Method for separating and identifying nafamostat mesylate and impurities thereof and measuring content of nafamostat mesylate and impurities thereof

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for separating and identifying nafamostat mesylate and impurities thereof and determining the content of nafamostat mesylate. The impurities comprise DMAP, an impurity NAA-Z1, an impurity NAA-Z1b, an impurity NAA-X1e, an impurity A, an impurity B, an impurity C, an impurity D, an impurity E, an impurity F, an impurity G, an impurity I, an impurity J and / or an impurity K. The method comprises the following steps: taking octadecylsilane chemically bonded silica as a chromatographic column filler; carrying out gradient elution by taking a sodium heptanesulfonate solution as a mobile phase A and acetonitrile as a mobile phase B, and then detecting by adopting an ultraviolet detector; and determining the nature of each impurity according to the chromatogram, and calculating the content of each impurity by adopting a principal component self-contrast method multiplied by a correction factor. The method can effectively determine 14 known impurities in nafamostat mesylate, and has the advantages of strong specificity, high sensitivity, good reproducibility, strong durability, and simple and feasible 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 determining the content of nafamostat mesilate and impurities thereof. BACKGROUND

[0002] Nafamostat mesilate (NM) is a broad-spectrum and potent serine protease inhibitor, which has strong inhibitory effect on trypsin, thrombin, plasmin and various serine proteases such as complement components. Nafamostat mesilate is developed by Toris Pharma Co., Ltd. in Japan. It is initially used as a prophylactic and therapeutic drug for acute pancreatitis, and then applied to anticoagulation in blood purification extracorporeal circulation due to its strong anticoagulant activity. At present, it is mainly used in the treatment of acute pancreatitis, acute exacerbation of chronic pancreatitis, acute pancreatitis after pancreatography, traumatic pancreatitis, postoperative acute pancreatitis, disseminated intravascular coagulation (DIC) and anticoagulation during blood extracorporeal circulation.

[0003] In the research of the synthesis process of nafamostat mesilate, it is found that the following 14 known impurities are easily introduced in the synthesis process: 4-dimethylaminopyridine (DMAP), impurity NAA-Z1, impurity NAA-Z 1b , impurity NAA-X 1e , impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity I, impurity J and impurity K. The structural formulas of nafamostat mesilate, DMAP, impurity NAA-Z1, impurity NAA-Z 1b , impurity NAA-X 1e , impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity I, impurity J and impurity K are shown in formulas 1-15, respectively. If the content of these impurities exceeds a certain limit, it will affect the quality of nafamostat mesilate bulk drug and subsequent preparation products.

[0004]

[0005]

[0006] The query found that only three components, impurity A, impurity B and main component nafamostat mesylate, were separated and determined in the nafamostat mesylate related substance method of Japanese Pharmacopoeia 18th edition (JP18). In the prior art, only CN117247335A discloses a method for detecting nafamostat mesylate intermediates, which uses Agilent 1260DAD type (or Shimadzu LC-2030) liquid chromatograph for detection, an octadecylsilane bonded silica gel chromatographic column as the filler, gradient elution for separation; the detector is an ultraviolet detector, the detection wavelength is 254 nm; the flow rate is 1.0 mL per minute; the injection volume is 10 μl; the patent determines the test solution by automatic integration method, and calculates the purity of the test product according to the peak area. The method does not disclose the key chromatographic conditions such as mobile phase, and cannot simultaneously determine the above-mentioned 14 known impurities of nafamostat mesylate.

[0007] Therefore, in order to ensure the controllable quality of nafamostat mesylate bulk drug and subsequent drugs, it is necessary to establish a new method to separate and determine the above-mentioned 14 known impurities and other single impurities. SUMMARY

[0008] Therefore, one of the purposes of the present application is to provide a method for separating nafamostat mesylate and related impurities by high performance liquid chromatography, which can effectively separate nafamostat mesylate and related impurities within 55 minutes or more than 55 minutes, providing support for the identification and quantification of subsequent impurities.

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

[0010] The method for separating nafamostat mesylate and related impurities by high performance liquid chromatography comprises: using octadecylsilane bonded silica gel as the filler of the chromatographic column, using heptane sodium sulfonate solution as mobile phase A and acetonitrile as mobile phase B, and separating nafamostat mesylate and related impurities by gradient elution;

[0011] The related impurities include any one or more of DMAP, impurity NAA-Z1, impurity NAA-Z 1b , impurity NAA-X 1e , impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity I, impurity J and impurity K; the structural formula of nafamostat mesylate is shown as formula 1, the structural formula of DMAP is shown as formula 2, the structural formula of impurity NAA-Z1 is shown as formula 3, the structural formula of impurity NAA-Z 1b is shown as formula 4, and the structural formula of impurity NAA-X 1eFormula 5, the structural formula of the impurity A is shown as Formula 6, the structural formula of the impurity B is shown as Formula 7, the structural formula of the impurity C is shown as Formula 8, the structural formula of the impurity D is shown as Formula 9, the structural formula of the impurity E is shown as Formula 10, the structural formula of the impurity F is shown as Formula 11, the structural formula of the impurity G is shown as Formula 12, the structural formula of the impurity I is shown as Formula 13, the structural formula of the impurity J is shown as Formula 14, and the structural formula of the impurity K is shown as Formula 15.

[0012]

[0013]

[0014] Further, the sample solution to be tested contains nafamostat mesylate and the related impurities, which include DMAP, impurity NAA-Z1, impurity NAA-Z 1b , impurity NAA-X 1e , impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity I, impurity J and impurity K.

[0015] As preferred, the related impurities are any one or more of DMAP, impurity NAA-Z1, impurity NAA-Z 1b , impurity NAA-X 1e , impurity C, impurity D, impurity E, impurity F, impurity G, impurity I, impurity J and impurity K; or the related impurities are any one or more of DMAP, impurity NAA-Z1, impurity NAA-Z 1b , impurity NAA-X 1e , impurity C, impurity D, impurity E, impurity F, impurity G, impurity I, impurity J, impurity K, in combination with any one or more of impurity A and impurity B.

[0016] Further, impurity NAA-Z 1b , impurity A, DMAP, impurity B, impurity NAA-X 1e , impurity F, impurity D, impurity E, impurity I, impurity NAA-Z1, impurity C, nafamostat mesylate, impurity G, impurity J and / or impurity K are sequentially separated.

[0017] According to the sequence of separation, each component can be qualitatively determined.

[0018] Further, the running time is 55 minutes.

[0019] Further, the mobile phase A is a sodium heptanesulfonate solution with a concentration of 0.03 mol / L.

[0020] As preferred, the sodium heptanesulfonate solution is prepared by the following method: taking sodium heptanesulfonate in proper amount, adding proper amount of 0.6% glacial acetic acid solution to dissolve, and mixing uniformly to obtain.

[0021] Further, the gradient elution program is set as follows:

[0022] 0 minutes, the volume ratio of the mobile phase A and the mobile phase B is 75-85:25-15;

[0023] 10 minutes, the volume ratio of the mobile phase A and the mobile phase B is 75-85:25-15;

[0024] 35 minutes, the volume ratio of the mobile phase A and the mobile phase B is 65-75:35-25;

[0025] 45 minutes, the volume ratio of the mobile phase A and the mobile phase B is 45-55:55-45;

[0026] 46 minutes, the volume ratio of the mobile phase A and the mobile phase B is 75-85:25-15;

[0027] 55 minutes, the volume ratio of the mobile phase A and the mobile phase B is 75-85:25-15.

[0028] As preferred, the gradient elution program is set as follows:

[0029] 0 minutes, the volume ratio of the mobile phase A and the mobile phase B is 78-82:22-18;

[0030] 10 minutes, the volume ratio of the mobile phase A and the mobile phase B is 78-82:22-18;

[0031] 35 minutes, the volume ratio of the mobile phase A and the mobile phase B is 68-72:32-28;

[0032] 45 minutes, the volume ratio of the mobile phase A and the mobile phase B is 48-52:52-48;

[0033] 46 minutes, the volume ratio of the mobile phase A and the mobile phase B is 78-82:22-18;

[0034] 55 minutes, the volume ratio of the mobile phase A and the mobile phase B is 78-82:22-18.

[0035] As most preferred, the gradient elution program is set as follows:

[0036] 0 minutes, the volume ratio of the mobile phase A and the mobile phase B is 80:20;

[0037] After 10 minutes, the volume ratio of mobile phase A to mobile phase B is 80:20;

[0038] After 35 minutes, the volume ratio of mobile phase A to mobile phase B is 70:30;

[0039] After 45 minutes, the volume ratio of mobile phase A to mobile phase B is 50:50.

[0040] After 46 minutes, the volume ratio of mobile phase A to mobile phase B is 80:20;

[0041] After 55 minutes, the volume ratio of mobile phase A to mobile phase B is 80:20.

[0042] Furthermore, the flow rate of the mobile phase is 0.8 ml / min to 1.6 ml / min; the column temperature of the chromatographic column is 35℃ to 45℃; and the injector temperature is 15±2℃.

[0043] 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.

[0044] Preferably, the column temperature of the chromatographic column is 38℃~42℃, and most preferably 40℃.

[0045] Preferably, the temperature of the injector is 15°C.

[0046] Preferably, the chromatographic column has dimensions of 4.6 mm × 250 mm and a diameter of 5 μm.

[0047] As the preferred option, the chromatographic column is a ZORBAX Eclipse XDB-C18 4.6mm×250mm, 5μm, or a column with equivalent performance.

[0048] Preferably, the injection volume is 10 μl.

[0049] The second objective of this invention is to provide a method for identifying naphamostat mesylate and its related impurities. This method can effectively identify multiple impurities in naphamostat mesylate using a single chromatographic system, with a detection limit as low as 0.14 μg / ml to 0.15 μg / ml.

[0050] To achieve the above objectives, the present invention adopts the following technical solution:

[0051] The method for identifying nafamostat mesylate and its related impurities includes the following steps:

[0052] (1) Naphazoline mesylate and its related impurities were separated using the aforementioned separation method;

[0053] (2) Detection was performed using a detector with a detection wavelength of 240nm to 280nm to obtain a chromatogram.

[0054] Preferably, the detector is an ultraviolet detector.

[0055] Preferably, the detection wavelength is 260±2nm.

[0056] As the most preferred option, the detection wavelength is 260 nm.

[0057] Furthermore, by comparing the chromatograms of the test sample and the reference sample, the consistency of their chromatographic behavior is used to determine whether the test sample contains naphthostat mesylate and its related impurities.

[0058] Furthermore, using nafamostat mesylate as a reference peak, the relative retention time was 0.13 ± 0.2, which was determined to be the impurity NAA-Z. 1b The relative retention time was 0.16±0.2, and it was determined to be impurity A; the relative retention time was 0.19±0.2, and it was determined to be DMAP; the relative retention time was 0.38±0.2, and it was determined to be impurity B; the relative retention time was 0.46±0.2, and it was determined to be impurity NAA-X. 1e The following impurities were identified: F (relative retention time 0.54±0.2), D (relative retention time 0.79±0.2), E (relative retention time 0.82±0.2), I (relative retention time 0.85±0.2), NAA-Z1 (relative retention time 0.91±0.2), C (relative retention time 0.97±0.2), G (relative retention time 1.25±0.2), J (relative retention time 1.26±0.2), and K (relative retention time 1.30±0.2).

[0059] Furthermore, under optimal chromatographic conditions, the impurity NAA-Z had a retention time of 4.2 ± 0.5 min. 1b Impurity A has a retention time of 5.1 ± 0.5 min; DMAP has a retention time of 5.6 ± 0.5 min; impurity B has a retention time of 12.0 ± 0.5 min; and impurity NAA-X has a retention time of 14.4 ± 0.5 min. 1eImpurity F had a retention time of 17.0 ± 0.5 min; impurity D had a retention time of 24.9 ± 0.5 min; impurity E had a retention time of 25.7 ± 0.5 min; impurity I had a retention time of 26.7 ± 0.5 min; impurity NAA-Z1 had a retention time of 28.7 ± 0.5 min; impurity C had a retention time of 30.5 ± 0.5 min; naphthostat mesylate had a retention time of 31.5 ± 0.5 min; impurity G had a retention time of 39.4 ± 0.5 min; impurity J had a retention time of 39.8 ± 0.5 min; and impurity K had a retention time of 44.1 ± 0.5 min.

[0060] Impurities can be identified based on the relative retention time or retention time mentioned above.

[0061] The third objective of this invention is to provide a method for detecting the content of naphamostat mesylate and its related impurities. This method can effectively determine a variety of low-content impurities in naphamostat mesylate, with a limit of quantitation concentration as low as 0.2 μg / ml to 0.3 μg / ml.

[0062] To achieve the above objectives, the present invention adopts the following technical solution:

[0063] A method for determining the content of naphamostat mesylate and its related impurities includes the following steps:

[0064] (1) The aforementioned identification method was used to separate and detect naphalosporin mesylate and its related impurities, and chromatograms were obtained;

[0065] (2) Based on the chromatogram obtained in step (1), the content of each impurity is calculated by the principal component self-comparison method multiplied by the correction factor.

[0066] Furthermore, the impurity NAA-Z 1b The correction factor is 1.3, the correction factor for impurity A is 0.75, the correction factor for DMAP is 0.78, the correction factor for impurity B is 0.50, and the correction factor for impurity NAA-X is... 1e The correction factor for impurity F is 1.3, the correction factor for impurity D is 1.0, the correction factor for impurity E is 0.69, the correction factor for impurity I is 0.67, the correction factor for impurity NAA-Z1 is 2.5, the correction factor for impurity C is 1.4, the correction factor for impurity G is 0.60, the correction factor for impurity J is 1.3, the correction factor for impurity K is 0.72, and the correction factor for other individual impurities is 1.0.

[0067] Furthermore, before separation, the test solution is prepared using a mixed solution of water, acetonitrile, and glacial acetic acid as the solvent; the volume ratio of water, acetonitrile, and glacial acetic acid in the solvent is 650-800:250-350:0.05-0.2.

[0068] Preferably, the volume ratio of water, acetonitrile, and glacial acetic acid in the solvent is 700:300:0.1.

[0069] Furthermore, the test solution includes a sample solution and a control solution.

[0070] Furthermore, the concentration of the sample solution is preferably 1.0 mg / ml.

[0071] Furthermore, the preparation method of the control solution is as follows: 1) Take an appropriate amount of this product (naphthostat mesylate), accurately weigh it, dissolve and dilute it with solvent to prepare a solution containing about 1.0 mg per 1 ml, and obtain the sample solution; 2) Accurately measure 1 ml of the sample solution, place it in a 100 ml volumetric flask, dilute it to the mark with solvent, shake well, accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute it to the mark with solvent, shake well, and obtain the control solution.

[0072] Furthermore, the test solution may also include a mixed solution, a system suitability solution, or a sensitivity solution.

[0073] Furthermore, the method for preparing the system suitability solution is as follows: Take an appropriate amount of naphthostat mesylate system suitability reference standard (containing naphthostat mesylate, impurity A, impurity B, impurity C, impurity D, impurity G, impurity I, and impurity J), dissolve and dilute it with a solvent to prepare a solution containing approximately 1 mg per 1 ml.

[0074] Furthermore, the method for preparing the sensitivity solution is as follows: take 5 ml of the control solution, add 5 ml of solvent, and mix well.

[0075] Furthermore, the method for preparing the mixed solution is as follows:

[0076] 1) Take impurity NAA-Z1 and impurity NAA-Z 1b Appropriate amounts of impurities A, B, C, E, I, J, K, and 4-dimethylaminopyridine reference standard were dissolved in methanol by sonication and diluted to prepare a stock solution containing approximately 0.2 mg per ml.

[0077] 2) Take an appropriate amount of impurity G reference standard, add 0.1% glacial acetic acid methanol, sonicate to dissolve and dilute to prepare a stock solution 2 containing about 0.2 mg per ml;

[0078] 3) Take an appropriate amount of impurity D reference standard, add acetonitrile-water (50:50), sonicate to dissolve and dilute to prepare a stock solution containing approximately 0.2 mg per ml;

[0079] 4) Take the impurity NAA-X 1e Add an appropriate amount of reference standard, add approximately 10% of the total volume of dimethyl sulfoxide, sonicate to dissolve, and then dilute with water-acetonitrile (90:10) to prepare a stock solution containing approximately 0.2 mg per 1 ml;

[0080] 5) Take an appropriate amount of impurity F reference standard, add about 20% of the total volume of dimethyl sulfoxide, sonicate to dissolve, and then dilute with methanol to prepare a stock solution containing about 0.2 mg per 1 ml.

[0081] 6) Accurately measure appropriate amounts of the above stock solutions 1-5, take an appropriate amount of naphthostat mesylate, place them in the same volumetric flask, add solvent to dissolve and dilute to prepare a mixed solution containing approximately 1.0 mg of naphthostat mesylate and approximately 5 μg of other impurities per 1 ml.

[0082] Furthermore, the aforementioned method was used to detect whether the content of relevant impurities in naphthostat mesylate was up to standard; if the content of a single impurity was not higher than 0.10% and the total amount of impurities was not higher than 0.5%, the sample quality was considered up to standard; conversely, if the content of any single impurity was higher than 0.10% and / or the total amount of impurities was higher than 0.5%, the sample quality was considered down to standard.

[0083] The beneficial effects of this invention are as follows:

[0084] 1. This invention employs high-performance liquid chromatography (HPLC), and by considering the structural characteristics of the impurities to be studied and screening chromatographic conditions, establishes a set of methods capable of simultaneously determining 14 known impurities (impurity NAA-Z1, impurity NAA-Z) in nafamostat mesylate. 1b Impurities NAA-X 1e This method relates to 4-dimethylaminopyridine, impurities A, B, C, D, E, F, G, I, J, K, and other individual impurities. The method is highly specific, sensitive, reproducible, robust, and simple to operate.

[0085] 2. This method can simultaneously determine 14 known impurities in naphthostat mesylate within 55 minutes, with short separation time and good separation effect.

[0086] 3. This method is characterized by high sensitivity, and can effectively identify and determine the content of multiple impurities in naphthostat mesylate using only one chromatographic system. The detection limit is as low as 0.14 μg / ml to 0.15 μg / ml, and the quantitation limit is as low as 0.2 μg / ml to 0.3 μg / ml. Attached Figure Description

[0087] Figure 1 The chromatogram of the mixed solution in the specificity test;

[0088] Figure 2 The chromatogram of solution 1 is shown below.

[0089] Figure 3 The chromatogram of solution 2 is shown below.

[0090] Figure 4 The chromatogram of solution 1 is shown below the detection limit.

[0091] Figure 5 The chromatogram of solution 2 with the detection limit;

[0092] Figure 6 This is a chromatogram of the mixed solution under normal conditions during the durability test;

[0093] Figure 7 The chromatogram is of the mixed solution at a flow rate of 1.1 ml / min during the durability test.

[0094] Figure 8 The chromatogram is of the mixed solution at a flow rate of 1.3 ml / min during the durability test.

[0095] Figure 9 The chromatogram of the mixed solution in the durability test at a column temperature of 38°C is shown.

[0096] Figure 10 The chromatogram of the mixed solution in the durability test at a column temperature of 42°C;

[0097] Figure 11 The chromatogram of the mixed solution under the condition of an initial mobile phase ratio of A:B = 82:18 during the durability test;

[0098] Figure 12 The chromatogram of the mixed solution under the condition of an initial mobile phase ratio of A:B = 78:22 during the durability test;

[0099] Figure 13 The chromatogram of the mixed solution under the condition of a mobile phase gradient ratio A:B = 72:28 during the durability test;

[0100] Figure 14 The chromatogram of the mixed solution under the condition of a mobile phase gradient ratio A:B = 68:32 during the durability test;

[0101] Figure 15 The chromatogram of the mixed solution under the condition of a mobile phase gradient ratio A:B = 52:48 during the durability test;

[0102] Figure 16 This is a chromatogram of the mixed solution under the condition of a mobile phase gradient ratio of A:B = 48:52 during the durability test. Detailed Implementation

[0103] 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.

[0104] In this invention, the test sample refers to a solution whose main component is naphthostat mesylate. The test sample solution contains naphthostat mesylate, DMAP, impurity NAA-Z1, and impurity NAA-Z. 1b Impurities NAA-X 1e Any one or more of the following: impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity I, impurity J, and impurity K.

[0105] The test sample solution may also contain other unspecified compounds, which refer to other impurities that may be introduced when preparing naphthostat mesylate using conventional methods known in the art.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] 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.

[0113] In the embodiments of the present invention, the structural formulas of the compounds involved are shown in Table 1.

[0114] Table 1. List of Compound Information

[0115]

[0116]

[0117] In this embodiment of the invention, the determination was strictly performed according to the high performance liquid chromatography method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512). The experiment was conducted in the dark.

[0118] In this embodiment of the invention, the solvent is a mixed solution of water, acetonitrile and glacial acetic acid, wherein the volume ratio of water, acetonitrile and glacial acetic acid is 700:300:0.1.

[0119] In this embodiment of the invention, the sodium heptanesulfonate solution with a concentration of 0.03 mol / L is prepared as follows: take 6.61 g of sodium heptanesulfonate, add 1000 ml of 0.6% glacial acetic acid solution to dissolve it, mix well, and the solution is obtained.

[0120] Example 1. Method for the separation and determination of related substances in naphthenic acid.

[0121] This invention establishes an accurate, reproducible, and robust HPLC method by screening chromatographic columns and conditions. This method can be used to separate and determine related substances in naphthenic acid: DMAP, impurity NAA-Z1, and impurity NAA-Z. 1b Impurities NAA-X 1e Impurities A, B, C, D, E, F, G, I, J, K, and other individual impurities. The specific methods are as follows:

[0122] 1. Prepare the test solution

[0123] Test solution: Take an appropriate amount of this product, accurately weigh it, dissolve and dilute it with solvent to prepare a solution containing about 1.0 mg per 1 ml.

[0124] Control solution: Accurately measure 1 ml of the test solution and place it in a 100 ml volumetric flask. Dilute to the mark with solvent and shake well. Accurately measure 1 ml of the test solution and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0125] System suitability solution: Take an appropriate amount of naphthostat mesylate system suitability reference standard (containing naphthostat mesylate, impurity A, impurity B, impurity C, impurity D, impurity G, impurity I, and impurity J), dissolve and dilute it with solvent to prepare a solution containing approximately 1 mg per 1 ml.

[0126] Sensitivity solution: Take 5 ml of the control solution, add 5 ml of solvent, and mix well.

[0127] Stock solution 1: Take impurity NAA-Z1 and impurity NAA-Z 1b Appropriate amounts of impurities A, B, C, E, I, J, K, and 4-dimethylaminopyridine reference standard were dissolved in methanol by sonication and diluted to prepare a stock solution containing approximately 0.2 mg per ml.

[0128] Stock solution 2: Take an appropriate amount of impurity G reference standard, add 0.1% glacial acetic acid methanol, sonicate to dissolve and dilute to prepare stock solution 2 containing about 0.2 mg per ml;

[0129] Stock solution 3: Take an appropriate amount of impurity D reference standard, add acetonitrile-water (50:50), sonicate to dissolve and dilute to prepare stock solution 3 containing about 0.2 mg per ml;

[0130] Stock solution 4: Take impurity NAA-X 1e Add an appropriate amount of reference standard, add approximately 10% of the total volume of dimethyl sulfoxide, sonicate to dissolve, and then dilute with water-acetonitrile (90:10) to prepare a stock solution containing approximately 0.2 mg per 1 ml;

[0131] Stock solution 5: Take an appropriate amount of impurity F reference standard, add about 20% of the total volume of dimethyl sulfoxide, sonicate to dissolve, and then dilute with methanol to prepare stock solution 5 containing about 0.2 mg per 1 ml;

[0132] Mixed solution: Accurately measure appropriate amounts of stock solutions 1-5 above, take an appropriate amount of naphthostat mesylate, place them in the same volumetric flask, add solvent to dissolve and dilute to prepare a mixed solution containing approximately 1.0 mg of naphthostat mesylate and approximately 5 μg of other impurities per 1 ml.

[0133] 2. Chromatographic conditions

[0134] The column was packed with octadecylsilane-bonded silica gel (ZORBAX Eclipse XDB-C18 4.6mm×250mm, 5μm, or a column with equivalent performance). The mobile phase was 0.03mol / L sodium heptanesulfonate 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 40℃. The detector was a UV detector with a detection wavelength of 260nm. The injector temperature was 15℃. The injection volume was 10μl.

[0135] Table 2. Linear Gradient Elution Table

[0136] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 10 80 20 35 70 30 45 50 50 46 80 20 55 80 20

[0137] 3. System Applicability Requirements

[0138] In the chromatogram of the mixed solution, the impurity NAA-Z 1b Impurity A, DMAP, Impurity B, Impurity NAA-X 1e Impurities F, D, E, I, NAA-Z1, C, naphthalmosat, G, J, and K should elute sequentially. The resolution between impurity C and naphthalmosat should meet the requirements, as should the resolution between impurity G and J. In the sensitivity solution chromatogram, the signal-to-noise ratio of the main component peak should be no less than 30.

[0139] 4. Sample testing

[0140] Accurately measure the test solution and the control solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0141] Limits: In the chromatogram of the test sample solution, excluding solvent peaks and gradient elution peaks, the content of each impurity, calculated by the principal component self-comparison method multiplied by the correction factor, should comply with the provisions of Table 3, and the total amount of impurities should not exceed 0.5%. Chromatographic peaks with a content of less than 0.05% can be ignored.

[0142] Table 3. Impurity Limits

[0143] Principal and impurity names Relative retention time Correction factor Impurity limit Impurities NAA-Z 1b ]] 0.13 1.3 0.10％ Impurity A 0.16 0.75 0.10％ DMAP 0.19 0.78 0.10％ Impurity B 0.38 0.50 0.10％ Impurity NAA-X 1e ]] 0.46 1.3 0.10％ Impurity F 0.54 1.0 0.10％ Impurity D 0.79 1.0 0.10％ Impurity E 0.82 0.69 0.10％ Impurity I 0.85 0.67 0.10％ Impurity NAA-Z1 0.91 2.5 0.10％ Impurity C 0.97 1.4 0.10％ Naphthomoxur mesylate 1.00 —— —— Impurity G 1.25 0.60 0.10％ Impurity J 1.26 1.3 0.10％ Impurity K 1.30 0.72 0.10％ Other individual impurities —— 1.0 0.10％

[0144] Example 2. Specificity

[0145] This embodiment addresses the potential presence of DMAP, impurity NAA-Z1, and impurity NAA-Z in nafamostat mesylate raw material. 1b Impurities NAA-X 1e The separation of 14 known impurities, namely impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity I, impurity J, and impurity K, was investigated. The specific experimental methods are as follows:

[0146] 1. Prepare the test solution

[0147] Impurity NAA-Z1 stock solution: Weigh approximately 20 mg of impurity NAA-Z1 reference standard accurately, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well.

[0148] Impurity NAA-X 1e Stock solution: Take impurity NAA-X 1e Weigh approximately 20 mg of the reference standard accurately and place it in a 100 ml volumetric flask. Add 10 ml of dimethyl sulfoxide and sonicate to dissolve. Dilute to the mark with water-acetonitrile (90:10) and shake well.

[0149] Impurity NAA-Z 1b Stock solution: Take impurity NAA-Z 1b Weigh approximately 20 mg of the reference standard accurately, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with acetonitrile, and shake well.

[0150] 4-Dimethylaminopyridine stock solution: Weigh approximately 20 mg of 4-dimethylaminopyridine reference standard accurately, place it in a 100 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well.

[0151] Impurity A stock solution: Accurately weigh approximately 12 mg of impurity A reference standard, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well. (Impurity A is hydrochloride, and its concentration, calculated based on base, is 0.2 mg / ml).

[0152] Impurity B stock solution: Accurately weigh approximately 15 mg of impurity B reference standard, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well. (Impurity B is methanesulfonate, with a concentration of 0.2 mg / ml based on base).

[0153] Impurity C stock solution: Accurately weigh approximately 11 mg of impurity C reference standard, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well. (Impurity C is hydrochloride, with a concentration of 0.2 mg / ml based on base; refrigerate after preparation and use within 24 hours).

[0154] Impurity D stock solution: Accurately weigh approximately 13 mg of impurity D reference standard, place it in a 50 ml volumetric flask, add 50% acetonitrile, sonicate to dissolve and dilute to the mark, and shake well. (Impurity D is trifluoroacetate, with a concentration of 0.2 mg / ml based on base).

[0155] Impurity E stock solution: Accurately weigh approximately 13 mg of impurity E reference standard, place it in a 50 ml volumetric flask, dissolve and dilute to the mark with methanol, and shake well. (Impurity E is trifluoroacetate, with a concentration of 0.2 mg / ml based on base).

[0156] Impurity F stock solution: Weigh approximately 10 mg of impurity F reference standard accurately, place it in a 50 ml volumetric flask, add 10 ml of dimethyl sulfoxide, sonicate to dissolve, dilute to the mark with methanol, and shake well.

[0157] Impurity G stock solution: Accurately weigh approximately 28 mg of impurity G reference standard, place it in a 100 ml volumetric flask, add 0.1% glacial acetic acid methanol, sonicate to dissolve and dilute to the mark, and shake well. (Impurity G is methanesulfonate, concentration based on base, 0.2 mg / ml).

[0158] Impurity I stock solution: Accurately weigh approximately 26 mg of impurity I reference standard, place it in a 100 ml volumetric flask, add methanol, sonicate to dissolve and dilute to the mark, and shake well. (Impurity I consists of methanesulfonate and hydrochloride, with a concentration of 0.2 mg / ml based on base).

[0159] Impurity J stock solution: Accurately weigh approximately 28 mg of impurity J reference standard, place it in a 100 ml volumetric flask, add methanol, sonicate to dissolve and dilute to the mark, and shake well. (Impurity J is methanesulfonate, concentration based on base, 0.2 mg / ml).

[0160] Impurity K stock solution: Accurately weigh approximately 22 mg of impurity K reference standard, place it in a 100 ml volumetric flask, add methanol, sonicate to dissolve and dilute to the mark, and shake well. (Impurity K is hydrochloride, and its concentration is 0.2 mg / ml based on base).

[0161] Impurity location solution: Measure 0.5 ml of each of the above impurity stock solutions, place them in 10 ml volumetric flasks respectively, dilute to the mark with solvent, and shake well.

[0162] Test solution: Weigh approximately 25 mg of the test sample (naphthostat mesylate) accurately, place it in a 25 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0163] Mixed solution: Accurately weigh approximately 25 mg of the test sample (naphthostat mesylate), place it in a 25 ml volumetric flask, dissolve it in solvent, and separately measure impurity NAA-Z1 and impurity NAA-Z.1b Impurities NAA-X 1e 0.25 ml each of the stock solutions of 4-dimethylaminopyridine, impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity I, impurity J, and impurity K were placed in the same volumetric flask, diluted to the mark with solvent, and shaken well.

[0164] 2. Detection

[0165] Take 10 μl of each of the blank solution (solvent), each impurity localization solution, the test solution, and the mixed solution, and inject them into the chromatographic conditions of Example 1. Record the chromatograms.

[0166] The measurement results are shown in Tables 4 and 5. Figure 1 As shown, the blank solution does not interfere with the detection of impurities, and the separation of each impurity is good, indicating that the method of the present invention has good specificity.

[0167] Table 4. Results of Specificity Test

[0168]

[0169] Table 5. Figure 1 Integral Results Table

[0170]

[0171] Example 3. Limit of Quantification and Limit of Detection

[0172] 1. Prepare the test solution

[0173] Naphamostat mesylate stock solution: Weigh approximately 25 mg of naphamostat mesylate accurately, place it in a 25 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0174] Mixed stock solution 1: Accurately measure 1 ml of naphthox mesylate stock solution, 5 ml each of impurity A, impurity B, impurity C, impurity D, impurity E, impurity F, impurity G, impurity I, impurity J, and impurity K, place them in the same 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0175] Mixed stock solution 2: Accurately measure impurity NAA-Z 1b Stock solution, impurities 4-dimethylaminopyridine stock solution, impurities NAA-X 1e Place 5 ml each of the stock solution and the impurity NAA-Z1 stock solution into the same 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0176] Limit of Quantification Solution 1: Accurately measure 1.5 ml of Mixed Stock Solution 1, place it in the same 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0177] Limit of Quantification Solution 2: Measure 1.5 ml of Mixed Stock Solution 2, place it in the same 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0178] Detection limit solution 1: Accurately measure 1.5 ml of the quantitation limit solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0179] Detection limit solution 2: Accurately measure 5 ml of quantitation limit solution 2, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0180] 2. Detection

[0181] Take the above-mentioned limit of quantitation solution 1 and limit of quantitation solution 2, inject them 6 times consecutively, record the chromatograms, and calculate the ratio of the peak height of the main component to the noise (S / N); then take the above-mentioned limit of detection solution 1 and limit of detection solution 2, inject them 3 times consecutively, record the chromatograms, and calculate the ratio of the peak height of the main component to the noise (S / N).

[0182] The results of the limit of quantitation test are shown in Tables 6 to 8. Figures 2-3 Data shows that the impurity NAA-Z 1b The limit of quantitation (LOQ) concentration was 0.2969 μg / ml, expressed as 0.030% of the concentration present in the sample, with a peak area RSD of 1.9% and an average S / N ratio of 78.4. The limit of quantitation (LOQ) concentration for the impurity 4-dimethylaminopyridine was 0.3038 μg / ml, expressed as 0.030% of the concentration present in the sample, with a peak area RSD of 2.4% and an average S / N ratio of 104.1. The impurity NAA-X... 1eThe limit of quantitation (LOQ) concentration for impurity NAA-Z1 was 0.2887 μg / ml, expressed as 0.029% of the concentration in the sample, with a peak area RSD of 4.1% and an average S / N ratio of 28.2. The LQ concentration for impurity NAA-Z1 was 0.3063 μg / ml, expressed as 0.031% of the concentration in the sample, with a peak area RSD of 7.2% and an average S / N ratio of 13.1. The LQ concentration for impurity A was 0.3061 μg / ml, expressed as 0.031% of the concentration in the sample, with a peak area RSD of 1.0% and an average S / N ratio of 201.3. The LQ concentration for impurity B was 0.2 μg / ml. Impurity F had a limit of quantitation of 957 μg / ml, expressed as 0.030% in the sample, with a peak area RSD of 0.4% and an average S / N ratio of 142.1; Impurity F had a limit of quantitation of 0.2822 μg / ml, expressed as 0.028% in the sample, with a peak area RSD of 1.1% and an average S / N ratio of 67.1; Impurity D had a limit of quantitation of 0.2548 μg / ml, expressed as 0.025% in the sample, with a peak area RSD of 1.2% and an average S / N ratio of 79.6; Impurity E had a limit of quantitation of 0.3063 μg / ml, expressed as 0.030% in the sample, with a peak area RSD of 0.4% and an average S / N ratio of 142.1; Impurity D had a limit of quantitation of 0.2822 μg / ml, expressed as 0.028% in the sample, with a peak area RSD of 1.2% and an average S / N ratio of 79.6; Impurity E had a limit of quantitation of 0.3063 μg / ml, expressed as 0.030% in the sample, with a peak area RSD of 0.4% and an average S / N ratio of 142.1. The concentration of impurity I was 0.2936 μg / ml, expressed as 0.029% of the concentration in the sample, with a peak area RSD of 1.7% and an average S / N of 133.2; the concentration of impurity C was 0.2875 μg / ml, expressed as 0.029% of the concentration in the sample, with a peak area RSD of 1.7% and an average S / N of 26.8; the concentration of impurity G was 0.3023 μg / ml, expressed as 0.030% of the concentration in the sample, with a peak area RSD of 0.5% and an average S / N of 174.6; the concentration of impurity G was 0.031% of the concentration in the sample, with a peak area RSD of 0.5% and an average S / N of 174.6; the concentration of impurity C was 0.2875 μg / ml, expressed as 0.029% of the concentration in the sample, with a peak area RSD of 1.7% and an average S / N of 26.8; the concentration of impurity G was 0.3023 μg / ml, expressed as 0.030% of the concentration in the sample, with a peak area RSD of 0.031% and an average S / N of 174.6. The peak area RSD was 1.7%, and the average S / N was 52.1; the quantitation limit concentration of impurity J was 0.3039 μg / ml, which is 0.030% as the concentration present in the sample, with a peak area RSD of 2.0% and an average S / N of 43.7; the quantitation limit concentration of impurity K was 0.3031 μg / ml, which is 0.030% as the concentration present in the sample, with a peak area RSD of 0.4% and an average S / N of 74.4; the quantitation limit concentration of the main component was 0.3052 μg / ml, with a peak area RSD of 1.0% and an average S / N of 32.9. All quantitation limit tests met the requirements.

[0183] The results of the detection limit test are shown in Tables 9 to 11. Figures 4-5 Data shows that the impurity NAA-Z 1bThe detection limit concentration was 0.1484 μg / ml, expressed as 0.015% in the sample, with an average S / N ratio of 42.0; the detection limit concentration of impurity 4-dimethylaminopyridine was 0.1519 μg / ml, expressed as 0.015% in the sample, with an average S / N ratio of 54.3; impurity NAA-X 1e The detection limit concentration was 0.1443 μg / ml, expressed as 0.014% in the sample, with an average S / N ratio of 15.8; the detection limit concentration of impurity NAA-Z1 was 0.1531 μg / ml, expressed as 0.015% in the sample, with an average S / N ratio of 8.7; the detection limit concentration of impurity A was 0.1531 μg / ml, expressed as 0.015% in the sample, with an average S / N ratio of 114.4; the detection limit concentration of impurity B was... The limit of detection (LOD) for impurity F was 0.1479 μg / ml, expressed as 0.015% in the sample, with an average S / N of 84.2; the LOD for impurity F was 0.1411 μg / ml, expressed as 0.014% in the sample, with an average S / N of 34.0; the LOD for impurity D was 0.1274 μg / ml, expressed as 0.013% in the sample, with an average S / N of 44.0; and the LOD for impurity E was 0.1532 μg / ml. The concentration of impurity I was 0.015% as expressed in the sample, with an average S / N of 97.3; the detection limit concentration of impurity I was 0.1468 μg / ml, expressed as 0.015% as expressed in the sample, with an average S / N of 72.2; the detection limit concentration of impurity C was 0.1438 μg / ml, expressed as 0.014% as expressed in the sample, with an average S / N of 14.3; the detection limit concentration of impurity G was 0.1511 μg / ml, expressed as... The detection limit concentration of impurity J was 0.015%, with an average S / N of 31.3; the detection limit concentration of impurity K was 0.1519 μg / ml, expressed as 0.015% in the sample, with an average S / N of 22.8; the detection limit concentration of impurity K was 0.1515 μg / ml, expressed as 0.015% in the sample, with an average S / N of 35.8; and the detection limit concentration of the principal component was 0.1526 μg / ml, with an average S / N of 17.5. All detection limit tests met the requirements. These results demonstrate that the method of this invention has high sensitivity.

[0184] Table 6. Results of Limit of Quantitation Determination

[0185]

[0186]

[0187]

[0188] Table 7. Integral Results of the Graph

[0189]

[0190] Table 8. Figure 3 Integral Results Table

[0191]

[0192] Table 9. Results of Limit of Detection (LOD) determination

[0193]

[0194]

[0195] Table 10. Figure 4 Integral Results Table

[0196]

[0197]

[0198] Table 11. Figure 5 Integral Results Table

[0199]

[0200] Example 4. Chromatographic condition robustness

[0201] Take the mixed solution under “Example 2. Specificity” and use the normal mobile phase. Test different column flow rates, column temperatures, initial gradient ratios of mobile phase and gradient ratios of mobile phase as planned. After the instrument system stabilizes, test and record the resolution between each peak.

[0202] The test results are shown in Tables 12 to 23. Figures 6-16 Data shows that when chromatographic conditions are slightly changed (flow rate ±0.1 ml / min, column temperature ±2℃, initial mobile phase ratio ±2%, gradient ratio ±2%), the resolution between each component is greater than 1.5, and the minimum resolution is 2.12, indicating that the method robustness meets the requirements.

[0203] Table 12. Results of the robustness test for changes in chromatographic conditions (resolution)

[0204]

[0205]

[0206] Table 13. Figure 6 Integral Results Table

[0207]

[0208] Table 14.Figure 7 Integral Results Table

[0209]

[0210]

[0211] Table 15. Figure 8 Integral Results Table

[0212]

[0213] Table 16. Figure 9 Integral Results Table

[0214]

[0215] Table 17. Figure 10 Integral Results Table

[0216]

[0217]

[0218] Table 18. Figure 11 Integral Results Table

[0219]

[0220] Table 19. Figure 12 Integral Results Table

[0221]

[0222]

[0223] Table 20. Figure 13 Integral Results Table

[0224]

[0225] Table 21. Figure 14 Integral Results Table

[0226]

[0227]

[0228] Table 22. Figure 15 Integral Results Table

[0229]

[0230] Table 23. Figure 16 Integral Results Table

[0231]

[0232]

Claims

1. A method for separating naphthostat mesylate and its related impurities by high performance liquid chromatography, characterized in that, The high performance liquid chromatography method includes: using an octadecylsilane-bonded silica gel column as the packing material, using sodium heptanesulfonate solution as mobile phase A and acetonitrile as mobile phase B, and separating naphthalenemole mesylate and its related impurities by gradient elution; The relevant impurities include DMAP, impurity NAA-Z1, and impurity NAA-Z. 1b Impurities NAA-X 1e Impurities A, B, C, D, E, F, G, I, J, and K are any one or more of the following: Naphazoline mesylate has the structural formula shown in Formula 1; DMAP has the structural formula shown in Formula 2; and impurity NAA-Z1 has the structural formula shown in Formula 3. 1b The structural formula is shown in Formula 4, and the impurity NAA-X 1e The structural formulas of impurity A, impurity B, and impurity C are shown in Formula 5, respectively. The structural formulas of impurity A, B, C, D, E, F, and K are shown in Formula 10, 12, 13, 14, and 15, respectively.

2. The method according to claim 1, characterized in that, The mobile phase A is a sodium heptanesulfonate solution with a concentration of 0.03 mol / L.

3. The method according to claim 1, characterized in that, The gradient elution procedure is set as follows: At 0 minutes, the volume ratio of mobile phase A to mobile phase B is 75-85:25-15; After 10 minutes, the volume ratio of mobile phase A to mobile phase B is 75-85:25-15; For 35 minutes, the volume ratio of mobile phase A to mobile phase B is 65-75:35-25; For 45 minutes, the volume ratio of mobile phase A to mobile phase B is 45-55:55-45; After 46 minutes, the volume ratio of mobile phase A to mobile phase B is 75-85:25-15; After 55 minutes, the volume ratio of mobile phase A to mobile phase B is 75-85:25-15.

4. The method according to claim 1, characterized in that, The flow rate of the mobile phase is 0.8 ml / min to 1.6 ml / min; the column temperature is 35℃ to 45℃; and the injector temperature is 15±2℃.

5. A method for identifying naphthostat mesylate and its related impurities, characterized in that, Includes the following steps: (1) The method described in any one of claims 1-4 is used to separate naphthostat mesylate and its related impurities; (2) Detection was performed using a detector with a detection wavelength of 240nm to 280nm to obtain a chromatogram.

6. The method according to claim 5, characterized in that, By comparing the chromatograms of the test sample and the reference sample, the consistency of their chromatographic behavior is used to determine whether the test sample contains naphthostat mesylate and its related impurities.

7. The method according to claim 5, characterized in that, Using nafamostat mesylate as a reference peak, the relative retention time was 0.13 ± 0.2, which was determined to be the impurity NAA-Z. 1b The relative retention time was 0.16±0.2, and it was determined to be impurity A; the relative retention time was 0.19±0.2, and it was determined to be DMAP; the relative retention time was 0.38±0.2, and it was determined to be impurity B; the relative retention time was 0.46±0.2, and it was determined to be impurity NAA-X. 1e The following impurities were identified: 0.54±0.2 relative retention time, identified as impurity F; 0.79±0.2 relative retention time, identified as impurity D; 0.82±0.2 relative retention time, identified as impurity E; 0.85±0.2 relative retention time, identified as impurity I; 0.91±0.2 relative retention time, identified as impurity NAA-Z1; and 0.97±0.2 relative retention time, identified as impurity C. A relative retention time of 1.25±0.2 is identified as impurity G; a relative retention time of 1.26±0.2 is identified as impurity J; and a relative retention time of 1.30±0.2 is identified as impurity K.

8. A method for determining the content of naphthostat mesylate and its related impurities, characterized in that, Includes the following steps: (1) The method described in any one of claims 4-7 is used to separate and detect naphthalenesulfonic acid and its related impurities, and a chromatogram is obtained; (2) Based on the chromatogram obtained in step (1), the content of each impurity is calculated by the principal component self-comparison method multiplied by the correction factor.

9. The method according to claim 8, characterized in that, The impurity NAA-Z 1b The correction factor is 1.3, the correction factor for impurity A is 0.75, the correction factor for DMAP is 0.78, the correction factor for impurity B is 0.50, and the correction factor for impurity NAA-X is... 1e The correction factor for impurity F is 1.3, the correction factor for impurity D is 1.0, the correction factor for impurity E is 0.69, the correction factor for impurity I is 0.67, the correction factor for impurity NAA-Z1 is 2.5, the correction factor for impurity C is 1.4, the correction factor for impurity G is 0.60, the correction factor for impurity J is 1.3, the correction factor for impurity K is 0.72, and the correction factor for other individual impurities is 1.

0.

10. The method according to claim 8, characterized in that, Before separation, the test solution is prepared using a mixed solution of water, acetonitrile and glacial acetic acid as the solvent; the volume ratio of water, acetonitrile and glacial acetic acid in the solvent is 650-800:250-350:0.05-0.2.

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