tetrahydrofuran-water homogeneous mixture.

5. The quantitative detection method according to claim 1 or 4, characterized in that: In the liquid chromatography method, the elution gradient of the mobile phase is 0→3 minutes, mobile phase A: mobile phase B: mobile phase C = 90:8:2; 3→15 minutes, mobile phase A: mobile phase B: mobile phase C = 0:85:15; 15→40 minutes, mobile phase A: mobile phase B: mobile phase C = 0:85:15; 40→42 minutes, mobile phase A:mobile phase B:mobile phase C=90:8:2; 42→50 minutes, mobile phase A:mobile phase B:mobile phase C=90:8:

2.

6. The quantitative detection method as claimed in claim 1, characterized in that: In the liquid chromatography method, the column temperature is maintained at 45±5°C.

7. The quantitative detection method as claimed in claim 1, characterized in that: In the liquid chromatography method, the detection wavelength is 262±5 nm.

8. The quantitative detection method as claimed in claim 1, characterized in that: In this liquid chromatography method, the injection volume is 10±5 μL.

9. The quantitative detection method as claimed in claim 1, characterized in that: In the liquid chromatography method, the flow rate of the mobile phase is 1.1±0.2 ml / min.

10. The quantitative detection method as claimed in claim 1, characterized in that: In the liquid chromatography method, the sample diluent is an acetonitrile-water mixture.

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