A method for detecting indobufen impurities and its application

Abstract

The present invention discloses an indobufen impurity detection method, which adopts high performance liquid chromatography, wherein the chromatographic column adopts amidoalkylsilane bonded silica gel as a filler; the mobile phase A adopts a phosphate buffer solution, and the mobile phase B adopts a mixed solvent containing tetrahydrofuran, which achieves the technical effect of simultaneously separating and detecting impurities 2, impurity 3, impurity 6 and the like. Therefore, the indobufen impurity detection method provided by the present invention can be used for quality control in the research of indobufen raw materials or preparations.

Description

Technical Field

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

[0002] Indobufen is an isoindolinylphenylbutyric acid derivative, with the chemical name (±) 2-[4-(1-oxo-2-isoindolinyl)phenyl]butyric acid, and the structural formula is shown below. Indobufen is an antiplatelet aggregation drug developed by Farmitalia CarloErba (Pharmacia, now Pfizer), which can block platelet aggregation by reversibly inhibiting platelet cyclooxygenase and reducing the generation of thromboxane A2. It can be used to prevent and treat ischemic cardiovascular disease, ischemic cerebrovascular disease, venous thrombosis, etc. caused by arteriosclerosis.

[0003]

[0004] The synthesis process of indobufen involves multiple steps such as hydrogenation reaction, cyclization reaction, zinc powder reduction reaction, and crude product purification, which are prone to produce a variety of process impurities: hydrogenation reaction product (recorded as impurity 1), cyclization reaction product (recorded as impurity 2), zinc powder reduction reaction by-product (recorded as impurities 3 to 6) and crude product purification reaction by-product (7 to 8).

[0005]

[0006] 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. Research on Quality Control 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.

[0007] The prior art also discloses a variety of impurity detection methods: the national drug standard for indobufen (WS1-(X-069)-2006Z) discloses a detection method for related substances: using octadecylsilane bonded silica gel as a filler and methanol-water-triethylamine (65:35:0.5) (adjusting the pH value to 3.0 with phosphoric acid) as a mobile phase. However, this detection method is not suitable for the simultaneous separation and detection of multiple process impurities.

[0008] Patent CN114594168A discloses a method for detecting indobufen impurities, using octadecylsilane bonded silica gel as filler, potassium dihydrogen phosphate buffer (pH adjusted to 4.0 with phosphoric acid) as mobile phase A, and acetonitrile as mobile phase B for gradient elution. Four impurities can be separated and detected simultaneously: YDBF-IM-A (i.e., impurity 2), YDBF-IM-C, YDBF-IM-F (i.e., impurity 8), and YDBF-IM-I.

[0009] Patent CN114624339A discloses a method for detecting indobufen impurities, using octadecylsilane bonded silica gel as filler, potassium dihydrogen phosphate buffer (pH adjusted to 3.0 with phosphoric acid) as mobile phase A, and 0.1% acetonitrile phosphoric acid as mobile phase B, for gradient elution. Seven impurities can be separated and detected simultaneously: YDBF-IM-A (i.e. impurity 2), YDBF-IM-B (i.e. impurity 6), YDBF-IM-C, YDBF-IM-E, YDBF-IM-F (i.e. impurity 8), YDBF-IM-H, and YDBF-IM-I.

[0010] However, there is no prior art that discloses a separation and detection method for impurity 3 or impurity 7, nor does it provide relevant technical inspiration.

[0011] In summary, how to optimize the existing detection method of indobufen so as to achieve the simultaneous separation and detection of impurity 2 required by the national drug standard, impurity 3 and impurity 6 which are by-products easily produced by the zinc powder reduction reaction; further, how to simultaneously separate and detect impurities such as impurity 7 and impurity 8 which are by-products easily produced by the crude product purification reaction, is a technical problem that has not yet been solved by those skilled in the art. Summary of the invention

[0012] The technical problem to be solved by the present invention is to provide an indobufen impurity detection method, which can simultaneously separate and detect multiple impurities such as impurity 2, impurity 3, impurity 6, etc., for quality control of indobufen raw materials and preparations.

[0013] The above technical problems to be solved are achieved through the following technical solutions:

[0014] The invention provides an indobufen impurity detection method, which adopts a high performance liquid chromatography method, wherein a chromatographic column adopts amidoalkylsilane bonded silica gel as a filler; a mobile phase A adopts a phosphate buffer solution, and a mobile phase B adopts a mixed solvent containing tetrahydrofuran.

[0015] The present invention optimizes the chromatographic column:

[0016] Preferably, the chromatographic column is an amidohexadecylsilane bonded silica gel column or an amidooctadecylsilane bonded silica gel column.

[0017] More preferably, the chromatographic column is ZORBAX Bonus-RP 4.6mm×250mm, 3.5μm, ZORBAX Bonus-RP4.6mm×150mm, 3.5μm, or RP 4.6×150mm, 5μm or equivalent performance column.

[0018] Most preferably, the chromatography column is ZORBAX Bonus-RP 4.6 mm×250 mm, 3.5 μm.

[0019] The chromatographic column filler is amide alkylsilane bonded silica gel. The polar amide group embedded in the long alkyl chain is the key group for separating the decarboxylation 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.

[0020] The present invention is also optimized for mobile phase:

[0021] 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.

[0022] More preferably, mobile phase A is potassium dihydrogen phosphate buffer solution.

[0023] Preferably, the concentration of mobile phase A is 10 to 25 mmol.

[0024] Preferably, the pH of mobile phase A is 2-4.

[0025] More preferably, the pH of mobile phase A is 2.5 to 3.2.

[0026] Most preferably, the pH of mobile phase A is 2.8-3.0.

[0027] 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.

[0028] More preferably, mobile phase B is methanol-acetonitrile-tetrahydrofuran, methanol-tetrahydrofuran or acetonitrile-tetrahydrofuran.

[0029] Most preferably, mobile phase B is acetonitrile-tetrahydrofuran.

[0030] Preferably, the content of tetrahydrofuran in mobile phase B is 15-40%.

[0031] More preferably, the content of tetrahydrofuran in mobile phase B is 35-40%.

[0032] Adding tetrahydrofuran to mobile phase B to adjust the ability of the mobile phase to accept protons 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 needs of actual detection.

[0033] The present invention also optimizes the elution gradient of the mobile phase:

[0034] Preferably, the mobile phase adopts gradient elution, including the following gradient:

[0035]

[0036] More preferably, the gradient includes:

[0037]

[0038]

[0039] Most preferably, the gradient includes:

[0040]

[0041] The present invention also optimizes the column temperature:

[0042] Preferably, the column temperature is 30-50°C.

[0043] More preferably, the column temperature is 40-50°C.

[0044] Most preferably, the column temperature is 40°C.

[0045] The present invention is also optimized for the detection wavelength:

[0046] Preferably, the detection wavelength is 208-248 nm.

[0047] More preferably, the detection wavelength is 228 nm.

[0048] The present invention is also optimized for flow rate:

[0049] Preferably, the flow rate is 0.8-1.2 ml / min.

[0050] More preferably, the flow rate is 1.0 ml / min.

[0051] 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.

[0052] Specifically, it can be used to separate and detect all or part of the indobufen impurities in impurities 1 to 8.

[0053] The aforementioned indobufen impurities include but are not limited to

[0054] One or more of .

[0055] Preferably, the indobufen impurity detection method provided by the present invention can 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.

[0056] 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 and eight impurities must include impurities 2, impurity 3 and impurity 6). Examples include but are not limited to: when four impurities are separated at the same time, except for impurities 2, impurity 3 and impurity 6, the remaining one impurity can be selected from one of impurities 1, 4, 5, 7 and 8; similarly, when five impurities are separated at the same time, except for 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 at the same time, except for 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 at the same time, except for 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 at the same time, they are impurities 1 to 8.

[0057] Furthermore, as an internal quality control standard with higher requirements, it can also be implemented by separating impurity 2, impurity 3, impurity 6 and impurity 7 at the same time, or separating impurity 2, impurity 3, impurity 6, impurity 7 and impurity 8 at the same time.

[0058] Compared with the prior art, the indobufen impurity detection method provided by the present invention has the following beneficial effects:

[0059] 1) The indobufen impurity detection method provided by the present invention uses a chromatographic column with amide alkylsilane bonded silica gel as a filler, which improves the separation degree of impurity 3 and impurity 6 compared with the chromatographic column with octadecylsilane bonded silica gel as a filler used in CN114594168A and CN114624339A, so that the two can be separated and detected simultaneously.

[0060] 2) The indobufen impurity detection method provided by the present invention solves the problem that the main peak and impurity 2 are difficult to be effectively separated in the amidoalkylsilane bonded silica gel column by optimizing the mobile phase B to be a mixed solvent containing tetrahydrofuran.

[0061] 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.

[0062] 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.

[0063] 3) The indobufen impurity detection method provided by the present invention can simultaneously separate and detect indobufen impurity 2, impurity 3, impurity 6, impurity 7 and impurity 8 by optimizing the type of chromatographic column, the type of mobile phase B and gradient elution;

[0064] On this basis, by further optimizing the pH range of 2.5 to 3.2 (even pH 2.8 to 3.0) and other conditions, impurities such as impurity 1, impurity 4 and impurity 5 can be further separated and detected simultaneously, all of which can be used for the quality control of indobufen raw materials and preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 The test results of the suitability of the indobufen impurity detection system in Example 1;

[0066] Figure 2 The test results of the suitability of the indobufen impurity detection system in Example 2;

[0067] Figure 3 For comparative example 1, refer to the test results of the impurity detection method in CN114594168A;

[0068] Figure 4 This is the result of investigating the types of chromatographic columns based on the impurity detection method in CN114594168A in comparative example 2. DETAILED DESCRIPTION

[0069] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention, but the protection scope 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 protection scope of the present invention.

[0070] 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.

[0071] Reagents:

[0072] Indobufen reference substance (purity: 99.9%, manufacturer: China Food and Drug Inspection Institute)

[0073] Indobufen API (homemade or commercially available)

[0074] instrument:

[0075] High performance liquid chromatography (model: Ultimate 3000, manufacturer: Thermo Fisher )

[0076] Example 1

[0077] A method for detecting indobufen impurities comprises the following steps:

[0078] 1) Sample treatment: Take appropriate amount of indobufen, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, and impurity 8 reference substances, dissolve and dilute with acetonitrile-water (50:50) solvent to make 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 as the system suitability solution.

[0079] 2) High performance liquid chromatography detection: Take the sample solution in step 1) and inject it. The detection conditions are as follows:

[0080] Chromatographic column: ZORBAX Bonus-RP 4.6mm×250mm, 3.5μm or other column with equivalent performance

[0081] Mobile phase: Use phosphate buffer (weigh 1.36 g potassium dihydrogen phosphate, add 1000 ml water to dissolve, and adjust the pH value to 3.0 with phosphoric acid) as mobile phase A, acetonitrile-tetrahydrofuran (60:40) as mobile phase B, and perform linear gradient elution according to the following table:

[0082]

[0083] The detection wavelength is 228nm;

[0084] The column temperature was 40°C;

[0085] Flow rate: 1.0ml / min;

[0086] Injection volume: 10 μl.

[0087] Test results such as Figure 1 As shown in Table 1:

[0088] Table 1. Results of suitability test of indobufen impurity detection system

[0089] Components Retention time (min) Separation Impurity 1 6.685 / Impurity 4 18.036 49.41 Impurity 5 18.610 2.75 Indobufen 21.609 13.80 Impurity 2 22.946 5.17 Impurity 8 27.654 21.64 Impurity 3 29.337 9.05 Impurity 7 29.808 2.62 Impurity 6 31.360 8.58

[0090] 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.

[0091] Example 2

[0092] A method for detecting indobufen impurities comprises the following steps:

[0093] 1) Sample treatment: Take appropriate amount of indobufen, impurity 1, impurity 2, impurity 3, impurity 4, impurity 5, impurity 6, impurity 7, and impurity 8 reference substances, dissolve and dilute with acetonitrile-water (50:50) solvent to make 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 as the system suitability solution.

[0094] 2) High performance liquid chromatography detection: Take the sample solution in step 1) and inject it. The detection conditions are as follows:

[0095] Chromatographic column: ZORBAX Bonus-RP 4.6mm×250mm, 3.5μm or other column with equivalent performance

[0096] Mobile phase: Use phosphate buffer (weigh 1.36 g potassium dihydrogen phosphate, add 1000 ml water to dissolve, and adjust the pH value to 3.0 with phosphoric acid) as mobile phase A, acetonitrile-tetrahydrofuran (65:35) as mobile phase B, and perform linear gradient elution according to the following table:

[0097]

[0098] The detection wavelength is 228nm;

[0099] The column temperature was 40°C;

[0100] Flow rate: 1.0ml / min;

[0101] Injection volume: 10 μl.

[0102] Test results such as Figure 2 As shown in Table 2:

[0103] Table 2. Results of suitability test of indobufen impurity detection system

[0104]

[0105]

[0106] 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.

[0107] It should be noted that in the above-mentioned Examples 1 and 2, the pH value of mobile phase A can be adjusted to 2.8-3.0, or 2.5-3.2, etc., in addition to being adjusted to 3.0, without affecting the effective separation of impurities 1-8.

[0108] Example 3-5 Investigation of the type of mobile phase B

[0109] On the basis of Example 1, the influence of the types of mobile phase B on the separation of impurities was investigated: acetonitrile (Example 3), acetonitrile-methanol mixed solvent (Example 4), acetonitrile-methanol-tetrahydrofuran mixed solvent (Example 5), acetonitrile-tetrahydrofuran (Example 1). The investigation process is as follows:

[0110] A method for detecting indobufen impurities comprises the following steps:

[0111] 1) Sample processing: same as in Example 1;

[0112] 2) High performance liquid chromatography detection: Take the sample solution in step 1) and inject it. The detection conditions are as follows:

[0113] Mobile phase: Example 3: Phosphate buffer (weigh 1.36 g potassium dihydrogen phosphate, dissolve in 1000 ml of water, and adjust the pH value to 3.0 with phosphoric acid) is used as mobile phase A, and acetonitrile is used as mobile phase B;

[0114] Mobile phase: Example 4: Phosphate buffer (weigh 1.36 g potassium dihydrogen phosphate, dissolve in 1000 ml water, and adjust the pH value to 3.0 with phosphoric acid) is used as mobile phase A, and acetonitrile-methanol (90:10) is used as mobile phase B;

[0115] Mobile phase: Example 5: Phosphate buffer (weigh 1.36 g potassium dihydrogen phosphate, dissolve in 1000 ml water, and adjust the pH value to 3.0 with phosphoric acid) is used as mobile phase A, and acetonitrile-methanol-tetrahydrofuran (60:25:15) is used as mobile phase B;

[0116] The chromatographic column, elution gradient, detection wavelength, column temperature, flow rate and injection volume are the same as those in Example 1.

[0117] The results of the investigation are shown in Table 3:

[0118] Table 3. Results of investigation on mobile phase B types for indobufen impurity detection

[0119]

[0120]

[0121] 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.

[0122] Example 6-7 Investigation of the proportion of mobile phase B

[0123] On the basis of Example 1, the effect of the ratio of mobile phase B on the separation of impurities was investigated: acetonitrile-tetrahydrofuran (85:15) (Example 6), acetonitrile-tetrahydrofuran (75:25) (Example 7), acetonitrile-tetrahydrofuran (60:40) (Example 1). The investigation process is as follows:

[0124] A method for detecting indobufen impurities comprises the following steps:

[0125] 1) Sample processing: same as in Example 1;

[0126] 2) High performance liquid chromatography detection: Take the sample solution in step 1) and inject it. The detection conditions are as follows:

[0127] Mobile phase: Example 6: Phosphate buffer (weigh 1.36 g potassium dihydrogen phosphate, add 1000 ml of water to dissolve, and adjust the pH value to 3.0 with phosphoric acid) is used as mobile phase A, and acetonitrile-tetrahydrofuran (85:15) is used as mobile phase B;

[0128] Mobile phase: Example 7: Phosphate buffer (1.36 g potassium dihydrogen phosphate was weighed, dissolved in 1000 ml of water, and the pH value 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;

[0129] The chromatographic column, elution gradient, detection wavelength, column temperature, flow rate and injection volume are the same as those in Example 1.

[0130] The results of the investigation are shown in Table 4:

[0131] Table 4. Results of the investigation on the proportion of mobile phase B for indobufen impurity detection

[0132]

[0133]

[0134] The results of the investigation show that when the content of tetrahydrofuran in mobile phase B is 15-40%, the separation degree between the main peak and impurity 2 meets the requirements, and the baseline separation effect can be further improved.

[0135] Example 8-9 Elution Gradient Investigation

[0136] Based on Example 2, the effect of elution gradient on impurity separation was investigated. The investigation process is as follows:

[0137] A method for detecting indobufen impurities comprises the following steps:

[0138] 1) Sample processing: same as in Example 2;

[0139] 2) High performance liquid chromatography detection: Take the sample solution in step 1) and inject it. The detection conditions are as follows:

[0140] Elution gradient: Example 8: Linear gradient elution was performed according to the following table:

[0141]

[0142] Elution gradient: Example 9: Linear gradient elution was performed according to the following table:

[0143]

[0144] The chromatographic column, mobile phase, detection wavelength, column temperature, flow rate and injection volume are the same as those in Example 2.

[0145] The results of the investigation are shown in Table 5:

[0146] Table 5. Results of the gradient adjustment ratio test for indobufen impurities

[0147]

[0148] The results of the investigation show that in the process of gradient change of mobile phase A from 60-62% to 55-62%, impurities 1, 4 and 5 are eluted and separated; in the process of gradient change of mobile phase A from 55-62% to 40-45%, impurity 2 is eluted and separated; in the process of gradient change of mobile phase A from 40-45% to 20-37%, impurities 8, 3, 7 and 6 are eluted and separated. Those skilled in the art can appropriately change the setting of the gradient according to the linear slope of the mobile phase gradient change process to achieve the purpose of separating and detecting multiple impurities at the same time.

[0149] Example 10: Detection of impurities in indobufen bulk drug

[0150] A method for detecting indobufen impurities comprises the following steps:

[0151] 1) Sample treatment: Take an appropriate amount of indobufen raw material, dissolve it in acetonitrile-water (50:50) solvent and dilute it to make a solution containing about 0.5 mg per 1 ml as the test solution.

[0152] 2) HPLC detection: Same as Example 1.

[0153] The test results are shown in Table 6:

[0154] Table 6. Indobufen API impurity test results

[0155]

[0156]

[0157] 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 raw materials.

[0158] Comparative Example 1: Reference to CN114594168A impurity detection method

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

[0160] 1) Sample treatment: Take appropriate amount of indobufen, impurity 2, impurity 3, impurity 6, impurity 7, and impurity 8 reference substances, dissolve and dilute 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 as the system suitability solution.

[0161] 2) High performance liquid chromatography detection: Take the sample solution in step 1) and inject it. The detection conditions are as follows:

[0162] Chromatographic column: Waters XTerra RP18 4.6mm×250mm, 3.5μm or equivalent

[0163] Mobile phase: Use phosphate buffer (weigh 1.36 g potassium dihydrogen phosphate, add 1000 ml water to dissolve, and adjust the pH value to 4.0 with phosphoric acid) as mobile phase A and acetonitrile as mobile phase B. Perform gradient elution according to the following table:

[0164]

[0165] Detection wavelength: 228nm;

[0166] Column temperature: 30°C;

[0167] Flow rate: 1.0ml / min;

[0168] Injection volume: 10 μl.

[0169] Test results such as Figure 3 As shown in Table 7:

[0170] Table 7. Test results of impurity detection method according to CN114594168A

[0171]

[0172] The test results show that indobufen, impurity 2, impurity 8, impurity 6 (impurity 3 and impurity 6 are retained as the same), and impurity 7 appear in sequence, and the separation degree between the main peak and impurity 2 is >1.5, which meets the separation degree requirements. However, impurity 3 and impurity 6 appear together, making it difficult to effectively separate and detect them.

[0173] Comparative Example 2: Investigation of chromatographic column types

[0174] Based on the impurity detection method of CN114594168A, the influence of chromatographic column type on impurity separation was investigated: chromatographic column with amido hexadecylsilane bonded silica gel as filler. The investigation process is as follows:

[0175] A method for detecting indobufen impurities comprises the following steps:

[0176] 1) Sample treatment: same as Comparative Example 1.

[0177] 2) High performance liquid chromatography detection: Take the sample solution in step 1) and inject it. The detection conditions are as follows:

[0178] Chromatographic column: ZORBAX Bonus-RP 4.6mm×250mm, 3.5μm or equivalent column;

[0179] Mobile phase, elution gradient, detection wavelength, column temperature, flow rate, injection volume: same as Comparative Example 1.

[0180] The results of the investigation are as follows Figure 4 As shown in Table 8:

[0181] Table 8. Results of investigation on chromatographic column types based on the impurity detection method of CN114594168A

[0182]

[0183]

[0184] 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.

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; The indobufen impurities include 2. The method for detecting indobufen impurities according to claim 1, characterized in that: The chromatographic column is an amide hexadecylsilane bonded silica gel column or an amide octadecylsilane bonded silica gel column.

3. The method for detecting indobufen impurities according to claim 2, characterized in that: The chromatographic column is ZORBAX Bonus-RP 4.6mm×250mm, 3.5μm, or ZORBAX Bonus-RP 4.6mm×150mm, 3.5μm, or RP 4.6×150mm, 5μm.

4. 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.

5. The method for detecting indobufen impurities according to claim 4, characterized in that: The mobile phase B is methanol-acetonitrile-tetrahydrofuran, methanol-tetrahydrofuran or acetonitrile-tetrahydrofuran.

6. 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%.

7. The method for detecting indobufen impurities according to claim 6, characterized in that: The content of tetrahydrofuran in the mobile phase B is 35-40%.

8. The method for detecting indobufen impurities according to claim 1, characterized in that: The mobile phase A is one or more of potassium dihydrogen phosphate buffer solution, sodium dihydrogen phosphate buffer solution, and ammonium dihydrogen phosphate buffer solution.

9. The method for detecting indobufen impurities according to claim 1, characterized in that: The concentration of the mobile phase A is 10-25 mmol.

10. The method for detecting indobufen impurities according to claim 1, characterized in that: The pH of the mobile phase A is 2-4.

11. The method for detecting indobufen impurities according to claim 10, characterized in that: The pH of the mobile phase A is 2.5-3.

2.

12. The method for detecting indobufen impurities according to claim 11, characterized in that: The pH of the mobile phase A is 2.8-3.

0.

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

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

15. The method for detecting indobufen impurities according to claim 1, characterized in that: The high performance liquid chromatography method also includes using a column temperature of 30 to 50°C.

16. The method for detecting indobufen impurities according to claim 15, characterized in that: The column temperature is 40-50°C.

17. The method for detecting indobufen impurities according to claim 1, characterized in that: The high performance liquid chromatography method also includes using a detection wavelength of 208 to 248 nm.

18. The method for detecting indobufen impurities according to claim 1, characterized in that: The high performance liquid chromatography method also includes using a flow rate of 0.8 to 1.2 ml / min.

19. The method for detecting indobufen impurities according to any one of claims 1 to 18, characterized in that: The indobufen impurities are also selected from One or more of .

20. Use of the indobufen impurity detection method according to any one of claims 1 to 19 in the quality control of indobufen bulk drug or preparation.

21. The use according to claim 20, characterized in that The indobufen impurity detection method is used to simultaneously separate impurity 3 and impurity 6, or to simultaneously separate impurity 2, impurity 3 and impurity 6.

22. The use according to claim 20, characterized in that The indobufen impurity detection method is used to simultaneously separate at least four of the eight impurities 1 to 8, and the three impurities must be impurity 2, impurity 3 and impurity 6.

23. The use according to claim 21 or 22, characterized in that The indobufen impurity detection method adopts high performance liquid chromatography, and its chromatographic conditions are: The chromatographic column was ZORBAX Bonus-RP 4.6 mm × 250 mm, 3.5 μm; Mobile phase A was potassium dihydrogen phosphate buffer solution at pH 3.0; Mobile phase B is a mixed solvent of acetonitrile and tetrahydrofuran; The column temperature was 40°C; The detection wavelength is 228nm; Flow rate: 1.0 ml / min; Gradient elution using the following gradient: or

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