Method for separating, identifying and quantifying nafamostat mesylate Z1 and impurities thereof

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating, identifying and quantifying nafamostat mesylate Z1 and impurities thereof. And the nafamostat mesylate Z1 is 6-cyano-2-naphthol. According to the method, octadecylsilane chemically bonded silica is adopted as a chromatographic column filler, a potassium hexafluorophosphate solution is adopted as a mobile phase A, acetonitrile is adopted as a mobile phase B, an impurity Z1b, an impurity Z1c, nafamostat mesylate Z1, an impurity Z1h, an impurity Z1a, an impurity Z1g, an impurity Z1d, an impurity SM1, an impurity SM1i, an impurity Z1f and / or an impurity SM1j are sequentially separated through linear gradient elution, and the separated impurities enter a detector for detection; and finally, calculating the content of each impurity by adopting a correction factor-added principal component self-contrast method. The method can effectively separate 6-cyano-2-naphthol and related impurities thereof within 60 minutes, and has the advantages of high sensitivity, strong specificity, good durability and simplicity and feasibility in operation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating, identifying and quantifying nafamostat mesylate Z1 and impurities thereof. BACKGROUND

[0002] Nafamostat mesylate is a non-peptide protease inhibitor developed by Japan Tobacco Company, and was first marketed in Japan in 1986. It is clinically used for the treatment of acute pancreatitis. With the deepening of clinical research, the indications of nafamostat mesylate have gradually expanded, and it is used for the treatment of more diseases related to protease abnormalities, including acute exacerbation of chronic pancreatitis, postoperative acute pancreatitis, post-pancreatic ductography acute pancreatitis, acute traumatic pancreatitis, disseminated intravascular coagulation (DIC) and the like. In addition, nafamostat mesylate is also used as an anticoagulant in hemodialysis.

[0003] Nafamostat mesylate Z1 is a key intermediate in the synthesis of nafamostat mesylate, and its chemical name is 6-cyano-2-naphthol, and its structural formula is shown as formula I. According to its process route, there may be a total of 10 impurities: impurity SM1, impurity SM 1i , impurity Z 1j , impurity Z 1a , impurity Z 1b , impurity Z 1c , impurity Z 1d , impurity Z 1f , impurity Z 1g , impurity Z 1h , and their structural formulas are shown as formulas II to XI, respectively.

[0004]

[0005] Further research on the above-mentioned 10 impurities generated in the synthesis process of nafamostat mesylate Z1 has great significance for the quality control of nafamostat mesylate Z1 and subsequent nafamostat mesylate preparation products, but at present, there is no analysis method for detecting the above-mentioned 10 related substances in nafamostat mesylate Z1.

[0006] In the prior art, the patent CN117247335A discloses a chromatographic condition for detecting nafamostat intermediate, which is carried out by using Agilent 1260DAD type (or Shimadzu LC-2030) liquid chromatograph, specifically including a chromatographic column with octadecylsilane bonded silica gel as the filler; the detector is an ultraviolet detector with a wavelength of 254 nm; the flow rate is 1.0 mL per minute; gradient elution is adopted, and the test sample solution is determined by automatic integration method, and the purity of the test sample is calculated according to the peak area. However, the invention does not disclose the key mobile phase conditions and the information of the separated impurities, so it is impossible to effectively determine the above-mentioned 10 kinds of impurities in nafamostat mesylate Z1.

[0007] Therefore, it is necessary to study the determination method of the related impurities in nafamostat mesylate Z1. SUMMARY

[0008] Therefore, one of the purposes of the present application is to provide a method for separating nafamostat mesylate Z1 and its related impurities by HPLC method, which can separate nafamostat mesylate Z1 and its related impurities within 60 minutes or more than 60 minutes, providing support for subsequent qualitative and quantitative detection of each impurity.

[0009] To achieve the above-mentioned purposes, the present application adopts the following technical solutions:

[0010] The method for separating nafamostat mesylate Z1 and its related impurities by HPLC method, the related impurities include any one or more of impurity SM1, impurity SM 1i , impurity Z 1j , impurity Z 1a , impurity Z 1b , impurity Z 1c , impurity Z 1d , impurity Z 1f , impurity Z 1g , impurity Z 1h The method includes using octadecylsilane bonded silica gel as the chromatographic column filler, using potassium hexafluorophosphate solution as the mobile phase A, and acetonitrile as the mobile phase B, and sequentially separating impurity Z 1b , impurity Z 1c , nafamostat mesylate Z1, impurity Z 1h , impurity Z 1a , impurity Z 1g , impurity Z 1d , impurity SM1, impurity SM 1i , impurity Z 1f and / or impurity SM 1j by linear gradient elution.

[0011] The structural formula of the nafamostat mesylate Z1 is shown as Formula I; the structural formula of the impurity SM1 is shown as Formula II, the structural formula of the impurity SM 1i is shown as Formula III, the structural formula of the impurity SM 1j is shown as Formula IV, the structural formula of the impurity Z 1a is shown as Formula V, the structural formula of the impurity Z 1b is shown as Formula VI, the structural formula of the impurity Z 1c is shown as Formula VII, the structural formula of the impurity Z 1d is shown as Formula VIII, the structural formula of the impurity Z 1f is shown as Formula IX, the structural formula of the impurity Z 1g is shown as Formula X, the structural formula of the impurity Z 1h is shown as Formula XI.

[0012]

[0013] Further, the separated components include nafamostat mesylate Z1, and any one or more of the impurity SM1, the impurity SM 1i , the impurity SM 1j , the impurity Z 1a , the impurity Z 1b , the impurity Z 1c , the impurity Z 1d , the impurity Z 1f , the impurity Z 1g , the impurity Z 1h .

[0014] Further, the gradient elution includes:

[0015] 0 min, the volume ratio of the mobile phase A and the mobile phase B is 65-85: 35-15;

[0016] 10 min, the volume ratio of the mobile phase A and the mobile phase B is 65-85: 35-15;

[0017] 25 min, the volume ratio of the mobile phase A and the mobile phase B is 55-75: 45-25;

[0018] 40 min, the volume ratio of the mobile phase A and the mobile phase B is 35-55: 65-45;

[0019] 45 min, the volume ratio of the mobile phase A and the mobile phase B is 35-15: 65-85;

[0020] 50 min, the volume ratio of the mobile phase A and the mobile phase B is 35-15: 65-85;

[0021] 51 min, the mobile phase A and the mobile phase B are in a volume ratio of 73-77 : 27-23;

[0022] 60 min, the mobile phase A and the mobile phase B are in a volume ratio of 73-77 : 27-23.

[0023] As preferred, the gradient elution comprises:

[0024] 0 min, the mobile phase A and the mobile phase B are in a volume ratio of 75 : 25;

[0025] 10 min, the mobile phase A and the mobile phase B are in a volume ratio of 75 : 25;

[0026] 25 min, the mobile phase A and the mobile phase B are in a volume ratio of 65 : 35;

[0027] 40 min, the mobile phase A and the mobile phase B are in a volume ratio of 45 : 55;

[0028] 45 min, the mobile phase A and the mobile phase B are in a volume ratio of 25 : 75;

[0029] 50 min, the mobile phase A and the mobile phase B are in a volume ratio of 25 : 75;

[0030] 51 min, the mobile phase A and the mobile phase B are in a volume ratio of 73-77 : 27-23;

[0031] 60 min, the mobile phase A and the mobile phase B are in a volume ratio of 73-77 : 27-23.

[0032] As most preferred, the gradient elution comprises:

[0033] 0 min, the mobile phase A and the mobile phase B are in a volume ratio of 75 : 25;

[0034] 10 min, the mobile phase A and the mobile phase B are in a volume ratio of 75 : 25;

[0035] 25 min, the mobile phase A and the mobile phase B are in a volume ratio of 65 : 35;

[0036] 40 min, the mobile phase A and the mobile phase B are in a volume ratio of 45 : 55;

[0037] 45 min, the mobile phase A and the mobile phase B are in a volume ratio of 25 : 75;

[0038] For 50 minutes, the volume ratio of mobile phase A to mobile phase B is 25:75.

[0039] At 51 min, the volume ratio of mobile phase A to mobile phase B was 75:25;

[0040] After 60 minutes, the volume ratio of mobile phase A to mobile phase B is 75:25.

[0041] Furthermore, the concentration of the potassium hexafluorophosphate solution is 0.005 mol / L to 0.02 mol / L, and the pH is 2.5 to 5.

[0042] Preferably, the pH of the potassium hexafluorophosphate solution is 3.3 to 3.7, and most preferably 3.5.

[0043] Preferably, the concentration of the potassium hexafluorophosphate solution is 0.01 mol / L.

[0044] Furthermore, the flow rate of the mobile phase is 1.0 ml / min to 1.5 ml / min, and the column temperature of the chromatographic column is 20℃ to 30℃.

[0045] Preferably, the flow rate of the mobile phase is 1.1 ml / min to 1.3 ml / min, and most preferably 1.2 ml / min.

[0046] Preferably, the column temperature of the chromatographic column is 23℃~27℃, and most preferably 25℃.

[0047] Furthermore, the chromatographic column specifications are 4.6mm × 250mm, 5μm.

[0048] Preferably, the chromatographic column is a ZORBAX SB-C18 4.6mm×250mm, 5μm, or a column with equivalent performance.

[0049] Preferably, the injection volume is 15 μl.

[0050] The second objective of this invention is to provide a method for qualitatively identifying naphthostat mesylate Z1 and its related impurities.

[0051] To achieve the above objectives, the present invention adopts the following technical solution:

[0052] A method for qualitative identification of naphthostat mesylate Z1 and its related impurities is provided, wherein naphthostat mesylate Z1 and its related impurities are separated using the aforementioned method and detected by a detector to obtain a chromatogram; the detection wavelength of the detector is 235±2nm.

[0053] Preferably, the detection wavelength of the detector is 235 nm.

[0054] Furthermore, the presence of relevant impurities in the test sample can be determined by comparing the chromatographic behavior of the test sample and the reference sample.

[0055] Furthermore, the separation order from first to last is as follows: Impurity Z 1b Impurity Z 1c Naphthostat mesylate Z1, impurities Z 1h Impurity Z 1a Impurity Z 1g Impurity Z 1d Impurity SM1, Impurity SM 1i Impurity Z 1f Impurities SM 1j .

[0056] Furthermore, using naphthostat mesylate Z1 as a reference peak, with a relative retention time of 0.16 ± 0.2, it was identified as impurity Z. 1b The relative retention time was 0.33 ± 0.2, and it was identified as impurity Z. 1c The relative retention time was 1.05 ± 0.2 seconds, and it was identified as impurity Z. 1h The relative retention time was 1.47 ± 0.2 seconds, and it was identified as impurity Z. 1a The relative retention time was 1.57 ± 0.2, and it was identified as impurity Z. 1g The relative retention time was 1.61 ± 0.2 seconds, and it was identified as impurity Z. 1d The relative retention time was 1.64±0.2, and it was identified as impurity SM1; the relative retention time was 1.92±0.2, and it was identified as impurity SM. 1i The relative retention time was 1.98 ± 0.2 seconds, and it was identified as impurity Z. 1f The relative retention time was 2.13 ± 0.2 seconds, and it was identified as impurity SM. 1j .

[0057] Furthermore, under optimal chromatographic conditions, the retention time was 3.6 ± 0.5, and it was identified as impurity Z. 1b The retention time was 7.7 ± 0.5 seconds, and it was identified as impurity Z. 1c The retention time was 22.9 ± 0.5 seconds, and it was identified as naphthostat mesylate Z1; the retention time was 24.0 ± 0.5 seconds, and it was identified as impurity Z. 1h The retention time was 33.6 ± 0.5 seconds, and it was identified as impurity Z. 1a The retention time was 36.0 ± 0.5 seconds, and it was identified as impurity Z. 1g The retention time was 36.8 ± 0.5 seconds, and it was identified as impurity Z. 1d The retention time was 37.5 ± 0.5 seconds, and it was identified as impurity SM1; the retention time was 44.0 ± 0.5 seconds, and it was identified as impurity SM. 1i The retention time was 45.3 ± 0.5 seconds, and it was identified as impurity Z.1f The retention time was 48.8 ± 0.5 seconds, and it was identified as impurity SM. 1j .

[0058] Each impurity can be qualitatively identified based on the relative retention time, or the retention time, or the order in which the components are separated.

[0059] The third objective of this invention is to provide a method for quantitatively detecting naphthostat mesylate Z1 and its related impurities.

[0060] To achieve the above objectives, the present invention adopts the following technical solution:

[0061] A method for quantitative detection of naphthostat mesylate Z1 and its related impurities is proposed. Naphthostat mesylate Z1 and its related impurities are separated and detected using the aforementioned method to obtain chromatograms. Based on the obtained chromatograms, the content of each impurity is calculated using the principal component self-comparison method with correction factors.

[0062] Furthermore, before separation, the test solution was prepared using an aqueous solution of acetonitrile as the solvent.

[0063] Preferably, the solvent is prepared by mixing acetonitrile and water in a volume ratio of 50:50.

[0064] Preferably, the test solution includes a sample solution and a control solution.

[0065] The concentration of the sample solution was 0.1 mg / ml.

[0066] Furthermore, impurity Z 1b The correction factor is 1.3, and the impurity Z 1c The correction factor is 1.2, and the impurity Z 1h The correction factor is 1.5, and the impurity Z 1a The correction factor is 1.7, and the impurity Z 1g The correction factor is 1.1, and the impurity Z 1d The correction factor is 1.2, the correction factor for impurity SM1 is 1.1, and the correction factor for impurity SM is 1.1. 1i The correction factor is 1.3, and the impurity Z 1f The correction factor is 1.2, and the impurity SM 1j The correction factor is 1.2, and the correction factor for other individual impurities is 1.0.

[0067] Furthermore, the method specifically includes the following steps:

[0068] 1. Take an appropriate amount of this product, accurately weigh it, add solvent to dissolve and dilute it to prepare a solution with a concentration of about 0.1 mg / ml, as the sample solution;

[0069] 2. Accurately measure 0.5 ml of the test solution and place it in a 100 ml volumetric flask. Dilute to the mark with solvent and shake well to obtain the control solution.

[0070] 3. Take naphthostat mesylate Z1 system suitability reference standard, and take impurity Z... 1b Impurity Z 1c Impurity Z 1h Impurity Z 1a Impurity Z 1g Impurity Z 1d Impurity SM1, Impurity SM 1i Impurity Z 1f Impurities SM 1j An appropriate amount of reference standard was dissolved in acetonitrile by sonication and diluted to a concentration containing approximately Z impurities per 1 ml. 1b Impurity Z 1c Impurity Z 1h Impurity Z 1a Impurity Z 1g Impurity Z 1d Impurity SM1, Impurity SM 1i Impurities SM 1j 0.2mg, impurity Z 1f Prepare a 0.1 mg stock solution; accurately measure an appropriate amount of the stock solution, take an appropriate amount of naphamostat mesylate Z1, place them in the same volumetric flask, add solvent to dissolve and dilute to prepare a solution containing approximately 0.1 mg of naphamostat mesylate Z1, approximately 0.3 μg of impurity SM1, and impurity Z1 per ml. 1c Impurity Z 1a Impurities SM 1i Impurities SM 1j Approximately 0.2 μg each, impurity Z 1b Approximately 7.0 μg, impurity Z 1d Approximately 1.0 μg, impurity Z 1h Impurity Z 1f Approximately 2.0 μg each, impurity Z 1g Approximately 0.6 μg of the mixed solution was used as a system suitability solution.

[0071] 4. Take the above system suitability solution, sample solution and control solution respectively, inject them sequentially for detection, and obtain chromatograms; then use the principal component self-comparison method with correction factor to calculate the content of each impurity.

[0072] Furthermore, in the system suitability solution chromatogram, impurity Z... 1d The separation between the main peak and the impurity SM1 peak should meet the requirements. The separation between the main peak and impurity Z... 1h The separation degree should meet the requirements.

[0073] The beneficial effects of this invention are as follows:

[0074] 1. This invention, through screening chromatographic columns and conditions, establishes a self-developed HPLC method that can effectively remove impurity Z. 1b Impurity Z 1c Impurity Z 1h Impurity Z 1a Impurity Z 1g Impurity Z 1d Impurity SM1, Impurity SM 1i Impurity Z 1f Impurities SM 1j This invention effectively separates 6-cyano-2-naphthol from 6-cyano-2-naphthol, solving the problem of separating and determining known impurities in 6-cyano-2-naphthol that has been unresolved by existing technologies. The method of this invention is highly sensitive, specific, reproducible, robust, and simple to operate.

[0075] 2. The detection method of this invention is highly efficient and durable, and can effectively separate 6-cyano-2-naphthol and impurity Z within 60 minutes. 1b Impurity Z 1c Impurity Z 1h Impurity Z 1a Impurity Z 1g Impurity Z 1d Impurity SM1, Impurity SM 1i Impurity Z 1f and impurities SM 1j .

[0076] 3. This method has the advantage of high sensitivity, enabling effective identification and quantification of low-concentration impurities in 6-cyano-2-naphthol. Impurity Z 1b The limit of detection (LOD) was 0.0194 μg / ml, and the limit of quantitation (LOQ) was 0.0387 μg / ml; impurity Z 1c The limit of detection (LOD) was 0.0203 μg / ml, and the limit of quantitation (LOQ) was 0.0406 μg / ml; the LOD of nafamostat mesylate Z1 was 0.0206 μg / ml, and the LOQ was 0.0411 μg / ml; impurity Z 1h The limit of detection (LOD) was 0.0196 μg / ml, and the limit of quantitation (LOQ) was 0.0391 μg / ml; impurity Z 1a The limit of detection (LOD) is 0.0202 μg / ml, and the limit of quantitation (LOQ) is 0.0405 μg / ml; impurity Z 1g The limit of detection (LOD) was 0.0219 μg / ml, and the limit of quantitation (LOQ) was 0.0438 μg / ml; impurity Z 1d The limit of detection (LOD) was 0.0210 μg / ml, and the limit of quantitation (LOQ) was 0.0420 μg / ml; the LOD of impurity SM1 was 0.0208 μg / ml, and the LOD of quantitation was 0.0417 μg / ml; impurity SM 1iThe limit of detection (LOD) was 0.0211 μg / ml, and the limit of quantitation (LOQ) was 0.0422 μg / ml; impurity Z 1f The limit of detection (LOD) was 0.0223 μg / ml, and the limit of quantitation (LOQ) was 0.0446 μg / ml; impurity SM 1j The limit of detection was 0.0205 μg / ml, and the limit of quantitation was 0.0409 μg / ml. Attached Figure Description

[0077] Figure 1 The chromatogram is of the mixed solution;

[0078] Figure 2 The chromatogram of solution 1 is shown below.

[0079] Figure 3 The chromatogram of solution 2 is shown below.

[0080] Figure 4 The chromatogram of solution 1 is shown below the detection limit.

[0081] Figure 5 The chromatogram of solution 2 with the detection limit;

[0082] Figure 6 This is a chromatogram of the mixed solution under pH 3.7 conditions during the durability test;

[0083] Figure 7 This is a chromatogram of the mixed solution under pH 3.3 conditions during the durability test;

[0084] Figure 8 The chromatogram of the mixed solution under the condition of initial mobile phase ratio A:B = 77:23 during the durability test;

[0085] Figure 9 The chromatogram of the mixed solution under the condition of an initial mobile phase ratio of A:B = 73:27 during the durability test;

[0086] Figure 10 The chromatogram of the mixed solution under the condition of initial mobile phase ratio A:B = 67:33 during the durability test;

[0087] Figure 11 The chromatogram of the mixed solution under the condition of initial mobile phase ratio A:B = 63:37 during the durability test;

[0088] Figure 12 The chromatogram of the mixed solution under the condition of mobile phase gradient ratio A:B = 23:73 during the durability test;

[0089] Figure 13 The chromatogram of the mixed solution under the condition of a mobile phase gradient ratio A:B = 27:77 during the durability test;

[0090] Figure 14 The chromatogram of the mixed solution under the condition of mobile phase gradient ratio A:B = 47:53 during the durability test;

[0091] Figure 15 The chromatogram of the mixed solution under the condition of mobile phase gradient ratio A:B = 43:57 during the durability test;

[0092] Figure 16 The chromatogram of the mixed solution during the durability test at a column temperature of 23°C is shown.

[0093] Figure 17 The chromatogram of the mixed solution during the durability test at a column temperature of 27°C is shown.

[0094] Figure 18 The chromatogram is of the mixed solution at a flow rate of 1.1 ml / min during the durability test;

[0095] Figure 19 The chromatogram is of the mixed solution at a flow rate of 1.3 ml / min during the durability test;

[0096] Figure 20 This is the chromatogram of the mixed solution under LC441 column conditions during the durability test. Detailed Implementation

[0097] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0098] In this invention, the test sample is a solution of naphthostat mesylate Z1 as the main component, and the test sample contains naphthostat mesylate Z1, as well as impurity SM1 and impurity SM2. 1i Impurities SM 1j Impurity Z 1a Impurity Z 1b Impurity Z 1c Impurity Z 1d Impurity Z 1f Impurity Z 1g Impurity Z 1h Any one or more of the following.

[0099] The sample to be tested may also contain other unspecified compounds, which refer to other impurities that may be introduced when naphthostat mesylate Z1 is prepared using conventional methods known in the art.

[0100] Unless otherwise stated, all numerical values ​​of parameters and similar expressions used in this invention are to be understood as being modified by the term "approximately." That is, these values ​​are allowed a reasonable deviation or error range in practical applications and do not need to strictly conform to the given precise values. Unless otherwise indicated, the numerical parameters set forth in this invention are approximate values ​​that can be varied to obtain the desired performance.

[0101] Unless otherwise stated, the terms or phrases used in this invention should not be considered uncertain or unclear unless specifically defined, but should be understood in accordance with their ordinary meaning in the art.

[0102] In this invention, the term "high-performance liquid chromatography" (HPLC), also known as "high-pressure liquid chromatography," "high-speed liquid chromatography," "high-resolution liquid chromatography," or "modern column chromatography," is a chromatographic analysis technique based on classical liquid chromatography and incorporating gas chromatography theory. HPLC uses a liquid as the mobile phase and employs a high-pressure delivery system to pump a single solvent or a mixture of solvents and buffer solutions with different polarities into a chromatographic column packed with a stationary phase. After separation within the column, the components are detected by a detector, thus enabling the analysis of the sample. This method can be used to separate, identify, and quantify various complex samples.

[0103] In this invention, specificity, also known as selectivity or particularity, refers to the ability of an analytical method to accurately and selectively identify the analyte when other components may be present in the sample. Specificity is a measure of mutual interference when a method is used to analyze complex samples. This invention verifies whether the HPLC method has sufficient specificity to ensure accurate and error-free determination of the target analyte in complex samples.

[0104] In this invention, the limit of detection (LOD) refers to the minimum concentration or content of a target compound that an analytical method can reliably detect. Determining the detection sensitivity of an HPLC method, i.e., its ability to identify the lowest concentration of the target analyte in a sample, helps evaluate the reliability of the method at extremely low concentrations.

[0105] In this invention, the limit of quantitation (LOQ) refers to the lowest concentration or content of a target compound that an analytical method can reliably quantify. The LOQ reflects the sensitivity and quantitative detection capability of an analytical method, determining at what concentration an HPLC method can accurately and reliably quantify the target analyte.

[0106] In this invention, robustness refers to the degree to which the measurement results are unaffected by small changes in the measurement conditions. It is mainly used to test the ability of an analytical method to resist interference from variable test factors.

[0107] In this embodiment of the invention, the compound information is shown in Table 1.

[0108] Table 1. Compound Information Table

[0109]

[0110]

[0111] In this embodiment of the invention, all tests were performed according to high performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512).

[0112] In this embodiment of the invention, the preparation method of mobile phase A (potassium hexafluorophosphate solution with a concentration of 0.01 mol / L) is as follows: take 1.84 g of potassium hexafluorophosphate, add 1000 ml of water to dissolve it, and adjust the pH value to 3.5 with phosphoric acid.

[0113] Example 1. Analysis Method

[0114] (1) Preparation of the test solution

[0115] Solvent: 50% acetonitrile aqueous solution, prepared by mixing acetonitrile and water in a volume ratio of 50:50.

[0116] Test solution: Take an appropriate amount of this product, accurately weigh it, add solvent to dissolve and dilute it to prepare a solution containing about 0.1 mg per ml.

[0117] Control solution: Accurately measure 0.5 ml of the test solution and place it in a 100 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0118] System suitability solution: Take naphthostat mesylate Z1 system suitability reference standard, and take impurity Z... 1b Impurity Z 1c Impurity Z 1h Impurity Z 1a Impurity Z 1g Impurity Z 1d Impurity SM1, Impurity SM 1i Impurity Z 1f Impurities SM 1j An appropriate amount of reference standard was dissolved in acetonitrile by sonication and diluted to a concentration containing approximately Z impurities per 1 ml. 1b Impurity Z 1c Impurity Z 1h Impurity Z 1a Impurity Z 1gImpurity Z 1d Impurity SM1, Impurity SM 1i Impurities SM 1j 0.2mg, impurity Z 1f Accurately measure an appropriate amount of the 0.1 mg stock solution, add an appropriate amount of naphamostat mesylate Z1, place them in the same volumetric flask, dissolve and dilute with solvent to prepare a solution containing approximately 0.1 mg of naphamostat mesylate Z1 and approximately 0.3 μg of impurity SM1 per ml. 1c Impurity Z 1a Impurities SM 1i Impurities SM 1j Approximately 0.2 μg each, impurity Z 1b Approximately 7.0 μg, impurity Z 1d Approximately 1.0 μg, impurity Z 1h Impurity Z 1f Approximately 2.0 μg each, impurity Z 1g (Approximately 0.6 μg of mixed solution).

[0119] (2) Chromatographic conditions

[0120] The column was packed with octadecylsilane-bonded silica gel (ZORBAX SB-C18 4.6mm×250mm, 5μm, or a column with equivalent performance). The mobile phase was 0.01mol / L potassium hexafluorophosphate solution as mobile phase A and acetonitrile as mobile phase B. Linear gradient elution was performed according to Table 2. The flow rate was 1.2ml per minute. The column temperature was 25℃. The detection wavelength was 235nm. The injection volume was 15μl.

[0121] Table 2. Gradient Elution Table

[0122] Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 10 75 25 25 65 35 40 45 55 45 25 75 50 25 75 51 75 25 60 75 25

[0123] (3) Measurement

[0124] System suitability requirements: In the system suitability solution chromatogram, impurity Z 1d The separation between the main peak and the impurity SM1 peak should meet the requirements. The separation between the main peak and impurity Z... 1h The separation degree should meet the requirements.

[0125] Assay: Accurately measure the test solution and the control solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0126] Limits: In the chromatogram of the test solution, excluding solvent peaks and gradient elution peaks, the content of each impurity, calculated using the principal component self-comparison method with correction factors, should comply with the provisions of Table 3 below, and the total amount of impurities should not exceed 6.0%. Chromatographic peaks with a content less than 0.05% are negligible.

[0127] Table 3. Limits for Impurity Content

[0128]

[0129]

[0130] Example 2. Specificity

[0131] Possible impurities in naphthostat mesylate intermediate Z1: Impurity Z 1b Impurity Z 1c Impurity Z 1h Impurity Z 1a Impurity Z 1g Impurity Z 1d Impurity SM1, Impurity SM 1i Impurity Z 1f Impurities SM 1j 10 impurities. In this method, impurity Z... 1b Impurity Z 1c Impurity Z 1h Impurity Z 1a Impurity Z 1g Impurity Z 1d Impurity SM1, Impurity SM 1i Impurity Z 1f Impurities SM 1j The separation between principal components was examined.

[0132] (1) Preparation of solution

[0133] Solvent: 50% acetonitrile aqueous solution, prepared by mixing acetonitrile and water in a volume ratio of 50:50.

[0134] Impurity SM1 stock solution: Weigh approximately 20 mg of impurity SM1 reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well.

[0135] Impurities SM 1i Stock solution: Take impurities SM 1i Weigh approximately 20 mg of the reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well.

[0136] Impurities SM 1j Stock solution: Take impurities SM 1j Weigh approximately 20 mg of the reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile, sonicate to dissolve and dilute to the mark, and shake well.

[0137] Impurity Z 1a Stock solution: Take impurity Z 1a Weigh approximately 20 mg of the reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile, sonicate to dissolve and dilute to the mark, and shake well.

[0138] Impurity Z 1b Stock solution: Take impurity Z 1b Weigh approximately 20 mg of the reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile, sonicate to dissolve and dilute to the mark, and shake well.

[0139] Impurity Z 1c Stock solution: Take impurity Z 1c Weigh approximately 20 mg of the reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile, sonicate to dissolve and dilute to the mark, and shake well.

[0140] Impurity Z 1d Stock solution: Take impurity Z 1d Weigh approximately 20 mg of the reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile, sonicate to dissolve and dilute to the mark, and shake well.

[0141] Impurity Z 1f Stock solution: Take impurity Z 1f Weigh approximately 10 mg of the reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile, sonicate to dissolve and dilute to the mark, and shake well.

[0142] Impurity Z 1g Stock solution: Take impurity Z 1g Accurately weigh approximately 20 mg of the reference standard, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well.

[0143] Impurity Z 1h Stock solution: Take impurity Z 1h Weigh approximately 20 mg of the reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile, sonicate to dissolve and dilute to the mark, and shake well.

[0144] Impurity localization solution: Measure out the above impurity Z respectively 1f and impurity Z 1h Place 1.0 ml of each stock solution and 0.5 ml of each other impurity stock solution into 100 ml volumetric flasks, dilute to the mark with solvent, and shake well.

[0145] Test solution: Weigh approximately 10 mg of the test sample accurately, place it in a 100 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0146] Mixed solution: Accurately weigh approximately 10 mg of the test sample, place it in a 100 ml volumetric flask, dissolve it in solvent, and then measure 0.15 ml of impurity SM1 stock solution and impurity SM 1i Impurities SM 1j Impurity Z 1a Impurity Z 1c 0.1 ml of each stock solution, and Z impurity. 1b 3.5 ml of stock solution, Z impurities1d 0.5 ml of stock solution, Z impurities 1g 0.3 ml of stock solution, Z impurities 1f Impurity Z 1h Place 1.0 ml of each stock solution into the same 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0147] (2) Detection

[0148] Inject 20 μl of each of the blank solvent, each impurity localization solution, the test solution, and the mixed solution, and record the chromatograms.

[0149] The results are shown in Tables 4 and 5. Figure 1 As shown, the blank solution does not interfere with the detection of known impurities and the main component; the minimum resolution between the main peak and adjacent impurity peaks, and between each known impurity peak in the mixed solution, is 2.76, which is greater than 1.5, and meets the specificity requirements.

[0150] Table 4. Results of Specificity Test

[0151]

[0152] Table 5. Figure 1 Integral Results Table

[0153]

[0154] Example 3. Limit of Quantification

[0155] (1) Solution preparation

[0156] Naphthostat mesylate Z1 stock solution: Weigh approximately 20 mg of the test sample accurately, place it in a 100 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0157] Limit of Quantitation Stock Solution 1: Measure naphthostat mesylate Z1, impurity SM1, and impurity SM. 1i Impurities SM 1j Impurity Z 1a Impurity Z 1c Impurity Z 1b Impurity Z 1d Impurity Z 1g 2.5 ml each of stock solution and impurity Z 1f Add 5.0 ml of the stock solution to the same 100 ml volumetric flask, dilute to the mark with solvent, and mix well. (5%)

[0158] Limit of Quantity Stock Solution 2: Measure naphthostat mesylate Z1 and impurity Z. 1h Combine 2.5 ml of each stock solution in the same 100 ml volumetric flask, dilute to the mark with solvent, and mix well. (5%)

[0159] Limit of Quantification Solution 1: Measure 0.8 ml of Limit of Quantification Stock Solution 1, place it in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0160] Limit of Quantification Solution 2: Measure 0.8 ml of Limit of Quantification Stock Solution 2, place it in a 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0161] (2) Detection

[0162] Take 15 μl of each of the above limit of quantitation solution 1 and limit of quantitation solution 2, inject them 6 times consecutively and record the chromatograms, and calculate the peak height to noise ratio (S / N) and peak area RSD of each component.

[0163] The test results are shown in Tables 6 to 8. Figures 2-3 Impurity Z 1b The limit of quantitation was 0.0387 μg / ml, the peak area RSD was 4.8% (N=6), the average S / N ratio was 19.4, and the concentration in the sample was expressed as 0.039%; impurity Z 1c The limit of quantitation (LOQ) concentration was 0.0406 μg / ml, the peak area RSD was 8.0% (N=6), the average S / N ratio was 13.5, and the concentration in the sample was expressed as 0.041%; the LOQ concentration of naphamostat mesylate Z1 was 0.0411 μg / ml, the peak area RSD was 7.8% (N=6), the average S / N ratio was 12.7; impurity Z 1h The limit of quantitation (LOQ) was 0.0391 μg / ml, the peak area RSD was 6.9% (N=6), less than 10.0%; the average S / N ratio was 19.0, which, expressed as a concentration of 0.039% in the sample, was [missing information]. Impurity Z [missing information]. 1a The limit of quantitation was 0.0405 μg / ml, the peak area RSD was 9.1% (N=6), the average S / N was 10.5, and the concentration in the sample was expressed as 0.041%; impurity Z 1g The limit of quantitation was 0.0438 μg / ml, the peak area RSD was 3.0% (N=6), the average S / N ratio was 17.1, and the concentration in the sample was expressed as 0.044%; impurity Z 1d The limit of quantitation (LOQ) concentration was 0.0420 μg / ml, the peak area RSD was 5.9% (N=6), the average S / N ratio was 18.8, and the concentration expressed in the sample was 0.042%. The LQ concentration of impurity SM1 was 0.0417 μg / ml, the peak area RSD was 4.6% (N=6), the average S / N ratio was 16.1, and the concentration expressed in the sample was 0.042%. 1i The limit of quantitation was 0.0422 μg / ml, the peak area RSD was 4.4% (N=6), the average S / N ratio was 19.1, and the concentration in the sample was expressed as 0.042%; impurity Z1f The limit of quantitation (LOQ) was 0.0446 μg / ml, the peak area RSD was 3.4% (N=6), the average S / N ratio was 28.1, and the concentration in the sample was expressed as 0.045%; impurity SM 1j The limit of quantitation was 0.0409 μg / ml, the peak area RSD was 3.5% (N=6), the average S / N ratio was 18.0, and the concentration in the sample was expressed as 0.041%.

[0164] Table 6. Results of Limit of Quantitation Determination

[0165]

[0166]

[0167]

[0168] Table 7. Figure 2 Integral Results Table

[0169]

[0170] Table 8. Figure 3 Integral Results Table

[0171]

[0172] Example 4. Detection Limit

[0173] Detection limit solution 1: Measure 5.0 ml of quantitation limit solution 1, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0174] Detection limit solution 2: Measure 5.0 ml of quantitation limit solution 2, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0175] Take 15 μl of the detection limit solution and inject it three times consecutively. Record the chromatogram and calculate the ratio of peak height of each impurity to noise (signal-to-noise ratio).

[0176] The test results are shown in Tables 9 to 11 below. Figures 4-5 Impurity Z 1b The detection limit was 0.0194 μg / ml, the average S / N ratio was 8.2, and the concentration present in the sample was expressed as 0.019%; impurity Z 1c The detection limit concentration was 0.0203 μg / ml, the average S / N ratio was 5.5, and the concentration present in the sample was expressed as 0.020%; the detection limit concentration of naphamostat mesylate Z1 was 0.0206 μg / ml, the average S / N ratio was 5.5; impurity Z 1hThe detection limit was 0.0196 μg / ml, the average S / N ratio was 9.3, and the concentration present in the sample was expressed as 0.020%; impurity Z 1a The detection limit was 0.0202 μg / ml, the average S / N ratio was 4.4, and the concentration present in the sample was expressed as 0.020%; impurity Z 1g The detection limit was 0.0219 μg / ml, the average S / N ratio was 7.2, and the concentration present in the sample was expressed as 0.022%; impurity Z 1d The detection limit concentration was 0.0210 μg / ml, the average S / N ratio was 7.8, and the concentration present in the sample was expressed as 0.021%; the detection limit concentration of impurity SM1 was 0.0208 μg / ml, the average S / N ratio was 6.8, and the concentration present in the sample was expressed as 0.021%; impurity SM 1i The detection limit was 0.0211 μg / ml, the average S / N ratio was 8.1, and the concentration present in the sample was expressed as 0.021%; impurity Z 1f The detection limit was 0.0223 μg / ml, the average S / N ratio was 11.8, and the concentration present in the sample was expressed as 0.022%; impurity SM 1j The detection limit was 0.0205 μg / ml, the average S / N ratio was 8.0, and the concentration in the sample was expressed as 0.021%.

[0177] Table 9. Results of Limit of Detection (LOD) determination

[0178]

[0179]

[0180] Table 10. Figure 4 Integral Results Table

[0181]

[0182] Table 11. Figure 5 Integral Results Table

[0183]

[0184] Example 5. Durability of chromatographic conditions

[0185] Take the mixed solution under “Example 2. Specificity” and test it under normal conditions and with adjustments to each experimental condition (flow rate ±0.1 ml / min, column temperature ±2℃, pH value ±0.2, initial proportion of mobile phase B ±2%, gradient proportion ±2%, and different batches of the same manufacturer and model of chromatographic column) until the instrument system is stable. Record the chromatograms and examine the changes in the resolution between the peaks of each component.

[0186] The test results are shown in Tables 12 to 28. Figures 6-20 When there are minor changes in chromatographic conditions such as flow rate, column temperature, mobile phase pH, initial mobile phase ratio, gradient, and column, the minimum resolution between the main component and adjacent components, as well as between each impurity peak, is 2.09. The relative retention time and area normalized content of each component are basically consistent, all greater than 1.5; the method has good robustness.

[0187] Table 12. Results of the durability test for changes in chromatographic conditions

[0188]

[0189]

[0190] Table 13. Results of the durability test for changes in chromatographic conditions

[0191]

[0192] Table 14. Figure 6 Integral Results Table

[0193]

[0194]

[0195] Table 15. Figure 7 Integral Results Table

[0196]

[0197] Table 16. Figure 8 Integral Results Table

[0198]

[0199] Table 17. Figure 9 Integral Results Table

[0200]

[0201]

[0202] Table 18. Figure 10 Integral Results Table

[0203]

[0204] Table 19. Figure 11 Integral Results Table

[0205]

[0206]

[0207] Table 20. Figure 12 Integral Results Table

[0208]

[0209] Table 21. Figure 13 Integral Results Table

[0210]

[0211] Table 22. Figure 14 Integral Results Table

[0212]

[0213] Table 23. Figure 15 Integral Results Table

[0214]

[0215] Table 24. Figure 16 Integral Results Table

[0216]

[0217]

[0218] Table 25. Figure 17 Integral Results Table

[0219]

[0220] Table 26. Figure 18 Integral Results Table

[0221]

[0222] Table 27. Figure 19 Integral Results Table

[0223]

[0224] Table 28. Figure 20 Integral Results Table

[0225]

Claims

1. A method for separating naphthostat mesylate Z1 and its related impurities using HPLC, characterized in that, The relevant impurities include impurity SM1 and impurity SM. 1i Impurities SM 1j Impurity Z 1a Impurity Z 1b Impurity Z 1c Impurity Z 1d Impurity Z 1f Impurity Z 1g Impurity Z 1h Any one or more of the following; the method includes using octadecylsilane-bonded silica gel as the chromatographic column packing material, potassium hexafluorophosphate solution as mobile phase A, acetonitrile as mobile phase B, and sequentially removing impurities Z by linear gradient elution. 1b Impurity Z 1c Naphthostat mesylate Z1, impurities Z 1h Impurity Z 1a Impurity Z 1g Impurity Z 1d Impurity SM1, Impurity SM 1i Impurity Z 1f and / or impurities SM 1j Separation is performed; the structural formula of the naphthostat mesylate Z1 is shown in Formula I; The structural formula of the impurity SM1 is shown in Formula II. 1i The structural formula is shown in Formula III, and the impurity SM 1j The structural formula is shown in Formula IV, and the impurity Z 1a The structural formula is shown in Formula V, and the impurity Z 1b The structural formula is shown in Formula VI, and the impurity Z 1c The structural formula is shown in formula VII, and the impurity Z 1d The structural formula is shown in formula VIII, and the impurity Z 1f The structural formula is shown in formula IX, and the impurity Z 1g The structural formula is shown in Formula X, and the impurity Z 1h The structural formula is shown in equation XI; 2. The method according to claim 1, characterized in that, The gradient elution includes: At 0 min, the volume ratio of mobile phase A to mobile phase B is 65-85:35-15; For 10 minutes, the volume ratio of mobile phase A to mobile phase B is 65-85:35-15; For 25 minutes, the volume ratio of mobile phase A to mobile phase B is 55-75:45-25; For 40 minutes, the volume ratio of mobile phase A to mobile phase B is 35-55:65-45; After 45 minutes, the volume ratio of mobile phase A to mobile phase B is 35-15:65-85. For 50 minutes, the volume ratio of mobile phase A to mobile phase B is 35-15:65-85. 51 min, the volume ratio of mobile phase A to mobile phase B is 65-85:35-15; After 60 minutes, the volume ratio of mobile phase A to mobile phase B is 65-85:35-15.

3. The method according to claim 1, characterized in that, The concentration of the potassium hexafluorophosphate solution is 0.005 mol / L to 0.02 mol / L. pH is 2.5–5.

4. The method according to claim 1, characterized in that, The flow rate of the mobile phase is 1.0 ml / min to 1.5 ml / min, and the column temperature of the chromatographic column is 20℃ to 30℃.

5. A method for qualitative identification of naphthostat mesylate Z1 and its related impurities, characterized in that, Naphthostat mesylate Z1 and its related impurities are separated by the method according to any one of claims 1-4 and detected by a detector to obtain a chromatogram; the detection wavelength of the detector is 235±2nm.

6. The method according to claim 5, characterized in that, The presence of relevant impurities in the test sample is determined by comparing the chromatographic behavior of the test sample and the reference sample.

7. The method according to claim 5, characterized in that, Using naphthostat mesylate Z1 as a reference peak, the relative retention time was 0.16 ± 0.2, and it was identified as impurity Z. 1b The relative retention time was 0.33 ± 0.2, and it was identified as impurity Z. 1c ; The relative retention time was 1.05 ± 0.2, and it was identified as impurity Z. 1h ; The relative retention time was 1.47 ± 0.2, and it was identified as impurity Z. 1a ; The relative retention time was 1.57 ± 0.2, and it was identified as impurity Z. 1g ; The relative retention time was 1.61 ± 0.2 seconds, and it was identified as impurity Z. 1d ; The relative retention time was 1.64 ± 0.2, and it was identified as impurity SM1; The relative retention time was 1.92 ± 0.2, and it was identified as impurity SM. 1i ; The relative retention time was 1.98 ± 0.2, and it was identified as impurity Z. 1f ; The relative retention time was 2.13 ± 0.2 seconds, and it was identified as impurity SM. 1j .

8. A method for quantitatively detecting naphthostat mesylate Z1 and its related impurities, characterized in that, Naphthostat mesylate Z1 and its related impurities are separated and detected by the method described in any one of claims 5-7, and a chromatogram is obtained; based on the obtained chromatogram, the content of each impurity is calculated by the principal component self-comparison method with correction factor.

9. The method according to claim 8, characterized in that, Before separation, the test solution is prepared using an aqueous solution of acetonitrile.

10. The method according to claim 8, characterized in that, Impurity Z 1b The correction factor is 1.3, and the impurity Z 1c The correction factor is 1.2, and the impurity Z 1h The correction factor is 1.5, and the impurity Z 1a The correction factor is 1.7, and the impurity Z 1g The correction factor is 1.1, and the impurity Z 1d The correction factor is 1.2, the correction factor for impurity SM1 is 1.1, and the correction factor for impurity SM is 1.

1. 1i The correction factor is 1.3, and the impurity Z 1f The correction factor is 1.2, and the impurity SM 1j The correction factor is 1.2, and the correction factor for other individual impurities is 1.0.

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