Method for detecting related substances of robericoxib injection

Abstract

The invention discloses a method for detecting related substances in robericoxib injection, and relates to the technical field of medicine detection. A high performance liquid chromatography (HPLC) is adopted, cyanopropyl silane bonded silica gel is used as a chromatographic column of a filler, a 0.04%-0.06% trifluoroacetic acid solution is used as a mobile phase A, methyl alcohol is used as a mobile phase B, linear gradient elution is carried out, and the flow velocity of the mobile phases is 0.9-1.1 ml / min; the column temperature of the chromatographic column is 28-32 DEG C; the detection method can be used for effectively separating and determining the related substances of the robericoxib injection, under a prepared chromatographic condition, each impurity peak is completely separated, the linear relationship between the concentration and the peak area in a limit range is good (r is greater than or equal to 0.999), and the recovery rate is 90.0-110.0%. The analysis method is easy and convenient to operate, good in specificity, high in accuracy, stable, reliable and suitable for determination of related substances of the robericoxib injection.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a method for detecting related substances of Robenacoxib injection. Background Art

[0002] Robenacoxib, as a highly effective nonsteroidal anti-inflammatory drug (NSAIDs), has been proven in many foreign studies to be effective in controlling pain and inflammatory responses after surgery in cats. Robenacoxib injection (Onsiorinjection) was approved for marketing in Europe in December 2008, and was approved for marketing by the FDA in August 2011, May 2015, and May 2016. It is also available in Australia, South Africa, and other countries. The structural formula of Robenacoxib is as follows:

[0003]

[0004] After searching, it was found that there are literature reports on the determination of the content of Robenacoxib in preparations in China, but no method for detecting related substances of Robenacoxib Injection was found. Therefore, a method for detecting related substances of Robenacoxib Injection was developed, which is easy to operate, accurate in quantitative determination, strong in specificity and high in sensitivity, and is of great significance to the quality control of Robenacoxib Injection. Summary of the invention

[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to fill the gaps in the prior art and provide a method for determining related substances in Robenacoxib injection by reversed-phase high performance liquid chromatography and its application.

[0006] The technical solution of the present invention is as follows:

[0007] In the first aspect of the present invention, a method for determining related substances of Robenacoxib injection by reverse phase high performance liquid chromatography is provided;

[0008] The related substances are the degradation product cyclic impurity (with the structural formula shown in the following formula (I)), impurity R9 (with the structural formula shown in the following formula (II)) and other related substances;

[0009]

[0010] The method comprises the following steps: taking an appropriate amount of Robenacoxib injection, accurately weighing it, dissolving it with 50% (v / v, the same below, no further description) methanol and quantitatively diluting it to prepare a solution containing 0.8-1.2 mg of Robenacoxib per 1 ml as a test solution; accurately measuring the test solution according to the dilution ratio of 1→100, placing it in a volumetric bottle, diluting it to the scale with 50% methanol, shaking it well, and preparing it as a control solution; injecting the test solution and the control solution into a high performance liquid chromatograph, and using an ultraviolet detector as a detector to record a chromatogram; if there is an impurity peak in the chromatogram of the test solution, calculate it according to the following formula:

[0011]

[0012] Total impurity content (%) = the sum of the content of ring-closing impurities and other impurities

[0013] The chromatographic conditions were as follows:

[0014] Chromatographic column: A chromatographic column using cyanopropylsilane bonded silica gel as a filler;

[0015] Mobile phase: 0.04% to 0.06% (v / v) trifluoroacetic acid solution as mobile phase A, methanol as mobile phase B, linear gradient elution;

[0016] The gradient elution conditions are as follows:

[0017] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 65 35 8 53 47 28 53 47 45 20 80 50 20 80 51 65 35 58 65 35

[0018] Furthermore, the mobile phase A is 0.05% (v / v) trifluoroacetic acid solution.

[0019] Furthermore, the flow rate of the mobile phase is 0.9-1.1 ml / min, preferably 1.0 ml / min.

[0020] Furthermore, the temperature of the chromatographic column is 28-32°C, preferably 30°C.

[0021] Furthermore, the method further comprises a detection wavelength, and the detection wavelength is 245-255 nm, preferably 250 nm.

[0022] Furthermore, the method also includes an injection volume, which is 5 to 20 μl, preferably 10 μl.

[0023] Furthermore, in the method, the preparation method of each solution is as follows:

[0024] Test solution: Take an appropriate amount of Robenacoxib injection, accurately weigh it, and dilute it with 50% methanol to prepare a sample solution containing 1 mg of Robenacoxib per 1 ml.

[0025] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with 50% methanol, shake well, and use it as the control solution.

[0026] Furthermore, in a preferred embodiment of the present invention,

[0027] The HPLC conditions are as follows:

[0028] Chromatographic column: a chromatographic column filled with cyanopropylsilane bonded silica gel (250 mm × 4.6 mm, 3 μm);

[0029] Mobile phase A: 0.05% (v / v) trifluoroacetic acid solution; Mobile phase B: methanol;

[0030] Flow rate: 1.0ml / min;

[0031] Column temperature: 30°C;

[0032] Detection wavelength: 250nm;

[0033] The gradient elution program is as follows:

[0034] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 65 35 8 53 47 28 53 47 45 20 80 50 20 80 51 65 35 58 65 35

[0035] In the second aspect of the present invention, the use of the method for determining the related substances of Robenacoxib injection by reversed-phase high performance liquid chromatography as described in the first aspect in controlling the quality of Robenacoxib injection is provided, and the method is used to separate and determine the related substances of Robenacoxib injection. The related substances are degradation product cyclic impurities and other related substances.

[0036] Compared with the prior art, the method of the present invention has the following advantages and effects:

[0037] 1) The present invention adopts reversed-phase high performance liquid chromatography to determine the related substances of Robenacoxib injection, and can accurately determine the degradation product cyclized impurities and other related substances of Robenacoxib. Under the chromatographic conditions provided by the present invention, the main peak and the adjacent impurity peaks, and the adjacent impurity peaks are completely separated, and the linear relationship between the concentration and the peak area is good within the limit (r≥0.999), and the recovery rate is between 90.0% and 110.0%. The analytical method is simple to operate, has good specificity, high accuracy, stability and reliability, and is suitable for the determination of related substances of Robenacoxib injection.

[0038] 2) The method of the present invention is applicable to the determination of related substances of Robenacoxib Injection within the limits of the quality standard, ensuring the quality control of Robenacoxib Injection. The limits of the quality standard are: the ring impurities shall not exceed 1.0%, the other single impurities shall not exceed 1.0%, and the total amount of impurities shall not exceed 5.0%. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 is the chromatogram of the control solution in Example 1;

[0040] Figure 2 is the chromatogram of the test solution in Example 1;

[0041] Figure 3 is the chromatogram of the acid-destroyed test solution in Example 3;

[0042] Figure 4 is the chromatogram of the alkali-destroyed test solution in Example 3;

[0043] Figure 5 is the chromatogram of the oxidative destruction of the test solution in Example 3;

[0044] Figure 6 is a chromatogram of the light-damaged test solution in Example 3;

[0045] Figure 7 is a chromatogram of the test solution destroyed by high temperature in Example 3;

[0046] Figure 8 is a chromatogram of the light-damaged test solution in Comparative Example 1;

[0047] Fig. 9 It is the chromatogram of the light-damaged test solution in Comparative Example 2. DETAILED DESCRIPTION

[0048] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0049] Instruments and reagents

[0050] (1) Instruments: Waters Arc 2998 high performance liquid chromatograph; XSR205DU Mettler Toledo analytical balance; Raman PHSJ-3F pH meter.

[0051] (2) Reagents: Methanol was of chromatographic grade, trifluoroacetic acid was of analytical grade, and water was of homemade Milli-Q grade.

[0052] (3) Reference substances: cyclic impurity, content: 99.0%, batch number: LB-P5-7-20230804, source: Wuhan Huisheng Technology Co., Ltd.; impurity R9, content: 98.2%, batch number: impurity R9-20240913, source: Wuhan Huisheng Technology Co., Ltd.; Robenacoxib, content: 99.5%, batch number: LB-PS-17-20230814, source: Wuhan Huisheng Technology Co., Ltd.

[0053] (4) Test sample: Robenacoxib injection, source: Wuhan Huisheng Technology Co., Ltd., specification: 20 ml: 0.4 g.

[0054] (5) Blank auxiliary materials: provided by Wuhan Huisheng Technology Co., Ltd.

[0055] Example 1: Analysis method establishment

[0056] 1. Solution preparation

[0057] (1) Blank solution / diluent: 50% methanol.

[0058] (2) Blank excipient solution: Take about 490 mg of blank excipient, accurately weigh it, place it in a 10 ml volumetric flask, add 50% methanol to dilute to the scale, shake well, and obtain the solution.

[0059] (3) Test solution: Take about 0.5 g of the test sample (containing about 10 mg of robenacoxib), accurately weigh it, place it in a 10 ml volumetric flask, dilute it to the mark with 50% methanol, and shake well to prepare the test solution.

[0060] (4) Control solution: Accurately measure 1 ml of the test solution and place it in a 100 ml volumetric flask. Dilute to the mark with 50% methanol and shake well. This is used as the control solution.

[0061] 2. Chromatographic conditions

[0062] Column: Ultimate XB-CN, 250 mm × 4.6 mm, 3 μm;

[0063] Mobile phase A: 0.05% (v / v) trifluoroacetic acid solution; Mobile phase B: methanol;

[0064] Flow rate: 1.0ml / min;

[0065] Column temperature: 30°C;

[0066] Detection wavelength: 250nm;

[0067] The gradient elution program is as follows:

[0068]

[0069]

[0070] 3. Determination method

[0071] Take 10 μl of each of the control solution and the test solution, inject them into the high performance liquid chromatograph, use the above chromatographic conditions to determine the related substances of Robenacoxib in Robenacoxib injection, and record the chromatogram. Use the main component self-reference method with correction factors to calculate the content of each impurity, among which the cyclic impurities shall not exceed 1.0%, other impurities shall not exceed 1.0%, and the total impurities shall not exceed 5.0%.

[0072] 4. Measurement results

[0073] The chromatogram of the control solution is as follows Figure 1 The chromatogram of the test solution is shown in Figure 2 As shown. Figure 1-2 It can be seen that Robenacoxib and adjacent impurities, and adjacent impurities are well separated without interference, and the method of the present invention can be used to determine the content of related substances in Robenacoxib injection.

[0074] Next, the detection method of the related substances of the Robenacoxib injection of the present invention is investigated.

[0075] Example 2: System suitability test

[0076] Take 10 μl of the control solution in Example 1 and inject it into the high performance liquid chromatograph. According to the chromatographic conditions in Example 1, inject the sample 5 times continuously, record the peak area and calculate the RSD value. The results of the system suitability test are shown in Table 1.

[0077] Table 1 Summary of system suitability test results

[0078]

[0079]

[0080] As can be seen from Table 1, the RSD of the main peak area in the 5-pronged reference solution was 0.3%, which was less than 10.0%, and the system suitability met the requirements.

[0081] Example 3: Specificity test

[0082] Acid-destroyed test sample solution: Take about 0.5 g of the test sample, place it in a 10 ml volumetric flask, add 1 ml of 1 mol / L hydrochloric acid solution, shake for 1 minute, leave it at room temperature for 3 days, add 1 ml of 1 mol / L sodium hydroxide solution, neutralize to neutrality, dissolve it with 50% methanol and dilute to the scale, shake well, and use it as the acid-destroyed test sample solution.

[0083] Alkali-destroyed test solution: Take about 0.5 g of the test sample, place it in a 10 ml volumetric flask, add 1 ml of 1 mol / L sodium hydroxide solution, shake for 1 minute, leave it at room temperature for 10 days, add 1 ml of 1 mol / L hydrochloric acid solution, neutralize to neutrality, dissolve it with 50% methanol and dilute to the scale, shake well, and use it as the alkali-destroyed test solution.

[0084] Oxidative damage test sample solution: Take about 0.5g of the test sample, place it in a 10ml volumetric flask, add 1ml of 3% hydrogen peroxide solution, shake for 1 minute, leave it at room temperature for 10 days, dissolve it in 50% methanol and dilute to the scale, shake well, and use it as the oxidative damage test sample solution.

[0085] Light damage to the test solution: Take about 0.5g of the test sample, place it in a 10ml transparent volumetric bottle, and place it in a light test box (5000lx, 200μw / cm 2 ) for 10 days, then dissolve and dilute to scale with 50% methanol, shake well, and use as the light-destroyed test solution.

[0086] High temperature destruction test sample solution: Take about 0.5g of the test sample, put it in a 10ml volumetric flask, place it at 60℃ for 10 days, dissolve it in 50% methanol and dilute it to the scale, shake it well, and use it as the high temperature destruction test sample solution.

[0087] Take 10 μl of each of the above-mentioned destruction test sample solutions, inject them into a high performance liquid chromatograph, detect them according to the chromatographic conditions in Example 1, and record the chromatogram. The above research results are shown in Table 2, and the corresponding chromatograms are shown in Figure 3 to Figure 7 .

[0088] Table 2 Summary of Robenacoxib forced degradation results

[0089]

[0090] It can be seen from Table 2 that under each degradation condition, the main peak purity angle is less than the purity threshold, and the minimum separation between the main peak and the adjacent peak is greater than 1.5, and the detection method has good specificity.

[0091] Example 4: Linear range and correction factor

[0092] Take about 10 mg each of the Robenacoxib reference substance, the cyclic impurity reference substance and the impurity R9 reference substance, accurately weigh them, put them in the same 100 ml volumetric flask, add 50% methanol to dissolve and dilute to the scale, shake well, and use them as the mixed reference substance stock solution a.

[0093] Accurately pipette an appropriate amount of the mixed reference substance stock solution a to prepare a series of linear solutions of different concentrations. Take 10 μl of each linear solution of different concentrations, inject it into a high performance liquid chromatograph, detect it according to the chromatographic conditions in Example 1, and record the chromatogram. Perform linear regression analysis with concentration (μg / ml) as the abscissa and peak area as the ordinate to obtain the linear regression equations of the cyclic impurity, impurity R9 and Robenacoxib.

[0094] Table 3 Linear regression equations of Robenacoxib and cyclic impurities

[0095] Compound Name Linear range Linear equations Linear correlation coefficient Robenacoxib 0.50~20.24μg / ml y=21630x-908.07 0.999 Ring-closing impurities 0.52~20.46μg / ml y=30897x+1010.7 0.999 Impurity R9 0.48~19.26μg / ml y=21174x-908.07 0.999

[0096] According to the linear regression equation of the cyclic impurity, impurity R9 and Robenacoxib, the correction factor of the cyclic impurity f=21630 / 30897=0.7 and the correction factor of the impurity R9 f=21630 / 21174=1.0 can be obtained. Therefore, the impurity content of the cyclic impurity is calculated according to the self-control method with the correction factor added, and the impurity content of the impurity R9 is calculated according to the self-control method without the correction factor added. In practical applications, it is not necessary to reconfirm the correction factor each time.

[0097] Example 5: Limit of Detection and Limit of Quantitation

[0098] Quantitative limit solution: Take about 10 mg each of the robenacoxib reference substance, the cyclic impurity reference substance, and the impurity R9 reference substance, accurately weigh them, dissolve them in 50% methanol and gradually dilute them to a solution with a concentration of about 0.50 μg / ml, shake well, and obtain.

[0099] Detection limit solution: Accurately measure 5 ml of the quantitative limit solution, place it in a 10 ml volumetric flask, dilute to the scale with 50% methanol, shake well, and obtain.

[0100] 10 μl of the quantitative limit solution and the detection limit solution were taken and injected into a high performance liquid chromatograph, and the detection was performed according to the chromatographic conditions in Example 1, and the chromatogram was recorded. The quantitative limit results are shown in Table 4, and the detection limit results are shown in Table 5.

[0101] Table 4 Quantitation limit results

[0102] Components Concentration (μg / ml) Equivalent to the concentration of the test solution (%) Signal-to-Noise Ratio Ring-closing impurities 0.52 0.052 24.2 Impurity R9 0.48 0.048 13.4 Robenacoxib 0.50 0.050 11.8

[0103] Table 5 Detection limit results

[0104]

[0105]

[0106] As can be seen from Tables 4-5, the quantitative limits of the cyclic impurities, impurity R9 and Robenacoxib were 0.52 μg / ml, 0.48 μg / ml and 0.50 μg / ml, respectively, and the detection limits were 0.26 μg / ml, 0.24 μg / ml and 0.25 μg / ml, respectively, indicating that the detection method of the present invention has good sensitivity.

[0107] Example 6: Repeatability

[0108] Test solution: Take about 0.5 g of the test sample, accurately weigh it, place it in a 10 ml volumetric flask, dissolve it in 50% methanol and dilute it to the scale, shake well, and use it as the test solution.

[0109] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with 50% methanol, shake well, and use it as the control solution.

[0110] Six test solutions and control solutions were prepared in parallel, 10 μl of each was taken and injected into a high performance liquid chromatograph. The detection was performed according to the chromatographic conditions in Example 1. The content of the degradation product cyclic impurity was calculated by the peak area according to the main component self-control method with a correction factor, and the content of other impurities was calculated by the peak area according to the main component self-control method without a correction factor. The total impurity content was the sum of the cyclic impurity and other impurity contents. The calculation results are shown in Table 6.

[0111] Table 6 Repeatability results

[0112]

[0113] As can be seen from Table 6, the RSD of the cyclic impurity content in the 6 test solutions was 1.3%, the RSD of the impurity R9 content was 0.7%, and the RSD of the total impurity content was 0.6%, all of which were less than 10.0%, indicating that the method had good repeatability.

[0114] Example 7: Accuracy

[0115] Preparation of impurity stock solution: Take about 10 mg of the cyclic impurity reference substance and the impurity R9 reference substance respectively, accurately weigh them, put them in the same 100 ml volumetric flask, add 50% methanol to dissolve and dilute to the scale, shake well, and obtain.

[0116] Preparation of impurity reference solution: Pipette 1 ml of impurity stock solution into a 10 ml volumetric flask, dilute to the mark with 50% methanol, shake well, and obtain.

[0117] Preparation of accuracy background solution: Take about 0.5 g of the test sample of Robenacoxib injection, accurately weigh it in a 10 ml volumetric flask, dilute it to the scale with 50% methanol, shake well, and prepare 2 more in parallel using the same method.

[0118] Preparation of accuracy solution a: Take about 0.5 g of the test sample of Robenacoxib injection, accurately weigh it in a 10 ml volumetric flask, pipette 0.5 ml of the impurity stock solution into it, dilute to the scale with 50% methanol, shake well, and prepare 3 copies in parallel using the same method.

[0119] Preparation of accuracy solution b: Take about 0.5 g of the test sample of Robenacoxib injection, accurately weigh it in a 10 ml volumetric flask, pipette 1.0 ml of the impurity stock solution into it, dilute to the scale with 50% methanol, shake well, and prepare 3 more in parallel using the same method.

[0120] Preparation of accuracy solution c: Take about 0.5 g of the test sample of Robenacoxib injection, accurately weigh it in a 10 ml volumetric flask, pipette 1.5 ml of the impurity stock solution into it, dilute to the scale with 50% methanol, shake well, and prepare 3 copies in parallel using the same method.

[0121] Accurately pipette 10 μl of the above impurity reference solution and each accuracy solution, inject into the high performance liquid chromatograph, detect according to the chromatographic conditions in Example 1, calculate the impurity recovery rate in each accuracy solution, and the calculation results are shown in Table 7.

[0122] Table 7 Accuracy test results

[0123]

[0124]

[0125] As can be seen from Table 7, the recoveries of the cyclized impurities and impurity R9 in the solutions of each accuracy were all in the range of 90.0% to 110.0%, and the RSD of the 9 recoveries was less than 10.0%, so the accuracy of this method was good.

[0126] Example 8: Solution Stability

[0127] Test solution: Take about 0.5 g of Robenacoxib injection test sample, accurately weigh it in a 10 ml volumetric flask, dilute it to the scale with 50% methanol, shake well, and use it as the test solution.

[0128] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with 50% methanol, shake well, and use it as the control solution.

[0129] At 0 hours, 12 hours, 24 hours, 36 hours and 48 hours, 10 μl of the test solution and the control solution were accurately pipetted and injected into the high performance liquid chromatograph. The detection was carried out according to the chromatographic conditions in Example 1. The measured impurity content, the change value compared with the 0h content and the change value of the peak area of ​​the control solution were calculated. The calculation results are shown in Table 8.

[0130] Table 8 Solution stability

[0131]

[0132] As can be seen from Table 8, within 48 hours, the change value of the content of each impurity in the test solution compared with 0 hour was less than 10%, and no new impurities appeared; the change value of the peak area of ​​the control solution within 48 hours was less than 2%, indicating that the test solution and the control solution had good stability within 48 hours.

[0133] Example 9: Durability

[0134] In order to verify the degree to which the measurement results remain unaffected when slight changes occur in the method conditions, tests were conducted according to Table 9 to examine the durability of the chromatographic conditions of the method for detecting related substances.

[0135] Test solution: Take about 0.5 g of Robenacoxib injection test sample, accurately weigh it in a 10 ml volumetric flask, dilute it to the scale with 50% methanol, shake well, and use it as the test solution.

[0136] Control solution: Accurately measure 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the scale with 50% methanol, shake well, and use it as the control solution.

[0137] Accurately measure 10 μl of the test solution and the control solution respectively, inject them into the high performance liquid chromatograph, detect according to the chromatographic conditions in Example 1 and the chromatographic conditions in Table 9, record the chromatogram, and the test results are shown in Table 10.

[0138] Table 9 Durability of relevant substance inspection methods

[0139]

[0140] Table 10 Durability test results of relevant material inspection methods

[0141]

[0142]

[0143] It can be seen from Table 10 that the changes in trifluoroacetic acid concentration, column temperature, flow rate and detection wavelength within a certain range do not affect the detection of related substances, and there are no special requirements for the chromatographic column model, indicating that the method has good durability.

[0144] Comparative Example 1

[0145] The invention name is "A method for detecting the content of Robenacoxib" (application number is 202110852798.2), which uses high performance liquid chromatography to detect the content of Robenacoxib. This comparative example uses the chromatographic conditions to determine the related substances of Robenacoxib injection.

[0146] Light damage to the test solution: Take about 0.5g of the test sample, place it in a 10ml transparent volumetric bottle, and place it in a light test box (5000lx, 200μw / cm 2 ) for 10 days, then dissolve and dilute to scale with 50% methanol, shake well, and use as the light-destroyed test solution.

[0147] Accurately measure 10 μl of the light-damaged test sample solution, inject it into the liquid chromatograph, and detect it according to the chromatographic conditions in Table 11.

[0148] Table 11 Chromatographic conditions of Comparative Example 1

[0149]

[0150] The chromatogram of the light-destroyed test solution detected is as follows Figure 8 As shown in the figure, it can be seen that the main peak and the adjacent impurity peaks are not baseline separated, which affects the detection of Robenacoxib-related substances in the test solution.

[0151] Comparative Example 2

[0152] In this comparative example, a conventional C18 column was used to determine the related substances of Robenacoxib injection.

[0153] Light damage to the test solution: Take about 0.5g of the test sample, place it in a 10ml transparent volumetric bottle, and place it in a light test box (5000lx, 200μw / cm 2 ) for 10 days, then dissolve and dilute to scale with 50% methanol, shake well, and use as the light-destroyed test solution.

[0154] Accurately measure 10 μl of the light-damaged test solution, inject it into the liquid chromatograph, and perform the test according to the following chromatographic conditions:

[0155] Chromatographic column: Inertsil ODS-3 (4.6*250mm, 5μm);

[0156] Mobile phase A: 0.05% (v / v) trifluoroacetic acid solution; Mobile phase B: methanol;

[0157] Flow rate: 1.0ml / min;

[0158] Column temperature: 30°C;

[0159] Detection wavelength: 250nm;

[0160] The gradient elution program is as follows:

[0161] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 65 35 8 53 47 28 53 47 45 20 80 50 20 80 51 65 35 58 65 35

[0162] The chromatogram of the light-destroyed test solution is as follows: Fig. 9As shown. Fig. 9 It can be seen that there is no baseline separation between the main peak and the adjacent impurity peaks, and between the adjacent impurity peaks. Under this chromatographic condition, accurate detection of related substances in Robenacoxib Injection cannot be achieved.

[0163] The above are only some embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention.

Claims

1. A high performance liquid chromatography method for detecting related substances in Robenacoxib, characterized in that: The chromatographic conditions were as follows: Chromatographic column: cyanopropylsilane bonded silica gel as filler; Mobile phase: Use 0.04% to 0.06% trifluoroacetic acid solution as mobile phase A and methanol as mobile phase B. Perform linear gradient elution according to the following elution procedure: Mobile phase flow rate: 0.9~1.1ml / min; Chromatographic column temperature: 28~32℃; Detection wavelength: 245~255nm; Injection volume: 5~20μl.

2. The detection method according to claim 1, characterized in that: The chromatographic column is filled with cyanopropylsilane bonded silica gel and has a specification of 250mm×4.6mm×3μm; the mobile phase A is a trifluoroacetic acid solution with a volume percentage of 0.05%; the flow rate is 1.0ml / min; the column temperature of the chromatographic column is 30°C; the detection wavelength is 250nm; and the injection volume is 10μl.

3. The detection method according to claim 1 or 2, characterized in that: The related substances include degradation product ring-closed impurities and / or impurity R9.

4. The detection method according to claim 1, 2 or 3, characterized in that: The Robenacoxib drug is Robenacoxib injection.

5. The detection method according to claim 4, characterized in that: The detection method is used for qualitative and quantitative analysis of Robenacoxib and its related substances.

6. The detection method according to claim 5, characterized in that: The quantitative analysis further comprises the following steps: Take Robenacoxib injection, accurately weigh it, dissolve it in 50% by volume methanol and quantitatively dilute it to make a solution containing 0.8-1.2 mg of Robenacoxib per 1 ml as the test solution; According to the dilution ratio of 1→100, accurately measure the test solution, put it into a volumetric bottle, dilute it to the scale with 50% methanol by volume, shake well, and use it as the control solution; Inject the test solution and the reference solution into the HPLC, record the chromatogram, and calculate the content of each impurity by the main component self-reference method.

7. The detection method according to claim 6, characterized in that: The content of each impurity is calculated using the following formula: Total impurity content (%) = the sum of the contents of closed-ring impurities and other impurities.

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