Method for separating and determining key initial material of clarcodone and related compounds thereof by HPLC (High Performance Liquid Chromatography) method

Abstract

The invention relates to a method for separating and determining a key starting material of clarcosinone and related compounds thereof by HPLC (High Performance Liquid Chromatography), belonging to the technical field of analytical chemistry. 17-hydroxy-21-acetoxyprogesterone and related compounds A-F of the key starting material of clarcosinone are separated and detected by liquid chromatography; a chromatographic column with octadecylsilane chemically bonded silica as a filler is adopted, an acetonitrile aqueous solution is used as a mobile phase A, an acetonitrile methanol solution is used as a mobile phase B, gradient elution is carried out, detection and analysis are carried out, and the content of each compound is calculated. According to the present invention, the key initial material of the clarcosinone can be effectively separated from the related impurity compounds, and the method has advantages of high sensitivity, high separation degree, good repeatability, good durability, simple operation and stable and reliable result, and has important significance on the realization of the clarcosinone drug quality control.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, and more specifically relates to a method for separating and determining key starting materials and related compounds of clacoate ketone by HPLC. Background Technology

[0002] Acne vulgaris is a common chronic inflammatory condition caused by blockage of the hair follicles and sebaceous glands. Clinical manifestations include open and closed comedones, papules, pustules, nodules, and cysts, and further deterioration can easily lead to scarring. Acne vulgaris is influenced by multiple factors, and its pathogenesis is not yet fully understood, but it is mainly related to excessive keratinization of the hair follicle, excessive sebum secretion, and inflammatory response. Excessive keratinization of the hair follicle and excessive sebum secretion are usually closely related to hormone levels in the body.

[0003] Treatments for eczema mainly include antibiotics, retinoids, and hormone medications. Clascoterone (brand name WINLEVI) is one such medication. ® As an androgen receptor antagonist, it is the first topical acne treatment with a completely new mechanism of action since isotretinoin in 1982. On August 26, 2020, the U.S. Food and Drug Administration (FDA) approved it for the treatment of acne vulgaris in children aged 12 years and older or adults.

[0004] Currently, the quality standards for clacoate ketone are not included in the United States Pharmacopeia, the European Pharmacopeia, the Japanese Pharmacopeia, or the Chinese Pharmacopeia, and there are few reports on relevant analytical methods for its key starting materials. Generally, the total impurity content of a drug should be less than 1.0%, and the content of a single impurity should be less than 0.1%. Impurities generated or related substances introduced during the preparation of clacoate ketone, whether in the active pharmaceutical ingredient or the finished product, require strict control.

[0005] Acetyldeoxycortisone is a key starting material for clacoate ketone, and the quality control of this compound plays an important role in the preparation of high-quality clacoate ketone active pharmaceutical ingredient. Therefore, developing an efficient and accurate analytical method for quality analysis is of great significance for the subsequent preparation of high-purity clacoate ketone products.

[0006] In the preparation of the key starting material acetyldeoxycortisone, some impurities are inevitably introduced, including 17α-hydroxyprogesterone, 17,21-diacetoxy-4-pregnen-3,20-dione, 17α-hydroxyprogesterone acetate, androstenedione, 16-dehydroprogesterone, and (8R,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecanoic acid-1H-cyclopentan[a]phenanthrene-17-carboxylic acid ethyl ester. It is of paramount importance to avoid the influence of these compounds on the main component, improve the purity of the main component, and control the synthetic route or reaction process of the key starting material clacoate ketone by analyzing the content of these compounds. Summary of the Invention

[0007] In view of the above-mentioned technical problems, the present invention provides a method for separating and determining key starting materials and related compounds of clacoate ketone by HPLC. The aim is to effectively separate key starting materials of clacoate ketone and related impurity compounds using the method of the present invention, which is expected to have high sensitivity and resolution, good repeatability and robustness, simple operation, and stable and reliable results.

[0008] To achieve the above objectives, in a first aspect, the present invention provides a method for separating and determining key starting materials and related compounds of clacoate ketone by HPLC. The key starting material of clacoate ketone is (3,5-dichloro-4-methoxyphenyl)(1,1-benzo[d]thiazol-3(2H)-yl) methyl ketone, and the related compounds include one or more of compounds A, B, C, D, E, and F, wherein compounds A, B, and F are respectively:

[0009] .

[0010] The key starting material for the synthesis of clacoate ketone, chemically named (3,5-dichloro-4-methoxyphenyl)(1,1-benzo[d]thiazol-3(2H)-yl) methyl ketone, also known as acetyldeoxycortisone, is referred to in this invention as "Compound of Formula I" or "Key Starting Material for Clacoate Ketone," and its chemical formula is C 22 H 32 O5, structural formula as shown in formula I:

[0011]

[0012] Ⅰ

[0013] In some preferred embodiments, the method includes the following steps:

[0014] S1. Preparation of test solution: Dissolve the test sample in diluent to prepare a solution containing 0.5±0.05mg per ml.

[0015] S2. Preparation of control solution: Take an appropriate amount of the test solution and dilute it with the diluent to prepare a solution containing 2.5±0.05μg per ml, which shall be used as the control solution;

[0016] S3. Preparation of System Suitability Solution: Accurately weigh appropriate amounts of this product and impurity reference standard, dissolve and dilute with diluent to prepare a solution containing approximately 0.5 ± 0.05 mg of this product and approximately 2.5 ± 0.05 μg of impurity A per 1 ml. Impurity A is one or more related compounds including compound A, compound B, compound C, compound D, compound E, and compound F. The diluent is acetonitrile.

[0017] S4. Inject the system suitability solution prepared in step S3 into a sample and perform high-performance liquid chromatography (HPLC) analysis. Record the chromatogram and determine the resolution of the analytical method for the key starting material of clacoate ketone. Then, take the test solution from step S1 and the control solution from step S2 into separate samples and perform HPLC analysis. Record the chromatograms and calculate the content of each relevant compound in the test sample using the principal component self-comparison method. The relevant compounds corresponding to each chromatographic peak on the chromatogram are determined based on the chromatographic peaks obtained from the individual injection and detection of each relevant compound reference standard.

[0018] In these embodiments, by setting a specific concentration ratio of the test solution (0.5 mg / ml) to the control solution (2.5 μg / ml), the sensitivity requirements for impurity quantification can be met, effectively detecting trace amounts of related substances. By preparing a system suitability solution containing a specific concentration of impurities, the resolution of the chromatographic method can be directly verified, ensuring the method's effectiveness and reliability. Quantification using the main component self-reference method eliminates the need to rely on impurity reference standards to calculate content, reducing detection costs and making it suitable for routine quality control.

[0019] In some other preferred embodiments, in step S4, when performing high performance liquid chromatography analysis, the mobile phase used for elution includes mobile phase A and mobile phase B, wherein mobile phase A includes an aqueous solution of acetonitrile and mobile phase B includes a mixed solution of acetonitrile and an organic solvent.

[0020] In these embodiments, a mixed solution of acetonitrile aqueous solution and acetonitrile containing organic solvent is used as a dual mobile phase system. Gradient elution effectively adjusts the elution intensity, enabling baseline separation of structurally similar key starting materials and their related compounds. The addition of organic solvent effectively improves peak tailing, enhances peak symmetry, and improves the accuracy and reproducibility of quantification. This reasonable combination of mobile phases can shorten analysis time and improve detection efficiency while ensuring resolution.

[0021] More preferably, the volume percentage of acetonitrile in the acetonitrile aqueous solution is 8-12%, and the mobile phase B is a mixed solution of acetonitrile and methanol, wherein the volume percentage of acetonitrile in the mixed solution of acetonitrile and methanol is 38-42%. This specific ratio range achieves optimal separation of structurally similar impurity compounds, ensuring complete baseline separation of each chromatographic peak. The optimized ratio ensures that both the main component and impurity compounds elute within a reasonable time, avoiding overlap due to excessively short retention and preventing efficiency losses due to excessively long retention.

[0022] More preferably, the elution is gradient elution, and the elution gradient includes:

[0023] .

[0024] This multi-stage linear gradient design continuously changes the polarity of the mobile phase, ensuring complete baseline separation of structurally diverse compounds from the principal component within a reasonable timeframe, with no overlapping peaks. A high proportion of organic phase at the end rapidly washes away strongly retained substances, avoiding residual interference; finally, the initial proportions are returned to equilibrium, ensuring consistent injection conditions and good method reproducibility.

[0025] In a more preferred embodiment, the elution gradient is:

[0026] .

[0027] According to an embodiment of the present invention, the flow rate of the mobile phase during gradient elution is 0.8-1.2 mL / min. This specific gradient ratio ensures that the six structurally similar related compounds are completely baseline separated from the main component, without co-elution interference, and with good peak purity. The elution gradient changes smoothly and reasonably, each component elutes at the optimal solvent strength, and the chromatographic peaks have good symmetry, which is beneficial for accurate quantification.

[0028] According to an embodiment of the present invention, in step S4, when performing high performance liquid chromatography analysis, the chromatographic column used is filled with octadecylsilane-bonded silica gel, and the particle size of the filler is 2-5 μm.

[0029] And / or, the column temperature of the chromatographic column is 30-45℃;

[0030] And / or, the wavelength for detection by high performance liquid chromatography is 240±5nm.

[0031] An octadecylsilane-bonded silica gel column (C18) with a particle size of 2-5 µm was used, providing suitable column efficiency and separation capabilities to ensure good separation of six structurally similar related compounds from the main component. Column temperature control within the range of 30-45℃ reduced mobile phase viscosity, stabilized retention time, and improved the reproducibility and accuracy of analytical results. The detection wavelength was set to 240±5 nm, which covers the characteristic absorption of the target compounds (such as androstenedione and progesterone derivatives), ensuring high and relatively consistent UV absorption for both the main component and related compounds. This avoids underestimation or missed detection of certain impurities due to inappropriate wavelength selection. Furthermore, this wavelength is above the UV cutoff wavelength of commonly used solvents such as acetonitrile and methanol (acetonitrile cutoff wavelength 190 nm, methanol 205 nm), effectively avoiding interference from solvent peaks on early eluted impurities and ensuring baseline stability.

[0032] According to an embodiment of the present invention, in step S4, the formula for calculating the content of each relevant compound in the test sample using the principal component self-comparison method is as follows:

[0033] Related compounds (%) = ×100%;

[0034] In the formula: The peak area of ​​the relevant compounds in the key starting material of clacoate in the test solution;

[0035] The peak area of ​​the main peak in the control solution;

[0036] F is the correction factor, which is determined based on the slope of the linear equations of the key starting material of clacoate and each related compound in the linear validation results. Specifically, it is obtained by dividing the slope of the linear equation of the key starting material of clacoate by the slope of the linear equation of each related compound.

[0037] V represents the dilution factor of the control solution.

[0038] In a second aspect, the present invention provides an application of the method described in the first aspect of the present invention in the detection of key starting materials of clacoate ketone and their impurities, wherein the impurities include one or more of compounds A, B, C, D, E, and F.

[0039] Unlike existing technologies, the above technical solution has the following beneficial effects:

[0040] The present invention provides an HPLC method for separating and determining key starting materials and related compounds of clacoate ketone. This method can effectively separate key starting materials of clacoate ketone and related impurity compounds. For the first time, an HPLC method capable of simultaneously separating and determining the key starting material and six related compounds (compound AF) has been established, solving the problem of lack of targeted analytical methods in the prior art. Moreover, this method has high sensitivity and resolution, good repeatability and robustness, simple operation, and stable and reliable results.

[0041] Simultaneously, the method of this invention separates the key starting materials of clacoate ketone from related impurity compounds, and performs reverse quality control of the synthetic route by analyzing the content of related compounds, thereby further reducing the formation of related compounds. Therefore, the analysis and research of the key starting materials of clacoate ketone and its related impurity compounds in this invention plays a crucial role in controlling the synthetic reaction and improving its quality, and also directly affects the quality of the finished clacoate ketone product. This is of paramount importance for achieving quality control of the key starting materials of clacoate ketone and clacoate ketone itself.

[0042] The above description of the invention is merely an overview of the technical solution of this application. In order to enable those skilled in the art to better understand the technical solution of this application and to implement it based on the description and drawings, and to make the above-mentioned objectives and other objectives, features and advantages of this application easier to understand, the following description is provided in conjunction with the specific embodiments and drawings of this application. Attached Figure Description

[0043] The accompanying drawings are only used to illustrate the principles, implementation methods, applications, features, and effects of specific embodiments of this application and other related content, and should not be considered as limitations on this application.

[0044] In the accompanying drawings of the instruction manual:

[0045] Figure 1 This is a chromatogram of the system suitability solution used in the specificity verification of Example 3;

[0046] Figure 2 The chromatogram of the system suitability solution for Comparative Example 1;

[0047] Figure 3 The chromatogram of the system suitability solution for Comparative Example 2;

[0048] Figure 4 The chromatogram is for the system suitability solution of Comparative Example 3. Detailed Implementation

[0049] To illustrate the possible application scenarios, technical principles, implementable specific solutions, and achievable objectives and effects of this application in detail, the following description, in conjunction with the listed specific embodiments and accompanying drawings, provides a detailed explanation. The embodiments described herein are merely illustrative of the technical solutions of this application and are therefore intended to limit the scope of protection of this application.

[0050] In this document, the term "embodiment" means that a specific feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The term "embodiment" appearing in various places throughout the specification does not necessarily refer to the same embodiment, nor does it specifically limit its independence or connection with other embodiments. In principle, in this application, as long as there are no technical contradictions or conflicts, the technical features mentioned in each embodiment can be combined in any way to form corresponding implementable technical solutions.

[0051] Unless otherwise defined, the technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the use of related terms herein is merely for the purpose of describing particular embodiments and is not intended to limit this application.

[0052] In the description of this application, the term "and / or" is used to describe the logical relationship between objects, indicating that three relationships can exist. For example, A and / or B means: A exists, B exists, and A and B exist simultaneously. Additionally, the character " / " in this document generally indicates that the preceding and following objects have an "or" logical relationship.

[0053] In this application, terms such as “first” and “second” are used only to distinguish one entity or operation from another, and do not necessarily require or imply any actual quantity, hierarchy or order relationship between these entities or operations.

[0054] Unless otherwise specified, the use of terms such as “comprising,” “including,” “having,” or other similar expressions in this application is intended to cover non-exclusive inclusion, which does not exclude the presence of additional elements in a process, method, or product that includes the stated elements, such that a process, method, or product that includes a list of elements may include not only those defined elements but also other elements not expressly listed, or elements inherent to such a process, method, or product.

[0055] As understood in the Examination Guidelines, in this application, expressions such as "greater than," "less than," and "exceeding" are understood to exclude the stated number; expressions such as "above," "below," and "within" are understood to include the stated number. Furthermore, in the description of the embodiments in this application, "multiple" means two or more (including two), and similar expressions related to "multiple" are also understood in this way, such as "multiple groups" and "multiple times," unless otherwise explicitly specified.

[0056] Acne is a common chronic inflammatory skin disease closely related to follicular keratosis and hormone levels. Clavotene, as the first topical acne medication with a novel mechanism of action among androgen receptor antagonists in nearly forty years, has attracted considerable attention since its FDA approval in 2020. However, its quality standards are not currently included in the pharmacopoeias of various countries, and research on analytical methods for its key starting materials is limited.

[0057] Acetyldeoxycortisone is a key starting material for the synthesis of clacoate ketone, and its purity directly affects the quality of the active pharmaceutical ingredient. However, during the preparation process, various structurally similar impurities are inevitably introduced, posing challenges to subsequent process control. For example, compounds A, B, C, D, E, and F have very similar structures to (3,5-dichloro-4-methoxyphenyl)(1,1-benzo[d]thiazol-3(2H)-yl) methyl ketone (also known as acetyldeoxycortisone), a key starting material for the preparation of clacoate ketone. If the reaction is not controlled in the step of obtaining (3,5-dichloro-4-methoxyphenyl)(1,1-benzo[d]thiazol-3(2H)-yl) methyl ketone, and the reaction continues, it will be difficult to remove these impurity compounds in the subsequent synthesis of clacoate ketone. Therefore, strict monitoring of each impurity compound is required in this step of the reaction. Furthermore, to ensure the stringent standards of total impurity content in the active pharmaceutical ingredient being less than 1.0% and individual impurities being less than 0.1%, it is urgent to develop an efficient and accurate analytical method to achieve effective separation and determination of key starting materials and related substances, thus providing an important guarantee for the production of high-quality clacoate ketone.

[0058] In this invention, the "main component self-comparison method" refers to the main component self-comparison method with correction factors used in the General Chapters, Part III of the 2020 edition of the Chinese Pharmacopoeia. Specifically, it involves measuring the correction factors of impurities and main components, multiplying the peak area of ​​the impurities by the correction factor, and then comparing it with the peak area of ​​the main components.

[0059] For reagents and instruments used in this invention whose manufacturers are not specified, all can be obtained through commercial purchases.

[0060] Example 1

[0061] Patent applications EP4071160A1 and CN115073546A disclose a synthetic route for clacoate ketone. This route uses a compound of formula I as a starting material, undergoing hydrolysis, cyclization, and ring-opening to obtain crude clacoate ketone, which is then purified to obtain the final product. In this synthetic route, the key starting material for clacoate ketone, (3,5-dichloro-4-methoxyphenyl)(1,1-benzo[d]thiazolyl) methyl ketone (trade name acetyldeoxycortisone), is the compound of formula I shown below:

[0062]

[0063] I

[0064] The related compounds of acetyldeoxycortisone include one or more of compounds A, B, C, D, E, and F. Information on compound AF is shown in Table 1.

[0065] Table 1 List of relevant compounds

[0066] .

[0067] The structural formulas of compound AF in Table 1 are shown in formulas II-VII below:

[0068]

[0069] II III

[0070]

[0071] IV V

[0072]

[0073] VI VII

[0074] Example 2

[0075] 2.1 Solution Preparation

[0076] Accurately weigh approximately 10 mg of compound A reference standard and place it in a 200 ml volumetric flask. In this example, the diluent is acetonitrile. Tests showed that acetonitrile has good solubility in all compounds and does not interfere with chromatographic determination. Dissolve the compound A reference standard in acetonitrile by sonication and dilute to the mark, then shake well. This solution serves as the stock solution for compound A reference standard. Accurately weigh an appropriate amount of the test sample (with clacoate ketone as the main starting material), add an appropriate amount of the compound A reference standard stock solution, dissolve in acetonitrile, and quantitatively dilute to prepare a solution containing approximately 0.5 mg of the main component and approximately 2.5 μg of compound A per 1 ml. This solution serves as the system suitability solution.

[0077] 2.2 Detection Method

[0078] (1) Accurately measure the system-suitable solution for injection, perform high performance liquid chromatography analysis, record the chromatogram, and determine the resolution;

[0079] Then, accurately measure the test solution and the control solution, and perform high-performance liquid chromatography analysis on them respectively, and record the chromatograms.

[0080] The relevant compounds corresponding to each chromatographic peak on the chromatograms of the test solution and the control solution are determined based on the chromatographic peaks obtained from the individual injection and detection of each relevant compound reference standard.

[0081] (2) The chromatographic conditions for HPLC are as follows:

[0082] Chromatographic column: Titank C18, a conventional C18 column packed with octadecylsilane-bonded silica gel, 150 mm × 4.6 mm, 3 μm. Mobile phase A: acetonitrile-water (10:90), mobile phase B: acetonitrile-methanol (40:60), gradient elution was performed, and the gradient elution settings are shown in Table 2 below.

[0083] Table 2 Elution gradient table

[0084]

[0085] Flow rate: 1 ml / min, column temperature: 35℃, detection wavelength: 245nm, injection volume: 10µl.

[0086] (3) Calculate the content of each relevant compound in the test sample using the principal component self-comparison method with correction factors. The calculation formula is as follows:

[0087] Related compounds (%) = ×100%;

[0088] In the formula: The peak area of ​​the relevant compounds in the key starting material of clacoate in the test solution;

[0089] The peak area of ​​the main peak in the control solution;

[0090] F is the correction factor;

[0091] V represents the dilution factor of the control solution.

[0092] The correction factor F is determined based on the slope of the linear equations of the key starting material of clacoate ketone and each related compound in the linear validation results. Specifically, it is obtained by dividing the slope of the linear equation of the key starting material of clacoate ketone by the slope of the linear equation of each related compound.

[0093] Taking compound A as an example, if the peak area of ​​compound A in the test solution is 5000, the main peak area in the control solution is 200000, the correction factor F=0.82, and the dilution factor of the control solution V=200 (because the test solution is diluted 200 times to obtain the control solution), then the content of compound A (%) = (5000 / 200000) × 0.82 × 200 × 100% = 0.41%.

[0094] Example 3

[0095] The key starting materials for clacoate ketone and the relevant AF reference compounds used in the following validations are shown below:

[0096] (1) Clavotene key starting material: acetyldeoxycortisone, CAS No. 640-87-9, purchased from Shandong Junrui Pharmaceutical Technology Co., Ltd.

[0097] (2) Compound A: 17α-hydroxyprogesterone, CAS No. 68-96-2, purchased from Shandong Saituo Biotechnology Co., Ltd.

[0098] (3) Compound B: 21-bromo-17α-hydroxy-4-pregnen-3,20-dione, CAS number 20380-17-0, self-made.

[0099] (4) Compound C: 17,21-diacetoxy-4-pregnen-3,20-dione, CAS number 1807-15-4, prepared in-house.

[0100] (5) Compound D: 17α-hydroxyprogesterone acetate, purchased from Shanghai Huayuan Biochemical Technology Co., Ltd.

[0101] (6) Compound E: 16-dehydroprogesterone, CAS No. 1096-38-4. Purchased from Shanghai Hanhong Technology Co., Ltd.

[0102] (7) Compound F: (8R,9S,10R,13S,14S,17R)-17-hydroxy-10,13-dimethyl-3-oxo-2,3,6,7,8,9,10,11,12,13,14,15,16,17-tetradecano-1H-cyclopentan[a]phenanthrene-17-carboxylic acid ethyl ester, prepared in-house.

[0103] The following verifications were performed in this embodiment:

[0104] 3.1 Specificity

[0105] Blank solution: solvent.

[0106] Impurity stock solution: Accurately weigh approximately 25 mg of reference compound A, approximately 25 mg of compound B, approximately 25 mg of compound C, approximately 25 mg of compound D, approximately 25 mg of compound E, and approximately 12 mg of compound F. Place each in a 25 ml volumetric flask, dissolve and dilute to the mark with solvent, and shake well.

[0107] Mixed impurity stock solution: Accurately measure 5 ml of each of the above impurity stock solutions, place them in the same 100 ml volumetric flask, dilute to the mark with solvent, and shake well.

[0108] System suitability solution: Weigh approximately 10 mg of this product accurately and place it in a 20 ml volumetric flask. Dissolve the product in an appropriate amount of solvent. Accurately measure 1 ml of the impurity mixed stock solution and place it in the same volumetric flask. Dilute to the mark with an appropriate amount of solvent and shake well.

[0109] Accurately measure 10 μl of the system suitability solution and the stock solutions of each relevant compound reference standard, inject them into the liquid chromatograph, and record the chromatograms using HPLC. The chromatogram results for the system suitability solution are shown in the appendix. Figure 1 Based on the chromatograms of the relevant compound reference stock solutions, determine the chromatographic peaks of each relevant compound on the chromatogram of the system suitability solution.

[0110] The elution order of the system suitability solution was as follows: compound D (retention time Rt 27.547 min), compound A (retention time Rt 31.397 min), compound I (retention time Rt 34.193 min), compound C (retention time Rt 39.717 min), compound B (retention time Rt 40.485 min), compound F (retention time Rt 41.835 min), and compound E (retention time Rt 43.361 min). In the system suitability solution, the resolution between the key starting material compound I of clacoate ketone and the adjacent related compound peaks was greater than 1.5, with a retention time Rt of 34.193 min.

[0111] 3.2 Repeatability

[0112] Take appropriate amounts of the key starting material of clacoate ketone and its related compound reference standards, dissolve and dilute with acetonitrile to prepare a solution containing approximately 0.5 mg of compound I, approximately 2.5 μg of compound A, approximately 2.5 μg of compound B, approximately 2.5 μg of compound C, approximately 2.5 μg of compound D, approximately 2.5 μg of compound E, and approximately 1.0 μg of compound F per 1 ml, as the spiked test solution; accurately measure 1 ml of the spiked test solution and place it in a 200 ml volumetric flask, dilute with acetonitrile to prepare a solution containing approximately 2.5 μg of compound I per 1 ml, as the control solution. Accurately measure the above spiked test solution and control solution and inject them, recording the chromatograms. Calculate the RSD (relative standard deviation) of the content of each related compound in the six spiked test solutions using the principal component self-comparison method with correction factor. The repeatability test results are shown in Table 3. The RSD of the content of each related compound detected in the six spiked test solutions is less than 10.0%, which meets the requirements of high performance liquid chromatography for the detection of related substances.

[0113] Table 3 Repeatability test results

[0114]

[0115] 3.3 Limits of Detection and Limits of Quantification

[0116] Take appropriate amounts of the key starting materials of clacoate ketone and its related compound reference standards, dissolve and dilute them in acetonitrile to prepare a solution containing approximately 5 μg of compound I, approximately 5 μg of compound A, approximately 5 μg of compound B, approximately 5 μg of compound C, approximately 1 μg of compound D, approximately 5 μg of compound E, and approximately 2 μg of compound F per 1 ml. This solution serves as the limit of quantitation (S / N ≥ 10). Take 3 ml of the limit of quantitation solution, place it in a 10 ml volumetric flask, dissolve and dilute it to the mark with solvent, and shake well. This solution serves as the limit of detection (S / N ≥ 3). The limits of quantitation and limits of detection for the key starting materials of clacoate ketone and its related compounds are shown in Table 4.

[0117] Table 4 Results of Limit of Quantitation and Limit of Detection

[0118]

[0119] 3.4 Linear

[0120] Take appropriate amounts of the key starting material of clacoate ketone and its related compound reference standards, dissolve and dilute with acetonitrile to prepare a solution containing approximately 5 μg of compound I, approximately 5 μg of compound A, approximately 5 μg of compound B, approximately 5 μg of compound C, approximately 1 μg of compound D, approximately 5 μg of compound E, and approximately 2 μg of compound F per 1 ml, as a linearity stock solution. Measure 1 ml of the linearity stock solution into a 10 ml volumetric flask, 1 ml into a 25 ml volumetric flask, 1 ml into a 20 ml volumetric flask, 3 ml into a 50 ml volumetric flask, 1 ml into a 10 ml volumetric flask, and 5 ml into a 20 ml volumetric flask, respectively, and dilute to the mark with acetonitrile and mix well. Separately, take the limit of quantitation solution as the linearity test solution. Perform linear regression on peak area against concentration to obtain the linear equation (as shown in Table 5). The key starting material of clacoate ketone and its related compounds exhibit good linearity within the linear range. Based on the linearity determination results, the correction factor for each related compound is obtained by dividing the slope of the linear equation of the principal component I compound by the slope of the linear equation of each related compound (as shown in Table 6).

[0121] Table 5. Results of linearity determination

[0122]

[0123] Table 6 Correction Factor Table

[0124]

[0125] 3.5 Accuracy

[0126] Take appropriate amounts of reference standards for compounds A, B, C, D, E, and F, dissolve and dilute them separately with acetonitrile to prepare solutions containing approximately 1000 μg of compound A, 1000 μg of compound B, 1000 μg of compound C, 1000 μg of compound D, 1000 μg of compound E, and 400 μg of compound F per 1 ml, as stock solutions for each relevant compound. Accurately measure 5 ml of each relevant compound stock solution into a 100 ml volumetric flask, dilute to the mark with acetonitrile, and mix well to prepare a mixed reference stock solution. Accurately weigh approximately 10 mg of clacoate ketone starting material, place it in a 20 ml volumetric flask, add an appropriate amount of the mixed reference stock solution, place it into each of the above volumetric flasks, dilute to the mark with acetonitrile, and mix well to prepare accuracy solutions (limit of quantitation level, 50%, 100%, and 200% concentration levels) for recovery testing, in triplicate. The recoveries and RSDs of each relevant compound were calculated using the principal component self-comparison method with correction factors, and the results are shown in Table 7 below. The results indicate that the recoveries of each relevant compound at various concentrations were between 80.0% and 120.0%, and the RSDs were all less than 10.0%, meeting the requirements for the detection of related substances by high-performance liquid chromatography.

[0127] Table 7 Accuracy Test Results

[0128]

[0129] 3.6 Conclusion

[0130] The results above show that under the chromatographic conditions described, the key starting material of clacoate ketone and its related compounds can be completely separated. This method is highly specific, accurate, sensitive, reproducible, and has good system applicability, meeting the technical requirements of drug quality research standards. The results obtained are stable and reliable.

[0131] Comparative Example 1

[0132] 1. Chromatographic conditions:

[0133] Chromatographic column: Titank C18 (150 mm × 4.6 mm, 3.5 μm), mobile phase A: acetonitrile-water (10:90), mobile phase B: acetonitrile, gradient elution was performed, and the gradient elution settings are shown in Table 8 below:

[0134] Table 8 Elution Gradient Table

[0135]

[0136] Flow rate: 1.0 ml / min, column temperature: 35℃, detection wavelength: 245 nm, injection volume: 10 µl.

[0137] 2. Method: Accurately weigh appropriate amounts of the key starting material of clacotone, compound I, and compounds A, B, C, D, E, and F. Place them in the same volumetric flask, dissolve and dilute with acetonitrile to prepare a solution containing approximately 0.5 mg of compound I, approximately 2.5 μg of compound A, approximately 2.5 μg of compound B, approximately 2.5 μg of compound C, approximately 2.5 μg of compound D, approximately 2.5 μg of compound E, and approximately 2.5 μg of compound F per ml. This solution is used as the system suitability solution. Accurately inject 10 μl into the liquid chromatograph and record the chromatogram. The chromatogram of the system suitability solution for this comparative example is shown in the appendix. Figure 2 .

[0138] 3. Conclusion: From Figure 2 The results show that under these chromatographic conditions, the resolution between compound C (retention time Rt 29.824 min) and compound B (retention time Rt 30.018 min) is 1.1, which is not baseline separated and is less than the requirement of the Chinese Pharmacopoeia (the resolution should be greater than 1.5), thus affecting the quantitative results and qualitative judgment of compounds C and B.

[0139] Comparative Example 2

[0140] 1. Chromatographic conditions:

[0141] Chromatographic column: Superfex AQ-C18 (4.6×250mm, 5μm), mobile phase A: acetonitrile-water (10:90), mobile phase B: acetonitrile, gradient elution was performed, and the gradient elution settings are shown in Table 9 below:

[0142] Table 9 Elution Gradient Table

[0143]

[0144] Flow rate: 1.0 ml / min, column temperature: 35℃, detection wavelength: 245 nm, injection volume: 10 µl.

[0145] 2. The method is the same as Comparative Example 1. The system suitability solution chromatogram of this comparative example is attached. Figure 3 .

[0146] 3. Conclusion: From Figure 3It can be seen that under these chromatographic conditions, the separation between the main component peak and the impurities is good. The separation degree between compound A (retention time Rt 20.026 min) and the unknown impurity (retention time 20.457 min) is 1.0, which is less than the requirement of the Chinese Pharmacopoeia (the separation degree should be greater than 1.5). Compound C (retention time Rt 26.277 min) and compound B (retention time Rt 26.813 min) did not reach baseline separation, with a separation degree of 1.2, which is less than the requirement of the Chinese Pharmacopoeia (the separation degree should be greater than 1.5). This affects the quantitative results and qualitative judgment of compounds A, B and C.

[0147] Comparative Example 3

[0148] 1. Chromatographic conditions:

[0149] Chromatographic column: Titank C18 (150 mm × 4.6 mm, 3.5 μm), mobile phase A: acetonitrile-water (10:90), mobile phase B: acetonitrile-methanol (40:60), gradient elution was performed, and the gradient elution settings are shown in Table 10 below:

[0150] Table 10 Elution Gradient Table

[0151]

[0152] Flow rate: 1.0 ml / min, column temperature: 35℃, detection wavelength: 245 nm, injection volume: 10 µl.

[0153] 2. The method is the same as Comparative Example 1. The system suitability solution chromatogram of this comparative example is attached. Figure 4 .

[0154] 3. Conclusion: Under these chromatographic conditions, the separation between the main component peak and the impurities was good. The separation degree between compound A (retention time Rt 31.752 min) and the unknown impurity (retention time 32.493 min) was 1.4, which is less than the requirement of the Chinese Pharmacopoeia (the separation degree should be greater than 1.5), thus affecting the quantitative results and qualitative judgment of compound A.

[0155] In summary, this invention, through the use of a specific C18 column and a dual mobile phase system consisting of acetonitrile-water and acetonitrile-methanol, along with an optimized gradient elution program, successfully achieved complete baseline separation of the key starting material of clacoate ketone and its six related compounds. The resolution between adjacent peaks was greater than 1.5, fully meeting the requirements of the Chinese Pharmacopoeia and ensuring the accuracy of qualitative and quantitative analysis of each impurity. Furthermore, the method of this invention exhibits high specificity (no blank interference), high sensitivity (detection limit as low as 0.03 μg / ml), good repeatability (RSD < 3.0%), good linearity (r ≥ 0.9999), and high accuracy (recovery rates of each impurity were between 80.0% and 120.0%), fully meeting the technical requirements for drug quality research, and the results are stable and reliable. Compared to the problems in Comparative Examples 1-3 where the separation degree between compound A and unknown impurities, and between compound B and compound C, was less than 1.5 due to improper mobile phase composition or gradient settings, this invention solves the technical problem of the difficulty in effectively separating multiple structurally similar impurity compounds simultaneously, and provides a better technical solution for the quality control of key starting materials for clacoate ketone.

[0156] Finally, it should be noted that although the above embodiments have been described in the text and drawings of this application, this should not limit the scope of patent protection of this application. Any technical solutions that are based on the essential concept of this application and utilize the content described in the text and drawings of this application, resulting in equivalent structural or procedural substitutions or modifications, as well as the direct or indirect application of the technical solutions of the above embodiments to other related technical fields, are all included within the scope of patent protection of this application.

Claims

1. A method for separating and determining key starting materials of clavatone and related compounds by HPLC, characterized in that, The key starting material for clacotone is (3,5-dichloro-4-methoxyphenyl)(1,1-benzo[d]thiazo-3(2H)-yl) methyl ketone, and the related compounds include one or more of compounds A, B, C, D, E, and F, wherein compounds A, B, C, D, E, and F are respectively: 。 2. The method of claim 1, wherein, Includes the following steps: S1. Preparation of test solution: Dissolve the test sample in diluent to prepare a solution containing 0.5±0.05mg per ml. S2. Preparation of control solution: Take an appropriate amount of the test solution and dilute it with the diluent to prepare a solution containing 2.5±0.05μg per ml, which shall be used as the control solution; S3. Preparation of System Suitability Solution: Accurately weigh appropriate amounts of this product and impurity reference standard, dissolve and dilute with diluent to prepare a solution containing approximately 0.5 ± 0.05 mg of this product and approximately 2.5 ± 0.05 μg of impurity A per 1 ml. Impurity A is one or more related compounds including compound A, compound B, compound C, compound D, compound E, and compound F. The diluent is acetonitrile. S4. Inject the system suitability solution prepared in step S3 into a sample and perform high performance liquid chromatography analysis. Record the chromatogram and determine the resolution of the analytical method for key starting materials of clacoate ketone. Then, take the test solution from step S1 and the control solution from step S2 into a sample and perform high performance liquid chromatography analysis. Record the chromatogram and calculate the content of each relevant compound in the test sample according to the principal component self-comparison method.

3. The method of claim 2, wherein, In step S4, when performing high performance liquid chromatography analysis, the mobile phase used for elution includes mobile phase A and mobile phase B. Mobile phase A includes an aqueous solution of acetonitrile, and mobile phase B includes a mixed solution of acetonitrile and an organic solvent.

4. The method of claim 3, wherein, The volume percentage of acetonitrile in the acetonitrile aqueous solution is 8-12%, and the mobile phase B is a mixed solution of acetonitrile and methanol, wherein the volume percentage of acetonitrile in the mixed solution of acetonitrile and methanol is 38-42%.

5. The method of claim 3, wherein, The elution is gradient elution, and the elution gradient includes: 。 6. The method of claim 5, wherein, The elution gradient is: 。 7. The method of claim 6, wherein, When performing gradient elution, the flow rate of the mobile phase is 0.8-1.2 mL / min.

8. The method of claim 2, wherein, In step S4, when performing high performance liquid chromatography analysis, the chromatographic column used is filled with octadecylsilane-bonded silica gel, and the particle size of the filler is 2-5 μm. And / or, the column temperature of the chromatographic column is 30-45℃; And / or, the wavelength for detection by high performance liquid chromatography is 240±5nm.

9. The method of claim 2, wherein, In step S4, the formula for calculating the content of each relevant compound in the test sample using the principal component self-comparison method is as follows: Related compounds (%) x 100%; In the formula: The peak area of ​​the relevant compounds in the key starting material of clacoate in the test solution; Peak area of main peak in control solution; F is the correction factor; V represents the dilution factor of the control solution.

10. The application of the method according to any one of claims 1-9 in the detection of key starting materials of clacoate ketone and their impurities, said impurities comprising one or more of compounds A, B, C, D, E, and F.

Images