Method for analyzing related substances of sildenafil citrate oral soluble film

Abstract

The invention provides a method for analyzing related substances of a sildenafil citrate oral-soluble film, which comprises the following steps: dissolving the sildenafil citrate oral-soluble film in an acetonitrile-water solution, and uniformly shaking to obtain a test solution; the method comprises the following steps: respectively dissolving a sildenafil citrate reference substance and related substance A, B and F reference substances in an acetonitrile-water solution, and uniformly shaking to obtain a reference substance solution; detecting the test solution and the reference solution by adopting a high performance liquid chromatography, wherein the detection adopts gradient elution; wherein the mobile phase A is a monopotassium phosphate solution and acetonitrile, and the mobile phase B is an acetonitrile-water solution. According to the scheme, a high performance liquid chromatography method is adopted, automatic operation can be achieved, repeatability is good, accuracy is high, sensitivity is high, the content of all components in a sample can be effectively detected, detection of related substances can also be taken into consideration, the detection time needed during quality control is shortened, and the analysis efficiency is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug analysis, and in particular to a method for analyzing substances related to sildenafil citrate orally dissolving film. Background Art

[0002] Sildenafil citrate orodissolving film is a fast-dissolving oral preparation used to treat erectile dysfunction (ED). It is a phosphodiesterase type 5 (PDE5) inhibitor. Its active ingredient is the same as in standard tablets, but it is rapidly absorbed through the oral mucosa, eliminating the need for water.

[0003] ‌ Related substances in sildenafil citrate orally disintegrating film refer to substances other than the main components of the orally disintegrating film in the drug. They may be other raw materials, reagents introduced during the production process according to the established process, intermediates, by-products and stereoisomers produced during the production process, or degradation products and polymers produced during normal storage and transportation due to the influence of environmental conditions. These related substances not only reduce the efficacy of the drug, but also have an extremely close relationship with people's health and life safety. An important part of drug safety evaluation is the content control of related substances in the drug. ‌ There are three common related substances in sildenafil citrate orally disintegrating film. The specific names and structures are as follows: The structure of related substance A of sildenafil citrate orally disintegrating film is: , Chemical formula: C 23 H 32 N6O4S, relative molecular mass: 488.61, chemical name: 5-[2-ethoxy-5-[(4-methylpiperazin-1-yl)sulfonyl]phenyl]-1-methyl-3-(2-methylpropyl)-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one; The structure of the related substance B of sildenafil citrate orally disintegrating film is: , Chemical formula: C 22 H 30 N6O5S, relative molecular mass: 490.58, chemical name: 1-methyl-4-[[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)phenyl]sulfonyl]piperazine 1-oxide; The structure of the related substance F of sildenafil citrate orally disintegrating film is: , Chemical formula: C 21 H 28N6O4S, relative molecular mass: 460.55, chemical name: 5-[2-ethoxy-5-(piperazin-1-ylsulfonyl)phenyl]-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one.

[0004] In order to strictly control the quality of sildenafil citrate orally disintegrating film and ensure the safety and effectiveness of clinical medication, it is necessary to establish an analytical method for the above-mentioned related substances. However, the existing technology lacks an analysis method for sildenafil citrate orally disintegrating film and its related substances. Summary of the Invention

[0005] The main purpose of the present invention is to propose a method for analyzing related substances of sildenafil citrate orally dissolving film, aiming to solve the technical problem that the existing technology lacks analysis of related substances of sildenafil citrate orally dissolving film.

[0006] To achieve the above objectives, the present invention provides a method for analyzing related substances in sildenafil citrate orally dissolving film. The chemical structures of the related substances are shown below: Related substance A ; Related substance B ; Related substance F ; The analytical method comprises the following steps: Dissolve the sildenafil citrate orally disintegrating film in an acetonitrile-water solution and shake well to obtain the test solution; Dissolve the sildenafil citrate reference substance and related substances A, B, and F reference substances in an acetonitrile-water solution and shake well to obtain a reference substance solution; The test solution and the reference solution are detected by high performance liquid chromatography, wherein the detection adopts gradient elution; Among them, mobile phase A is potassium dihydrogen phosphate solution and acetonitrile, and mobile phase B is acetonitrile-water solution.

[0007] Optionally, the elution conditions of the gradient elution are as follows: At 0 min, mobile phase A was 80% and mobile phase B was 20%; At 15 min, mobile phase A was 65% and mobile phase B was 35%; At 30 min, mobile phase A was 36% and mobile phase B was 64%; At 40 min, mobile phase A was 36% and mobile phase B was 64%; At 45 min, mobile phase A was 80% and mobile phase B was 20%; At 50 min, mobile phase A was 80% and mobile phase B was 20%.

[0008] Optionally, the volume ratio of potassium dihydrogen phosphate solution to acetonitrile in the mobile phase A is 80:20.

[0009] Optionally, the pH of the mobile phase A is 6.4-6.6.

[0010] Optionally, the volume ratio of acetonitrile to water solution in the mobile phase B is 80:20.

[0011] Optionally, the chromatographic column for detection uses octadecylsilane bonded silica gel as a filler.

[0012] Optionally, the column temperature of the detection is 38~42°C.

[0013] Optionally, the detection wavelength is 288~292nm.

[0014] Optionally, the flow rate of the mobile phase for the detection is 0.8~1.2mL / min.

[0015] Optionally, the injection volume of the detection is 10 μL.

[0016] The technical solution of the present invention utilizes a high-performance liquid chromatography (HPLC) method, which is automated and offers excellent repeatability, high accuracy, and high sensitivity. This method can effectively detect the content of each component in a sample while also allowing for the inspection of related substances, thereby reducing the testing time required for quality control and improving analysis efficiency. The target related substances A, B, and F in the component content can be effectively separated, and impurities in the sample can be effectively detected using this method, providing a reasonable basis for establishing improved quality standards for preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. 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 the structures shown in these drawings without paying any creative work.

[0018] Figure 1 The figure is a flow chart of an embodiment of a method for analyzing related substances of sildenafil citrate orally dissolving film provided by the present invention.

[0019] Figure 2 This is a chromatogram of the system suitability-sensitivity solution in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 3 1. The chromatogram of the system suitability-system suitability solution in the first embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 4 This is a chromatogram of the system suitability of the reference solution 01 in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 5 This is a chromatogram of the system suitability-reference solution 02 in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 6 This is a chromatogram of the system suitability of the reference solution 03 in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 7 This is a chromatogram of the system suitability-reference solution 04 in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 8 This is a chromatogram of the system suitability-reference solution 05 in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 9 This is a chromatogram of the system suitability-reference solution 06 in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 10 This is a chromatogram of a specific blank solution in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 11 This is a chromatogram of the specificity-24010203 blank excipient film solution in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 12 This is a chromatogram of the specificity-impurity A localization solution in the first embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 13 This is a chromatogram of the specificity-impurity B localization solution in the first embodiment of the analysis method for related substances of sildenafil citrate orally dissolving film provided by the present invention; Figure 14 This is a chromatogram of the specificity-impurity F localization solution in the first embodiment of the analysis method for related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 15 This is a chromatogram of a specific citric acid solution in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 16 This is a chromatogram of the linear-linear solution 01 in the first embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 17This is a chromatogram of the linear-linear solution 02 in the first embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 18 This is a chromatogram of the linear-linear solution 03 in the first embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 19 This is a chromatogram of the linear-linear solution 04 in the first embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 20 This is a chromatogram of the linear-linear solution 05 in the first embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 21 is the linear-sildenafil linear regression equation in the first embodiment of the analysis method for related substances of sildenafil citrate orally dissolving film provided by the present invention; Figure 22 is the linear regression equation of the linear-impurity A in the first embodiment of the analysis method for related substances of sildenafil citrate orally dissolving film provided by the present invention; Figure 23 is the linear regression equation of the linearity-impurity B in the first embodiment of the analysis method for related substances of sildenafil citrate orally dissolving film provided by the present invention; Figure 24 is the linear regression equation of the linear-impurity F in the first embodiment of the analysis method for related substances of sildenafil citrate orally dissolving film provided by the present invention; Figure 25 This is a chromatogram of the solution stability of the reference solution from 01 to 9 hours in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 26 This is a chromatogram of the solution stability of the reference solution from 01 to 23 hours in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 27 This is a chromatogram of the solution stability of the reference solution from 01 to 32 hours in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 28 This is a chromatogram of the solution stability of the reference solution from 01 to 38 hours in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 29 This is a chromatogram of the solution stability of the reference solution from 01 to 62 hours in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 30This is a chromatogram of the solution stability of the reference solution from 01 to 85 hours in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 31 This is a chromatogram of the solution stability of the 25240101E test solution at 0 h in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 32 This is a chromatogram of the solution stability of the 25240101E test solution for 14 hours in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 33 This is a chromatogram of the solution stability of the 25240101E test solution over 22.5 hours in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 34 This is a chromatogram of the solution stability of the 25240101E test solution over 29 hours in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 35 This is a chromatogram of the solution stability of the 25240101E test solution for 53 hours in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; Figure 36 This is a chromatogram of the solution stability of the 25240101E test solution over 76 hours in an embodiment of the method for analyzing related substances of the sildenafil citrate orally dissolving film provided by the present invention; The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0020] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0021] It should be noted that, in the embodiments, those without specifying specific conditions, are carried out according to conventional conditions or the conditions recommended by the manufacturer. Those for reagents or instruments used that do not specify the manufacturer are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes. Taking "A and / or B" as an example, it includes schemes A, B, or A and B that meet the same time. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on those of ordinary skill in the art. When the combination of the technical solutions is contradictory or cannot be realized, it should be considered that the combination of such technical solutions does not exist and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work belong to the scope of protection of the present invention.

[0022] The technical scheme of the present invention is further described in detail below with reference to specific examples and accompanying drawings. It should be understood that the following examples are merely illustrative of the present invention and are not intended to limit the present invention. Information on the instruments, reference substances, materials, reagents, test solutions, chromatographic columns, chromatographic conditions, etc. used in this example is shown in Tables 1-4 below: Table 1 Instruments and equipment

[0023] Table 2 Reagents and materials

[0024] Table 3 Chromatographic columns

[0025] Table 4 Chromatographic conditions 1. Materials

[0026] Diluent: acetonitrile-water solution (volume ratio 20:80).

[0027] Blank solution: Take the diluent, place it in a 100mL volumetric flask, add the diluent to the mark to obtain a blank solution.

[0028] Test solution: Cut one piece of Sildenafil Citrate Orally Dissolving Film (50 mg, manufactured by Shanghai Xinfeng Pharmaceutical Co., Ltd.) into pieces and place in a 100 ml volumetric flask; cut one piece of Sildenafil Citrate Orally Dissolving Film (25 mg, manufactured by Shanghai Xinfeng Pharmaceutical Co., Ltd.) into pieces and place in a 50 ml volumetric flask. Add an appropriate amount of diluent, vortex and oscillate to dissolve the film, then add diluent to dilute to the mark and shake well.

[0029] Reference solution: Accurately weigh approximately 14 mg of sildenafil citrate reference substance (equivalent to approximately 10 mg of sildenafil) and place in a 100 ml volumetric flask. Add diluent to dissolve and dilute to the mark, then shake well. Accurately measure 1 ml and place in a 100 ml volumetric flask. Add diluent to dilute to the mark, then shake well to obtain the sildenafil citrate reference solution.

[0030] Take about 10 mg of impurity A reference substance, accurately weigh it, place it in a 100 mL volumetric flask, add diluent to dissolve it, add diluent to the scale, and shake well to obtain impurity A stock / fixing solution; Take about 10 mg of impurity B reference substance, accurately weigh it, place it in a 100 ml volumetric flask, add diluent to dissolve it, add diluent to the scale, and shake well to obtain impurity B stock / fixing solution; Take about 10 mg of impurity F reference substance, accurately weigh it, place it in a 100 ml volumetric flask, add diluent to dissolve it, add diluent to the scale, and shake well to obtain impurity F stock / fixing solution; Accurately pipette 10 mL of each impurity stock solution prepared above into a 100 mL volumetric flask, add diluent to dilute and dissolve to the scale, and shake well to obtain the impurity mixed stock solution.

[0031] System suitability solution: Weigh approximately 17.5 mg of sildenafil citrate reference substance into a 25 mL volumetric flask, accurately add 2.5 mL of the impurity mixed stock solution, add diluent to dissolve to the mark, and shake well.

[0032] Sensitivity solution: Accurately pipette 2.5 ml of sildenafil citrate reference solution into a 10 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0033] Determine according to high-performance liquid chromatography (Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0512). Accurately measure the test sample solution and each reference solution, inject them into the liquid chromatograph, and record the chromatogram. Organize all records from the analysis of related substances in sildenafil citrate orally disintegrating film, and analyze and evaluate the recorded data. 2. Results Analysis 2.1 System Applicability

[0034] Combine Figure 2-9 ,Throughout the entire verification process, the system applicability met the requirements.,The experimental results are shown in Table 5.

[0035] Table 5 System suitability results

[0036] Result analysis: 1) System suitability requirements: The signal-to-noise ratio of the sildenafil peak in the sensitivity solution is 16, greater than 10; impurity B, impurity F, sildenafil, and impurity A elute in the system suitability solution in sequence, with a resolution of 12.9 for impurity A and sildenafil, greater than 5.0; and a resolution of 21.3 for impurity F and sildenafil, greater than 10.0; 2) The reference solution was injected six times, and the RSD of the main component peak area was 0.5%, not greater than 5.0%. The RSD of the main component peak area of all repeated injections of the reference solution were 0.5%, 1.2%, 1.1%, 2.2%, 2.2%, and 2.9%, respectively, all of which were not greater than 5.0%.

[0037] Therefore, the system suitability meets the protocol requirements. 2.2 Exclusivity

[0038] Take blank solution, system suitability solution, blank excipient, each impurity location solution (impurity stock solution), test solution, and reference solution, inject them into liquid chromatograph, and record the chromatogram. Figures 10-15 , the experimental results are shown in Table 6.

[0039] Table 6 Specificity results

[0040] Result analysis: 1) In the blank solution chromatogram, there is no interference with the determination of sildenafil peak and impurity peak.

[0041] 2) In the chromatogram of the blank excipient, there is no interference with the determination of the sildenafil peak and impurity peak.

[0042] 3) The retention time of the impurity location solution is consistent with the retention time of the impurity peak in the system suitability solution.

[0043] The specificity meets the requirements of the plan. 2.3 Specificity (Forced Degradation)

[0044] One injection of each of the undamaged solution, each damaged solution, and the corresponding blank excipient solution was performed, and the chromatograms were recorded. The experimental results are shown in Table 7.

[0045] Table 7 Forced degradation results

[0046] Result analysis: 1) Strong acid: 3 ml of 1 mol / L hydrochloric acid was placed at room temperature for 4 hours. Compared with the undamaged sample, no other impurities were found, no impurities were found near the main peak, the purity of the main peak was 1000.0, and the material balance was 99.6%; 2) Strong base: 3 ml of 1 mol / L sodium hydroxide was placed at room temperature for 4 hours. Compared with the undamaged sample, no other impurities were found, no impurities were found near the main peak, the main peak purity was 1000.0, and the material balance was 99.6%; 3) Oxidation: 5 ml of 30% hydrogen peroxide was placed at room temperature for 5 hours. Compared with the undamaged sample, the impurity B increased significantly. The area-normalized impurity B content was 7.36%, and the degradation rate was 7.4%. The other impurities did not change significantly. There were no impurities near the main peak. The purity of the main peak was 1000.0, and the material balance was 99.5%; 4) Lighting: A. Liquid: 4500Lx±500Lx, irradiated under UV conditions of 90w / m2 for 10 days. Compared with the undamaged sample, more unknown impurities appeared after degradation. Impurity F, impurity B, and impurity A increased significantly, with a degradation rate of 3.3%. The remaining impurities showed no significant changes. The separation degree of impurities near the main peak was 1.5 and not less than 1.5. The purity of the main peak was 1000.0, and the material balance was 96.8%. B. Solid: 4500Lx±500Lx, irradiated under UV conditions of 90w / m2 for 10 days. Compared with the undamaged sample, three unknown impurities appeared after degradation, all of which were far below the limit. The degradation rate was 0.1%. The remaining impurities showed no significant changes. There was no impurity peak near the main peak. The main peak purity was 1000.0, and the material balance was 97.2%. 5) High temperature: A. Liquid: After being placed in a 90℃ water bath for 3 days, the impurities B and F increased significantly compared to the undamaged sample, the degradation rate was 3.0%, there were no impurities near the main peak, the purity of the main peak was 1000.0, and the material balance was 99.4%; B. Solid: After being placed in a constant temperature box at 90℃ for 5 days, no impurities were degraded compared with the undamaged sample, no impurities were found near the main peak, the purity of the main peak was 1000.0, and the material balance was 97.9%.

[0047] In summary, the material balance of sildenafil citrate orally disintegrating film was between 95.0% and 105.0% under all decomposition conditions, the main peak purity was >990, and the resolution between each chromatographic peak and the main peak was no less than 1.5. The orally disintegrating film showed no significant degradation under either strong acid or strong base conditions. Impurities A, B, and F were produced in the solution under both high-temperature and light-induced decomposition, with impurity B being the most readily degraded product. The solid sample was stable under both high temperature and light conditions and was not easily degraded. It was also stable under both strong acid and strong base conditions and was not easily degraded. No impurities were detected in any of the blank excipient solutions. The specificity (strong degradation) met the protocol requirements. 2.4 Precision (Repeatability and Intermediate Precision)

[0048] Different experimenters injected blank solutions, system suitability solutions, reference solutions, and test solutions into the liquid chromatograph and recorded the chromatograms. The RSDs of the impurity content in the six replicate solutions were calculated. The RSDs of the impurity content in the 12 replicates (repeatability and intermediate precision) of the test solution were also calculated. The results are shown in Table 8.

[0049] Table 8 Precision determination results

[0050] Result analysis: 1) Repeatability: Impurity B and impurity F were detected in all 6 reproducible samples with good reproducibility. The detection amount of each impurity was less than 0.05%, and RSD was not required. The RSD of total impurity was 2.7%.

[0051] 2) Intermediate precision: Impurity B and impurity F were detected in all 6 reproducible samples with good reproducibility. The detection amount of each impurity was less than 0.05%, and RSD was not required. The RSD of total impurity was 1.8%.

[0052] In the 12 precision solutions, the impurity content was less than 0.05%, and RSD was not required. The RSD of total impurity was 21.5%, not more than 40%.

[0053] Therefore, the precision met the protocol requirements. 2.5 Limit of Quantitation and Limit of Detection

[0054] The quantification limit solution was injected six times continuously, and the chromatogram was recorded to obtain the RSD% of the peak area for the six injections. The detection limit solution was injected once. The results are shown in Table 9.

[0055] Table 9 Results of determination of limit of quantitation and limit of detection:

[0056]

[0057]

[0058]

[0059] Result analysis: Limit of Quantitation: The signal-to-noise ratio of impurity A was not less than 10, and the RSD of peak area was 2.1%; the signal-to-noise ratio of impurity B was not less than 10, and the RSD of peak area was 1.0%; the signal-to-noise ratio of impurity F was not less than 10, and the RSD of peak area was 1.4%; the signal-to-noise ratio of sildenafil was not less than 10, and the RSD of peak area was 3.9%.

[0060] Detection limit: The signal-to-noise ratio of impurity A, impurity B, impurity F, and sildenafil is not less than 3.

[0061] Therefore, the limits of quantification / detection were within the protocol requirements. 2.6 Linearity and Range (Impurity Relative Response Factor)

[0062] Inject each linear solution separately, record the chromatogram, make a linear regression equation, calculate the correlation coefficient, the ratio of the Y-axis intercept to the 100% response value and the residual sum of squares, and calculate the response factor. Figures 16 to 24 , the results are shown in Table 10.

[0063] Table 10 Linearity:

[0064]

[0065]

[0066]

[0067]

[0068] Result analysis: For each impurity and main component, the concentration within the limit of 20% to 200% (theoretical 0.2 μg / ml to 2 μg / ml) was plotted on the horizontal axis and the peak area on the vertical axis. The correlation coefficient r was ≥ 0.995. The Y-axis intercept / 100% response value was no greater than 0.025. The linear equation for impurity A was Y = 16.783X - 0.3495; the linear equation for impurity B was Y = 14.45X - 0.203; the linear equation for impurity F was Y = 14.211X - 0.3527; and the linear equation for the main component was Y = 15.894X + 0.0.094. The relative response factors for each impurity were: impurity A (1.1), impurity B (0.9), and impurity F (0.9), all ranging from 0.9 to 1.1, and therefore calculated as 1.0.

[0069] Therefore, the linear range (relative response factors of impurities) meets the protocol requirements. 2.7 Accuracy

[0070] Take the blank solution, test solution, and accuracy solution, inject them into the liquid chromatograph, and record the chromatogram. Calculate the recovery and relative standard deviation of each impurity at different concentration levels. The results are shown in Table 11.

[0071] Table 11 Accuracy

[0072]

[0073]

[0074] Result analysis: The recoveries at each concentration level were between 97.0% and 114.8%, and the RSDs of the recovery results were all ≤4.8%.

[0075] Therefore, the accuracy meets the protocol requirements. 2.8 Solution stability

[0076] Take blank solution, system suitability solution, reference solution, and test solution respectively, inject them into liquid chromatograph, and record the chromatogram. Observe the reference solution and test solution at different time points for comparison. Figure 25-36 , the results are shown in Table 12.

[0077] Table 12 Solution stability results

[0078]

[0079] Result analysis: 1) The reference solution, at 15 degrees Celsius, has a ratio of no more than 108.4% to its 0-hour value within 85 hours. The reference solution is stable within 85 hours.

[0080] 2) Test solution: At 15°C, the difference in impurity content between the 0-hour and 76-hour values is no greater than 0.015%, no greater than 0.03%, and no new impurities greater than 0.05% appear within 76 hours. The test solution is stable within 76 hours.

[0081] Therefore, the solution stability met the protocol requirements. 2.9 Durability

[0082] Change the wavelength (±2nm, i.e. 288nm, 292nm), flow rate (±0.2mL·min -1 , i.e. 0.8 mL min -1 、1.2mL·min -1 ), column temperature (±2℃, i.e. 42℃, 38℃), buffer salt pH value (±0.1, i.e. 6.4, 6.6), take blank solution, sensitivity solution, system suitability solution, reference solution, and test solution respectively, inject them into liquid chromatograph, record the chromatogram and calculate the content of each impurity in the test sample, and calculate the difference compared with the original conditions. The results are shown in Table 13.

[0083] Table 13 Durability results

[0084]

[0085] Result analysis: Change the wavelength (±2nm, i.e. 288nm, 292nm), flow rate (±0.2mL·min -1 , i.e. 0.8 mL min -1 、1.2mL·min -1 ), column temperature (±2°C, i.e. 42°C, 38°C), buffer salt pH value (±0.1, i.e. 6.4, 6.6): 1) System applicability complies with regulations 2) The absolute value of the difference between the impurity test results of the test solution under each condition and the sample solution results under the target parameters was no more than 0.02%, and no new impurities ≥ 0.05% appeared, indicating that slight changes in the method did not affect the impurity test results and the method was robust. 3. Verification process deviation

[0086] N / A 4. Conclusions and Recommendations

[0087] The results of the validation experiment of the analytical method for related substances in sildenafil citrate orally disintegrating film were in compliance with the regulations, proving that the analytical method was stable and reliable and suitable for the detection of related substances in sildenafil citrate orally disintegrating film.

[0088] The technical features of the above embodiments can be combined arbitrarily. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0089] The above embodiments merely represent several implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art could make various modifications and improvements without departing from the spirit of the present application, all of which fall within the scope of protection of the present application. Therefore, the scope of protection of the present patent application shall be determined by the appended claims.

Claims

1. A method for analyzing related substances of sildenafil citrate orally dissolving film, characterized in that: The chemical structures of the substances in question are shown below: Related substance A ; Related substance B ; Related substance F ; The analytical method comprises the following steps: Dissolve the sildenafil citrate orally disintegrating film in an acetonitrile-water solution and shake well to obtain the test solution; Dissolve the sildenafil citrate reference substance and related substances A, B, and F reference substances in acetonitrile-water solution respectively and shake well to obtain reference substance solutions; The test solution and the reference solution are detected by high performance liquid chromatography, wherein the detection adopts gradient elution; Among them, mobile phase A is potassium dihydrogen phosphate solution and acetonitrile, and mobile phase B is acetonitrile-water solution.

2. The method for analyzing related substances of sildenafil citrate orally dissolving film according to claim 1, wherein: The elution conditions of the gradient elution are as follows: At 0 min, mobile phase A was 80% and mobile phase B was 20%; At 15 min, mobile phase A was 65% and mobile phase B was 35%; At 30 min, mobile phase A was 36% and mobile phase B was 64%; At 40 min, mobile phase A was 36% and mobile phase B was 64%; At 45 min, mobile phase A was 80% and mobile phase B was 20%; At 50 min, mobile phase A was 80% and mobile phase B was 20%.

3. The method for analyzing related substances of sildenafil citrate orally dissolving film according to claim 1, wherein: The volume ratio of potassium dihydrogen phosphate solution to acetonitrile in the mobile phase A is 80:

20.

4. The method for analyzing related substances of sildenafil citrate orally dissolving film according to claim 1, wherein: The pH of the mobile phase A is 6.4-6.

6.

5. The method for analyzing related substances of sildenafil citrate orally dissolving film according to claim 1, wherein: The volume ratio of acetonitrile to water solution in the mobile phase B is 80:

20.

6. The method for analyzing related substances of sildenafil citrate orally dissolving film according to claim 1, wherein: The chromatographic column used in the detection uses octadecylsilane bonded silica gel as a filler.

7. The method for analyzing related substances of sildenafil citrate orally dissolving film according to claim 1, wherein: The column temperature of the detection is 38~42°C.

8. The method for analyzing related substances of sildenafil citrate orally dissolving film according to claim 1, wherein: The detection wavelength of the detection is 288~292nm.

9. The method for analyzing related substances of sildenafil citrate orally dissolving film according to claim 1, wherein: The flow rate of the mobile phase for the detection is 0.8~1.2mL / min.

10. The method for analyzing related substances of sildenafil citrate orally dissolving film according to claim 1, wherein: The injection volume for the assay was 10 μL.

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