Indobufen impurity, detection method therefor, and use thereof

Abstract

An indobufen impurity with a brand-new structure, a preparation method therefor, and a use thereof, and an indobufen impurity detection method, using a high-performance liquid chromatography method, wherein the chromatographic column uses amidoalkylsilane bonded silica gel as a filler; a phosphate buffer solution is used for a mobile phase A and a mixed solvent containing tetrahydrofuran is used for a mobile phase B; the technical effect of simultaneously separating and detecting impurities such as impurity 2, impurity 3 and impurity 6 is achieved. The indobufen impurity detection method can thus be used for quality control or the establishment of quality standards in indobufen raw material or formulation research.

Description

Indobufen impurity and its detection method and application Technical Field

[0001] The present invention belongs to the technical field of indobufen detection methods, and particularly relates to an indobufen impurity and a preparation method, a detection method and an application thereof. Background Art

[0002] Indobufen is an isoindolinylphenylbutyric acid derivative, chemically known as (±)2-[4-(1-oxo-2-isoindolinyl)phenyl]butyric acid, with the structural formula shown below. Developed by Farmitalia Carlo Erba (now Pfizer), indobufen is an antiplatelet aggregation drug. It reversibly inhibits platelet cyclooxygenase, reducing the production of thromboxane A2, thereby blocking platelet aggregation. It is used to prevent and treat ischemic cardiovascular disease, ischemic cerebrovascular disease, and venous thrombosis caused by arteriosclerosis.

[0003] The synthesis process of indobufen involves multiple steps including hydrogenation, cyclization, zinc powder reduction, and crude product purification, which can easily produce a variety of process impurities: hydrogenation reaction product (denoted as impurity 1), cyclization reaction product (denoted as impurity 2), zinc powder reduction reaction by-products (denoted as impurities 3 to 6), and crude product purification reaction by-products (7 to 8).

[0004] The prior art has disclosed a variety of indobufen impurities: ① The national drug standard for indobufen discloses indobufen impurity I (i.e., impurity 2); ② Document 1 (Yu Li et al. Characterization of an Unknown Impurity in Indobufen Tablets by HPLC-Q-TOF MS and NMR [J]. Current Pharmaceutical Analysis, 2021 (17): 885-891) discloses a new impurity (i.e., impurity 4); ③ Document 2 (Zhao Huan. Quality Control Research of Indobufen [D]. Chengdu University, 2023.) discloses impurities B (i.e., impurity 1), impurity C (i.e., impurity 2), impurity F (i.e., impurity 8), impurity G (i.e., impurity 6), and impurity TM-3 (i.e., impurity 5). However, no prior art discloses impurities 3 and 7.

[0005] Existing technologies also disclose various impurity detection methods. The National Drug Standard for Indobufen (WS1-(X-069)-2006Z) discloses a method for detecting related substances: using octadecylsilane-bonded silica gel as a filler and a mobile phase of methanol-water-triethylamine (65:35:0.5) (pH adjusted to 3.0 with phosphoric acid). However, this method is not suitable for the simultaneous separation and detection of multiple process impurities.

[0006] Patent CN114594168A discloses a method for detecting indobufen impurities. The method uses octadecylsilane bonded silica gel as a filler, potassium dihydrogen phosphate buffer (pH adjusted to 4.0 with phosphoric acid) as mobile phase A, and acetonitrile as mobile phase B, using gradient elution. Four impurities can be simultaneously separated and detected: YDBF-IM-A (i.e., impurity 2), YDBF-IM-C, YDBF-IM-F (i.e., impurity 8), and YDBF-IM-I.

[0007] Patent CN114624339A discloses a method for detecting indobufen impurities. The method uses octadecylsilane bonded silica gel as a filler, potassium dihydrogen phosphate buffer (pH adjusted to 3.0 with phosphoric acid) as mobile phase A, and 0.1% acetonitrile phosphate as mobile phase B, using gradient elution. Seven impurities can be simultaneously separated and detected: YDBF-IM-A (impurity 2), YDBF-IM-B (impurity 6), YDBF-IM-C, YDBF-IM-E, YDBF-IM-F (impurity 8), YDBF-IM-H, and YDBF-IM-I.

[0008] However, there is no prior art that discloses a method for separating and detecting impurity 3 or impurity 7, nor is there any related technical inspiration.

[0009] In summary, how to optimize the existing indobufen detection method to achieve the simultaneous separation and detection of impurity 2 required by national drug standards, impurity 3 and impurity 6, which are byproducts easily produced by the zinc powder reduction reaction; and further, how to simultaneously separate and detect impurities such as impurity 7 and impurity 8, which are byproducts easily produced by the crude product purification reaction, are technical difficulties that have not yet been solved by those skilled in the art. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to provide a method for detecting indobufen impurities, which can simultaneously separate and detect multiple impurities such as impurity 2, impurity 3, impurity 6, etc., for use in the quality control of indobufen raw materials and preparations.

[0011] The above technical problems to be solved are achieved through the following technical solutions:

[0012] The present invention provides a method for detecting indobufen impurities, which adopts a high performance liquid chromatography method, wherein a chromatographic column adopts amidoalkylsilane bonded silica gel as a filler; mobile phase A adopts a phosphate buffer solution, and mobile phase B adopts a mixed solvent containing tetrahydrofuran.

[0013] The present invention is optimized for the chromatographic column:

[0014] Preferably, the chromatographic column is an amidohexadecylsilane bonded silica gel column or an amidooctadecylsilane bonded silica gel column.

[0015] More preferably, the chromatographic column is ZORBAX Bonus-RP 4.6mm×250mm, 3.5μm, ZORBAX Bonus-RP 4.6mm×150mm, 3.5μm, or RP 4.6×150mm, 5μm or equivalent performance column.

[0016] Most preferably, the chromatography column is ZORBAX Bonus-RP 4.6 mm×250 mm, 3.5 μm.

[0017] The chromatographic column filler is amidoalkylsilane bonded silica gel. The polar amide group embedded in the long alkyl chain is the key group for separating the decarboxylated impurity 3. Those skilled in the art can routinely screen the specific manufacturer and specifications of the chromatographic column to meet the actual detection needs.

[0018] The present invention is also optimized for mobile phase:

[0019] The mobile phase A described in the present invention is preferably a phosphate buffer solution, including but not limited to one or more of potassium dihydrogen phosphate buffer solution, sodium dihydrogen phosphate buffer solution, and ammonium dihydrogen phosphate buffer solution.

[0020] More preferably, mobile phase A is potassium dihydrogen phosphate buffer solution.

[0021] Preferably, the concentration of mobile phase A is 10 to 25 mmol.

[0022] Preferably, the pH of mobile phase A is 2-4.

[0023] More preferably, the pH of mobile phase A is 2.5 to 3.2.

[0024] Most preferably, the pH of mobile phase A is 2.8-3.0.

[0025] The mobile phase B of the present invention preferably contains one or more of methanol and acetonitrile in addition to tetrahydrofuran in its mixed solvent.

[0026] More preferably, mobile phase B is methanol-acetonitrile-tetrahydrofuran, methanol-tetrahydrofuran or acetonitrile-tetrahydrofuran.

[0027] Most preferably, mobile phase B is acetonitrile-tetrahydrofuran.

[0028] Preferably, the content of tetrahydrofuran in mobile phase B is 15-40%.

[0029] More preferably, the content of tetrahydrofuran in mobile phase B is 35-40%.

[0030] Adding tetrahydrofuran to mobile phase B to adjust the proton-accepting ability of the mobile phase is a key factor in separating impurity 2 under the conditions of amide alkylsilane bonded silicon column. Those skilled in the art can select conventional liquid chromatography organic solvents such as methanol and / or acetonitrile to form a mixed solvent with tetrahydrofuran to meet the actual detection requirements.

[0031] The present invention also optimizes the elution gradient of the mobile phase:

[0032] Preferably, the mobile phase adopts gradient elution, including the following gradient:

[0033] More preferably, the gradient includes:

[0034] Most preferably, the gradient includes:

[0035] or

[0036] The present invention also optimizes the column temperature:

[0037] Preferably, the column temperature is 30-50°C.

[0038] More preferably, the column temperature is 40-50°C.

[0039] Most preferably, the column temperature is 40°C.

[0040] The present invention is also optimized for detection wavelength:

[0041] Preferably, the detection wavelength is 208-248 nm.

[0042] More preferably, the detection wavelength is 228 nm.

[0043] The present invention is also optimized for flow rate:

[0044] Preferably, the flow rate is 0.8-1.2 ml / min.

[0045] More preferably, the flow rate is 1.0 ml / min.

[0046] The present invention also provides an application of the above-mentioned indobufen impurity detection method in the quality control of indobufen bulk drug or preparation.

[0047] Specifically, it can be used to separate and detect all or part of the indobufen impurities in impurities 1 to 8.

[0048] The aforementioned indobufen impurities include but are not limited to One or more of .

[0049] Preferably, the indobufen impurity detection method provided by the present invention can be used to simultaneously separate at least two of the eight impurities 1 to 8, that is, to separate at least impurity 3 and impurity 6.

[0050] More preferably, the indobufen impurity detection method provided by the present invention can also be used to simultaneously separate at least three of the eight impurities 1 to 8, that is, to separate at least impurity 2, impurity 3, and impurity 6.

[0051] More preferably, the indobufen impurity detection method provided by the present invention can also be used to simultaneously separate at least four, five, six, seven or eight of the eight impurities 1 to 8 (and the four, five, six, seven or eight impurities must include impurity 2, impurity 3 and impurity 6). Examples include but are not limited to: when four impurities are separated simultaneously, in addition to impurities 2, impurity 3 and impurity 6, the remaining impurity can be selected from one of impurities 1, 4, 5, 7 and 8; similarly, when five impurities are separated simultaneously, in addition to impurities 2, impurity 3 and impurity 6, the remaining two impurities can be selected from two of impurities 1, 4, 5, 7 and 8; similarly, when six impurities are separated simultaneously, in addition to impurities 2, impurity 3 and impurity 6, the remaining three impurities can be selected from three of impurities 1, 4, 5, 7 and 8; similarly, when seven impurities are separated simultaneously, in addition to impurities 2, impurity 3 and impurity 6, the remaining four impurities can be selected from four of impurities 1, 4, 5, 7 and 8; similarly, when eight impurities are separated simultaneously, they are impurities 1 to 8.

[0052] Furthermore, as an internal quality control standard with higher requirements, it can also be implemented according to the simultaneous separation of impurity 2, impurity 3, impurity 6 and impurity 7, or the simultaneous separation of impurity 2, impurity 3, impurity 6, impurity 7 and impurity 8.

[0053] In addition, the present invention also provides some new impurities with structures not disclosed in the prior art, including impurity 7 and impurity 3, etc., whose structural formula is:

[0054] Preferably, the impurity 7 provided by the present invention is obtained by esterification reaction of indobufen and isopropyl alcohol.

[0055] Preferably, the present invention also provides a method for preparing impurity 7, comprising the following steps: esterification reaction of indobufen and isopropyl alcohol under acidic conditions, cooling and crystallization after the reaction, filtering with an alkaline solution, extracting with an organic solvent, washing, concentrating, and drying to obtain impurity 7.

[0056] Preferably, in the preparation method of the aforementioned impurity 7, the acidic conditions include but are not limited to concentrated sulfuric acid.

[0057] Preferably, in the preparation method of the aforementioned impurity 7, the alkaline solution includes but is not limited to sodium hydroxide solution, more preferably 3% sodium hydroxide solution.

[0058] Preferably, in the preparation method of the aforementioned impurity 7, the organic solvent includes but is not limited to dichloromethane.

[0059] Furthermore, the present invention also provides applications of indobufen impurity 7 in the pharmaceutical field, including but not limited to applications for impurity detection and quality control of indobufen APIs or preparations.

[0060] Preferably, the above-mentioned indobufen preparation is selected from solid preparations, semisolid preparations and liquid preparations.

[0061] Preferably, the above-mentioned indobufen solid preparation is selected from indobufen tablets.

[0062] The impurity 7 provided by the present invention can be used as a key quality control impurity (whether a standard or a reference substance) in the preparation process of dobufen raw materials or preparations, and can be used for quality control of dobufen raw materials or preparations, or for establishing quality standards.

[0063] When impurity 7 is used in the quality control of dobufen drug substance or drug product, its impurity limit should be <1.0%, more preferably <0.5%.

[0064] The impurity 7 provided by the present invention can be used for impurity detection in indobufen raw materials or preparations; preferably, high performance liquid chromatography is used to detect indobufen impurities.

[0065] Furthermore, the present invention also provides a method for detecting impurity 7, which, as described above, mainly adopts high performance liquid chromatography, wherein the chromatographic column uses amidoalkylsilane bonded silica gel as a filler; mobile phase A uses a phosphate buffer solution, and mobile phase B uses a mixed solvent containing tetrahydrofuran.

[0066] Preferably, in addition to the above chromatographic conditions, the mobile phase can be gradient elution, including the following gradient:

[0067] More preferably, the gradient includes:

[0068] Most preferably, the gradient includes:

[0069] or

[0070] Preferably, in addition to the above chromatographic conditions, the column temperature can be 30-50°C; more preferably, the column temperature is 40-50°C; more preferably, the column temperature is 40°C.

[0071] Preferably, in addition to the above chromatographic conditions, the detection wavelength can be 208-248 nm; more preferably, the detection wavelength is 2428 nm.

[0072] Preferably, in addition to the above chromatographic conditions, the flow rate can be 0.8-1.2 ml / min; more preferably, the flow rate is 1.0 ml / min.

[0073] In summary, the present invention provides a method for detecting a specific indobufen impurity 7, as well as a method for simultaneously detecting indobufen impurity 7 and other known impurities; the above-mentioned detection method can be widely applied to indobufen raw materials, and various solid preparations, semi-solid preparations or liquid preparations of indobufen. As long as the preparation contains impurity 7, it can be detected, and the quality control of the indobufen raw materials or its preparations can be carried out, or quality standards can be established to improve the stability of the raw materials or preparations, ensure that the impurities do not exceed the limit, and comply with the drug review regulations.

[0074] Compared with the prior art, the indobufen impurity detection method provided by the present invention has the following beneficial effects:

[0075] 1) The indobufen impurity detection method provided by the present invention uses a chromatographic column filled with amide alkylsilane bonded silica gel. Compared with the chromatographic columns filled with octadecylsilane bonded silica gel used in CN114594168A and CN114624339A, the separation degree of impurity 3 and impurity 6 is improved, so that both impurities can be separated and detected simultaneously.

[0076] 2) The indobufen impurity detection method provided by the present invention solves the problem of the difficulty in effectively separating the main peak and impurity 2 in the amidoalkylsilane bonded silica gel column by optimizing the mobile phase B to a mixed solvent containing tetrahydrofuran.

[0077] Therefore, the above scheme adopts a specific chromatographic column filler and mobile phase B, which can achieve the technical effect of simultaneously separating and detecting impurity 2 (an impurity that must be detected according to the pharmacopoeia), impurity 3 and impurity 6, solving the primary technical problem of the present invention.

[0078] Furthermore, by optimizing the content of tetrahydrofuran in mobile phase B to 15-40%, the separation effect of the main peak and impurity 2 can be further improved.

[0079] 3) The indobufen impurity detection method provided by the present invention can simultaneously separate and detect indobufen impurities 2, 3, 6, 7, and 8 by optimizing the type of chromatographic column, mobile phase B, and gradient elution;

[0080] On this basis, further optimization of conditions such as the pH range of 2.5-3.2 (even pH 2.8-3.0) can further simultaneously separate and detect impurities such as impurity 1, impurity 4 and impurity 5, all of which can be used for quality control of indobufen raw materials and preparations.

[0081] 4) The present invention also discovered new impurities (impurity 7, impurity 3, etc.) whose structures were not disclosed in the prior art, confirmed the structures of these impurities, provided methods for their preparation, and their applications in the medical field.

[0082] These uses can be used as standard substances or reference substances for quality control or establishment of quality standards of indobufen raw materials or preparations; detection methods for these impurities can also be developed for related substance control and content determination of raw materials or drugs, etc.; especially for indobufen tablets currently under development on the market, they have extremely important quality control guidance significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIG1 is a diagram showing the applicability test results of the indobufen impurity detection system of Example 1;

[0084] FIG2 is the applicability test results of the indobufen impurity detection system in Example 2;

[0085] Figure 3 is the test results of the impurity detection method of comparative example 1 with reference to CN114594168A;

[0086] Figure 4 shows the results of the chromatographic column type investigation based on the impurity detection method of CN114594168A in Comparative Example 2.

[0087] FIG5 is a high-resolution mass spectrum of impurity 7.

[0088] FIG6 is the H NMR spectrum of impurity 7.

[0089] FIG7 is the carbon NMR spectrum of impurity 7. DETAILED DESCRIPTION

[0090] The following are specific embodiments of the present invention to further describe the technical solution of the present invention, but the scope of protection of the present invention is not limited to these embodiments. Any changes or equivalent substitutions that do not deviate from the concept of the present invention are protected within the scope of protection of the present invention.

[0091] The reagents and instruments used in the examples are all conventional reagents and instruments in the art and can be purchased through conventional channels. The following is only for illustration.

[0092] Reagents:

[0093] Indobufen reference substance (purity: 99.9%, manufacturer: China Food and Drug Administration)

[0094] Indobufen API (homemade or commercially available)

[0095] instrument:

[0096] High-performance liquid chromatography (model: Ultimate 3000, manufacturer: Thermo Fisher )

[0097] Example 1

[0098] A method for detecting indobufen impurities comprises the following steps:

[0099] 1) Sample Preparation: Take appropriate amounts of indobufen, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, and impurity 8 reference substances, dissolve and dilute them with acetonitrile-water (50:50) solvent to prepare a solution containing approximately 0.5 mg of indobufen and 0.75 μg each of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, and impurity 8 per 1 ml, which serves as the system suitability solution.

[0100] 2) High Performance Liquid Chromatography Detection: The sample solution in step 1) was measured and injected. The detection conditions were as follows:

[0101] Chromatographic column: ZORBAX Bonus-RP 4.6mm×250mm, 3.5μm or equivalent performance column

[0102] Mobile phase: Use phosphate buffer (weigh 1.36 g of potassium dihydrogen phosphate, dissolve in 1000 ml of water, and adjust the pH to 3.0 with phosphoric acid) as mobile phase A and acetonitrile-tetrahydrofuran (60:40) as mobile phase B. Perform linear gradient elution as shown in the table below:

[0103] The detection wavelength is 228 nm;

[0104] The column temperature was 40°C;

[0105] Flow rate: 1.0 ml / min;

[0106] The injection volume was 10 μl.

[0107] The test results are shown in Figure 1 and Table 1:

[0108] Table 1. Results of suitability test of indobufen impurity detection system

[0109] The test results show that impurity 1, impurity 4, impurity 5, indobufen, impurity 2, impurity 8, impurity 3, impurity 7, and impurity 6 appear in sequence, and the separation between each impurity meets the requirements and is effectively separated from the baseline, and can be used to simultaneously separate and detect the above 8 impurities.

[0110] Example 2

[0111] A method for detecting indobufen impurities comprises the following steps:

[0112] 1) Sample Preparation: Take appropriate amounts of indobufen, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, and impurity 8 reference substances, dissolve and dilute them with acetonitrile-water (50:50) solvent to prepare a solution containing approximately 0.5 mg of indobufen and 0.75 μg each of impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, and impurity 8 per 1 ml, which serves as the system suitability solution.

[0113] 2) High Performance Liquid Chromatography Detection: The sample solution in step 1) was measured and injected. The detection conditions were as follows:

[0114] Chromatographic column: ZORBAX Bonus-RP 4.6mm×250mm, 3.5μm or equivalent performance column

[0115] Mobile phase: Use phosphate buffer (weigh 1.36 g potassium dihydrogen phosphate, dissolve in 1000 ml of water, and adjust the pH to 3.0 with phosphoric acid) as mobile phase A and acetonitrile-tetrahydrofuran (65:35) as mobile phase B. Perform linear gradient elution according to the following table:

[0116] The detection wavelength is 228 nm;

[0117] The column temperature was 40°C;

[0118] Flow rate: 1.0 ml / min;

[0119] The injection volume was 10 μl.

[0120] The test results are shown in Figure 2 and Table 2:

[0121] Table 2. Results of suitability test of indobufen impurity detection system

[0122] The test results show that impurity 1, impurity 4, impurity 5, indobufen, impurity 2, impurity 8, impurity 3, impurity 7, and impurity 6 appear in sequence, and the separation between each impurity meets the requirements and is effectively separated from the baseline, and can be used to simultaneously separate and detect the above 8 impurities.

[0123] It should be noted that in the aforementioned Examples 1 and 2, the pH value of mobile phase A may be adjusted to 2.8-3.0, or 2.5-3.2, in addition to 3.0, without affecting the effective separation of impurities 1-8.

[0124] Example 3-5 Investigation of the type of mobile phase B

[0125] Based on Example 1, the effects of the mobile phase B types on impurity separation were investigated: acetonitrile (Example 3), an acetonitrile-methanol mixed solvent (Example 4), an acetonitrile-methanol-tetrahydrofuran mixed solvent (Example 5), and acetonitrile-tetrahydrofuran (Example 1). The investigation process is as follows:

[0126] A method for detecting indobufen impurities comprises the following steps:

[0127] 1) Sample processing: same as in Example 1;

[0128] 2) High Performance Liquid Chromatography Detection: The sample solution in step 1) was measured and injected. The detection conditions were as follows:

[0129] Mobile phase: Example 3: Phosphate buffer (1.36 g potassium dihydrogen phosphate was weighed and dissolved in 1000 ml of water, and the pH was adjusted to 3.0 with phosphoric acid) was used as mobile phase A, and acetonitrile was used as mobile phase B;

[0130] Mobile phase: Example 4: Phosphate buffer (1.36 g of potassium dihydrogen phosphate was weighed and dissolved in 1000 ml of water, and the pH was adjusted to 3.0 with phosphoric acid) was used as mobile phase A, and acetonitrile-methanol (90:10) was used as mobile phase B;

[0131] Mobile phase: Example 5: Phosphate buffer (1.36 g of potassium dihydrogen phosphate was weighed and dissolved in 1000 ml of water, and the pH was adjusted to 3.0 with phosphoric acid) was used as mobile phase A, and acetonitrile-methanol-tetrahydrofuran (60:25:15) was used as mobile phase B;

[0132] The chromatographic column, elution gradient, detection wavelength, column temperature, flow rate, and injection volume were the same as those in Example 1.

[0133] The results of the investigation are shown in Table 3:

[0134] Table 3. Results of investigation on mobile phase B types for indobufen impurity detection

[0135] The investigation results show that when the mobile phase B is acetonitrile or acetonitrile-methanol mixed solvent, the main peak and impurity 2 are merged or almost merged, and the separation degree does not meet the requirements; when the mobile phase B is acetonitrile-tetrahydrofuran mixed solvent or methanol-acetonitrile-tetrahydrofuran mixed solvent, the separation degree between the main peak and impurity 2 meets the requirements.

[0136] Example 6-7 Investigation of the proportion of mobile phase B

[0137] Based on Example 1, the effects of the mobile phase B ratios of acetonitrile-tetrahydrofuran (85:15) (Example 6), acetonitrile-tetrahydrofuran (75:25) (Example 7), and acetonitrile-tetrahydrofuran (60:40) (Example 1) on impurity separation were investigated. The investigation process was as follows:

[0138] A method for detecting indobufen impurities comprises the following steps:

[0139] 1) Sample processing: same as in Example 1;

[0140] 2) High Performance Liquid Chromatography Detection: The sample solution in step 1) was measured and injected. The detection conditions were as follows:

[0141] Mobile phase: Example 6: Phosphate buffer (1.36 g potassium dihydrogen phosphate was weighed and dissolved in 1000 ml of water, and the pH was adjusted to 3.0 with phosphoric acid) was used as mobile phase A, and acetonitrile-tetrahydrofuran (85:15) was used as mobile phase B;

[0142] Mobile phase: Example 7: Phosphate buffer (1.36 g of potassium dihydrogen phosphate was weighed and dissolved in 1000 ml of water, and the pH was adjusted to 3.0 with phosphoric acid) was used as mobile phase A, and acetonitrile-tetrahydrofuran (75:25) was used as mobile phase B;

[0143] The chromatographic column, elution gradient, detection wavelength, column temperature, flow rate, and injection volume were the same as those in Example 1.

[0144] The results of the investigation are shown in Table 4:

[0145] Table 4. Results of investigation on the proportion of mobile phase B for indobufen impurity detection

[0146] The results of the investigation showed that when the content of tetrahydrofuran in mobile phase B was 15-40%, the separation degree between the main peak and impurity 2 met the requirements, and the baseline separation effect could be further improved.

[0147] Example 8-9 Elution Gradient Investigation

[0148] Based on Example 2, the effect of the elution gradient on impurity separation was investigated. The investigation process is as follows:

[0149] A method for detecting indobufen impurities comprises the following steps:

[0150] 1) Sample processing: same as in Example 2;

[0151] 2) High Performance Liquid Chromatography Detection: The sample solution in step 1) was measured and injected. The detection conditions were as follows:

[0152] Elution gradient: Example 8: Linear gradient elution was performed according to the following table:

[0153] Elution gradient: Example 9: Linear gradient elution was performed according to the following table:

[0154] The chromatographic column, mobile phase, detection wavelength, column temperature, flow rate and injection volume were the same as those in Example 2.

[0155] The results of the investigation are shown in Table 5:

[0156] Table 5. Results of gradient adjustment ratio investigation for indobufen impurity detection

[0157] The results showed that when the mobile phase A gradient changed from 60-62% to 55-62%, impurities 1, 4, and 5 were eluted and separated; when the mobile phase A gradient changed from 55-62% to 40-45%, impurity 2 was eluted and separated; and when the mobile phase A gradient changed from 40-45% to 20-37%, impurities 8, 3, 7, and 6 were eluted and separated. Those skilled in the art can appropriately change the gradient setting according to the linear slope of the mobile phase gradient change process to achieve the purpose of simultaneously separating and detecting multiple impurities.

[0158] Example 10: Detection of impurities in indobufen bulk drug

[0159] A method for detecting indobufen impurities comprises the following steps:

[0160] 1) Sample preparation: Take an appropriate amount of indobufen API, dissolve it in acetonitrile-water (50:50) solvent and dilute it to make a solution containing approximately 0.5 mg per 1 ml, which is used as the test solution.

[0161] 2) HPLC detection: same as in Example 1.

[0162] The test results are shown in Table 6:

[0163] Table 6. Impurity test results of indobufen API

[0164] The test results showed that the content of impurity 8 in the homemade indobufen raw material was 0.010%, other single impurities were 0.032%, and total impurities were 0.10%, which met the quality standards of the raw material.

[0165] Comparative Example 1: Reference CN114594168A impurity detection method

[0166] A method for detecting indobufen impurities comprises the following steps:

[0167] 1) Sample Preparation: Take appropriate amounts of indobufen, impurity 2, impurity 3, impurity 6, impurity 7, and impurity 8 reference substances, dissolve and dilute them with acetonitrile-water (50:50) solvent to make a solution containing approximately 0.5 mg of indobufen and 0.75 μg each of impurity 2, impurity 3, impurity 6, impurity 7, and impurity 8 per 1 ml, which serves as the system suitability solution.

[0168] 2) High Performance Liquid Chromatography Detection: The sample solution in step 1) was measured and injected. The detection conditions were as follows:

[0169] Chromatographic column: Waters XTerra RP18 4.6mm×250mm, 3.5μm or equivalent performance column

[0170] Mobile phase: Use phosphate buffer (weigh 1.36 g potassium dihydrogen phosphate, dissolve in 1000 ml of water, and adjust the pH to 4.0 with phosphoric acid) as mobile phase A and acetonitrile as mobile phase B. Perform gradient elution as shown in the table below:

[0171] Detection wavelength: 228nm;

[0172] Column temperature: 30°C;

[0173] Flow rate: 1.0 ml / min;

[0174] Injection volume: 10 μl.

[0175] The test results are shown in Figure 3 and Table 7:

[0176] Table 7. Test results of impurity detection method according to CN114594168A

[0177] The test results showed that indobufen, impurity 2, impurity 8, impurity 6 (impurity 3 and impurity 6 remained the same), and impurity 7 appeared in sequence, and the separation degree between the main peak and impurity 2 was >1.5, which met the separation degree requirements. However, impurity 3 and impurity 6 appeared together, making it difficult to effectively separate and detect them.

[0178] Comparative Example 2: Investigation of chromatographic column types

[0179] Based on the impurity detection method in CN114594168A, the effect of chromatographic column type on impurity separation was investigated using a column filled with amidohexadecylsilane bonded silica gel. The investigation process is as follows:

[0180] A method for detecting indobufen impurities comprises the following steps:

[0181] 1) Sample treatment: same as Comparative Example 1.

[0182] 2) High Performance Liquid Chromatography Detection: The sample solution in step 1) was measured and injected. The detection conditions were as follows:

[0183] Chromatographic column: ZORBAX Bonus-RP 4.6 mm × 250 mm, 3.5 μm or equivalent performance column;

[0184] Mobile phase, elution gradient, detection wavelength, column temperature, flow rate, injection volume: the same as Comparative Example 1.

[0185] The results of the investigation are shown in Figure 4 and Table 8:

[0186] Table 8. Results of the investigation of chromatographic column types based on the impurity detection method in CN114594168A

[0187] The results of the investigation showed that impurities 1, 4, 5, indobufen, 2, 8, 3, 7 and 6 appeared in sequence, and the separation degree between impurity 3 and 6 was >1.5, which met the separation degree requirements. However, the main peak almost overlapped with impurity 2, making it difficult to effectively separate and detect them.

[0188] As mentioned above, impurity 7 is not disclosed in the prior art and is an unknown impurity with a completely new structure. Therefore, the present invention also discloses a method for synthesizing impurity 7 and the related structural confirmation.

[0189] The synthetic route of impurity 7 is as follows:

[0190] Specific operation of the synthetic route of impurity 7:

[0191] 5.01 g of indobufen was added to 30 ml of isopropyl alcohol (IPA) and 2 ml of concentrated sulfuric acid, and the mixture was reacted at 55°C. 2 ml of concentrated sulfuric acid and 30 ml of isopropyl alcohol were added, and the temperature was raised to 85°C for 4 h to terminate the reaction. The reaction solution was cooled and crystallized, filtered, and the filter cake was taken. 100 g of 3% sodium hydroxide solution was added, stirred, filtered, and dried to obtain 4.37 g of a crude product.

[0192] Take 1.16g of the crude product, 0.54g of sodium hydroxide, 10ml of water, and 10ml of dichloromethane, add them to a reaction vessel, stir to dissolve, let stand and separate the layers, separate the dichloromethane layer, add 10ml of dichloromethane to extract the aqueous layer, combine the dichloromethane layers, add 10ml of water to wash, and concentrate the dichloromethane layer to obtain a white solid. Take it out and continue to dry to obtain 0.93g of impurity 7.

[0193] In the above-mentioned synthesis method, indobufen can be purchased commercially or prepared by existing technologies (such as CN106631974B, PL172906B1, CN101914055A, CN104744339A, etc., all of which are conventional processes).

[0194] The spectra related to the structure confirmation of impurity 7 are shown in Figures 5, 6 and 7.

[0195] Among them, Figure 5 is the high-resolution mass spectrometry (MS) spectrum of impurity 7, and the analysis results are:

[0196] Figure 6 is the H-NMR spectrum of impurity 7, and the analysis results are:

[0197] 1 The H NMR spectrum shows 13 groups of proton peaks, corresponding to a total of 23 hydrogen protons in the structure, which is consistent with the structure of impurity 7. Figure 7 is the carbon nuclear magnetic resonance (C-NMR) spectrum of impurity 7, and the analysis results are:

[0198] 13 There are 19 groups of carbon signals in the C NMR spectrum, corresponding to 21 carbon atoms in the structure, which is consistent with the structure of impurity 7.

Claims

1. A method for detecting indobufen impurities, using high performance liquid chromatography, characterized in that: In the high performance liquid chromatography method, the chromatographic column uses amidoalkylsilane bonded silica gel as a filler; the mobile phase A uses a phosphate buffer solution, and the mobile phase B uses a mixed solvent containing tetrahydrofuran.

2. The method for detecting indobufen impurities according to claim 1, characterized in that: The chromatographic column is an amido hexadecylsilane bonded silica gel column or an amido octadecylsilane bonded silica gel column; preferably, the chromatographic column is ZORBAX Bonus-RP 4.6mm×250mm, 3.5μm, ZORBAX Bonus-RP 4.6mm×150mm, 3.5μm, or RP 4.6×150mm, 5μm or equivalent performance column.

3. The method for detecting indobufen impurities according to claim 1, characterized in that: The mobile phase B further comprises one or more of methanol and acetonitrile; preferably, the mobile phase B is methanol-acetonitrile-tetrahydrofuran, methanol-tetrahydrofuran or acetonitrile-tetrahydrofuran.

4. The method for detecting indobufen impurities according to claim 1, characterized in that: The content of tetrahydrofuran in the mobile phase B is 15-40%; preferably, the content of tetrahydrofuran in the mobile phase B is 35-40%.

5. The method for detecting indobufen impurities according to claim 1, characterized in that: The mobile phase A includes, but is not limited to, one or more of a potassium dihydrogen phosphate buffer solution, a sodium dihydrogen phosphate buffer solution, and an ammonium dihydrogen phosphate buffer solution.

6. The method for detecting indobufen impurities according to claim 1, characterized in that: The pH of the mobile phase A is 2-4; preferably, the pH of the mobile phase A is 2.5-3.2; more preferably, the pH of the mobile phase A is 2.8-3.

0.

7. The method for detecting indobufen impurities according to claim 1, characterized in that: The mobile phase adopts gradient elution, including the following gradient: ; More preferably, comprising the following gradient:

8. The method for detecting indobufen impurities according to claim 1, characterized in that: The column temperature is 30-50°C; preferably, the column temperature is 40-50°C.

9. The method for detecting indobufen impurities according to any one of claims 1 to 8, characterized in that: The indobufen impurity is selected from One or more of .

10. Use of the indobufen impurity detection method according to any one of claims 1 to 9 in the quality control of indobufen bulk drug or preparation.

11. An indobufen impurity, which is a compound having the following structural formula:

12. The indobufen impurity according to claim 11, characterized in that The invention comprises the following preparation method: the indobufen is obtained by esterification reaction of indobufen and isopropanol.

13. The use of the indobufen impurity in the medical field according to claim 11, characterized in that: Including but not limited to use in impurity detection, quality control of indobufen API or preparation.

14. The use according to claim 13, characterized in that Used for detecting impurities in indobufen API or preparations; preferably, high performance liquid chromatography is used to detect indobufen impurities; Alternatively, it can be used as a standard or reference substance for indobufen impurity 7, for quality control of indobufen raw materials or preparations, or for the establishment of quality standards.

15. A method for detecting the indobufen impurity 7 as claimed in claim 11, using high performance liquid chromatography, characterized in that: In the high performance liquid chromatography method, the chromatographic column uses amidoalkylsilane bonded silica gel as a filler; the mobile phase A uses a phosphate buffer solution, and the mobile phase B uses a mixed solvent containing tetrahydrofuran.

16. The detection method according to claim 15, characterized in that: The mobile phase adopts gradient elution, including the following gradient: ; More preferably, comprising the following gradient:

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