HPLC method for separating and determining nafamostat mesylate X1 and impurities thereof

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to an HPLC (High Performance Liquid Chromatography) method for separating and determining nafamostat mesylate X1 and impurities thereof. The impurities comprise an impurity NAA-SM2, an impurity NAA-SM2a, an impurity NAA-SM2f, an impurity NAA-X1d, an impurity NAA-X1b, an impurity NAA-X1e, an impurity NAA-X1f and isopropyl p-aminobenzoate. According to the method, octadecyl silane bonded silica gel 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, and nafamostat mesylate X1 and impurities thereof are separated through linear gradient elution; and then calculating the content of each impurity by adopting an external standard method and / or a principal component self-contrast method multiplied by a correction factor according to a chromatogram. The method has the characteristics of good separation degree, good durability, high sensitivity and good reproducibility.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to an HPLC method for separating and determining naphthostat mesylate X1 and its impurities. Background Technology

[0002] Naphthostat mesylate, as a protease inhibitor, is widely used to treat a range of inflammatory diseases caused by protease abnormalities, such as acute pancreatitis, acute exacerbation of chronic pancreatitis, and postoperative acute pancreatitis. It can also be used to treat hemodialysis, coronary artery bypass grafting, disseminated intravascular coagulation (DIC), glomerulonephritis with systemic lupus erythematosus, or mixed cryoglobulinemia.

[0003] Naphthostat mesylate X1 is an important intermediate in the synthesis of naphthostat mesylate, and its structural formula is shown in Formula I.

[0004]

[0005] Studies have found that nafamostat X1 mesylate is prone to introducing the following eight impurities during its synthesis: impurity NAA-SM2, impurity NAA-SM... 2a Impurities NAA-SM 2f Impurities NAA-X 1d Impurities NAA-X 1b Impurities NAA-X 1e Impurities NAA-X 1f Isopropyl para-aminobenzoate. To ensure the quality of naphamostat mesylate X1 and subsequent formulations, the content of these eight impurities needs to be controlled. However, currently there are no established analytical methods or literature available to effectively separate naphamostat mesylate X1 and its eight known impurities.

[0006] In the prior art, patent CN117247335A discloses a method for detecting naphthostat intermediates, including (1) a chromatographic column packed with octadecylsilane-bonded silica gel; (2) a detector: an ultraviolet detector with a wavelength of 254 nm; (3) a flow rate: 1.0 mL per minute; (4) a run time: gradient elution; (5) a test solution: an appropriate amount of the sample is accurately weighed, dissolved in methanol, and quantitatively diluted to prepare a solution containing approximately 1 mg per mL; (6) an injection volume of 10 μl; the purity of the test solution is calculated by automatic integration method based on the peak area. This patent does not disclose important chromatographic conditions such as the mobile phase, and cannot achieve the separation and determination of naphthostat mesylate X1 and its related impurities.

[0007] Therefore, there is an urgent need to establish a new method to achieve quality control of naphthostat mesylate X1. Summary of the Invention

[0008] In view of this, one of the objectives of the present invention is to provide a method for separating naphthostat mesylate X1 and its impurities using HPLC. This method can simultaneously separate naphthostat mesylate X1 and its eight known impurities within 65 minutes or more, which is helpful for the subsequent qualitative and quantitative detection of each component.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A method for separating nafamostat X1 mesylate and its impurities using HPLC, wherein the impurities include impurity NAA-SM2 and impurity NAA-SM 2a Impurities NAA-SM 2f Impurities NAA-X 1d Impurities NAA-X 1b Impurities NAA-X 1e Impurities NAA-X 1f The method comprises: using an octadecylsilane-bonded silica gel column as the packing material, using potassium hexafluorophosphate solution as mobile phase A and acetonitrile as mobile phase B, and separating naphthalenemostat mesylate X1 and its impurities by linear gradient elution.

[0011] The structural formula of nafamostat mesylate X1 is shown in Formula I, and the structural formula of the impurity NAA-SM2 is shown in Formula II. 2a The structural formula is shown in Formula III, and the impurity NAA-SM 2f The structural formula is shown in Formula IV, and the impurity NAA-X 1d The structural formula is shown in Formula V, and the impurity NAA-X 1b The structural formula is shown in Formula VI, and the impurity NAA-X 1e The structural formula is shown in Formula VII, and the impurity NAA-X 1f The structural formula is shown in Formula VIII, and the structural formula of the isopropyl p-aminobenzoate is shown in Formula IX.

[0012]

[0013] Furthermore, the components separated from the sample included naphthostat mesylate X1, impurity NAA-SM2, and impurity NAA-SM. 2a Impurities NAA-SM 2f Impurities NAA-X 1d Impurities NAA-X 1b Impurities NAA-X 1e Impurities NAA-X 1f One or more of isopropyl p-aminobenzoate.

[0014] More preferably, the components separated from the sample include naphthostat mesylate X1, as well as impurities NAA-SM2 and NAA-SM4. 2a Impurities NAA-SM 2f Impurities NAA-X 1d Impurities NAA-X 1b Impurities NAA-X 1e Impurities NAA-X 1f Any one or more impurities in isopropyl p-aminobenzoate.

[0015] More preferably, the components separated in the sample to be tested are naphthostat mesylate X1 and impurity NAA-X. 1d Impurities NAA-X 1b Impurities NAA-X 1e Impurities NAA-X 1f One or more of the following: isopropyl p-aminobenzoate; or the separated component being nafamostat mesylate X1, impurity NAA-X. 1d Impurities NAA-X 1b Impurities NAA-X 1e Impurities NAA-X 1f Any one or more of the following, p-aminobenzoic acid isopropyl esters, and impurities NAA-SM2 and NAA-SM 2a Impurities NAA-SM 2f Any one or more combinations thereof.

[0016] Furthermore, the procedure for the linear gradient elution is as follows:

[0017] At 0 minutes, the volume ratio of mobile phase A to mobile phase B is set to 95-99:5-1;

[0018] At 15 minutes, the volume ratio of mobile phase A to mobile phase B is set to 95-99:5-1;

[0019] At 25 minutes, the volume ratio of mobile phase A to mobile phase B is set to 60-70:40-30;

[0020] At 55 minutes, the volume ratio of mobile phase A to mobile phase B is set to 40-50:60-50;

[0021] At 56 minutes, the volume ratio of mobile phase A to mobile phase B is set to 95-99:5-1;

[0022] At 65 minutes, the volume ratio of mobile phase A to mobile phase B is set to 95-99:5-1.

[0023] Preferably, the linear gradient elution procedure is as follows:

[0024] At 0 minutes, the volume ratio of mobile phase A to mobile phase B is set to 97-99:3-1;

[0025] At 15 minutes, the volume ratio of mobile phase A to mobile phase B is set to 97-99:3-1;

[0026] At 25 minutes, the volume ratio of mobile phase A to mobile phase B is set to 65:35;

[0027] At 55 minutes, the volume ratio of mobile phase A to mobile phase B is set to 45:55;

[0028] At 56 minutes, the volume ratio of mobile phase A to mobile phase B was set to 97-99:3-1;

[0029] At 65 minutes, the volume ratio of mobile phase A to mobile phase B is set to 97-99:3-1.

[0030] As the preferred option, the linear gradient elution procedure is as follows:

[0031] At 0 minutes, the volume ratio of mobile phase A to mobile phase B is set to 98:2;

[0032] At 15 minutes, the volume ratio of the mobile phase A to the mobile phase B is set to 98:2;

[0033] At 25 minutes, the volume ratio of mobile phase A to mobile phase B is set to 65:35;

[0034] At 55 minutes, the volume ratio of mobile phase A to mobile phase B is set to 45:55;

[0035] At 56 minutes, the volume ratio of mobile phase A to mobile phase B was set to 98:2;

[0036] At 65 minutes, the volume ratio of mobile phase A to mobile phase B is set to 98:2.

[0037] Furthermore, a potassium hexafluorophosphate solution with a concentration of 0.005 mol / L-0.02 mol / L and a pH of 2.0 ± 0.5 was used as the mobile phase A.

[0038] Preferably, a potassium hexafluorophosphate solution with a concentration of 0.01 mol / L and a pH of 2.0 ± 0.1 is used as the mobile phase A.

[0039] Furthermore, the flow rate was 0.5 ml / min-1.5 ml / min; the column temperature was 30℃-40℃.

[0040] Preferably, the flow rate is 0.7 ml / min-0.9 ml / min, and most preferably 0.8 ml / min.

[0041] Preferably, the column temperature is 33℃-37℃, and most preferably 35℃.

[0042] Preferably, the chromatographic column has dimensions of 4.6 mm × 250 mm and a diameter of 5 μm.

[0043] Furthermore, a ghost peak column was used before sample injection.

[0044] Furthermore, the injection volume was 15 μl.

[0045] As a preferred option, the running time is 65 minutes.

[0046] The second objective of this invention is to provide a method for identifying naphthostat mesylate X1 and its impurities. This method can simultaneously and effectively identify eight known impurities in naphthostat mesylate X1, with a detection limit concentration as low as 0.37 μg / ml-0.40 μg / ml.

[0047] To achieve the above objectives, the present invention adopts the following technical solution:

[0048] A method for identifying naphamostat mesylate X1 and its impurities is described, which involves separating naphamostat mesylate X1 and its impurities using a separate method, and then detecting them in a detector to obtain a chromatogram. By comparing the chromatographic characteristics of the test sample and the reference sample, it is determined whether the test sample contains naphamostat mesylate X1 and its impurities.

[0049] Furthermore, the detection wavelength of the detector is 200nm-260nm, more preferably 220nm.

[0050] Furthermore, using nafamostat mesylate X1 as a reference peak, a relative retention time of 0.66±0.2 was identified as impurity NAA-SM2; a relative retention time of 1.54±0.2 was also identified as impurity NAA-SM. 2a The relative retention time was 1.59 ± 0.2 seconds, which was determined to be the impurity NAA-SM. 2f The relative retention time was 0.17 ± 0.2, which was determined to be the impurity NAA-X. 1d The relative retention time was 1.50 ± 0.2 seconds, which was determined to be the impurity NAA-X. 1b The relative retention time was 1.36 ± 0.2 seconds, which was determined to be the impurity NAA-X. 1e The relative retention time was 1.63 ± 0.2 seconds, which was determined to be the impurity NAA-X. 1fThe relative retention time was 1.83 ± 0.2, which was determined to be the isopropyl para-aminobenzoate.

[0051] Furthermore, a retention time of 22.0 ± 0.5 min was identified as nafamostat mesylate X1; a retention time of 14.7 ± 0.5 min was identified as impurity NAA-SM2; and a retention time of 33.8 ± 0.5 min was identified as impurity NAA-SM. 2a The retention time was 34.9 ± 0.5 min, which was determined to be the impurity NAA-SM. 2f The retention time was 3.8 ± 0.5 min, which was determined to be the impurity NAA-X. 1d The retention time was 32.7 ± 0.5 min, which was determined to be the impurity NAA-X. 1b The retention time was 29.9 ± 0.5 min, which was determined to be the impurity NAA-X. 1e The retention time was 35.6 ± 0.5 min, which was determined to be the impurity NAA-X. 1f The retention time was 40.0 ± 0.5 min, which was determined to be the isopropyl p-aminobenzoate.

[0052] The components can be qualitatively identified based on the relative retention time and / or retention time mentioned above.

[0053] The third objective of this invention is to provide a method for determining the content of naphthostat mesylate X1 and its impurities. This method can quantify eight known impurities in naphthostat mesylate X1, with a quantification limit concentration as low as 0.75 μg / ml-0.80 μg / ml.

[0054] To achieve the above objectives, the present invention adopts the following technical solution:

[0055] The method for determining the content of naphthostat mesylate X1 and its impurities involves separating and identifying naphthostat mesylate X1 and its impurities using the aforementioned method for identifying naphthostat mesylate X1 and its impurities, and obtaining a chromatogram. Based on the obtained chromatogram, the content of each impurity is calculated using the external standard method and / or the principal component self-comparison method multiplied by the correction factor.

[0056] Furthermore, the impurity NAA-SM2 and the impurity NAA-X 1d The impurity NAA-SM 2a The impurity NAA-SM 2f And / or the content of the isopropyl p-aminobenzoate was calculated by peak area using the external standard method, and the impurity NAA-X 1e The impurity NAA-X 1f The NAA-X 1bThe content of and / or other individual impurities is calculated using the principal component self-comparison method multiplied by a correction factor.

[0057] Furthermore, the sample was prepared using an aqueous solution of acetonitrile.

[0058] Preferably, the solvent for sample preparation is a 10% acetonitrile aqueous solution, which is prepared by mixing water and acetonitrile in a volume ratio of 90:10.

[0059] Furthermore, before the separation step, the test solution is prepared using the sample preparation solvent, and the test solution includes a sample solution, a control solution, a reference solution, and / or a system suitability solution.

[0060] Preferably, the concentration of the sample solution is 2 mg / ml.

[0061] As a preferred technical solution, the method includes the following steps:

[0062] 1) Take an appropriate amount of this product, accurately weigh it, add solvent to dissolve it and quantitatively dilute it to prepare a solution containing about 2 mg per 1 ml, as the sample solution;

[0063] 2) Accurately measure 2 ml of the sample solution obtained in step 1), place it in a 100 ml volumetric flask, dilute it to the mark with solvent, and shake well; then accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute it to the mark with solvent, and shake well, as the control solution.

[0064] 3) Take an appropriate amount of impurity NAA-SM2 reference standard, accurately weigh it, add solvent and sonicate to dissolve it, and quantitatively dilute it to prepare a solution containing about 3 μg per 1 ml, as reference solution 1;

[0065] 4) Take the impurity NAA-X 1d Impurities NAA-SM 2a Impurities NAA-SM 2f Weigh an appropriate amount of isopropyl para-aminobenzoate, add water-acetonitrile (50:50), sonicate to dissolve and quantitatively dilute to prepare a stock solution 1 containing approximately 1.0 mg of each impurity per 1 ml. Then accurately measure an appropriate amount and dilute with solvent to prepare a solution containing approximately 2 μg of each impurity per 1 ml as a reference solution 2.

[0066] 5) Take the system suitability reference standard of naphamostat mesylate X1 (containing naphamostat mesylate X1, impurity NAA-SM2, and impurity NAA-X). 1e Impurities NAA-X 1f Take an appropriate amount of the reference solution obtained in step 4), and the impurity NAA-X. 1b An appropriate amount of reference standard was dissolved and diluted with solvent to prepare a solution containing approximately NAA-X impurities per 1 ml. 1d Impurities NAA-SM2a Impurities NAA-SM 2f Isopropyl para-aminobenzoate, impurity NAA-X 1b A mixed solution containing approximately 2 μg of each of the following substances and 2 mg of nafamostat X1 system suitability reference standard is used as the system suitability solution.

[0067] 6) Take the above system suitability solution, sample solution, control solution, reference solution 1 and reference solution 2, inject them into the liquid chromatograph respectively, and perform detection according to the aforementioned chromatographic conditions to obtain chromatograms.

[0068] 7) The external standard method was used to calculate impurity NAA-SM2 and impurity NAA-X based on peak area. 1d Impurities NAA-SM 2a Impurities NAA-SM 2f The content of isopropyl para-aminobenzoate was calculated using the principal component self-comparison method by multiplying by a correction factor, which is the impurity NAA-X. 1e Impurities NAA-X 1f NAA-X 1b And the content of other individual impurities.

[0069] The beneficial effects of this invention are as follows:

[0070] (1) Currently, there is no legally defined analytical method or literature to add 8 known impurities (NAA-X) to the main peak of naphthostat mesylate X1. 1d Impurities NAA-SM2, Naphazoline Mesylate X1, and NAA-X 1e Impurities NAA-X 1b Impurities NAA-SM 2a Impurities NAA-SM 2f Impurities NAA-X 1f This invention establishes a reverse HPLC method for the separation and determination of eight known impurities in naphthalmosta mesylate X1 by screening the mobile phase composition, column temperature, chromatographic conditions, chromatographic column, wavelength, etc. This liquid chromatography method can meet the requirements of resolution, sensitivity and solubility of the above impurities, and can ensure that the peak shape of the impurities is symmetrical and there is no solvent effect.

[0071] (2) The present invention uses a mobile phase system of dissociable potassium hexafluorophosphate and acetonitrile to replace the mobile phase system of ion-pair reagents. This not only solves the problem of mobile phase tolerance and reduces the damage of ion-pair reagents to the chromatographic column, but also improves the durability and applicability of the chromatographic column.

[0072] (3) This invention employs a single analytical method to separate eight known impurities and the main component in naphamostat mesylate X1, with a separation time of only 65 minutes. This invention successfully solves the research problem of known and potential impurities in naphamostat mesylate X1, while saving analytical costs and manpower.

[0073] (4) The present invention uses a common and widely applicable chromatographic column and a relatively simple mobile phase, which solves the problems of multiple impurities that are difficult to separate and solvent effects that have not been solved by the prior art. It has the characteristics of strong specificity, good reproducibility and simple and feasible operation.

[0074] (5) The method of this invention has the characteristic of high sensitivity. Impurity NAA-X 1d The limits of quantitation (LOQ) for the impurity NAA-SM2 were as low as 0.7919 μg / ml, and the limits of detection (LOD) for the impurity NAA-SM2 were as low as 0.8026 μg / ml, and the LOD for the impurity NAA-SM2 were as low as 0.4013 μg / ml. The limits of quantitation (LOQ) for nafamostat X1 mesylate were as low as 0.7981 μg / ml, and the LOD for the impurity NAA-SM2 were as low as 0.3991 μg / ml. 1e The limit of quantitation (LOQ) is as low as 0.7564 μg / ml, and the limit of detection (LOD) is as low as 0.3782 μg / ml; impurity NAA-X 1b The limit of quantitation (LOQ) is as low as 0.7899 μg / ml, and the limit of detection (LOD) is as low as 0.3950 μg / ml; impurity NAA-SM 2a The limit of quantitation (LOQ) is as low as 0.7808 μg / ml, and the limit of detection (LOD) is as low as 0.3904 μg / ml; impurity NAA-SM 2f The limit of quantitation (LOQ) is as low as 0.8009 μg / ml, and the limit of detection (LOD) is as low as 0.4004 μg / ml; impurity NAA-X 1f The limit of quantitation (LOQ) is as low as 0.8005 μg / ml, and the limit of detection (LOD) is as low as 0.4002 μg / ml; the limit of quantitation (LOQ) of isopropyl p-aminobenzoate is as low as 0.8014 μg / ml, and the LOD is as low as 0.4007 μg / ml. Attached Figure Description

[0075] Figure 1 The chromatogram is for a blank solution;

[0076] Figure 2 The chromatogram is of the mixed solution;

[0077] Figure 3 The chromatogram is for the LOD solution;

[0078] Figure 4 The chromatogram of the LOQ solution;

[0079] Figure 5This is a chromatogram of the mixed solution under normal conditions during the durability test;

[0080] Figure 6 The chromatogram is of the mixed solution at a flow rate of 0.7 ml / min during the durability test.

[0081] Figure 7 The chromatogram is of the mixed solution at a flow rate of 0.9 ml / min during the durability test.

[0082] Figure 8 The chromatogram of the mixed solution in the durability test at a column temperature of 33°C;

[0083] Figure 9 The chromatogram of the mixed solution in the durability test at a column temperature of 37°C;

[0084] Figure 10 The chromatogram of the mixed solution under the condition of an initial mobile phase ratio of A:B = 97:3 during the durability test;

[0085] Figure 11 The chromatogram of the mixed solution under the condition of an initial mobile phase ratio of A:B = 99:1 during the durability test;

[0086] Figure 12 This is a chromatogram of the mixed solution under the condition that the mobile phase pH is 1.9 during the durability test;

[0087] Figure 13 This is a chromatogram of the mixed solution under the condition of a mobile phase pH of 2.1 during the durability test;

[0088] Figure 14 The chromatograms are of the mixed solutions under different column conditions during the durability test. Detailed Implementation

[0089] 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.

[0090] In this invention, the test sample is a solution with nafamostat mesylate X1 as the main component, and the test sample contains nafamostat mesylate X1, impurity NAA-SM2, and impurity NAA-SM. 2a Impurities NAA-SM 2f Impurities NAA-X 1d Impurities NAA-X 1b Impurities NAA-X 1eImpurities NAA-X 1f One or more of isopropyl p-aminobenzoate.

[0091] The sample to be tested may also contain dimethyl sulfoxide or other unspecified compounds, which refer to other impurities that may be introduced when naphthostat mesylate X1 is prepared using conventional methods known in the art.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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.

[0098] 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.

[0099] In this embodiment of the invention, since the impurities studied by this system are all amphoteric compounds containing acid and base groups, ordinary mobile phase components exhibit main peaks at the solvent peak position, making accurate quantification of the relevant impurities impossible. Generally, ion-pairing reagent methods are established for the separation of these impurities. However, this invention uses a dissociable potassium hexafluorophosphate and acetonitrile as the mobile phase system. By adjusting the gradient elution ratio, the main impurity components were successfully separated from the individual impurities. The use of the potassium hexafluorophosphate system in this invention does not modify the chromatographic column, reducing damage to the column and increasing its applicability.

[0100] In this embodiment of the invention, because the related substances analysis method needs to separate a large number of impurities, and the impurities have significant differences in polarity and response, as well as significant differences in solubility between the impurities and the sample, it is necessary to elute from a low-proportion organic phase to a high-proportion organic phase. Furthermore, due to the significant differences in the responses of each impurity, to ensure accurate quantification of each impurity, the external standard method and the self-comparison method based on the composition multiplied by a correction factor are used to quantitatively calculate each known impurity and other individual impurities.

[0101] In this embodiment of the invention, information on naphthostat mesylate X1 and its related impurities is shown in Table 1.

[0102] Table 1. Compound Information Table

[0103]

[0104]

[0105] In this embodiment of the invention, the solvent is a 10% acetonitrile aqueous solution, which is prepared by mixing water and acetonitrile in a volume ratio of 90:10.

[0106] In this embodiment of the invention, the preparation method of mobile phase A is as follows: take 1.84g of potassium hexafluorophosphate, add 1000ml of water to dissolve it, and adjust the pH value to 2.0±0.1 with phosphoric acid.

[0107] Example 1

[0108] (1) Preparation of the test solution

[0109] Solvent: 10% acetonitrile aqueous solution.

[0110] Test solution: Take an appropriate amount of this product, accurately weigh it, add solvent to dissolve it and dilute quantitatively to prepare a solution containing about 2 mg per 1 ml.

[0111] Control solution: Accurately measure 2 ml of the test solution and place it in a 100 ml volumetric flask. Dilute to the mark with solvent and shake well. Then accurately measure 1 ml of the solution and place it in a 10 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0112] Reference solution 1: Take an appropriate amount of impurity NAA-SM2 reference standard, accurately weigh it, add solvent, sonicate to dissolve, and quantitatively dilute to prepare a solution containing about 3 μg per 1 ml.

[0113] Reference solution 2: Take impurity NAA-X 1d Impurities NAA-SM 2a Impurities NAA-SM 2f Weigh an appropriate amount of isopropyl para-aminobenzoate, add water-acetonitrile (50:50), sonicate to dissolve and quantitatively dilute to prepare a stock solution 1 containing approximately 1.0 mg of each impurity per 1 ml. Then accurately measure an appropriate amount and dilute with solvent to prepare a solution containing approximately 2 μg of each impurity per 1 ml.

[0114] System suitability solution: Take nafamostat X1 system suitability reference standard (containing nafamostat X1, impurity NAA-SM2, and impurity NAA-X). 1e Impurities NAA-X 1f Appropriate amount, measure 2 appropriate amounts of reference solution, and impurity NAA-X. 1b An appropriate amount of reference standard was dissolved and diluted with solvent to prepare a solution containing approximately NAA-X impurities per 1 ml. 1d Impurities NAA-SM 2a Impurities NAA-SM 2f Isopropyl para-aminobenzoate, impurity NAA-X 1b A mixed solution of approximately 2 μg each of naphthostat mesylate X1 system suitability reference standard and 2 mg of naphthostat mesylate X1 system suitability reference standard.

[0115] (2) Chromatographic conditions

[0116] The column was packed with octadecylsilane-bonded silica gel (Kromasil 100-5-C18, 4.6 mm × 250 mm, 5 μm, or a column with equivalent performance). A ghost peak column was used. The mobile phase A was potassium hexafluorophosphate solution with a concentration of 0.01 mol / L and a pH of 2.0 ± 0.1, and the mobile phase B was acetonitrile. Linear gradient elution was performed according to Table 2. The flow rate was 0.8 mL per minute. The column temperature was 35 °C. The detection wavelength was 220 nm. The injection volume was 15 μl.

[0117] Table 2. Gradient elution program table

[0118] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 98 2 15 98 2 25 65 35 55 45 55 56 98 2 65 98 2

[0119] (3) Measurement

[0120] A precise volume of the system suitability solution is injected into the liquid chromatograph, and the chromatogram is recorded. In the chromatogram of the system suitability solution, impurity NAA-X is present. 1d Impurities NAA-SM2, Naphazoline Mesylate X1, and NAA-X 1e Impurities NAA-X 1b Impurities NAA-SM 2a Impurities NAA-SM 2f Impurities NAA-X 1f Isopropyl p-aminobenzoate peaks should be emitted sequentially, and the separation between each impurity peak should meet the requirements. The separation between the main peak and impurity NAA-SM2 peak should also meet the requirements.

[0121] Accurately measure the test solution, reference solution, reference solution 1, and reference solution 2, and inject them separately into the liquid chromatograph, recording the chromatograms. In the chromatogram of the test solution, except for the solvent peak and gradient elution peak, calculate the impurities NAA-SM2 and NAA-X by peak area using the external standard method. 1d Impurities NAA-SM 2a Impurities NAA-SM 2f The content of isopropyl para-aminobenzoate was calculated using the principal component self-comparison method by multiplying by a correction factor, which is the impurity NAA-X. 1e Impurities NAA-X 1f NAA-X 1b The content of other individual impurities shall comply with the provisions of Table 3, and the total amount of impurities shall not exceed 2.0%. Chromatographic peaks with a content of less than 0.05% shall be ignored.

[0122] Table 3. Limits for Impurity Content

[0123] Main components and impurities Relative retention time Correction factor Impurity limits <![CDATA[Impurity NAA-X 1d > 0.17 —— 0.10％ <![CDATA[Nafamostat Mesilate X1]]> —— —— —— <![CDATA[Impurity NAA-SM2]]> 0.66 —— 0.15％ <![CDATA[Impurity NAA-X 1e > 1.36 1.0 1.0％ <![CDATA[Impurity NAA-X 1b > 1.50 1.2 0.10％ <![CDATA[Impurity NAA-SM 2a > 1.54 —— 0.10％ <![CDATA[Impurity NAA-SM 2f > 1.59 —— 0.10％ <![CDATA[Impurity NAA-X 1f > 1.63 1.6 0.15％ Isopropyl para-aminobenzoate 1.83 —— 0.10％ Other individual impurities —— 1.0 0.2％

[0124] Example 2. Specificity

[0125] This invention has revealed that the possible impurities in nafamostat mesylate X1 include: impurity NAA-SM2 and impurity NAA-SM. 2a Impurities NAA-SM 2f Impurities NAA-X 1d Impurities NAA-X 1b Impurities NAA-X 1e Impurities NAA-X 1f The sample contained eight impurities, including isopropyl para-aminobenzoate. This example examines the separation of the main peak and these eight impurities in specificity tests. The specific methods are as follows:

[0126] (1) Preparation of the test solution

[0127] Solvent: 10% acetonitrile aqueous solution.

[0128] Impurity NAA-SM2 stock solution: Weigh approximately 50 mg of impurity NAA-SM2 reference standard accurately, place it in a 50 ml volumetric flask, add solvent, sonicate to dissolve and dilute to the mark, and shake well.

[0129] Impurity NAA-SM 2a Stock solution: Take impurities NAA-SM 2a Weigh approximately 50 mg of the reference standard accurately and place it in a 50 ml volumetric flask. Add water-acetonitrile (50:50), sonicate to dissolve and dilute to the mark, and shake well.

[0130] Impurity NAA-SM 2f Stock solution: Take impurities NAA-SM 2f Weigh approximately 50 mg of the reference standard accurately and place it in a 50 ml volumetric flask. Add water-acetonitrile (50:50), sonicate to dissolve and dilute to the mark, and shake well.

[0131] Stock solution of p-aminobenzoic acid isopropyl ester: Weigh approximately 50 mg of p-aminobenzoic acid isopropyl ester reference standard accurately, place it in a 50 ml volumetric flask, add water-acetonitrile (50:50), sonicate to dissolve and dilute to the mark, and shake well.

[0132] Impurity NAA-X 1b Stock solution: Take impurity NAA-X 1b Accurately weigh approximately 58 mg of the reference standard and place it in a 50 ml volumetric flask. Add solvent, sonicate to dissolve, and dilute to the mark. Shake well. (Impurity NAA-X) 1b As a hydrochloride salt, all impurities involved in the validation of this analytical method are NAA-X. 1b The content of NAA-X in the quantitative calculations was all impurities. 1b (Report content × 207.23 / 243.69)

[0133] Impurity NAA-X 1dStock solution: Take impurity NAA-X 1d Weigh approximately 50 mg of the reference standard accurately, place it in a 50 ml volumetric flask, add solvent, sonicate to dissolve and dilute to the mark, and shake well.

[0134] 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 solvent and shake well.

[0135] Impurity NAA-X 1f Stock solution: Take impurity NAA-X 1f Weigh approximately 50 mg of the reference standard accurately, place it in a 50 ml volumetric flask, add solvent, sonicate to dissolve and dilute to the mark, and shake well.

[0136] Impurity localization solution (removing impurity NAA-X) 1e External): Measure 1.0 ml of each of the above impurity stock solutions, place them in separate 50 ml volumetric flasks, dilute to the mark with solvent, and shake well. (1%)

[0137] Impurity NAA-X 1e Positioning solution: Measure impurity NAA-X 1e Place 5.0 ml of the stock solution into a 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0138] Dimethyl sulfoxide positioning solution: Take 0.5 ml of dimethyl sulfoxide, place it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0139] Test solution: Weigh approximately 100 mg of the test sample accurately, place it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0140] Mixed solution: Accurately weigh approximately 100 mg of the test sample, place it in a 50 ml volumetric flask, add solvent to dissolve it, and then measure the amounts of impurity NAA-SM2 and impurity NAA-X. 1f 0.3 ml of each stock solution, NAA-X impurity 1d Impurities NAA-X 1b Impurities NAA-SM 2a Impurities NAA-SM 2f 0.2 ml each of isopropyl p-aminobenzoate stock solution and impurity NAA-X. 1e Place 2.5 ml of the stock solution into the same 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0141] (2) Measurement

[0142] Take 15 μl each of the blank solution, the localization solutions of each impurity, the mixed solution, and the test solution, inject them according to the method described in Example 1, and perform the chromatographic analysis. Record the chromatograms.

[0143] The test results are shown in Tables 4 and 5. Figures 1-2 The results showed that the blank solution and dimethyl sulfoxide solution did 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, was 3.41.

[0144] Table 4. Results of Specificity Test

[0145]

[0146] Table 5. Figure 2 Integral Results Table

[0147]

[0148]

[0149] Example 3. Limit of Quantification

[0150] (1) Preparation of the test solution

[0151] Each impurity stock solution: Prepare impurity NAA-SM2 and impurity NAA-SM according to the preparation method under "Example 2. Specificity". 2a Impurities NAA-SM 2f Isopropyl para-aminobenzoate, impurity NAA-X 1b Impurities NAA-X 1d Impurities NAA-X 1f The stock solution.

[0152] Test solution: Accurately weigh approximately 120 mg of naphamostat X1 mesylate, place it in a 50 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well. (Naphamostat X1 mesylate is a hydrochloride salt; in all quantitative calculations involving naphamostat X1 mesylate in this analytical method validation, the content is 100% × 179.18 / 215.64).

[0153] Naphamostat Mesylate X1 Linear Stock Solution: Measure 2.5 ml of the test solution and place it in a 50 ml volumetric flask. Dilute to the mark with solvent and shake well. (5.0%).

[0154] Linear Stock Solution 1: Measure 2.5 ml of the test solution, and add impurities NAA-SM2 and NAA-SM. 2a Impurities NAA-SM 2f Isopropyl para-aminobenzoate, impurity NAA-X 1b Impurities NAA-X1d Impurities NAA-X 1f Take 5.0 ml of each stock solution and place them in the same 50 ml volumetric flask. Dilute to the mark with solvent and shake well. (5.0%)

[0155] Linear stock solution 2: Impurity NAA-X as described in "Example 2. Specificity" 1e Prepare the stock solution as described above. (10%).

[0156] Limit of Quantitation (LOQ) Stock Solution: Measure 1.5 ml of linear stock solution 1 and 2.5 ml of linear stock solution 2, place them in a 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0157] Limit of Quantification Solution: Measure 4.0 ml of the limit of quantification stock solution, place it in a 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0158] (2) Measurement

[0159] Take the above-mentioned limit of quantitation solution, inject it 6 times consecutively, and detect it according to the chromatographic conditions of Example 1. Record the chromatogram, and calculate the peak height to noise ratio (S / N) and peak area RSD of each component.

[0160] The measurement results are shown in Table 6 and Figure 4 , Figure 4 The integration results are shown in Table 7, with impurity NAA-X. 1d The limit of quantitation (LOQ) concentration was 0.7919 μg / ml, the peak area RSD was 0.3% (N=6), the average S / N ratio was 312.5, and the concentration expressed in the sample was 0.040%. The LOQ concentration of impurity NAA-SM2 was 0.8026 μg / ml, the peak area RSD was 1.6% (N=6), the average S / N ratio was 51.3, and the concentration expressed in the sample was 0.040%. The LOQ concentration of nafamostat mesylate X1 was 0.7981 μg / ml, the peak area RSD was 2.5% (N=6), the average S / N ratio was 15.4, and the impurity NAA-X... 1e The limit of quantitation was 0.7564 μg / ml, the peak area RSD was 0.3% (N=6), the average S / N ratio was 81.2, and the concentration in the sample was expressed as 0.038%; impurity NAA-X 1b The limit of quantitation was 0.7899 μg / ml, the peak area RSD was 0.9% (N=6), the average S / N ratio was 48.7, and the concentration in the sample was expressed as 0.039%; impurity NAA-SM 2a The limit of quantitation was 0.7808 μg / ml, the peak area RSD was 1.9% (N=6), the average S / N ratio was 31.6, and the concentration in the sample was expressed as 0.039%; impurity NAA-SM 2fThe limit of quantitation was 0.8009 μg / ml, the peak area RSD was 0.9% (N=6), the average S / N ratio was 59.8, and the concentration in the sample was expressed as 0.040%; impurity NAA-X 1f The limit of quantitation (LOQ) for p-aminobenzoic acid is 0.8005 μg / ml, the peak area RSD is 2.3% (N=6), the average S / N is 29.5, and the concentration expressed in the sample is 0.040%. The limit of quantitation (LOQ) for p-aminobenzoic acid is 0.8014 μg / ml, the peak area RSD is 0.7% (N=6), the average S / N is 41.3, and the concentration expressed in the sample is 0.040%.

[0161] Table 6. Results of Limit of Quantitation Detection

[0162]

[0163]

[0164] Table 7. Figure 4 Integral Results Table

[0165]

[0166]

[0167] Example 4. Detection Limit

[0168] Detection limit solution: Accurately measure 5 ml of the quantitation limit solution, place it in a 10 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0169] Inject the detection limit solution three times consecutively, perform the detection under the chromatographic conditions of Example 1, record the chromatogram, and calculate the ratio of the main peak height to the noise (signal-to-noise ratio).

[0170] The test results are shown in Table 8 and Figure 3 , Figure 3 The integration results are shown in Table 9. The results show that the impurity NAA-X... 1d The detection limit concentration was 0.3960 μg / ml, the average S / N ratio was 197.7, and the concentration expressed in the sample was 0.020%; the detection limit concentration of impurity NAA-SM2 was 0.4013 μg / ml, the average S / N ratio was 33.6, and the concentration expressed in the sample was 0.020%; the detection limit concentration of nafamostat X1 mesylate was 0.3991 μg / ml, the average S / N ratio was 10.5; the detection limit concentration of impurity NAA-X was... 1e The detection limit was 0.3782 μg / ml, the average S / N ratio was 53.3, and the concentration in the sample was expressed as 0.019%; impurity NAA-X 1bThe detection limit was 0.3950 μg / ml, the average S / N ratio was 32.3, and the concentration in the sample was expressed as 0.020%; impurity NAA-SM 2a The detection limit was 0.3904 μg / ml, the average S / N ratio was 21.2, and the concentration present in the sample was expressed as 0.020%; impurity NAA-SM 2f The detection limit was 0.4004 μg / ml, the average S / N ratio was 39.5, and the concentration in the sample was expressed as 0.020%; impurity NAA-X 1f The detection limit concentration was 0.4002 μg / ml, the average S / N ratio was 19.5, and the concentration present in the sample was expressed as 0.020%. The detection limit concentration of isopropyl para-aminobenzoate was 0.4007 μg / ml, the average S / N ratio was 27.5, and the concentration present in the sample was expressed as 0.020%.

[0171] Table 8. Results of detection limit determination

[0172]

[0173]

[0174] Table 9. Figure 3 Integral Results Table

[0175]

[0176] Example. Durability of chromatographic conditions

[0177] Take the mixed solution under “Example 2. Specificity” and test it using the normal mobile phase, different column temperatures, column flow rates, pH values ​​of mobile phase A, and initial gradient ratios of the mobile phase. After the instrument system stabilizes, test it separately and record the separation degree between the main peak and adjacent peaks.

[0178] The test results are shown in Table 10. Figures 5-14 , Figures 5-14 The corresponding integration results are shown in Tables 11-20. The data show that when the chromatographic conditions fluctuate slightly, the minimum resolution between the main component and each known impurity and the adjacent components is 1.84, indicating good method robustness.

[0179] Table 10. Results of the robustness test for changes in chromatographic conditions (resolution)

[0180]

[0181] Table 11. Figure 5 Integral Results Table

[0182]

[0183] Table 12. Figure 6 Integral Results Table

[0184]

[0185]

[0186] Table 13. Figure 7 Integral Results Table

[0187]

[0188] Table 14. Figure 8 Integral Results Table

[0189]

[0190] Table 15. Figure 9 Integral Results Table

[0191]

[0192]

[0193] Table 16. Figure 10 Integral Results Table

[0194]

[0195] Table 17. Figure 11 Integral Results Table

[0196]

[0197]

[0198] Table 18. Figure 12 Integral Results Table

[0199]

[0200] Table 19. Figure 13 Integral Results Table

[0201]

[0202] Table 20. Figure 14 Integral Results Table

[0203]

[0204]

Claims

1. A method for separating naphthostat mesylate X1 and its impurities using HPLC, characterized in that, The impurities include impurity NAA-SM2 and impurity NAA-SM. 2a Impurities NAA-SM 2f Impurities NAA-X 1d Impurities NAA-X 1b Impurities NAA-X 1e Impurities NAA-X 1f The method comprises: using an octadecylsilane-bonded silica gel column as the packing material, using potassium hexafluorophosphate solution as mobile phase A and acetonitrile as mobile phase B, and separating naphthalenemostat mesylate X1 and its impurities by linear gradient elution. The structural formula of nafamostat mesylate X1 is shown in Formula I, and the structural formula of the impurity NAA-SM2 is shown in Formula II. 2a The structural formula is shown in Formula III, and the impurity NAA-SM 2f The structural formula is shown in Formula IV, and the impurity NAA-X 1d The structural formula is shown in Formula V, and the impurity NAA-X 1b The structural formula is shown in Formula VI, and the impurity NAA-X 1e The structural formula is shown in Formula VII, and the impurity NAA-X 1f The structural formula is shown in Formula VIII, and the structural formula of the isopropyl p-aminobenzoate is shown in Formula IX.

2. The method according to claim 1, characterized in that, The procedure for linear gradient elution is as follows: At 0 minutes, the volume ratio of mobile phase A to mobile phase B is set to 95-99:5-1; At 15 minutes, the volume ratio of mobile phase A to mobile phase B is set to 95-99:5-1; At 25 minutes, the volume ratio of mobile phase A to mobile phase B is set to 60-70:40-30; At 55 minutes, the volume ratio of mobile phase A to mobile phase B is set to 40-50:60-50; At 56 minutes, the volume ratio of mobile phase A to mobile phase B is set to 95-99:5-1; At 65 minutes, the volume ratio of mobile phase A to mobile phase B is set to 95-99:5-1.

3. The method according to claim 1, characterized in that, A mobile phase A was a potassium hexafluorophosphate solution with a concentration of 0.005 mol / L to 0.02 mol / L and a pH of 2.0 ± 0.

5.

4. The method according to claim 1, characterized in that, The flow rate was 0.5 ml / min to 1.5 ml / min; the column temperature was 30℃ to 40℃.

5. A method for identifying naphthostat mesylate X1 and its impurities, characterized in that, The method described in any one of claims 1-4 is used to separate naphthostat mesylate X1 and its impurities, and the samples are detected by a detector to obtain a chromatogram. By comparing the chromatogram characteristics of the test sample and the reference sample, it is determined whether the test sample contains naphthostat mesylate X1 and its impurities.

6. The method according to claim 5, characterized in that, The detector has a detection wavelength of 200nm-260nm.

7. The method according to claim 5, characterized in that, Using nafamostat mesylate X1 as a reference peak, a relative retention time of 0.66 ± 0.2 was identified as impurity NAA-SM2; a relative retention time of 1.54 ± 0.2 was also identified as impurity NAA-SM. 2a ; The relative retention time was 1.59 ± 0.2, which was determined to be the impurity NAA-SM. 2f ; The relative retention time was 0.17 ± 0.2, which was determined to be the impurity NAA-X. 1d The relative retention time was 1.50 ± 0.2 seconds, which was determined to be the impurity NAA-X. 1b ; The relative retention time was 1.36 ± 0.2 seconds, which was determined to be the impurity NAA-X. 1e The relative retention time was 1.63 ± 0.2 seconds, which was determined to be the impurity NAA-X. 1f The relative retention time was 1.83 ± 0.2, which was determined to be the isopropyl para-aminobenzoate.

8. A method for determining the content of naphthostat mesylate X1 and its impurities, characterized in that, The method described in any one of claims 5-7 is used to separate and identify naphthostat mesylate X1 and its impurities, and a chromatogram is obtained. Based on the obtained chromatogram, the content of each impurity is calculated by external standard method and / or principal component self-comparison method multiplied by correction factor.

9. The method according to claim 8, characterized in that, The impurity NAA-SM2, the impurity NAA-X 1d The impurity NAA-SM 2a The impurity NAA-SM 2f And / or the content of the isopropyl p-aminobenzoate was calculated by peak area using the external standard method, and the impurity NAA-X 1e The impurity NAA-X 1f The NAA-X 1b The content of and / or other individual impurities is calculated using the principal component self-comparison method multiplied by a correction factor.

10. The method according to claim 8, characterized in that, The solvent used to prepare the sample was an aqueous solution of acetonitrile.

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