Determination method for related substances in diclofenac diethylamine latex

Abstract

The invention provides a method for determining related substances in a diclofenac diethylamine latex agent. The method comprises the following steps: 1, preparing a diclofenac diethylamine test solution, a contrast solution, a sensitivity solution and a system applicability solution; 2, adopting a high performance liquid chromatography under the chromatographic conditions that a chromatographic column is an octadecyl silane bonded silica gel chromatographic column, a methanol-phosphate buffer solution is used as a mobile phase A, methanol is used as a mobile phase B, the detection wavelength is 260nm, gradient elution is carried out, and the process is as follows: the volume ratio of the mobile phase A to the mobile phase B is 100: 0 within 0-35min; within 35-65 min, the volume ratio of the mobile phase A to the mobile phase B is gradually changed to 8: 92 at a constant speed; 65-80 min, the volume ratio of the mobile phase A to the mobile phase B gradually changes to 100: 0 at a constant speed. By adopting the high performance liquid chromatography and the gradient elution mode, the separation degree and the detection sensitivity are remarkably improved, and a main peak and various degradation impurities and process impurities can be effectively separated in a wider time range.

Description

Technical Field

[0001] The invention relates to the technical field of chemical drug analysis, in particular to a method for determining related substances in diclofenac diethylamine emulsion. Background Art

[0002] Diclofenac diethylamine gel (according to the Chinese Pharmacopoeia: latex is a type of gel) is a new type of anti-inflammatory analgesic drug, mainly used to relieve mild to moderate pain in muscles, soft tissues and joints. It inhibits cyclooxygenase activity, thereby blocking the conversion of arachidonic acid into prostaglandins, achieving anti-inflammatory and analgesic effects. In order to ensure the quality and safety of diclofenac diethylamine latex, it is particularly important to detect related substances.

[0003] By consulting domestic and foreign pharmacopoeias, we know that the quality standards of diclofenac diethylamine gel are included in both the BP pharmacopoeia and the imported drug registration standards. Among them, the BP pharmacopoeia uses the high performance liquid chromatography (HPLC) method to detect the relevant substances, and the imported drug registration standards use the ultra-high performance liquid chromatography (UPLC) method to detect the relevant substances. The relevant standards are recorded in Table 1 below:

[0004] Table 1 Records of relevant testing standards

[0005]

[0006]

[0007] After reproducing the detection methods of the relevant substances in each standard, the inventors found that: each method could not simultaneously meet the detection and separation of the main peak, degradation impurities (impurity A, impurity B, impurity C, diclofenac isopropyl ester, diclofenac dodecyl ester) and process impurities (impurity D, impurity E, impurity F), so the methods included in the standards are not suitable for detecting the relevant substances in diclofenac diethylamine gel.

[0008] Therefore, it is urgent to optimize the related substance method in the standard to determine the related substances in diclofenac diethylamine emulsion. Summary of the invention

[0009] In view of this, the present invention proposes a method for determining related substances in diclofenac diethylamine emulsion. By optimizing the elution mode and setting a suitable elution program under the BP2024 chromatographic conditions, the full elution and effective separation of the main peak and the impurity peaks are achieved, and the method for determining related substances in diclofenac diethylamine emulsion is determined.

[0010] The present invention provides a method for determining related substances in a diclofenac diethylamine emulsion, the method comprising the following steps:

[0011] The first step is to prepare a diclofenac diethylamine test solution, a control solution, a sensitivity solution and a system suitability solution;

[0012] The second step is to use high performance liquid chromatography, and the chromatographic conditions include: the chromatographic column is an octadecylsilane bonded silica gel chromatographic column, methanol-phosphate buffer is used as mobile phase A, methanol is used as mobile phase B, the detection wavelength is 260nm, and gradient elution is performed;

[0013] The process of the gradient elution is as follows: within 0 to 35 minutes, the volume ratio of the mobile phase A to the mobile phase B is 100:0; within 35 to 65 minutes, the volume ratio of the mobile phase A to the mobile phase B changes gradually from 100:0 to 8:92 at a uniform speed; within 65 to 80 minutes, the volume ratio of the mobile phase A to the mobile phase B changes gradually from 8:92 to 100:0 at a uniform speed.

[0014] On the basis of the above technical scheme, preferably, a method for determining related substances in diclofenac diethylamine emulsion specifically comprises:

[0015] (1) preparing diclofenac diethylamine test solution, control solution, sensitivity solution and system suitability solution;

[0016] (2) using high performance liquid chromatography, wherein the chromatographic conditions include: using an octadecylsilane bonded silica gel column, using methanol-phosphate buffer as mobile phase A, using methanol as mobile phase B, and using a detection wavelength of 260 nm for gradient elution;

[0017] (3) using the chromatographic conditions in (2) to test the diclofenac diethylamine system suitability solution, and determining the peak positions of impurities A, B, C, diclofenac isopropyl ester (G), diclofenac dodecyl ester (H), and the main peak, and the separation of the impurity peaks from the main peak;

[0018] (4) using the chromatographic conditions in (2) to detect the diclofenac diethylamine test solution and the control solution, and determining whether the test solution contains known impurities A, B, C, G, H and other unknown impurities based on the peak positions, thereby determining the presence and / or amount of each impurity in the diclofenac sodium test solution;

[0019] (5) Using the chromatographic conditions in (2) to detect the diclofenac diethylamine sensitive solution, to determine the lowest detectable concentration of diclofenac diethylamine and its impurities for trace analysis.

[0020] Based on the above technical solution, the gradient elution program is shown in Table 2:

[0021] Table 2 Gradient elution program

[0022]

[0023]

[0024] In the present invention, as the system adaptability requirement: in the system suitability solution chromatogram, impurity A, impurity C, diclofenac diethylamine, impurity B, impurity G and impurity H are peaked in sequence, the separation between the diclofenac diethylamine peak and the impurity C peak should be not less than 2, and the theoretical plate number calculated based on the diclofenac diethylamine peak should be not less than 2000.

[0025] In the present invention, as a sensitivity requirement, in the sensitivity solution chromatogram, the signal-to-noise ratio of the main component peak height should be no less than 10.

[0026] The determination method of the present invention comprises the steps of precisely measuring a test solution and a control solution, injecting the solution into a liquid chromatograph respectively, recording a chromatogram, and determining the presence and amount of each impurity in the test sample based on the peak area and peak position in the chromatogram.

[0027] In the present invention, the limits (maximum allowable amount or range) of various impurities in diclofenac diethylamine emulsion include:

[0028] (1) The impurity A, impurity B, and impurity C calculated based on the corrected peak area shall not be greater than 1.5 times (0.3%) the main peak area of ​​the control solution;

[0029] (2) The peak areas of diclofenac isopropyl and diclofenac dodecyl ester calculated after correction shall not be greater than the main peak area of ​​the control solution (0.2%);

[0030] (3) The peak area of ​​other individual impurities shall not be greater than the main peak area of ​​the control solution (0.2%);

[0031] (4) Total impurities shall not exceed 1.0%;

[0032] Among them, the impurity peaks in the chromatogram of the test solution that are smaller than the main peak area of ​​the sensitivity solution can be ignored. The following calculation formula can be used as a reference:

[0033] Single impurity (%) = As × F / (Ar × Vr) × 100 formula (Ⅰ);

[0034] Total impurities (%) = the sum of all impurities above 0.05% (Formula (II));

[0035] In formula (I) and / or (II), Ar: main peak area in the reference solution; Vr: dilution factor (ml) of the reference solution to the test solution; As: peak area of ​​a single impurity in the test solution; F: correction factor.

[0036] Furthermore, the model of the chromatographic column is YMC-Pack Pro C18, the length is 250 mm, the inner diameter is 4.6 mm, and the particle size of the filler is 5 μm.

[0037] Furthermore, in the mobile phase A, the volume ratio of the methanol to the phosphate buffer is 66:34.

[0038] Furthermore, the phosphate buffer is prepared from phosphoric acid, sodium dihydrogen phosphate dihydrate and water in a mass-volume ratio of 0.5g:0.8g:1000ml.

[0039] Furthermore, the phosphate buffer is adjusted to a pH value of 2.5 using phosphoric acid.

[0040] Furthermore, the flow rate of the mobile phase is 1 ml / min.

[0041] Furthermore, the temperature of the chromatographic column is 30°C.

[0042] Furthermore, a guard column is included. The model of the guard column is a YMC-Pack Pro C18 guard column, the inner diameter is 4 mm, the length is 10 mm, and the particle size of the filler is 5 μm.

[0043] Furthermore, the filler is octadecylsilane bonded silica gel.

[0044] Furthermore, the related substances include degradation impurities and process impurities; the degradation impurities include impurities A, B, C, diclofenac isopropyl and diclofenac dodecyl ester; the process impurities include impurities D, E, and F.

[0045] The method for determining related substances in diclofenac diethylamine emulsion provided by the present invention has the following beneficial effects compared with the prior art:

[0046] (1) The determination method provided by the present invention significantly improves the separation degree and detection sensitivity by adopting high performance liquid chromatography (HPLC) and gradient elution mode, and can effectively separate the main peak and various degradation impurities and process impurities within a wide time range. The detection wavelength is set to 260nm, which enhances the absorption of diclofenac diethylamine and its impurities, ensuring that low-concentration impurities can also be accurately detected. The use of octadecylsilane bonded silica gel chromatographic column provides a sharper peak shape and higher resolution, ensuring the stability and reproducibility of the separation effect.

[0047] (2) The determination method of the present invention ensures the reliability and accuracy of the method through reasonable system suitability solution and sensitivity solution setting, shortens the analysis time, and improves the working efficiency of the laboratory. The present invention can comprehensively cover the main peak, degradation impurities (such as impurity A, impurity B, impurity C, diclofenac isopropyl, diclofenac dodecyl ester) and process impurities (such as impurity D, impurity E, impurity F) in diclofenac diethylamine, provides more accurate and reliable test results, and effectively ensures the quality and safety of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0049] Figure 1 The chromatogram overlay of the method for reproducing the related substances of BP2024 provided in Comparative Example 1 of the present invention;

[0050] Figure 2 The imported registered standard JX20220123 related substance method reproduction chromatogram overlay provided for comparative example 2 of the present invention;

[0051] Figure 3 It is a stability chromatographic analysis diagram of diclofenac diethylamine emulsion of the present invention;

[0052] Figure 4 This is a typical detection spectrum of Preparation Example 1 of the present invention when the detection wavelength is 254nm;

[0053] Figure 5 This is a typical spectrum of the related substances provided in Example 1 of the present invention under specific chromatographic conditions. DETAILED DESCRIPTION

[0054] The inventors referred to the pharmacopoeia and current standards, optimized the elution mode to gradient elution under the BP2024 chromatographic conditions and set a suitable elution program to achieve full elution and effective separation of the main peak and impurity peaks (including degradation impurities and process impurities), and finally determined the determination method of the relevant substances in this product.

[0055] In the present invention, since mobile phase A is determined to be a phosphate solution-methanol system with reference to BP2024, item B is preferentially determined to be methanol with reference to the organic phase (methanol) in phase A. Under the BP method of this product, there is no problem in separating the impurities, mainly because there are strongly retained substances that have not been eluted, so there is no need to introduce other organic phases to interfere with the separation of the impurities in the isocratic section. Here, the inventor only considers eluting the strongly retained impurities. Because the introduction of different organic phases may bring different separation mechanisms, when there is no problem with separation, other organic phases are not introduced to cause other problems that may arise. Therefore, the chromatographic conditions adopted are: methanol-pH2.5 phosphate buffer (66:34) as mobile phase A, methanol as mobile phase B, and gradient elution according to Table 1.

[0056] If the mobile phases A and B of the imported drug registration standard (JX20220123) are used and the experiment is carried out according to our elution procedure, since the mobile phase A of JX20220123 is a pure water phase, all impurities cannot be eluted under isocratic elution. The design of our elution procedure is to ensure that impurities A-impurity F can be completely eluted and well separated when the mobile phase A is isocratic elution is used for 35 minutes, and the proportion of mobile phase B is increased to elute diclofenac isopropyl ester and diclofenac dodecyl ester.

[0057] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0058] Analysis of the impurities in the British Pharmacopoeia Diclofenac Diethylamine API and Gel and Diclofenac Diethylamine Emulsion Import Drug Registration Standards shows that the process impurities of this product are impurity A, impurity B, impurity C, impurity D, impurity E, and impurity F, and the degradation impurities are impurity A, impurity B, impurity C, diclofenac isopropyl, and diclofenac dodecyl. The related substance method of this product needs to ensure the detection and separation of diclofenac diethylamine and the above 8 impurities. The specific process of establishing the related substance method is as follows.

[0059] Comparative Example 1

[0060] This comparative example demonstrates the reproduction process of the analysis method of related substances in diclofenac diethylamine using the BP2024 standard, including:

[0061] (1) Injection sequence (inject in the following order):

[0062] a. Blank solvent (diluent): methanol;

[0063] b. API + impurity mixed solution (containing about 1 mg / ml of diclofenac diethylamine and about 0.01 mg / ml of impurities A, B, C, D, E, and F);

[0064] c. Diclofenac isopropyl ester localized solution (about 0.03 mg / ml);

[0065] d. Diclofenac dodecyl ester localized solution (about 0.03 mg / ml).

[0066] (2) Chromatographic conditions (as shown in Table 3)

[0067] Table 3 Chromatographic conditions

[0068] method Method 1 (BP2024) Chromatography Waters ARC HPLC 2489 Chromatographic columns YMC-Pack Pro C18, 4.6mm×250mm, 5μm Mobile phase Methanol-pH 2.5 phosphate buffer (66:34) Elution method Isocratic Detection wavelength 254nm Injection volume 20μl Flow rate 1.0ml / min Column temperature 30℃

[0069] (3) Reproduce the results (such as Figure 1 (shown)

[0070] (4) Results analysis

[0071] Depend on Figure 1 It can be seen that diclofenac diethylamine and impurities A, B, C, D, E, and F all appeared within 60 minutes, and the peaks were well separated. Diclofenac isopropyl ester appeared at about 120 minutes, and diclofenac dodecyl ester did not appear within 120 minutes.

[0072] Theoretical analysis shows that diclofenac dodecyl ester has lower polarity than diclofenac isopropyl ester and is more strongly retained, so it is inferred that its retention time should be greater than 120 minutes. Since diclofenac dodecyl ester cannot be detected under this method and the retention time of diclofenac isopropyl ester is too long, this method is not applicable.

[0073] Comparative Example 2

[0074] This comparative example demonstrates the reproduction process of the analysis method of related substances in diclofenac diethylamine using the imported drug registration standard, including:

[0075] (1) Injection sequence (inject in the following order):

[0076] a. Blank solvent (diluent): 98% acetonitrile;

[0077] b. API + impurity mixed solution (containing about 1.16 mg / ml of diclofenac diethylamine, about 3.6 μg / ml of impurities A, B, and C, and about 2.4 μg / ml of impurities D, E, F, diclofenac isopropyl alcohol, and diclofenac dodecyl ester);

[0078] c. Reference preparation test solution (containing about 1.16 mg / ml of diclofenac diethylamine);

[0079] (2) Chromatographic conditions (as shown in Table 4)

[0080] Table 4 Chromatographic conditions

[0081]

[0082] (3) Reproduce the results (such as Figure 2 (shown)

[0083] (4) Results analysis

[0084] Depend on Figure 2 It can be seen that under the standard method for registration of imported drugs, the detection of the main peak and each impurity peak can be guaranteed, and the separation between the peaks is good. However, under this chromatographic condition, the reference excipient peak interferes with the detection of process impurity E, and the solvent peak interferes with the detection of impurity G, so the method is not applicable.

[0085] Theoretical analysis shows that: in the chromatogram of the reference preparation test solution, there is an excipient peak at a relative retention time RRT of about 0.04. According to the description of the limit in the imported drug registration standard (JX20220123), the chromatographic peak with RRT < 0.1 is the solvent and excipient peak. The RRT of impurity E in the figure is 0.04, so the reference excipient peak (RRT about 0.04) interferes with the detection of process impurity E.

[0086] Combining Comparative Examples 1 and 2, it can be seen that the methods in BP2024 and the imported drug registration standards are reproduced and it can be seen that both methods are not applicable. Diclofenac dodecyl ester cannot be detected under the BP2024 method, and the retention time of diclofenac isopropyl ester is too long, so this method is not applicable. The reference excipient peak of the imported drug registration standard interferes with the detection of process impurity E, and the solvent peak interferes with the detection of impurity G, so the method is not applicable. In summary, the methods for related substances included in the standards need to be optimized.

[0087] Combining practical operation and theoretical analysis, it can be seen that interference under the standard method for imported drug registration is difficult to eliminate (the interference peak and impurity peak are highly overlapped). Based on the BP2024 method, a high proportion of organic phase elution is added after isocratic elution to achieve the purpose of eluting diclofenac isopropyl and diclofenac dodecyl impurities. In summary, the optimization of the method for related substances is prioritized based on the BP2024 standard.

[0088] According to the results reproduced by BP2024, the main peak and impurity AF can be detected when the methanol-pH2.5 phosphate buffer (66:34) isocratic elution is performed for about 30 minutes. At about 45 minutes, an unknown impurity peak is detected. After 60 minutes of operation, diclofenac isopropyl ester and diclofenac dodecyl ester impurities cannot be eluted. In summary, after about 35 minutes of isocratic elution (in the process of conducting stability studies on diclofenac diethylamine latex preparations, chromatographic analysis of samples at different time points was performed, and no unknown impurity peak was detected at 45 minutes, such as Figure 3 As shown. This indicates that the peak at 45 minutes is not a degradation impurity peak generated during the storage or use of the preparation. Therefore, there is no need to monitor and analyze this peak specifically). A high-ratio organic phase gradient elution section is added to ensure the elution of diclofenac isopropyl and diclofenac dodecyl impurities with other strongly retained compounds.

[0089] The following are preparation examples of the present invention.

[0090] Preparation Example 1

[0091] Based on the above content, this preparation example provides a chromatographic condition for determining the related substances in diclofenac diethylamine emulsion, as shown:

[0092] (1) Chromatographic column: YMC-Pack Pro C18 (4.6×250 mm, 5 μm);

[0093] (2) Flow rate: 1.0 ml / min; column temperature: 30 °C; detection wavelength: 254 nm; injection volume: 20 μl;

[0094] (3) Mobile phase A: methanol-pH 2.5 phosphate buffer (66:34); Mobile phase B: 100% methanol; (4) Linear gradient elution according to Table 2:

[0095] Table 2 Gradient elution program

[0096] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 35 100 0 45 8 92 65 8 92 65.1 100 0 80 100 0

[0097] (5) The results are as follows Figure 4 As shown;

[0098] (6) Results analysis

[0099] Depend on Figure 3 It can be seen that: the determination method adopted in this embodiment has better peak shape of main peak and each impurity peak, and the minimum separation between each peak is 2.06, that is, each peak is well separated. At the same time, the blank solution does not interfere with the detection of the main peak and each impurity. In summary, it is confirmed that the chromatographic conditions are the chromatographic conditions of the determination method of related substances in diclofenac diethylamine emulsion.

[0100] Preparation Example 2

[0101] This preparation example is based on the chromatographic conditions determined in the above preparation example 1, and is used to confirm the detection wavelength of the determination method of this product. In this preparation example, the raw material drug solution and the impurity solution were scanned in the wavelength range of 210nm to 400nm by a diode array detector, as shown in the following Table 5. The detection wavelength of this product is confirmed according to the characteristic absorption wavelengths of the main component and the impurity.

[0102] Table 5 Maximum absorption wavelength and absorption spectrum of diclofenac diethylamine and specific impurities

[0103]

[0104] Since the absorption wavelength of methanol terminal is about 210nm, the absorption wavelength below 210nm is not listed in the table. It can be seen from Table 5 that the detection wavelength recommended in BP2024 is 254nm, and the wavelength recommended in the imported drug registration standard is 260nm. Full wavelength scanning of the main components and impurities found that the detection wavelength of 260nm is closer to the characteristic absorption wavelength of the main components and each impurity peak (except impurities A and B), so it is confirmed that the detection wavelength of this product is 260nm.

[0105] Combining Preparation Example 1 and Preparation Example 2, an embodiment of the present invention is obtained.

[0106] Example 1

[0107] This embodiment provides a method for determining related substances in diclofenac diethylamine emulsion, comprising:

[0108] (1) preparing diclofenac diethylamine test solution, control solution, sensitivity solution and system suitability solution;

[0109] Test solution: Take about 2.5g of this product (equivalent to 29mg of diclofenac diethylamine), put it in a 50ml volumetric flask, add about 30ml of methanol, vortex until the latex is evenly dispersed, add 5ml of saturated sodium chloride solution and shake until the flocculation of the auxiliary material no longer increases, dilute to the scale with methanol, shake well, centrifuge, filter the upper solution, discard 1ml of the initial filtrate, and take the subsequent filtrate.

[0110] Control solution: Accurately measure 11 ml of the test solution, place it in a 25 ml volumetric flask, dilute it to the mark with methanol, shake well, then accurately measure 1 ml of the solution, place it in a 20 ml volumetric flask, dilute it to the mark with methanol, shake well.

[0111] Sensitivity solution: Accurately measure an appropriate amount of the control solution, quantitatively dilute it with methanol to make a solution containing approximately 0.29 μg of diclofenac diethylamine per 1 ml, and shake well.

[0112] System suitability solution: Take appropriate amount of diclofenac diethylamine and impurity A, impurity B, impurity C, impurity G, and impurity H reference substances, dissolve in methanol and quantitatively dilute to make a mixed solution containing approximately 0.58 mg of diclofenac diethylamine, approximately 1.74 μg each of impurities A, impurity B, and impurity C, and approximately 1.16 μg each of impurity G and impurity H per 1 ml, and shake well.

[0113] (2) Chromatographic conditions

[0114] Octadecylsilane bonded silica gel (YMC-Pack Pro C18, 4.6mm×250mm, 5μm, with YMC-PackPro C18 guard column, 4mm×10mm, 5μm) was used as the filler; methanol-pH2.5 phosphate buffer (take 0.5g of phosphoric acid and 0.8g of sodium dihydrogen phosphate dihydrate, add 1000ml of water, stir to dissolve, and adjust the pH value to 2.5 with phosphoric acid) (66:34) was used as mobile phase A, methanol was used as mobile phase B, and gradient elution was performed according to Table 2; the detection wavelength was 260nm; the flow rate was 1.0ml per minute; the column temperature was 30℃; the injection volume was 20μl.

[0115] Table 2 Gradient elution program

[0116] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 35 100 0 45 8 92 65 8 92 65.1 100 0 80 100 0

[0117] The results of the test are as follows Figure 5 And as shown in Table 6.

[0118] Table 6 Test results of related substances

[0119]

[0120] Combination Figure 5 As shown in Table 6, no specific impurities and non-specific impurities were detected in the self-prepared preparation (diclofenac diethylamine emulsion prepared in this example) at 0 day and 1 month of accelerated preparation, indicating that the preparation of the present invention has good stability.

[0121] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for determining related substances in diclofenac diethylamine emulsion, characterized in that: The method comprises the following steps: The first step is to prepare a diclofenac diethylamine test solution, a control solution, a sensitivity solution and a system suitability solution; The second step is to use high performance liquid chromatography, and the chromatographic conditions include: the chromatographic column is an octadecylsilane bonded silica gel chromatographic column, methanol-phosphate buffer is used as mobile phase A, methanol is used as mobile phase B, the detection wavelength is 260nm, and gradient elution is performed; The process of the gradient elution is as follows: within 0 to 35 minutes, the volume ratio of the mobile phase A to the mobile phase B is 100:0; within 35 to 65 minutes, the volume ratio of the mobile phase A to the mobile phase B changes gradually from 100:0 to 8:92 at a uniform speed; within 65 to 80 minutes, the volume ratio of the mobile phase A to the mobile phase B changes gradually from 8:92 to 100:0 at a uniform speed.

2. The method according to claim 1, characterized in that The model of the chromatographic column is YMC-Pack Pro C18, with a length of 250 mm, an inner diameter of 4.6 mm, and a filler particle size of 5 μm.

3. The method according to claim 1, characterized in that In the mobile phase A, the volume ratio of the methanol to the phosphate buffer is 66:

34.

4. The method according to claim 3, characterized in that The phosphate buffer is prepared from phosphoric acid, sodium dihydrogen phosphate dihydrate and water in a mass-volume ratio of 0.5 g:0.8 g:1000 ml.

5. The method according to claim 4, characterized in that The phosphate buffer solution uses phosphoric acid to adjust the pH value to 2.

5.

6. The method according to claim 1, characterized in that The flow rate of the mobile phase is 1 ml / min.

7. The method according to claim 1, characterized in that The temperature of the chromatographic column was 30°C.

8. The method according to claim 1, characterized in that The invention also comprises a guard column, the model of which is a YMC-Pack Pro C18 guard column, with an inner diameter of 4 mm, a length of 10 mm, and a particle size of a filler of 5 μm.

9. The method according to claim 2 or 7, characterized in that: The filler is octadecylsilane bonded silica gel.

10. The method according to any one of claims 1 to 9, characterized in that: The related substances include degradation impurities and process impurities; The degradation impurities include impurities A, B, C, diclofenac isopropyl and diclofenac dodecyl ester; The process impurities include impurities D, E, and F.

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