Separation, identification and content detection method of nafamostat mesylate initial raw material SM1 and impurities of nafamostat mesylate initial raw material SM1

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and relates to a nafamostat mesylate starting material SM1 and impurity separation, identification and content detection method. The nafamostat mesylate initial raw material SM1 and the impurities jointly form the composition, and the impurities comprise any one or more of an impurity NAA-SM1a, an impurity NAA-SM1b, an impurity NAA-SM1c, an impurity NAA-SM1d, an impurity NAA-SM1e, an impurity NAA-SM1f, an impurity NAA-SM1h, an impurity NAA-SM1i, an impurity NAA-SM1j and phthalic acid; the method comprises the following steps: carrying out linear gradient elution by using an aqueous solution of phosphoric acid with a volume ratio of 0.1% as a mobile phase A, acetonitrile as a mobile phase B and octadecylsilane chemically bonded silica as a filler in a chromatographic column, and detecting in a detector to obtain a chromatogram; by comparing the chromatogram characteristics of the detection product and the reference product, whether the detection product contains the initial raw material SM1 of nafamostat mesylate and the impurities thereof is determined. The method has the advantages of high specificity, high sensitivity, high reproducibility, high durability and simplicity and feasibility in operation.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for separating SM1, the starting material of naphthostat mesylate, and its impurities. Background Technology

[0002] Naphtholimus mesylate is a synthetic serine protease inhibitor used as an anticoagulant in hemodialysis. It strongly inhibits membrane proteases, kallikrein, plasminogen lysine, fibrinolytic enzymes, and trypsin-like serine proteases such as Clr- and Cls- of the classical complement system pathway. It also inhibits phospholipase A2 and trypsin bound to α-macroglobulin in vitro, similarly to free trypsin. It is used to improve acute pancreatitis, acute exacerbations of chronic pancreatitis, postoperative acute pancreatitis, acute pancreatitis after pancreatography, and traumatic pancreatitis.

[0003] The synthetic routes for naprostol are mainly divided into two categories: one is obtained through the condensation of 4-guanidinobenzoic acid and 6-amidinyl-2-naphthol; the other is obtained through the reaction of 4-guanidinobenzoyl chloride and 6-amidinyl-2-naphthol. Among these, 6-amidinyl-2-naphthol is a key intermediate, and its synthetic methods are relatively few. Route 1 (Aoyama, Okutome, Nakayama T, et al.; Chemical and Pharmaceutical Bulletin, 33, 1985; 1458-1471) involves passing saturated HCl gas into an alcoholic solution of 6-cyano-2-naphthol, causing a Pinner reaction under acidic conditions to yield methyl 6-hydroxy-2-naphthylimine, followed by ammonolysis with ammonia gas to obtain 6-amidinyl-2-naphthol. This route uses highly corrosive hydrochloric acid gas, which can easily cause damage to the reaction vessel in actual industrial production, posing a high risk, and also results in an excessively long reaction time. Route 2 (CN103896809) is an improvement on Route 1, using acyl chloride to generate hydrochloric acid in situ to promote the Pinner reaction. Although this method avoids the use of hydrochloric acid gas, corrosion of the equipment under acidic conditions still exists. Route 3 (CN113999145) involves the alcoholysis of 6-cyano-2-naphthol under sodium ethoxide / ethanol conditions to obtain 6-hydroxy-2-naphthylimine methyl ester, followed by ammonium chloride as an ammonia source for ammonolysis to obtain 6-amidinyl-2-naphthol, but this method has a long reaction time. CN117247335A discloses a method using SM1 as a starting material, which is added to hydroxylamine to obtain intermediate M1. M1 is then reduced to 6-amidinyl-2-naphthol M2 through continuous or stepwise reactions, followed by condensation under the action of a condensing agent, and then salt formation with methanesulfonic acid to obtain naphthostat methanesulfonate.

[0004] In the synthesis reaction, SM1 serves as a starting material, and its quality control is crucial. We aim to establish a quality control method for future application in intelligent manufacturing. However, separating the dozens of impurities in this starting material, simultaneously determining their content, and establishing a set of judgment criteria presents a significant technical challenge. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for separating the starting material SM1 of naphthostat mesylate and its impurities, which can complete the separation of multiple substances in a short time.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A method for separating naphamostat mesylate starting material SM1 and its impurities using high-performance liquid chromatography, wherein the naphamostat mesylate starting material SM1 and the impurities together constitute a composition, and the impurities include: impurity NAA-SM. 1a Impurities NAA-SM 1b Impurities NAA-SM 1c Impurities NAA-SM 1d Impurities NAA-SM 1e Impurities NAA-SM 1f Impurities NAA-SM 1h Impurities NAA-SM 1i Impurities NAA-SM 1j The composition contains one or more of phthalic acid and phthalic acid; the structural formula of each component in the composition is shown in Table A. A 0.1% (v / v) aqueous phosphoric acid solution is used as mobile phase A, acetonitrile as mobile phase B, and octadecylsilane-bonded silica gel is used as the packing material for linear gradient elution.

[0008] The separated material can be used in the next stage of production.

[0009] The aforementioned impurities can be arranged and combined in various ways.

[0010] For example, combination 1: impurities NAA-SM1a, NAA-SM1b, NAA-SM1c, NAA-SM1d, NAA-SM1e, and NAA-SM1f. Combination 2: impurities NAA-SM1c, NAA-SM1d, NAA-SM1e, NAA-SM1f, NAA-SM1h, NAA-SM1i, NAA-SM1j, and phthalic acid. Combination 3: impurities NAA-SM1a, NAA-SM1b, and naphthenic acid starting material SM1.

[0011] All possible permutations and combinations will not be listed here. Theoretically, when the upper limit for the separation, identification, and / or content detection of this method is 11 substances, it can naturally detect 1 to 11 substances.

[0012] As a preferred embodiment, the linear gradient elution procedure is as follows:

[0013] Time - minutes Mobile phase A - volume ratio Mobile phase B - volume ratio 0 55±5 45±5 5±0.5 55±5 45±5 30±0.5 50±5 50±5 40±0.5 20±2 80±8 45±0.5 20±2 80±8 50±0.5 10±1 90±9 60±0.5 10±1 90±9 61±0.5 55±5 45±5 70±0.5 55±5 45±5

[0014] Preferably, the linear gradient elution procedure is as follows:

[0015] At 0 minutes, the volume ratio of mobile phase A to mobile phase B is set to 55:45;

[0016] At 5 minutes, the volume ratio of mobile phase A to mobile phase B is set to 55:45;

[0017] At 30 minutes, the volume ratio of mobile phase A to mobile phase B is set to 50:50;

[0018] At 40 minutes, the volume ratio of mobile phase A to mobile phase B is set to 20:80;

[0019] At 45 minutes, the volume ratio of mobile phase A to mobile phase B is set to 20:80;

[0020] At 50 minutes, the volume ratio of mobile phase A to mobile phase B is set to 10:90;

[0021] At 60 minutes, the volume ratio of mobile phase A to mobile phase B is set to 10:90;

[0022] At 61 minutes, the volume ratio of mobile phase A to mobile phase B was set to 55:45;

[0023] At 70 minutes, the volume ratio of mobile phase A to mobile phase B is set to 55:45.

[0024] As described above, the flow rate is 0.8-1.2 mL / min; the column temperature is 26℃-40℃. For example, the flow rate is 0.9 mL / min. Figure 7 ); or a flow rate of 1.1 mL / min ( Figure 8 For example, the column temperature is 28℃. Figure 9 ); or column temperature is 32℃ ( Figure 10 ).

[0025] The second objective of this invention is to provide a method for identifying SM1, the starting material of naphthostat mesylate, and its impurities. This method can identify multiple substances in a relatively short time.

[0026] To achieve the above objectives, the technical solution of the present invention is as follows:

[0027] A method for identifying naphthostat mesylate starting material SM1 and its impurities involves separating the composition using the method described above and detecting it 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 naphthostat mesylate starting material SM1 and its impurities.

[0028] As a preferred embodiment, in the method, the detection wavelength of the detector is 235 ± 10 nm. The ± 10 nm setting range is based on a comprehensive consideration of factors such as error tolerance, methodological superiority, and practical application requirements. This setting range helps ensure the reliability of the detection results, improves the repeatability and flexibility of the measurement, and meets the requirements of specific experiments.

[0029] As a preferred embodiment, the components of the composition can be identified, wherein the components of the composition, in ascending order, are phthalic acid, impurity NAA-SM. 1f NAA-SM 1a NAA-SM 1d Naphthostat mesylate starting material SM1, unknown impurities, impurity NAA-SM 1i Impurities NAA-SM 1b Impurities NAA-SM 1c Impurities NAA-SM 1j Impurities NAA-SM 1e Impurities NAA-SM 1h .

[0030] Relative retention time (R0) typically describes the relative retention of different components in a mixture on a chromatographic column. It is the ratio of the retention time of a particular component to the retention time of a reference component (usually the main peak or a known component). This ratio reflects the relative retention performance of different components on the column and is an important parameter used for localization, qualitative, and quantitative analysis in chromatographic analysis. R0 is calculated by dividing the retention time of the target component (tR) by the retention time of the reference component (tR0).

[0031] As a preferred method, using naphthenic acid starting material SM1 as a reference peak; a relative retention time of 0.12 indicates phthalic acid; a relative retention time of 0.28 indicates impurity NAA-SM. 1f The relative retention time was 0.49, indicating it was an impurity NAA-SM. 1a The relative retention time was 0.94, indicating it was an impurity NAA-SM. 1d The relative retention time was 1.59, indicating it was an impurity NAA-SM. 1iThe relative retention time was 1.63, indicating it was an impurity NAA-SM. 1b The relative retention time was 1.65, indicating it was an impurity NAA-SM. 1c The relative retention time was 1.86, indicating it was an impurity NAA-SM. 1j The relative retention time was 1.91, indicating it was an impurity, NAA-SM. 1e The relative retention time was 1.95, indicating it was an impurity NAA-SM. 1h The relative retention times of each component fluctuate within a range of 10%.

[0032] Besides relative retention time, retention time can also be used to identify components. Retention time refers to the time required for a sample to travel from entering the chromatographic column to being detected by the detector. This time is calculated based on the migration speed of the component on the chromatographic column, that is, the time interval from the start of injection to the chromatographic peak (maximum concentration) of a certain component. It is mainly used to determine the elution order and position of each component in the sample and is one of the basic data in chromatographic analysis. In quality control, changes in retention time can reflect factors such as the state of the chromatographic column, the stability of the mobile phase, and the performance of the instrument.

[0033] The third objective of this invention is to provide a method for determining the content of naphthostat mesylate starting material SM1 and its impurities. This method can complete the identification and content determination of multiple substances in a short time.

[0034] To achieve the above objectives, the present invention adopts the following technical solution:

[0035] A method for detecting the content of naphamostat mesylate starting material SM1 and its impurities is provided. The method described above is used to separate and identify naphamostat mesylate starting material SM1 and its impurities, obtaining a chromatogram. Based on the obtained chromatogram, when the impurity is one or more of impurities NAA-SM1a, NAA-SM1b, NAA-SM1c, NAA-SM1d, NAA-SM1e, NAA-SM1h, NAA-SM1i, and NAA-SM1j, the content is calculated using the principal component self-comparison method multiplied by a correction factor. When the impurity is phthalic acid, it is controlled by the limit method.

[0036] Content determination can further be used to determine whether the content of the starting material SM1 (naphalosporin mesylate) and impurities is within acceptable limits. If the impurity is NAA-SM... 1a Impurities NAA-SM 1b Impurities NAA-SM 1c Impurities NAA-SM 1d Impurities NAA-SM 1e Impurities NAA-SM 1fImpurities NAA-SM 1h Impurities NAA-SM 1i Impurities NAA-SM 1j If the peak area of ​​phthalic acid is larger than the peak area of ​​the corresponding impurity in the reference solution, it indicates that the impurity content is unqualified; conversely, if the impurity NAA-SM 1a Impurities NAA-SM 1b Impurities NAA-SM 1c Impurities NAA-SM 1d Impurities NAA-SM 1e Impurities NAA-SM 1f Impurities NAA-SM 1h Impurities NAA-SM 1i Impurities NAA-SM 1j If the peak area of ​​phthalic acid is not greater than the peak area of ​​the corresponding impurity in the reference solution, it indicates that the impurity content is within acceptable limits.

[0037] The aforementioned judgment method can serve as a drug quality assessment model and further as an indispensable key module in intelligent production processes. This model, through precise control of parameters such as mobile phase composition, flow rate, and column temperature, achieves accurate separation and quantitative analysis of active ingredients, impurities, and degradation products in drugs, providing a scientific basis for comprehensive drug quality assessment. In intelligent production systems, this model is seamlessly integrated, capable of receiving raw data from the production line in real time, automatically executing analysis tasks, and rapidly providing judgment results based on preset quality standards.

[0038] As a preferred option, the solvent for the sample to be tested is a mixture of water and acetonitrile.

[0039] As a preferred embodiment, the volume ratio of water to acetonitrile in the mixture is 50:50.

[0040] The beneficial effects of this invention are as follows:

[0041] According to the synthesis route of naphamostat mesylate starting material SM1, the possible impurities in naphamostat mesylate starting material SM1 include: impurity NAA-SM. 1a Impurities NAA-SM 1b Impurities NAA-SM 1c Impurities NAA-SM 1d Impurities NAA-SM 1h Impurities NAA-SM 1i Impurities NAA-SM 1j Impurities NAA-SM 1e Impurities NAA-SM 1fThere are 10 known impurities, including phthalic acid. Therefore, this method separates and determines these 10 known impurities, as well as other individual impurities, to ensure the quality control of the naphthalenemostat mesylate starting material SM1.

[0042] This method employs high-performance liquid chromatography (HPLC). Based on the structural characteristics of the impurities under investigation, chromatographic conditions are selected, and an octadecylsilane-bonded silica gel column is used as the chromatographic column. The mobile phase is 0.1% phosphoric acid aqueous solution (A), and acetonitrile (B). Linear gradient elution is used for separation. An ultraviolet (UV) detector is employed to simultaneously detect 10 known impurities (NAA-SM) in the naphthalenemostat mesylate starting material SM1. 1a Impurities NAA-SM 1b Impurities NAA-SM 1c Impurities NAA-SM 1d Impurities NAA-SM 1h Impurities NAA-SM 1i Impurities NAA-SM 1j Impurities NAA-SM 1e Impurities NAA-SM 1f Phthalic acid and other individual impurities. This method is highly specific, sensitive, reproducible, robust, and simple to operate. Attached Figure Description

[0043] Figure 1 The chromatogram is of the mixed solution;

[0044] Figure 2 The chromatogram is for a blank solution;

[0045] Figure 3 The chromatogram is for the solution at the limit of quantitation.

[0046] Figure 4 Chromatogram of the solution at the detection limit;

[0047] Figure 5 The chromatogram is for LOD (phthalic acid);

[0048] Figure 6 Chromatogram for robustness-mixed solution-normal conditions;

[0049] Figure 7 Chromatogram for robustness-mixed solution flow rate of -0.9 ml / min;

[0050] Figure 8 Chromatogram for robustness-mixed solution flow rate of -1.1 ml / min;

[0051] Figure 9 Chromatogram for durability-mixed solution-28°C;

[0052] Figure 10 Chromatogram for durability-mixed solution-32°C;

[0053] Figure 11 Chromatogram of acetonitrile 43 for durability-mixed solution-mobile phase initial ratio gradient;

[0054] Figure 12 Chromatogram of acetonitrile 47 as a durable-mixed solution-mobile phase initial ratio gradient. Detailed Implementation

[0055] 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.

[0056] Supplementary tables to the accompanying drawings in the specification. Included in this patent. Figure 1-12 Visual aids are provided for understanding and interpretation. In case of any ambiguity, users should refer to the corresponding numbered tables (Tables 1-12) for more detailed information. Conversely, if any information that may cause misunderstanding or ambiguity is found during the review of Tables 1-12, the content of the corresponding numbered figures should be taken as the standard. The above guidelines aim to ensure the correct interpretation of this document and the consistency of its information. Although some text overlaps in the spectra of this application, it is still clearly visible, and the integration results of each figure are described in detail in the specification. Furthermore, the numbers in the spectra do not affect the scope of protection of the claims or the full disclosure of the technical solutions in the specification.

[0057] Table 1

[0058]

[0059] Table 2

[0060]

[0061] Table 3

[0062]

[0063] Table 4

[0064]

[0065] Table 5

[0066]

[0067] Table 6

[0068]

[0069] Table 7

[0070]

[0071] Table 8

[0072]

[0073] Table 9

[0074]

[0075] Table 10

[0076]

[0077] Table 11

[0078]

[0079] Table 12

[0080]

[0081] To enhance understanding of the present invention, certain key technologies and scientific terms will be clearly defined below. Unless specifically defined herein, all other technical and scientific terms shall follow their generally accepted and understood meanings within the art to which this invention pertains. It should be emphasized that the scope of the present invention is not limited to the specific methods, reagents, compounds, or compositions described, but allows for reasonable variations and adjustments in these aspects. Furthermore, please understand that the terminology used herein is intended to illustrate specific embodiments and not to be construed as restrictive.

[0082] Furthermore, all references cited herein, including but not limited to patents, patent applications, academic papers, textbooks, and further citations therein, are considered to be incorporated into this document in their entirety through citation, unless directly cited, as a reference. If there are any inconsistencies or conflicts between the content of these cited references or similar materials and this application, particularly regarding terminology definitions, usage, or technical descriptions, the content of this application shall prevail. For any chromatographic conditions not mentioned, refer to the High Performance Liquid Chromatography (HPLC) method (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512) for determination.

[0083] the term

[0084] The limit of quantitation (LOQ) is the lowest amount of an analyte in a sample that can be quantitatively determined, and the measurement result should have a certain degree of accuracy and precision. In other words, the LQ is the lowest level at which an analytical method can accurately and reliably determine the concentration of the analyte in a sample. In HPLC, the determination of the LQ usually relies on the signal-to-noise ratio (S / N) method, that is, the concentration of the analyte corresponding to a certain level of signal-to-noise ratio is taken as the LQ. Determining the LQ is crucial for ensuring the accuracy and reliability of analytical results.

[0085] Chromatographic robustness refers to the ability of a chromatographic analysis system to maintain stable analytical performance and ensure that the results are not significantly affected by minor changes in measurement conditions. This robustness is crucial for ensuring the reliability, repeatability, and stability of analytical results.

[0086] The limit of detection (LOD) is the lowest concentration or amount of an analyte in a sample that can be detected. It reflects the sensitivity and noise level of the analytical method and instrument, and also indicates the level of the blank (background) value after sample processing.

[0087] A correction factor is a coefficient or parameter used to correct analytical results. It aims to improve data accuracy and reliability. In HPLC analysis, because the same detector responds differently to different substances, peak areas produced when the same mass of different substances passes through the detector may not be equal. To ensure that the peak area accurately reflects the content of the analyte, standard substances are used for correction, a correction factor is calculated, and this factor is applied to the measurement results of the sample.

[0088] The peak height to noise ratio (S / N, or signal-to-noise ratio) is used in high-performance liquid chromatography (HPLC) to evaluate the detection sensitivity and resolution of an instrument, and is an important indicator of instrument performance. Peak height refers to the signal value output by the detector when the analyte elutes from the column; noise refers to the fluctuation of the baseline signal, i.e., the signal value measured for a blank sample. The signal-to-noise ratio is the ratio of the signal measured for a sample of known concentration to the signal measured for a blank sample. A higher signal-to-noise ratio means that the instrument can more accurately separate and identify the target component when detecting samples, while also reducing interference from background noise.

[0089] Table A: Compound Information Table

[0090]

[0091] Table B Gradient Elution Procedure

[0092]

[0093]

[0094] Example 1

[0095] By screening chromatographic columns and conditions, an accurate, reproducible, and robust HPLC method was established, which can be used to detect SM1-related substances (impurities NAA-SM) in naphthenic acid. 1a Impurities NAA-SM 1b Impurities NAA-SM 1c Impurities NAA-SM 1d Impurities NAA-SM 1e Impurities NAA-SM 1f Impurities NAA-SM 1h Impurities NAA-SM 1i Impurities NAA-SM 1j For the separation and determination of phthalic acid and other individual impurities, please refer to [link to relevant documentation]. Figure 1 .

[0096] The solvent is water and acetonitrile in a volume ratio of 50:50.

[0097] Test solution: Take an appropriate amount of this product, accurately weigh it, add solvent to dissolve and dilute it to prepare a solution containing about 0.2 mg per ml.

[0098] 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.

[0099] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase (GL Sciences Inertsil ODS-HL, 4.6 mm × 250 mm, 5 μm, or a column with equivalent performance). Mobile phase A was 0.1% phosphoric acid aqueous solution (1000 ml of water was mixed with 1 ml of phosphoric acid). Acetonitrile was used as mobile phase B. Linear gradient elution was performed according to the gradient elution program in Table B. The flow rate was 1.0 ml / min; the column temperature was 30 °C; the detection wavelength was 235 nm; and the injection volume was 10 μl. Accurately measure the test solution and control solution, inject them separately into the liquid chromatograph, and record the chromatograms.

[0100] Limits: In the chromatogram of the test solution, except for the solvent peak and gradient elution peak, the impurities NAA-SM1a, NAA-SM1b, NAA-SM1c, NAA-SM1d, NAA-SM1e, NAA-SM1h, NAA-SM1i, and NAA-SM1j are calculated using the principal component self-comparison method multiplied by a correction factor; phthalic acid is controlled according to the limit method.

[0101] Table C

[0102]

[0103]

[0104] Example 2 Specificity Test

[0105] Solvent: Water-acetonitrile volume ratio is 50:50.

[0106] Impurity NAA-SM 1a Stock solution: Take impurities NAA-SM 1a Weigh approximately 20 mg of the reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well.

[0107] Impurity NAA-SM 1b Stock solution: Take impurities NAA-SM 1b Weigh approximately 20 mg of the reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well.

[0108] Impurity NAA-SM 1c Stock solution: Take impurities NAA-SM 1c Weigh approximately 20 mg of the reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well.

[0109] Impurity NAA-SM 1d Stock solution: Take impurities NAA-SM 1d Weigh approximately 20 mg of the reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well.

[0110] Impurity NAA-SM 1e Stock solution: Take impurities NAA-SM 1e Weigh approximately 20 mg of the reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well.

[0111] Impurity NAA-SM 1f Stock solution: Take impurities NAA-SM 1f Weigh approximately 20 mg of the reference standard accurately and place it in a 100 ml volumetric flask. Dissolve the standard in 10 ml of methanol first, then dilute with acetonitrile to the mark and shake well.

[0112] Impurity NAA-SM 1h Stock solution: Take impurities NAA-SM 1h Weigh approximately 20 mg of the reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well.

[0113] Impurity NAA-SM 1i Stock solution: Take impurities NAA-SM 1iWeigh approximately 20 mg of the reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile to dissolve and dilute to the mark, and shake well.

[0114] Impurity NAA-SM 1j Stock solution: Take impurities NAA-SM 1j Weigh approximately 20 mg of the reference standard accurately, place it in a 100 ml volumetric flask, add acetonitrile, sonicate to dissolve and dilute to the mark, and shake well.

[0115] Phthalic acid stock solution: Weigh approximately 20 mg of phthalic acid accurately, place it in a 100 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0116] Impurity location solution: Measure 1.0 ml of each of the above impurity stock solutions, place them in 100 ml volumetric flasks respectively, dilute to the mark with solvent, and shake well.

[0117] Test solution: Weigh approximately 20 mg of the test sample (naphthostat mesylate) accurately, place it in a 100 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0118] Mixed solution: Accurately weigh approximately 20 mg of the test sample (naphthostat mesylate), place it in a 100 ml volumetric flask, add solvent to dissolve, and measure the impurity NAA-SM. 1a Impurities NAA-SM 1b Impurities NAA-SM 1c Impurities NAA-SM 1d Impurities NAA-SM 1e Impurities NAA-SM 1f Impurities NAA-SM 1h Impurities NAA-SM 1j 0.5 ml each of phthalic acid stock solution and impurities NAA-SM 1i Place 1.0 ml of the stock solution in the same volumetric flask, dilute to the mark with solvent, and shake well.

[0119] Inject 10 μl each of the blank solution (solvent), each impurity localization solution, the test solution, and the mixed solution, record the chromatograms, and the results are shown in Table D. The original chromatograms can be found in [reference needed]. Figure 1-2 .

[0120] Table D

[0121]

[0122] Conclusion: Blank solution, impurity NAA-SM 1f Neither the main component nor the known impurities interfere with the detection of any known impurities. The minimum resolution between the main peak and adjacent component peaks, and between the known impurity peaks in the mixed solution, is 1.65, which meets the specificity requirements.

[0123] Example 3: Limit of Quantitation and Limit of Detection

[0124] Test solution: Weigh approximately 20 mg of naphthostat mesylate starting material SM1 accurately, place it in a 100 ml volumetric flask, add solvent to dissolve and dilute to the mark, and shake well.

[0125] Phthalic acid stock solution: Prepared according to the preparation method of phthalic acid stock solution under "1. Specificity".

[0126] Linear Stock Solution 1: Accurately measure the test solution and impurities NAA-SM 1a Impurities NAA-SM 1b Impurities NAA-SM 1c Impurities NAA-SM 1d Impurities NAA-SM 1e Impurities NAA-SM 1h Place 5 ml of each stock solution into the same 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0127] Linear stock solution 2: Accurately measure impurity NAA-SM 1i Impurities NAA-SM 1j Place 5 ml of each stock solution into the same 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0128] Limit of Quantitation (LOQ) Stock Solution: Measure 5.0 ml each of Linear Stock Solution 1 and Linear Stock Solution 2, place them in the same 50 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0129] 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.

[0130] 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.

[0131] Detection limit solution 2: Accurately measure 2 ml of phthalic acid stock 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 100 ml volumetric flask. Dilute to the mark with solvent and shake well.

[0132] Take the above-mentioned limit of quantitation solution, limit of detection solution and limit of detection solution 2, and inject them 6 times, 3 times and 3 times consecutively, respectively. Record the chromatograms and calculate the ratio of the peak height of the main component to the noise (S / N). The experimental results are shown in Table E and Table F.

[0133] Table E: Results of Limit of Quantitation Determination

[0134]

[0135]

[0136]

[0137] Conclusion: Impurity NAA-SM 1a The limit of quantitation was 0.0796 μg / ml, the peak area RSD was 0.6% (N=6), the average S / N ratio was 119.8, and the concentration in the sample was expressed as 0.040%; impurity NAA-SM 1d The limit of quantitation (LOQ) concentration was 0.0822 μg / ml, the peak area RSD was 1.5% (N=6), the average S / N ratio was 77.7, and the concentration in the sample was expressed as 0.041%. The LOQ concentration of the naphamostat mesylate starting material SM1 was 0.0836 μg / ml, the peak area RSD was 0.8% (N=6), and the average S / N ratio was 110.1. The impurity NAA-SM... 1i The limit of quantitation was 0.0828 μg / ml, the peak area RSD was 0.3% (N=6), the average S / N ratio was 140.1, and the concentration in the sample was expressed as 0.041%; impurity NAA-SM 1b The limit of quantitation was 0.0808 μg / ml, the peak area RSD was 0.4% (N=6), the average S / N ratio was 135.5, and the concentration in the sample was expressed as 0.040%; impurity NAA-SM 1c The limit of quantitation was 0.0788 μg / ml, the peak area RSD was 0.5% (N=6), the average S / N ratio was 130.9, and the concentration in the sample was expressed as 0.039%; impurity NAA-SM 1j The limit of quantitation was 0.0804 μg / ml, the peak area RSD was 0.3% (N=6), the average S / N ratio was 207.8, and the concentration in the sample was expressed as 0.040%; impurity NAA-SM 1h The limit of quantitation was 0.0846 μg / ml, the peak area RSD was 1.0% (N=6), the average S / N ratio was 97.4, and the concentration in the sample was expressed as 0.042%.

[0138] Table F shows the results of the detection limit determination.

[0139]

[0140]

[0141] The above data can be combined Figures 3-5 Let's interpret it.

[0142] Conclusion: The detection limit for phthalic acid was 0.0393 μg / ml, the average S / N ratio was 13.0, and the concentration present in the sample was expressed as 0.020%; impurity NAA-SM 1a The detection limit was 0.0398 μg / ml, the average S / N ratio was 58.8, and the concentration in the sample was expressed as 0.020%; impurity NAA-SM 1d The detection limit concentration was 0.0411 μg / ml, the average S / N ratio was 38.3, and the concentration present in the sample was expressed as 0.021%; the detection limit concentration of the naphamostat mesylate starting material SM1 was 0.0418 μg / ml, the average S / N ratio was 61.9; the impurity NAA-SM 1i The detection limit was 0.0414 μg / ml, the average S / N ratio was 68.4, and the concentration in the sample was expressed as 0.021%; impurity NAA-SM 1b The detection limit was 0.0404 μg / ml, the average S / N ratio was 66.7, and the concentration in the sample was expressed as 0.020%; impurity NAA-SM 1c The detection limit was 0.0394 μg / ml, the average S / N ratio was 64.1, and the concentration present in the sample was expressed as 0.020%; impurity NAA-SM 1j The detection limit was 0.0402 μg / ml, the average S / N ratio was 101.8, and the concentration in the sample was expressed as 0.020%; impurity NAA-SM 1e The detection limit was 0.0412 μg / ml, the average S / N ratio was 50.4, and the concentration in the sample was expressed as 0.021%; impurity NAA-SM 1h The detection limit was 0.0423 μg / ml, the average S / N ratio was 47.7, and the concentration in the sample was expressed as 0.021%.

[0143] Example 4: Chromatographic Condition Robustness

[0144] Take the mixed solution from the specificity experiment, and test different column flow rates, column temperatures, initial mobile phase gradient ratios, and mobile phase gradient ratios as planned. After the instrument system stabilizes, test each value separately and record the resolution between each peak. The experimental results are shown in Table G.

[0145] Table G

[0146]

[0147]

[0148] Conclusion: When there are minor changes in chromatographic conditions such as flow rate, column temperature, initial ratio of mobile phase, gradient, and column, the minimum resolution between the main component and adjacent component peaks, and between known impurity peaks (except between impurity NAA-SM1b peak and impurity NAA-SM1c peak) is 1.58, and the minimum resolution between impurity NAA-SM1b peak and impurity NAA-SM1c peak is 1.40. The relative retention times of each component are basically consistent with the normalized content, and the method has good robustness.

Claims

1. A method for separating SM1, the starting material of naphthostat mesylate, and its impurities based on high performance liquid chromatography, characterized in that, The naphthostat mesylate starting material SM1 and the impurities together constitute the composition, wherein the impurities include: impurity NAA-SM. 1a Impurities NAA-SM 1b Impurities NAA-SM 1c Impurities NAA-SM 1d Impurities NAA-SM 1e Impurities NAA-SM 1f Impurities NAA-SM 1h Impurities NAA-SM 1i Impurities NAA-SM 1j Any one or more of phthalic acid; the structural formula of each component in the composition is as follows: The mobile phase A was a 0.1% (v / v) phosphoric acid aqueous solution, and the mobile phase B was acetonitrile. The chromatographic column was packed with octadecylsilane-bonded silica gel, and linear gradient elution was performed.

2. The method according to claim 1, characterized in that, The procedure for linear gradient elution is as follows:

3. The method according to claim 2, characterized in that, The procedure for linear gradient elution is as follows:

4. The method according to claim 1, characterized in that, The flow rate was 0.8-1.2 mL / min; the column temperature was 26℃-40℃.

5. A method for identifying SM1, the starting material of naphthostat mesylate, and its impurities, characterized in that, The composition is separated by the method according to any one of claims 1-4 and 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 starting material SM1 and its impurities.

6. The method according to claim 5, characterized in that, The detector has a detection wavelength of 235±10nm.

7. The method according to claim 5, characterized in that, The components of the composition can be identified according to their relative retention times. The components of the composition, in ascending order, are phthalic acid, impurity NAA-SM, etc. 1f NAA-SM 1a NAA-SM 1d Naphthostat mesylate starting material SM1, unknown impurities, impurity NAA-SM 1i Impurities NAA-SM 1b Impurities NAA-SM 1c Impurities NAA-SM 1j Impurities NAA-SM 1e Impurities NAA-SM 1h .

8. The method according to claim 7, characterized in that, Using SM1, the starting material of naphthostat mesylate, as a reference peak; a relative retention time of 0.12 indicates phthalic acid; a relative retention time of 0.28 indicates impurity NAA-SM. 1f ; The relative retention time was 0.49, indicating it was an impurity NAA-SM. 1a ; The relative retention time was 0.94, indicating it was an impurity NAA-SM. 1d ; The relative retention time was 1.59, indicating it was an impurity NAA-SM. 1i ; The relative retention time was 1.63, indicating it was an impurity NAA-SM. 1b ; The relative retention time was 1.65, indicating it was an impurity NAA-SM. 1c ; The relative retention time was 1.86, indicating it was an impurity NAA-SM. 1j ; The relative retention time was 1.91, indicating it was an impurity, NAA-SM. 1e ; The relative retention time was 1.95, indicating it was an impurity, NAA-SM. 1h The relative retention times of each component fluctuate within a range of 10%.

9. A method for determining the content of naphthostat mesylate starting material SM1 and its impurities, characterized in that, The starting material SM1 of naphthostat mesylate and its impurities are separated and identified using the method described in any one of claims 5-7, and a chromatogram is obtained. According to the obtained chromatogram, when the impurity is one or more of impurity NAA-SM1a, impurity NAA-SM1b, impurity NAA-SM1c, impurity NAA-SM1d, impurity NAA-SM1e, impurity NAA-SM1h, impurity NAA-SM1i, and impurity NAA-SM1j, the impurity is calculated by the principal component self-comparison method multiplied by a correction factor; when the impurity is phthalic acid, it is controlled by the limit method.

10. The method according to claim 9, characterized in that, The solvent for the sample to be tested is a mixture of water and acetonitrile.

Images