Analysis and detection method of azonepan enantiomer

Abstract

The invention relates to a method for analyzing and detecting a non-azonetin enantiomer, which adopts normal-phase high performance liquid chromatography and takes normal hexane-ethanol-trifluoroacetic acid as a mobile phase to detect the non-azonetin enantiomer, and the specific chromatographic conditions are as follows: a chromatographic column is prepared by covalently bonding amylose-tri (3, 4, 6-trimethyl-1, 3-pentanediol) on the surface of silica gel; 2, 5-dichlorophenyl carbamate is used as a filling agent (IE, 4.6 * 250 mm, 5.0 [mu] m); a mobile phase: n-hexane: ethanol: trifluoroacetic acid = 500: 500: 1 (V / V / V); the flow rate is 1.0 ml / min; the column temperature is 30 DEG C; the sample size is 20 [mu] l; the detection wavelength is 280 nm; the elution gradient is isocratic elution. The invention provides a high performance liquid chromatography method for analyzing the enantiomer of the fiazonetin, the complete separation of the fiazonetin and the enantiomer of the fiazonetin can be realized by adopting the method, and the separation degree of a main component and the enantiomer is greater than 1.5.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical analysis, in particular to a high performance liquid chromatography method for analyzing and separating non-zanetan enantiomers. BACKGROUND

[0002] Fezolinetant is an oral selective neurokinin 3 receptor (NK3) antagonist for the treatment of moderate to severe vasomotor symptoms associated with menopause. It was approved for medical use in the United States in May 2023 and in the European Union in December 2023.

[0003] Fezolinetant has one chiral center. During chemical synthesis, fezolinetant is prone to racemization to obtain fezolinetant enantiomers. Fezolinetant enantiomers are ubiquitous in samples and are listed as process impurities of fezolinetant, affecting the quality of fezolinetant bulk drug and its preparation products. Therefore, the analysis and detection of this impurity play an important role in the quality control of the final product fezolinetant. In order to better control the quality of fezolinetant products, a simple, reliable, stable and effective method is needed to detect the enantiomers of the product. The chemical structural formula of fezolinetant and fezolinetant enantiomers is as follows:

[0004] SUMMARY

[0005] The purpose of the present application is to provide a method for analyzing and detecting fezolinetant enantiomers, specifically a high performance liquid chromatography method for analyzing fezolinetant enantiomers, so as to realize the determination of fezolinetant and its enantiomers.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] The present application adopts normal phase high performance liquid chromatography to detect fezolinetant enantiomers with n-hexane-ethanol-trifluoroacetic acid as the mobile phase, specifically:

[0008] A method for analyzing and detecting fezolinetant enantiomers, comprising the following steps:

[0009] (1) Preparation of system suitability solution: an appropriate amount of fezolinetant and its fezolinetant enantiomers are weighed and diluted with a diluent to prepare a mixed solution containing fezolinetant and fezolinetant enantiomers, which is used as the system suitability solution.

[0010] (2) Preparation of test sample solution: an appropriate amount of fezolinetant is weighed and diluted with a diluent to prepare a solution, which is used as the test sample solution.

[0011] (3) Determination: Take an appropriate amount of blank diluent, inject it into the liquid chromatograph, and record the chromatogram; take an appropriate amount of system suitability solution, inject it into the liquid chromatograph, and record the chromatogram; take an appropriate amount of test solution, inject it into the liquid chromatograph, and record the chromatogram;

[0012] (4) Content calculation: Based on the obtained chromatogram, the content of the enantiomer of fezonilant in the test sample was calculated by the area normalization method.

[0013] The structural formulas of the fezonatem and its enantiomers are as follows:

[0014]

[0015] In step (3), normal-phase high-performance liquid chromatography is used for determination; the optimal chromatographic conditions are: column: a column packed with covalently bonded linear starch-tris(3,5-dichlorophenylcarbamate) on silica gel surface; mobile phase: hexane-ethanol-trifluoroacetic acid as the mobile phase for elution.

[0016] The chromatographic conditions are specifically as follows:

[0017] Column: The stationary phase is composed of silica gel covalently bonded amylose-tris(3,5-dichlorophenylcarbamate). IE (4.6*250mm, 5.0μm);

[0018] Mobile phase: n-hexane: ethanol: trifluoroacetic acid = 500:500:1 (V / V / V);

[0019] Flow rate: 1.0 ml / min;

[0020] Column temperature: 30℃;

[0021] Injection volume: 20 μl;

[0022] Detection wavelength: 280nm;

[0023] Elution gradient: isocratic elution.

[0024] The diluent is ethanol.

[0025] The specific method of step (1) is as follows: Weigh appropriate amounts of fezonatem and its enantiomer, add diluent to prepare a mixed solution containing 1 mg / ml fezonatem and 10 μg / ml fezonatem enantiomer, as the system suitability solution.

[0026] The specific method for step (2) is as follows: Weigh an appropriate amount of fezonilant and add a diluent to prepare a 1 mg / ml solution as the test solution.

[0027] In step (3), the amounts of blank diluent, system suitability solution, and test solution are all 20 μl. Specifically, 20 μl of blank diluent is injected into the liquid chromatograph, and the chromatogram is recorded; 20 μl of system suitability solution is injected into the liquid chromatograph, and the chromatogram is recorded; 20 μl of test solution is injected into the liquid chromatograph, and the chromatogram is recorded.

[0028] Compared with the prior art, the advantages of the present invention are as follows:

[0029] This invention provides a high-performance liquid chromatography method for analyzing the enantiomers of fezolin. The method of this invention can achieve complete separation of fezolin and its enantiomers, and the resolution between the main component and the enantiomer is greater than 1.5.

[0030] This invention effectively ensures the quality control of enantiomers of the compound fezlonetine by detecting and analyzing its enantiomers, and provides data support and reference for the formulation of fezlonetine quality standards and the screening of preparation processes, which has important practical significance in production and quality control. Attached Figure Description

[0031] Figure 1 Example 2: Blank diluent chromatogram;

[0032] Figure 2 Example 2: Chromatogram of the determination of the fezonil test solution;

[0033] Figure 3 Example 3: Chromatogram of the system suitability solution determination;

[0034] Figure 4 Standard curve of fezonectin in Example 5.

[0035] Figure 5 Standard curves of the enantiomers of fezonine in Example 5. Detailed Implementation

[0036] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0037] Example 1: Optimization of chromatographic conditions

[0038] The fezonalant and its enantiomers were used to develop an analytical method for fezonalant. Preliminary optimization of the mobile phase yielded the following detection conditions. This method achieved a resolution greater than 1.5 on different chiral columns, effectively ensuring the quality control of fezonalant enantiomers in subsequent pharmaceutical processes.

[0039] The chromatographic conditions are specifically as follows:

[0040] Mobile phase: n-hexane: ethanol: trifluoroacetic acid = 500:500:1 (V / V / V);

[0041] Flow rate: 1.0 ml / min;

[0042] Column temperature: 30℃;

[0043] Injection volume: 20 μl;

[0044] Detection wavelength: 280nm;

[0045] Elution gradient: isocratic elution.

[0046] The diluent is ethanol.

[0047] (1) Solution preparation

[0048] System suitability solution: Accurately measure fezonatem and its enantiomer separately. Dilute with diluent to prepare a mixed solution containing approximately 1 mg of fezonatem and 10 μg of its enantiomer per 1 ml.

[0049] (2) Measurement

[0050] Determination: Take 20 μl of the prepared system suitability solution and perform determinations based on the above chromatographic conditions; investigate different chromatographic columns:

[0051] When the chromatographic column is packed with cellulose-tris(3,5-dichlorophenylcarbamate) covalently bonded to silica gel, The system suitability results for the IC (4.6*250mm, 5.0μm) are shown in the table below.

[0052]

[0053] The results showed that there was no interference from the blank, and the resolution between the main component and the enantiomer was greater than 1.5, indicating good specificity. When the chromatographic column was packed with silica gel covalently bonded amylose-tris(3,5-dichlorophenylcarbamate) as the packing material... The system applicability results for the IE (4.6*250mm, 5.0μm) are shown in the table below.

[0054]

[0055] The results showed that there was no interference from the blank, and the resolution between the main component and the enantiomer was greater than 1.5, indicating good specificity. Through the above investigation, it can be concluded that under these chromatographic conditions, the main component and the enantiomer maintained good resolution on different columns. Considering residual solvents in the sample and column robustness, the following conditions were determined as the detection chromatographic conditions:

[0056] Column: The stationary phase is composed of silica gel covalently bonded amylose-tris(3,5-dichlorophenylcarbamate). IE (4.6*250mm, 5.0μm);

[0057] Mobile phase: n-hexane: ethanol: trifluoroacetic acid = 500:500:1 (V / V / V);

[0058] Flow rate: 1.0 ml / min;

[0059] Column temperature: 30℃;

[0060] Injection volume: 20 μl;

[0061] Detection wavelength: 280nm;

[0062] Diluent: Ethanol;

[0063] Elution gradient: isocratic elution.

[0064] The method was robustly tested to ensure accurate detection of the fezonatetan enantiomer.

[0065] The specific operating procedure for the durability test is as follows:

[0066] (1) Solution preparation

[0067] System suitability solution: Accurately measure fezonatem and fezonatem enantiomer separately. Dilute with diluent to prepare a mixed solution containing approximately 1 mg of fezonatem and 10 μg of fezonatem enantiomer per 1 ml. (2) Determination: Take 20 μl of the prepared system suitability solution and perform the determination separately based on the above chromatographic conditions. Adjust the flow rate range during the determination process.

[0068] The results of the durability test are as follows:

[0069] The flow rate was 0.8 ml / min, the column temperature was 30℃, and the system suitability solution was determined. The system suitability results are shown in the table below.

[0070]

[0071] The flow rate was 1.0 ml / min, the column temperature was 30℃, and the system suitability solution was determined. The system suitability results are shown in the table below.

[0072]

[0073]

[0074] The flow rate was 1.2 ml / min, the column temperature was 30℃, and the system suitability solution was determined. The system suitability results are shown in the table below.

[0075]

[0076] The flow rate was 1.0 ml / min, the column temperature was 25℃, and the system suitability solution was determined. The system suitability results are shown in the table below.

[0077]

[0078] The flow rate was 1.0 ml / min, the column temperature was 35℃, and the system suitability solution was determined. The system suitability results are shown in the table below.

[0079]

[0080] Based on the above system suitability solution results, it can be seen that the method has good durability, and the following method can meet the needs of routine testing.

[0081] Column: Silica gel with covalently bonded amylose-tris(3,5-dichlorophenylcarbamate) as the packing material. IE (4.6*250mm, 5.0μm);

[0082] Mobile phase: n-hexane: ethanol: trifluoroacetic acid = 500:500:1 (V / V / V);

[0083] Flow rate: 0.8–1.2 ml / min;

[0084] Column temperature: 25~35℃;

[0085] Injection volume: 20 μl;

[0086] Detection wavelength: 280nm;

[0087] Diluent: Ethanol;

[0088] Elution gradient: isocratic elution.

[0089] In particular, the following conditions are optimal chromatographic conditions:

[0090] Column: Silica gel with covalently bonded amylose-tris(3,5-dichlorophenylcarbamate) as the packing material. IE (4.6*250mm, 5.0μm);

[0091] Mobile phase: n-hexane: ethanol: trifluoroacetic acid = 500:500:1 (V / V / V);

[0092] Flow rate: 1.0 ml / min;

[0093] Column temperature: 30℃;

[0094] Injection volume: 20 μl;

[0095] Detection wavelength: 280nm;

[0096] Diluent: Ethanol;

[0097] Elution gradient: isocratic elution.

[0098] These optimal chromatographic conditions will be used in subsequent experiments.

[0099] Example 2 Sample Determination

[0100] The test solution was determined using the optimized chromatographic conditions.

[0101] (1) System suitability solution: Accurately measure fezonatem and its enantiomer separately. Dilute with diluent to prepare a mixed solution containing approximately 1 mg of fezonatem and 10 μg of its enantiomer per 1 ml. (2) Preparation of test solution: Weigh an appropriate amount of fezonatem and add diluent to prepare a 1 mg / ml solution as the test solution. The diluent is ethanol.

[0102] (3) Determination: Inject 20 μl of blank diluent into the liquid chromatograph, record the chromatogram, equilibrate the instrument, and confirm the position of the solvent peak in the diluent. Next, inject 20 μl of system suitability solution and test solution into the liquid chromatograph, and record the chromatogram; wherein, the chromatographic conditions used in step (3) are as follows:

[0103] Column: The stationary phase is composed of silica gel covalently bonded amylose-tris(3,5-dichlorophenylcarbamate). IE (4.6*250mm, 5.0μm);

[0104] Mobile phase: n-hexane: ethanol: trifluoroacetic acid = 500:500:1 (V / V / V);

[0105] Flow rate: 1.0 ml / min;

[0106] Column temperature: 30℃;

[0107] Injection volume: 20 μl;

[0108] Detection wavelength: 280nm;

[0109] Diluent: Ethanol;

[0110] Elution gradient: isocratic elution.

[0111] The chromatogram obtained from the test is as follows Figures 1-2 As shown, where, Figure 1 This is the chromatogram of the blank diluent in Example 2. Figure 2 The chromatogram of the test sample in Example 2 shows that the blank has no interference, which meets the impurity control requirements.

[0112] Experiment Example 3: System Suitability Experiment

[0113] The blank solution, test solution, and system suitability solution were determined under the optimized chromatographic conditions.

[0114] (1) Solution preparation:

[0115] The diluent described below is ethanol.

[0116] Preparation of test solution: Weigh 25 mg of the test sample (fenzonalant) accurately, place it in a 25 ml volumetric flask, dilute to 25 ml with diluent, and shake well.

[0117] System suitability solution: Accurately measure fezonatem and fezonatem enantiomers separately. Dilute with diluent to prepare a mixed solution containing approximately 1 mg of fezonatem and 10 μg of fezonatem enantiomers per 1 ml. (2) Determination: Inject 20 μl each of blank diluent, test solution and system suitability solution into the high performance liquid chromatograph;

[0118] Experimental results show that the diluent does not interfere with the detection of each fezolidinedion enantiomer, the resolution between the main component and the isomers is greater than 1.5, and the analytical method has good specificity. The system suitability solution chromatogram is shown below. Figure 3 The system suitability index results are shown in Table 1.

[0119] Table 1. System Applicability Results

[0120]

[0121] Example 4: Limit of Detection and Limit of Quantification:

[0122] Solution preparation: Accurately weigh appropriate amounts of fezonatem and fezonatem enantiomers, and dilute them stepwise with diluent to prepare a series of solutions. Perform the determination under the chromatographic conditions described in Example 2. Based on the results, determine the limit of quantitation (LOQ) and limit of detection (LOD) of the product using the signal-to-noise ratio (S / N) method. Prepare six parallel LQ solutions for testing, and analyze them under the chromatographic conditions described in Example 2. Examine the RSD of the peak area to confirm the precision of the method at the LQ concentration.

[0123] The limit of quantitation is calculated based on a signal-to-noise ratio (S / N) ≥ 10:1; the limit of detection is calculated based on a signal-to-noise ratio (S / N) ≥ 3:1. The results are shown in Tables 2 and 3.

[0124] Table 2. Results of Limit of Quantitation

[0125]

[0126] Table 3. Detection Limit Results

[0127]

[0128] The results showed that the RSD of the peak area at the limit of quantitation (LOQ) for fezonelanthin enantiomers was no greater than 10.0%. Based on a sample concentration of 1 mg / mL, the LQs for fezonelanthin and its enantiomers were 0.015% and 0.015%, respectively, while the LQs for fezonelanthin and its enantiomers were 0.005% and 0.005%, respectively. These results demonstrate that the method is highly sensitive and can fully meet the requirements for the detection of fezonelanthin enantiomers.

[0129] Experiment Example 5: Linearity and Range Experiment

[0130] Diluent: Ethanol.

[0131] Solution preparation: Accurately weigh appropriate amounts of fezonalant and its enantiomers, dissolve and dilute with diluent to prepare a series of linear solutions containing each component at concentrations ranging from the limit concentration to 200% of the limit concentration. Accurately measure 20 μl of each of the above linear solutions and inject them into the liquid chromatograph. Analyze under the chromatographic conditions described in Example 2. Plot a standard curve with concentration (μg / ml) on the x-axis and peak area on the y-axis, and calculate the regression equation. The standard curve is shown below. Figure 4 , 5 As shown in the figure. The regression equation results are shown in Table 4.

[0132] Table 4. Results of the linear experiment

[0133] Name Range (pg / ml) Equivalent to limit concentration percentage Linear equation Correlation coefficient Fexofenadine enantiomers 0.153～20.368 1.53～200％ y = 39414.72x - 1226.52 0.9992 Fexofenadine 0.153～20.352 1.53～200％ y = 38920.12x + 1394.15 0.9993

[0134] The results showed that the peak area of ​​the enantiomers of fezonide and fezonide had a good linear relationship in the concentration range of 0.153 to 20.368 μg / ml, with correlation coefficients r not less than 0.995. The relative response factor of the enantiomers of fezonide and fezonide was 0.99, which can be considered as equivalent.

[0135] Example 6: Accuracy

[0136] Diluent: Ethanol.

[0137] Test solution: Weigh approximately 25 mg of fezonil and place it in a 25 ml volumetric flask. Dilute to 25 ml with solvent and shake well.

[0138] Enantiomer stock solution: Take an appropriate amount of fezonine enantiomer, accurately weigh it, place it in a volumetric flask, add diluent to dissolve and dilute it to prepare a solution containing about 250 μg of enantiomer per 1 ml, which is used as the enantiomer stock solution.

[0139] Enantiomer limit of quantitation stock solution: Take an appropriate amount of fezonilant enantiomer, accurately weigh it, place it in a volumetric flask, add diluent to dissolve and dilute to prepare a solution containing approximately 5.0 μg / ml of enantiomer per ml, as the enantiomer limit of quantitation stock solution.

[0140] LOQ% accuracy solution: Accurately weigh approximately 25 mg of fezoneltan and place it in a 25 ml volumetric flask. Accurately add 7.5 ml of the enantiomer limit of quantitation stock solution, dilute to 25 ml with diluent, and mix well. Prepare three aliquots using the same method.

[0141] 100% accuracy solution: Accurately weigh approximately 25 mg of fezoneltan and place it in a 25 ml volumetric flask. Accurately add 1 ml of the enantiomer stock solution, dilute to 25 ml with diluent, and mix well. Prepare three aliquots using the same method.

[0142] 200% accuracy solution: Accurately weigh approximately 25 mg of fezoneltan and place it in a 25 ml volumetric flask. Accurately add 2 ml of the enantiomer stock solution, dilute to 25 ml with diluent, and mix well. Prepare three aliquots using the same method.

[0143] Accurately measure 20 μl of each of the above solutions, inject them into the liquid chromatograph, and determine the chromatograms according to the chromatographic conditions of Example 2. The experimental results are shown in Table 5.

[0144] Table 5. Results of the recovery rate test

[0145]

[0146] The results showed that within the range of the limit of quantitation concentration to the limit concentration of 200% for the enantiomers of fezonine, the recoveries of the enantiomers were all between 80% and 120%, and the RSD of the recoveries were all less than 10.0%, indicating good method accuracy.

[0147] The above-described embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention to the scope of the described embodiments. The method of the present invention is not limited to the analysis and separation of fezonelanthan and its enantiomers; any method used to separate and determine fezonelanthan and its impurities falls within the scope of the present invention.

Claims

1. A method for the analysis and detection of enantiomers of fezolinate, characterized in that: Includes the following steps: (1) Preparation of system suitability solution: Weigh appropriate amounts of fezonatem and its enantiomers, add diluent to prepare a mixed solution containing fezonatem and its enantiomers, which is used as the system suitability solution. (2) Preparation of test solution: Weigh an appropriate amount of fezoniltan, add diluent to prepare a solution, and use it as the test solution. (3) Determination: Take an appropriate amount of blank diluent, inject it into the liquid chromatograph, and record the chromatogram; take an appropriate amount of system suitability solution, inject it into the liquid chromatograph, and record the chromatogram; take an appropriate amount of test solution, inject it into the liquid chromatograph, and record the chromatogram; (4) Content calculation: Based on the obtained chromatogram, the content of the enantiomer of fezonilant in the test sample was calculated by the area normalization method. The structural formulas of the fezonatem and its enantiomers are as follows: In step (3), normal-phase high-performance liquid chromatography was used for determination; the chromatographic conditions were as follows: chromatographic column: a column packed with covalently bonded linear starch-tris(3,5-dichlorophenylcarbamate) on the surface of silica gel; mobile phase: hexane-ethanol-trifluoroacetic acid was used as the mobile phase for elution.

2. The analytical detection method according to claim 1, characterized in that: The chromatographic conditions are specifically as follows: Column: Silica gel with covalently bonded amylose-tris(3,5-dichlorophenylcarbamate) as the packing material. IE (4.6*250mm, 5.0μm); Mobile phase: n-hexane: ethanol: trifluoroacetic acid = 500:500:1 (V / V / V); Flow rate: 1.0 ml / min; Column temperature: 30℃; Injection volume: 20 μl; Detection wavelength: 280nm; Elution gradient: isocratic elution.

3. The analytical detection method according to claim 1 or 2, characterized in that: The diluent is ethanol.

4. The analytical detection method according to claim 1, characterized in that: The specific method of step (1) is as follows: Weigh appropriate amounts of fezonatem and its enantiomer, add diluent to prepare a mixed solution containing 1 mg / ml fezonatem and 10 μg / ml fezonatem enantiomer, as the system suitability solution.

5. The analytical detection method according to claim 1, characterized in that: The specific method for step (2) is as follows: Weigh an appropriate amount of fezonilant and add a diluent to prepare a 1 mg / ml solution as the test solution.

6. The analytical detection method according to claim 1, characterized in that: In step (3), the amount of blank diluent, system suitability solution and test solution taken is 20 μl.

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