Determination method for related substances in diclofenac sodium gel

Abstract

The invention discloses a method for determining related substances in diclofenac sodium gel, which comprises the following steps: (1) preparing: preparing a test solution, a contrast solution, a system applicability solution and a sensitivity solution by taking methanol as a solvent; (2) determination: determining the content of diclofenac sodium and related substances in the diclofenac sodium gel by adopting a high performance liquid chromatography, and respectively feeding a test solution, a contrast solution, a system applicability solution and a sensitivity solution; chromatographic conditions are as follows: a chromatographic column takes octadecylsilane chemically bonded silica as a filler, the column temperature is 25-45 DEG C, the detection wavelength is 254 nm, the feeding amount is 10 [mu] L, the flow velocity is 0.8-1.2 mL / min, a glacial acetic acid solution is taken as a mobile phase A, and methanol is taken as a mobile phase B for gradient elution. The method solves the technical problems of precipitation after preparation of the test solution, poor impurity peak separation degree, detection interference by the chromatographic peak of the bacteriostatic agent, impurity peak cracking and the like.

Description

Technical Field

[0001] The invention relates to a method for determining related substances in diclofenac sodium gel, belonging to the technical field of drug analysis. Background Art

[0002] Diclofenac Sodium, chemically named 2-[(2,6-dichlorophenyl)amino]-phenylacetate sodium, is an antipyretic, analgesic, and non-steroidal anti-inflammatory drug. It exerts its antipyretic, analgesic, and anti-inflammatory effects by inhibiting cyclooxygenase, thereby reducing the synthesis of prostaglandins, and to a certain extent inhibiting lipoxygenase, thereby reducing the production of leukotrienes, bradykinin and other products.

[0003] At present, the national drug standard WS1-(X-360)-2004Z issued by the State Food and Drug Administration and the drug standards WS-319(X-262)-94(1), WS-319(X-262)-94(2), WS-737(X-595)-98, and WS-146(X-130)-97 issued by the Ministry of Health of the People's Republic of China do not include the determination methods of related substances of diclofenac sodium gel. Neither the Chinese Pharmacopoeia nor foreign pharmacopoeias include diclofenac sodium gel.

[0004] There are related substance determination methods under the diclofenac sodium raw materials and their suppositories, enteric-coated tablets, sustained-release capsules, sustained-release tablets, topical solutions, ointments, eye drops and other varieties included in the Chinese Pharmacopoeia and foreign pharmacopoeias. However, when using the above methods to detect the related substances of diclofenac sodium gel, there are situations such as the test sample cannot be completely dissolved, impurities are poorly separated, and antibacterial agents in the excipients interfere with the detection. Therefore, the methods cannot be used directly to detect the related substances of diclofenac sodium gel.

[0005] Other documents mention the method for related substances of diclofenac sodium gel, but the above situation also exists and it cannot be directly used to detect the related substances of diclofenac sodium gel. Summary of the invention

[0006] In view of at least one problem existing in the above-mentioned prior art, the present invention provides a method for determining related substances in diclofenac sodium gel, which solves the technical problems such as precipitation after preparation of the test solution, poor separation of impurity peaks, interference of chromatographic peaks of antibacterial agents in detection, and impurity peak splitting.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme: a method for determining related substances in diclofenac sodium gel, comprising the following steps:

[0008] (1) Preparation: Use methanol as solvent to prepare the test solution, control solution and sensitivity solution; use water as solvent to prepare the system suitability solution;

[0009] (2) Determination: Determine the content of diclofenac sodium and related substances in diclofenac sodium gel by high performance liquid chromatography, add test solution, reference solution, system suitability solution and sensitivity solution respectively, and record the chromatogram;

[0010] Chromatographic conditions: The chromatographic column was filled with octadecylsilane bonded silica gel, the column temperature was 25-45°C, the detection wavelength was 254nm, the feed volume was 10μL, the flow rate was 0.8-1.2mL / min, and the mobile phase was glacial acetic acid solution as mobile phase A and methanol as mobile phase B for gradient elution.

[0011] Preferably, in step (2), the gradient elution program is: 0min to 15min: the volume fraction of mobile phase A decreases from 90% to 35%, and the volume fraction of mobile phase B increases from 10% to 65%; 15min to 50min: the volume fraction of mobile phase A is maintained at 35%, and the volume fraction of mobile phase B is maintained at 65%; 50min to 50.1min: the volume fraction of mobile phase A is increased from 35% to 90%, and the volume fraction of mobile phase B is decreased from 65% to 10%; 50.1min to 60min: the volume fraction of mobile phase A is maintained at 90%, and the volume fraction of mobile phase B is maintained at 10%.

[0012] Preferably, in step (2), mobile phase A is 3.6% to 4.4% glacial acetic acid solution.

[0013] Preferably, mobile phase A is 4% glacial acetic acid solution.

[0014] Preferably, in step (2), the specification of the chromatographic column is 4.6 mm×150 mm, and the filler particle size is 5 μm.

[0015] Preferably, the chromatographic column is Agilent HC-C18(2), 150 mm×4.6 mm, 5 μm.

[0016] Preferably, in step (2), the column temperature is 35°C.

[0017] Preferably, in step (2), the flow rate is 1.0 mL / min.

[0018] Preferably, in step (1), the test solution is prepared from diclofenac sodium gel as a raw material and methanol as a solvent to prepare a test solution having a diclofenac sodium concentration of about 1 mg / mL;

[0019] The control solution is prepared with the test solution as the raw material and methanol as the solvent to prepare a control solution with a diclofenac sodium concentration of about 5 μg / mL;

[0020] The sensitivity solution is prepared by using the control solution as a raw material and methanol as a solvent to prepare a sensitivity solution having a diclofenac sodium concentration of about 0.5 μg / mL;

[0021] The system suitability solution is prepared with diclofenac sodium as the reference substance raw material and water as the solvent to prepare a system suitability solution with a diclofenac sodium concentration of about 1 mg / mL, and is exposed to ultraviolet light (254 nm) for 15 minutes.

[0022] Preferably, the specific preparation process of the test solution, control solution, system suitability solution and sensitivity solution is as follows:

[0023] The test solution: take about 2.5g of diclofenac sodium gel, accurately weigh it, put it into a 25mL volumetric flask, add methanol to dissolve and dilute to the scale, shake well, let it stand for 10min, filter it with a 0.45μm PTFE filter membrane, discard 2mL of the initial filtrate, and take the subsequent filtrate to obtain;

[0024] The control solution: accurately take 100 μl of the test solution into a 20 mL volumetric flask, dilute to the mark with methanol, and shake well;

[0025] Sensitivity solution: Accurately take 1 mL of the control solution into a 10 mL volumetric flask, add methanol to dilute to the mark, and shake well;

[0026] The system suitability solution: take about 10 mg of diclofenac sodium reference substance into a 10 mL volumetric bottle, add water to dissolve and dilute to the scale, and expose the solution to ultraviolet light (254 nm) for 15 minutes.

[0027] Preferably, the system suitability requirement is: in the chromatogram of the system suitability solution, the separation degree between the main peak and the chromatographic peak with a relative retention time of about 0.9 shall not be less than 6.0;

[0028] The sensitivity requirement is: in the chromatogram of the sensitivity solution, the signal-to-noise ratio of the main peak shall not be less than 10;

[0029] The calculation method is: self-control method with added correction factor.

[0030] Preferably, the related substances are: impurity A: 1-(2,6-dichlorophenyl)-1,3-dihydro-2H-indol-2-one; impurity B: 1-(2,6-dichlorophenylamino)benzaldehyde; impurity C: 1-(2,6-dichlorophenylamino)benzyl alcohol; impurity E: 2-indolone.

[0031] The limit requirements for related substances are: except for the chromatographic peak with a relative retention time of approximately 0.47, impurities A, B, C, and E shall not exceed 0.5%, other individual impurities shall not exceed 0.5%, and the total amount of impurities shall not exceed 1.0%; the relative retention times of impurities A, B, C, and E are approximately 0.78, 1.1, 0.90, and 0.39, respectively; the correction factors of impurities A, B, C, E and other individual impurities are 0.66, 0.58, 1.0, 0.28, and 1.0, respectively.

[0032] Compared with the prior art, the invention has the following beneficial effects: the method for determining related substances in diclofenac sodium gel of the invention adopts a gradient elution mode, reduces the injection amount, optimizes the test solution, solves the problem that using water as a solvent will cause diclofenac sodium to be unstable, and solves the problem that using methanol as a solvent will cause matrix precipitation in the test sample by increasing precipitation and filtration; at the same time, the problem that the separation difference between specific impurities and general impurities and between general impurities, and the problem that methylparaben in the auxiliary material interferes with detection are solved; the quality control and sensitivity requirements for impurities A, C, and E are increased; the relative retention time and correction factor of specific impurities are also increased, so that the detection result is more accurate; the method for determining related substances in diclofenac sodium gel of the invention fills the gap in the method for determining related substances in diclofenac sodium gel. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the liquid chromatogram of Example 1 of the present invention;

[0034] Figure 2 It is the liquid chromatogram of Example 2 of the present invention;

[0035] Figure 3 It is the liquid chromatogram of Example 3 of the present invention;

[0036] Figure 4 It is the acid destruction liquid phase chromatogram in Example 5 of the present invention;

[0037] Figure 5 This is a thermal destruction liquid chromatogram in Example 5 of the present invention;

[0038] Figure 6 This is a photo-destructed liquid chromatogram in Example 5 of the present invention;

[0039] Figure 7 is a linear graph of diclofenac sodium in Example 6 of the present invention;

[0040] Figure 8 is a linear graph of impurity A in Example 6 of the present invention;

[0041] Fig. 9 is a linear graph of impurity B in Example 6 of the present invention;

[0042] Fig.10 is a linear graph of impurity C in Example 6 of the present invention;

[0043] Fig.11 is a linear graph of impurity E in Example 6 of the present invention;

[0044] Fig.12 It is the liquid chromatogram of Comparative Example 1 of the present invention;

[0045] Fig.13 It is the liquid chromatogram of Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0046] The technical scheme in the implementation of the present invention is clearly and completely described below in conjunction with the accompanying drawings. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to the conditions recommended by the normal conditions or the manufacturers. If the manufacturers are not specified in the reagents, instruments, and components used, they are all conventional products that can be purchased commercially.

[0047] The diclofenac sodium gel in the present invention is sourced from Yaoda Pharmaceutical Co., Ltd., batch number 240505, specification 0.1% (15 ml: 0.15 g).

[0048] The diclofenac sodium reference substance in the present invention comes from: China Food and Drug Inspection Institute, batch number 100334-201803, and the content is 100.0%.

[0049] The impurity A reference substance in the present invention comes from: China Food and Drug Inspection Institute, batch number 100395-200301, the content is not indicated, it is calculated as 100%.

[0050] The source of the impurity B reference substance in the present invention is: China Food and Drug Inspection Institute, batch number 510123-201501, the content is not indicated, and it is calculated as 100%.

[0051] The impurity C reference substance in the present invention comes from: China Food and Drug Inspection Institutes, batch number 510124-201501, the content is not indicated, and is calculated as 100%.

[0052] The impurity E reference substance in the present invention comes from: China Food and Drug Inspection Institutes, batch number 510120-201501, the content is not indicated, and is calculated as 100%.

[0053] The methylparaben reference substance in the present invention comes from: China Food and Drug Inspection Institute, batch number 100278-202307, with a content of 100%.

[0054] Example 1

[0055] A method for determining related substances in diclofenac sodium gel, the specific steps are as follows:

[0056] (1) Preparation:

[0057] System suitability solution: Take about 10 mg of diclofenac sodium reference substance into a 10 mL volumetric bottle, add water to dissolve and dilute to the scale, expose the solution to ultraviolet light (254 nm) for 15 min, and use it as the diclofenac sodium reference substance positioning solution;

[0058] Location solutions of impurity A, impurity B, impurity C, and impurity E: weigh about 1 mg of impurity A reference substance, impurity B reference substance, impurity C reference substance, and impurity E reference substance, respectively, place them in 10 mL volumetric flasks, add methanol to dissolve and dilute to the scale, shake well, and obtain the stock solutions of impurity A, impurity B, impurity C, and impurity E; take 1 mL of each stock solution of impurity A, impurity B, impurity C, and impurity E, place them in 20 mL volumetric flasks, add methanol to dissolve and dilute to the scale, shake well, and use them as the location solutions of impurity A, impurity B, impurity C, and impurity E;

[0059] Methylparaben localizing solution: Take 5 mg of methylparaben reference substance, place it in a 250 mL volumetric flask, add methanol to dissolve and dilute to the mark, shake well, and use this as the methylparaben localizing solution;

[0060] Mixed solution: Take 4 mg of methylparaben to a 10 mL volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and use it as the methylparaben stock solution; take 1 mL of the methylparaben stock solution and 1 mL of each of the impurity A, B, C, and E stock solutions to the same 20 mL volumetric flask, then weigh about 2 g of diclofenac sodium gel, add methanol to dissolve and dilute to the scale, shake well, let stand for 10 minutes, filter with a 0.45 μm PTFE filter membrane, discard 2 mL of the initial filtrate, and take the subsequent filtrate to obtain a mixed solution.

[0061] (2) Determination: The content of diclofenac sodium and related substances in diclofenac sodium gel was determined by high performance liquid chromatography. The chromatographic conditions were as follows: the chromatographic column was Agilent HC-C18 (2), 150 mm × 4.6 mm, 5 μm, the column temperature was 30° C., the detection wavelength was 254 nm, the feed volume was 10 μL, the flow rate was 1 mL / min, and the mobile phase was 4% glacial acetic acid solution as mobile phase A and methanol as mobile phase B for gradient elution;

[0062] Gradient elution program:

[0063] Time / min Mobile phase A / (v)% Mobile phase B / (v)% 0 90 10 10 35 65 50 35 65 50.1 90 10 60 90 10

[0064] The diclofenac sodium reference substance positioning solution, impurity A positioning solution, impurity B positioning solution, impurity C positioning solution, impurity E positioning solution and methylparaben positioning solution were accurately measured for detection, and the retention times of diclofenac sodium, impurity A, impurity B, impurity C, impurity E and methylparaben were analyzed to be 19.782 min, 14.678 min, 22.356 min, 17.229 min, 7.866 min and 9.189 min, respectively;

[0065] Then accurately measure the mixed solution for testing and record the chromatogram. The results are shown in Figure 1 .

[0066] Example 2

[0067] A method for determining related substances in diclofenac sodium gel, the specific steps are as follows:

[0068] (1) Preparation: exactly the same as in Example 1;

[0069] (2) Determination: Partially the same as Example 1, except that the column temperature was 35°C;

[0070] The diclofenac sodium reference substance positioning solution, impurity A positioning solution, impurity B positioning solution, impurity C positioning solution, E positioning solution and methylparaben positioning solution were accurately measured for detection, and the retention times of diclofenac sodium, impurity A, impurity B, impurity C, impurity E and methylparaben were analyzed to be 24.115min, 18.901min, 26.487min, 21.669min, 9.333min and 11.310min, respectively;

[0071] Then accurately measure the mixed solution for testing and record the chromatogram. The results are shown in Figure 2 .

[0072] Example 3

[0073] A method for determining related substances in diclofenac sodium gel, the specific steps are as follows:

[0074] (1) Preparation: Exactly the same as in Example 1;

[0075] (2) Determination: Partially the same as Example 1; except that: column temperature and gradient elution procedure:

[0076] The column temperature was 40°C;

[0077] Elution gradient:

[0078] Time / min Mobile phase A / (v)% Mobile phase B / (v)% 0 90 10 20 35 65 50 35 65 50.1 90 10 60 90 10

[0079] The diclofenac sodium reference substance positioning solution, impurity A positioning solution, impurity B positioning solution, impurity C positioning solution, impurity E positioning solution and methylparaben positioning solution were accurately measured for detection, and the retention times of diclofenac sodium, impurity A, impurity B, impurity C, impurity E and methylparaben were analyzed to be 27.922 min, 19.786 min, 30.022 min, 24.073 min, 10.231 min and 12.454 min, respectively;

[0080] Then accurately measure the mixed solution for testing and record the chromatogram. The results are shown in Figure 3 .

[0081] Under the chromatographic conditions of Example 1, Example 2, and Example 3, the chromatographic peaks of diclofenac sodium, impurity A, impurity B, impurity C, impurity E, and methylparaben can be effectively distinguished, and the chromatographic peaks of diclofenac sodium, impurity A, impurity B, impurity C, impurity E and adjacent chromatographic peaks can achieve baseline separation, and the peak shape is good, which is conducive to identification and positioning, indicating that the chromatographic conditions of Example 1, Example 2, and Example 3 are all good in specificity.

[0082] Among them, Examples 1 to 3 are the most representative implementation methods of the method of the present invention. With reference to the 2020 edition of the Chinese Pharmacopoeia 9101 Analytical Method Validation Guidelines, the chromatographic conditions in Examples 1 to 3 were methodologically validated. The contents include:

[0083] Example 4 Specificity Test

[0084] Solution preparation:

[0085] System suitability solution: Take about 10 mg of diclofenac sodium reference substance into a 10 mL volumetric bottle, add water to dissolve and dilute to the scale, expose the solution to ultraviolet light (254 nm) for 15 min, and use it as the diclofenac sodium reference substance positioning solution;

[0086] Location solutions of impurity A, impurity B, impurity C, and impurity E: weigh about 1 mg of impurity A reference substance, impurity B reference substance, impurity C reference substance, and impurity E reference substance, respectively, place them in 10 mL volumetric flasks, add methanol to dissolve and dilute to the scale, shake well, and obtain the stock solutions of impurity A, impurity B, impurity C, and impurity E; take 1 mL of each stock solution of impurity A, impurity B, impurity C, and impurity E, place them in 20 mL volumetric flasks, add methanol to dissolve and dilute to the scale, shake well, and use them as the location solutions of impurity A, impurity B, impurity C, and impurity E;

[0087] Methylparaben localizing solution: Take 5 mg of methylparaben reference substance, place it in a 250 mL volumetric flask, add methanol to dissolve and dilute to the mark, shake well, and use this as the methylparaben localizing solution;

[0088] Mixed solution: Take 4 mg of methyl paraben to a 10 mL volumetric bottle, add methanol to dissolve and dilute to the mark, shake well, and use as the methyl paraben stock solution; take 1 mL of the methyl paraben stock solution and 1 mL of each of the impurity A, B, C, and E stock solutions to the same 20 mL volumetric bottle, then weigh about 2 g of diclofenac sodium gel, add methanol to dissolve and dilute to the mark, shake well, let stand for 10 min, filter with a 0.45 μm PTFE filter membrane, discard 2 mL of the initial filtrate, and take the subsequent filtrate to obtain a mixed solution;

[0089] Determination: Accurately measure each of the above-mentioned positioning solutions, and analyze the samples according to the chromatographic conditions selected in Example 1. The retention times of diclofenac sodium, impurity A, impurity B, impurity C, impurity E and methylparaben are 19.782 min, 14.678 min, 22.356 min, 17.229 min, 7.866 min, and 9.189 min, respectively;

[0090] Then accurately measure the above mixed solution, analyze it according to the chromatographic conditions selected in Example 1, and record the chromatogram. The results are as follows: Figure 1 shown.

[0091] The results show that the chromatographic conditions under this embodiment can effectively distinguish the chromatographic peaks of diclofenac sodium, impurity A, impurity B, impurity C, impurity E and methylparaben. The chromatographic peaks of diclofenac sodium, impurity A, impurity B, impurity C, impurity E and adjacent chromatographic peaks can achieve baseline separation, and the peak shape is good, which is conducive to identification and positioning, indicating that the specificity is good.

[0092] Example 5 Forced degradation test

[0093] Solution preparation:

[0094] Undamaged test solution: Take about 2.5 g of diclofenac sodium gel, weigh accurately, put into a 25 mL volumetric flask, add methanol to dissolve and dilute to the scale, shake well, let stand for 10 min, filter with a 0.45 μm PTFE filter membrane, discard 2 mL of the initial filtrate, and take the subsequent filtrate;

[0095] Acid destruction test solution: Take about 2.5g of diclofenac sodium gel, accurately weigh it, put it into a 25mL volumetric flask, add 10mL of methanol, then add 1mL of 1mol / L hydrochloric acid, shake well, let it stand at room temperature for 3h, add 1mL of 1mol / L sodium hydroxide to neutralize, add methanol to dilute to the scale, shake well, let it stand for 10min, filter it in the same way, and take the filtrate to obtain;

[0096] Alkali destruction test solution: take about 2.5g of diclofenac sodium gel, accurately weigh, put into a 25mL volumetric flask, add 10mL of methanol, add 1mL of 1mol / L sodium hydroxide, shake well, leave at room temperature for 1d, add 1mL of 1mol / L hydrochloric acid to neutralize, add methanol to dilute to the scale, shake well, filter in the same way, and take the filtrate;

[0097] Oxidative destruction of the test solution: Take about 2.5g of diclofenac sodium gel, accurately weigh it, put it into a 25mL volumetric flask, add 10mL of methanol, add 1mL of 3% hydrogen peroxide solution, shake well, let it stand at room temperature for 1d, add methanol to dilute to the scale, shake well, let it stand for 10min, filter it in the same way, and take the filtrate;

[0098] Heat-destroyed test solution: Take about 2.5 g of diclofenac sodium gel, accurately weigh it, put it into a 25 mL volumetric flask, place it at 80 ° C for 1 day, let it cool, add methanol to dissolve and dilute to the scale, shake well, let it stand for 10 minutes, filter it in the same way, and take the filtrate;

[0099] Photodestruction test solution: Take about 2.5g of diclofenac sodium gel, accurately weigh it, put it into a 25mL volumetric flask, add methanol to dissolve and dilute to the scale, shake well, and place it in a light box (visible light intensity 5000±500Lx; ultraviolet light intensity 90±5μW / cm 2 ) Leave it for 1 day, add methanol to dilute to the mark if necessary, shake well, filter in the same way, and take the filtrate;

[0100] Determination: Accurately measure each of the above solutions, analyze according to the chromatographic conditions selected in Example 1, and record the chromatogram. The results are as follows: Figure 4 (Acid Destruction Spectrum), Figure 5 (Thermal damage map), Figure 6 (Light damage spectrum) shown.

[0101] The results showed that diclofenac sodium gel was unstable mainly under acid and heat destruction conditions, and the main degradation product was diclofenac methyl ester, which was generated by reaction with the solvent methanol. Under light destruction conditions, photodegradable impurities with a retention time close to that of impurity C could be degraded in trace amounts. It could also be observed that in the chromatograms of each destruction solution, all degradation impurities could be detected, the separation between the peaks of each component should not be less than 1.5, the separation between the main peak and the adjacent impurity peak should not be less than 2.0, and the purity angle of the main peak was less than the purity threshold, indicating that this method has a good detection ability for potential degradation impurities.

[0102] Example 6 Strong Linearity Test and Correction Factors

[0103] Solution preparation:

[0104] Linear stock solution: Accurately weigh 10.09 mg, 10.03 mg, 9.74 mg, 10.05 mg, and 10.04 mg of diclofenac sodium, impurity A, impurity B, impurity C, and impurity E reference substances, place them in a 100 mL volumetric flask, add methanol to dissolve and dilute to the scale, and shake well;

[0105] 10%, 50%, 100%, 150%, 200% linear solutions: Accurately measure 0.1, 0.5, 1, 1.5, 2 mL of linear stock solution into the same 25 mL volumetric flask, add methanol to dilute to the scale, and shake well.

[0106] Determination: Accurately measure each of the above linear solutions, analyze them according to the chromatographic conditions selected in Example 1, and record the chromatogram.

[0107] The results are as follows Figure 7 , Figure 8 , Fig. 9 , Fig.10 , Fig.11 shown.

[0108] The results showed that diclofenac and impurity A, impurity B, impurity C and impurity E had good linearity in the ranges of 0.50μg / mL~10.09μg / mL, 0.50μg / mL~10.03μg / mL, 0.49μg / mL~9.74μg / mL, 0.50μg / mL~10.05μg / mL and 0.50μg / mL~10.04μg / mL, respectively, which met the validation requirements.

[0109] The correction factors of impurities A, B, C, and E were calculated based on the slopes of the linear regression equations of diclofenac and impurities A, B, C, and E, which were 0.66, 0.58, 1.04, and 0.28, respectively, meeting the validation requirements.

[0110] Example 7 Sensitivity Test

[0111] The 10% linear solution in the linear test of Example 6 was diluted step by step to obtain mixed reference solutions of different concentrations, as shown in Table 1.

[0112] Determination: Accurately measure the mixed reference solution of different concentrations, analyze the sample according to the chromatographic conditions selected in Example 1, and record the chromatogram; calculate the signal-to-noise ratio of each component peak, and take the signal-to-noise ratio not less than 10 as the quantitative limit and not less than 3 as the detection limit. The results are shown in Table 1

[0113] Table 1 Sensitivity test

[0114]

[0115] The results showed that the signal-to-noise ratio of the quantitative limit was not less than 10, and the signal-to-noise ratio of the detection limit was not less than 3, with high sensitivity, which met the verification requirements.

[0116] Example 8 Repeatability Test

[0117] Solution preparation:

[0118] Test solution: Take about 2.5 g of diclofenac sodium gel, weigh accurately, put it into a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, let stand for 10 minutes, filter with a 0.45 μm PTFE filter membrane, discard 2 ml of the initial filtrate, and take the subsequent filtrate; prepare 6 portions in parallel.

[0119] Control solution: Accurately take 1 ml of the test solution into a 200 ml volumetric flask, add methanol to dilute to the scale, and shake well.

[0120] Determination: Accurately measure the above test solution and control solution, analyze them according to the chromatographic conditions selected in Example 1, record the chromatogram, and calculate the impurity content, as shown in Table 2.

[0121] Table 2 Repeatability test

[0122]

[0123] The results showed that in the six test solutions, the RSDs of the individual impurity contents and the total amount of impurities detected were all lower than 10.0%, with good repeatability, meeting the validation requirements.

[0124] Example 9 Intermediate Precision Test

[0125] Two repeatability tests were performed according to Example 8 by different testers at different times. The results are shown in Table 3.

[0126] Table 3 Intermediate precision test

[0127]

[0128]

[0129] The results showed that in two repeatability tests, each with 6 test solutions, the RSDs of the detected individual impurity contents and the total amount of impurities were both lower than 10.0%, and the relative deviations of the means of the detected individual impurity contents and the total amount of impurities were both lower than 10.0%; in two repeatability tests, a total of 12 test solutions, the RSDs of the detected individual impurity contents and the total amount of impurities were both lower than 10.0%, the intermediate precision was good, and met the validation requirements.

[0130] Example 10 Accuracy Test

[0131] Solution preparation:

[0132] Mixed impurity stock solution: Accurately weigh about 10 mg of each of impurity A, impurity B, impurity C, and impurity E reference substances, place them in a 100 ml volumetric flask, add methanol to dissolve and dilute to the scale, and shake well.

[0133] 10%, 50%, 100%, 150% recovery rate solution: Take about 2.5g of diclofenac sodium gel, weigh accurately, put it into a 25ml volumetric flask, accurately add 0.1ml / 0.5ml / 1ml / 1.5ml mixed impurity stock solution, add methanol to dissolve and dilute to scale, shake well, let stand for 10min, filter with a 0.45μm PTFE filter membrane, discard 2ml of the initial filtrate, and take the subsequent filtrate. Prepare 3 copies of each recovery rate solution in parallel.

[0134] Control solution: Accurately take 1 ml of the recovery solution and put it into a 200 ml volumetric flask, add methanol to dilute to the scale, and shake well.

[0135] Determination: Accurately measure the above recovery solution and control solution, analyze the samples according to the chromatographic conditions selected in Example 1, record the chromatogram, and calculate the impurity recovery rate, as shown in Table 4, Table 5, Table 6, and Table 7.

[0136] Table 4 Recovery rate of impurity A

[0137]

[0138]

[0139] Table 5 Recovery rate of impurity B

[0140]

[0141] Table 6 Impurity C recovery rate

[0142]

[0143] Table 7 Recovery rate of impurity E

[0144]

[0145] The results showed that when the added amounts of impurities A, B, C and E were 10%, 50%, 100% and 150% of the limit, the recoveries were all within the range of 90% to 110%, and the RSD of the recoveries at each concentration was lower than 10%, with good accuracy, meeting the verification requirements.

[0146] Example 11 Stability Test

[0147] Solution preparation:

[0148] Test solution: Take about 2.5 g of diclofenac sodium gel, weigh accurately, put it into a 25 ml volumetric flask, add methanol to dissolve and dilute to the scale, shake well, let stand for 10 minutes, filter with a 0.45 μm PTFE filter membrane, discard 2 ml of the initial filtrate, and take the subsequent filtrate.

[0149] Place at room temperature for different time periods: 0d, 1d, 3d, and 5d.

[0150] Determination: Accurately measure the test solution at different times, analyze it according to the chromatographic conditions selected in Example 1, record the chromatogram, and calculate the impurity content according to the normalization method. The results are shown in Table 8.

[0151] Table 8 Stability test

[0152]

[0153]

[0154] The results show that when the test solution is placed at room temperature for 1, 3, and 5 days, the maximum relative deviations of the individual impurity content and the total impurity content compared with time 0 are all less than 10%. The test solution is stable at room temperature for at least 5 days, which meets the verification requirements.

[0155] Example 12 Durability Test

[0156] The chromatographic conditions provided in Example 1 were taken as standard conditions. On this basis, other parameters were kept unchanged, and the durability conditions were set as column temperature ±5°C, flow rate ±0.2ml / min, glacial acetic acid concentration ±10%, initial methanol ratio ±2%, detection wavelength ±2nm, and the use of chromatographic columns of different brands / models.

[0157] The same batch of diclofenac sodium gel was tested under standard conditions and various durability conditions. The test solution and the control solution were prepared according to the repeatability test of Example 8. The results are shown in Table 9.

[0158] Table 9 Durability test

[0159]

[0160] The results showed that when the related substances of the same batch of diclofenac sodium gel were determined under various durability conditions, the maximum relative deviations of the detected single impurity content and the total amount of impurities compared with the standard conditions were all less than 10%, with good durability and meeting the verification requirements.

[0161] Comparative Example 1

[0162] A method for determining related substances in diclofenac sodium gel adopts the method for related substances of diclofenac sodium in the 2020 edition of the Chinese Pharmacopoeia, and the specific steps are as follows:

[0163] (1) Preparation: exactly the same as in Example 1;

[0164] (2) Determination: The content of diclofenac sodium and related substances in diclofenac sodium gel was determined by high performance liquid chromatography. The chromatographic conditions were as follows: the chromatographic column was Agilent HC-C18 (2), 150 mm × 4.6 mm, 5 μm, the column temperature was 25° C., the detection wavelength was 254 nm, the feed volume was 10 μL, the flow rate was 1 mL / min, the mobile phase was 4% glacial acetic acid solution as mobile phase A and methanol as mobile phase B, and isocratic elution was performed at a volume ratio of mobile phase A:mobile phase B of 65:35;

[0165] Diclofenac sodium reference solution, impurity A solution, impurity B solution, impurity C solution, impurity E solution and methylparaben solution were added respectively, and the retention times of diclofenac sodium, impurity A, impurity B, impurity C, impurity E and methylparaben were detected to be 15.039min, 6.614min, 17.384min, 10.949min, 2.482min and 2.803min respectively;

[0166] Then add the mixed solution and record the chromatogram. The results are shown in Fig.12 .

[0167] from Fig.12 It can be seen that under the chromatographic conditions of Comparative Example 1, methylparaben and impurity E are poorly separated and baseline separation cannot be achieved. Moreover, due to isocratic elution, the peak shapes of the components are symmetrical, but the peaks are wide and the sensitivity is poor, so gradient elution is necessary.

[0168] Comparative Example 2

[0169] A method for determining related substances in diclofenac sodium gel, according to literature methods, the specific steps are as follows:

[0170] (1) Preparation:

[0171] System suitability solution: Take about 10 mg of diclofenac sodium reference substance into a 10 mL volumetric bottle, add ethanol to dissolve and dilute to the scale, expose the solution to ultraviolet light (254 nm) for 15 min, and use it as the diclofenac sodium reference substance positioning solution;

[0172] Positioning solution: Weigh about 1 mg of impurity A reference substance, impurity B reference substance, impurity C reference substance, and impurity E reference substance, respectively, place them in 10 mL volumetric flasks, add ethanol to dissolve and dilute to the scale, shake well, and obtain the stock solutions of impurity A, impurity B, impurity C, and impurity E; take 1 mL of each stock solution of impurity A, impurity B, impurity C, and impurity E, place them in 20 mL volumetric flasks, add ethanol to dissolve and dilute to the scale, shake well, and use them as the positioning solution;

[0173] Methylparaben localizing solution: Take 5 mg of methylparaben reference substance, place it in a 250 mL volumetric flask, add ethanol to dissolve and dilute to the mark, shake well; this is used as the methylparaben localizing solution;

[0174] Mixed solution: Take 4 mg of methylparaben and put it into a 10 mL volumetric flask, add ethanol to dissolve and dilute to the scale, shake well to obtain the methylparaben stock solution; take 1 mL of the methylparaben stock solution and 1 mL of the impurity A, B, C, and E stock solutions into the same 20 mL volumetric flask, then weigh about 2 g of diclofenac sodium gel, add ethanol to dissolve and dilute to the scale, shake well, let stand for 10 minutes, filter with a 0.45 μm PTFE filter membrane, discard 2 mL of the initial filtrate, and take the subsequent filtrate to obtain a mixed solution.

[0175] (2) Determination: The content of diclofenac sodium and related substances in diclofenac sodium gel was determined by high performance liquid chromatography. The chromatographic conditions were as follows: the chromatographic column was Waters XBridge C18, 250 mm × 4.6 mm, 5 μm, the sample plate was not temperature controlled, the detection wavelength was 254 nm, the feed volume was 5 μL, the flow rate was 1 mL / min, and the mobile phase was trifluoroacetic acid solution (1 L of pure water, added with trifluoroacetic acid to adjust to pH 2.0)-methanol (80:20) as mobile phase A and acetonitrile-methanol (80:20) as mobile phase B for gradient elution;

[0176] Gradient elution program:

[0177] Time min Mobile phase A / (v)% Mobile phase B / (v)% 0 70 30 10 70 30 14 45 55 20 45 55 25 30 70 30 30 70 35 10 90 45 10 90 45.1 70 30 60 70 30

[0178] Diclofenac sodium reference solution, impurity A solution, impurity B solution, impurity C solution, impurity E solution and methylparaben solution were added respectively, and the retention times of diclofenac sodium, impurity A, impurity B, impurity C, impurity E and methylparaben were detected to be 15.021min, 6.665min, 17.429min, 10.934min, 2.409min and 2.799min, respectively;

[0179] Then add the mixed solution and record the chromatogram. The results are shown in Fig.13 .

[0180] from Fig.13 It can be seen that under the chromatographic conditions of Comparative Example 2, the retention time of impurity E is too short, and the solvent peak interferes with it; the separation of the chromatographic peaks of diclofenac sodium, methylparaben, and impurity B is poor, and baseline separation cannot be achieved, and the chromatographic peaks of methylparaben and impurity B completely overlap; the chromatographic peaks of impurity A and impurity C are slightly tailed. The chromatographic conditions of this Comparative Example 2 cannot meet the detection requirements of related substances of diclofenac sodium gel.

[0181] In summary, 1. The existing methods cannot meet the detection needs of related substances of diclofenac sodium gel: (1) The current preparation standards do not include the related substance methods of diclofenac sodium gel, and foreign pharmacopoeias do not include diclofenac sodium gel preparation varieties; (2) The related substance methods of different dosage forms of diclofenac sodium included in the 2020 edition of the Chinese Pharmacopoeia are high-performance liquid chromatography methods with isocratic elution of a high proportion of organic phase, and the selectivity for impurity E is poor; (3) The related substance methods of diclofenac sodium gel reported in the literature use a mobile phase system and elution procedure different from that of the present invention, and the selectivity for impurities B and E is poor.

[0182] 2. The analytical method of the present invention can simultaneously detect impurities A, B, C, and E, achieve effective separation of related impurities from diclofenac sodium and methylparaben, and can further achieve qualitative and quantitative detection, which is beneficial to the quality control of diclofenac sodium gel.

[0183] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the present invention can be implemented in other specific forms without departing from the spirit and essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0184] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for determining related substances in diclofenac sodium gel, characterized in that: The following steps are involved: (1) Preparation: Use methanol as solvent to prepare the test solution, control solution and sensitivity solution, and use water as solvent to prepare the system suitability solution; (2) Determination: Determine the content of diclofenac sodium and related substances in diclofenac sodium gel by high performance liquid chromatography, add test solution, reference solution, system suitability solution and sensitivity solution respectively, and record the chromatogram; Chromatographic conditions: The chromatographic column was filled with octadecylsilane bonded silica gel, the column temperature was 25-45°C, the detection wavelength was 254nm, the feed volume was 10μL, the flow rate was 0.8-1.2mL / min, and the mobile phase was glacial acetic acid solution as mobile phase A and methanol as mobile phase B for gradient elution.

2. The method for determining related substances in diclofenac sodium gel according to claim 1, characterized in that: In step (2), the gradient elution program is: 0min to 15min: the volume fraction of mobile phase A is reduced from 90% to 35%, and the volume fraction of mobile phase B is increased from 10% to 65%; 15min to 50min: the volume fraction of mobile phase A is maintained at 35%, and the volume fraction of mobile phase B is maintained at 65%; 50min to 50.1min: the volume fraction of mobile phase A is increased from 35% to 90%, and the volume fraction of mobile phase B is reduced from 65% to 10%; 50.1min to 60min: the volume fraction of mobile phase A was maintained at 90%, and the volume fraction of mobile phase B was maintained at 10%.

3. The method for determining related substances in diclofenac sodium gel according to claim 1, characterized in that: In step (2), mobile phase A is 3.6% to 4.4% glacial acetic acid solution.

4. The method for determining related substances in diclofenac sodium gel according to claim 3, characterized in that: Mobile phase A was 4% glacial acetic acid solution.

5. The method for determining related substances in diclofenac sodium gel according to claim 1, characterized in that: In step (2), the specification of the chromatographic column is 4.6 mm×150 mm, and the filler particle size is 5 μm.

6. The method for determining related substances in diclofenac sodium gel according to claim 5, characterized in that: The chromatographic column is Agilent HC-C18(2), 150 mm×4.6 mm, 5 μm.

7. The method for determining related substances in diclofenac sodium gel according to claim 1, characterized in that: In step (2), the column temperature is 35°C.

8. The method for determining related substances in diclofenac sodium gel according to claim 1, characterized in that: In step (2), the flow rate is 1.0 mL / min.

9. The method for determining related substances in diclofenac sodium gel according to claim 1, characterized in that: In step (1), the test solution is prepared from diclofenac sodium gel as a raw material and methanol as a solvent to prepare a test solution with a diclofenac sodium concentration of about 1 mg / mL; The control solution is prepared with the test solution as the raw material and methanol as the solvent to prepare a control solution with a diclofenac sodium concentration of about 5 μg / mL; The sensitivity solution is prepared by using the control solution as a raw material and methanol as a solvent to prepare a sensitivity solution having a diclofenac sodium concentration of about 0.5 μg / mL; The system suitability solution is prepared with diclofenac sodium as the reference substance raw material and water as the solvent to prepare a system suitability solution with a diclofenac sodium concentration of about 1 mg / mL, and is exposed to ultraviolet light for 15 minutes.

10. The method for determining related substances in diclofenac sodium gel according to claim 1, characterized in that: In step (3), the related substances are: impurity A: 1-(2,6-dichlorophenyl)-1,3-dihydro-2H-indol-2-one; impurity B: 1-(2,6-dichlorophenylamino)benzaldehyde; impurity C: 1-(2,6-dichlorophenylamino)benzyl alcohol; impurity E: 2-indolone.

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