A method for detecting polymer impurity content in Nicorandil tablets

Abstract

The present invention relates to the technical field of chemical analysis, and discloses a method for detecting the content of polymer impurities in a Nicorandil tablet, wherein the detection method comprises: dissolving a sample to be tested with a diluent to obtain a sample solution, and adopting a high performance liquid chromatography method to detect the content of polymer impurities in the sample solution; wherein the diluent is a trifluoroacetic acid-water mixed solution; and the chromatographic conditions are: using octadecylsilane bonded silica gel as a filler; using a trifluoroacetic acid-water mixed solution as solution 1, using a triethylamine-tetrahydrofuran-methanol mixed solution as solution 2, mixing solution 1 and solution 2 as mobile phase A, and using methanol as mobile phase B; and performing gradient elution. The present invention can avoid the polymer impurities of carboxymethylcellulose calcium and Nicorandil trimer and Nicorandil tetramer from generating insoluble solids, thereby eliminating interference, accurately detecting polymer impurities, being easy to operate, and having good accuracy.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, and in particular to a method for detecting the content of polymer impurities in Nicorandil tablets. Background Art

[0002] The adverse reactions of drugs in clinical use are not only related to the pharmacological activity of the drugs themselves, but also to the drug impurities. Therefore, it is directly related to the quality and safety of marketed drugs to conduct impurity research in a standardized manner and control impurities within a safe and reasonable limit.

[0003] Nicorandil, chemically known as N-(2-hydroxyethyl) nicotinamide nitrate, is the first ATP-sensitive potassium channel opener used in clinical practice, used to treat various types of angina pectoris. Nicorandil is very easy to polymerize under high temperature conditions to produce nicorandil polymers. The possible degradation impurities and degradation pathways are as follows:

[0004] The quality standard of Nicorandil Tablets is included in BP2021 and the bureau-issued standard WS-10001-(HD-0151)-2002. The BP2021 quality standard includes a method for detecting polymer impurities. The diluent used is 7% methanol solution, and Nicorandil dimer can be accurately determined. However, Nicorandil Tablets contain the excipient calcium carboxymethylcellulose. Calcium carboxymethylcellulose reacts with polymer impurities such as nicorandil trimer and nicorandil tetramer (all of which are ionized organic matter) to form insoluble solids, resulting in a decrease in the content of nicorandil trimer and nicorandil tetramer in the injection solution after centrifugation or filtration of the prepared test solution, making it impossible to accurately detect nicorandil trimer and nicorandil tetramer impurities. Summary of the invention

[0005] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a method for detecting the content of polymer impurities in nicorandil tablets. By selecting 0.1% trifluoroacetic acid solution (pH about 2.0) as a diluent, the polymer impurities of carboxymethylcellulose calcium and nicorandil trimer and nicorandil tetramer can be prevented from generating insoluble solids, thereby eliminating interference, accurately detecting polymer impurities, being easy to operate and having good accuracy.

[0006] Technical solution: The present invention provides a method for detecting the content of polymer impurities in Nicorandil tablets, the detection method comprising: dissolving a sample to be tested in a diluent to obtain a sample solution, and detecting the content of polymer impurities in the sample solution by high performance liquid chromatography; wherein the diluent is a trifluoroacetic acid-water mixture; the chromatographic conditions are: using octadecylsilane bonded silica gel as a filler; using a trifluoroacetic acid-water mixture as solution 1, using a triethylamine-tetrahydrofuran-methanol mixture as solution 2, mixing solution 1 and solution 2 as mobile phase A, and using methanol as mobile phase B; and performing gradient elution.

[0007] Furthermore, in the diluent, the pH of the mixture of trifluoroacetic acid and water is 1.5-2.5.

[0008] Furthermore, the mobile phase is subjected to linear gradient elution with the gradient shown in the following table:

[0009] In the solution 2, the volume ratio of triethylamine, tetrahydrofuran and methanol is 2:10:25; In the mobile phase A, the volume ratio of solution 1 to solution 2 is 1:1.

[0010] Furthermore, the chromatographic conditions also include: Flow rate: 1.0 ml / min; Detection wavelength: 254 nm; Column temperature: 25 °C; Sample chamber temperature: 5 °C; Injection volume: 10 μl.

[0011] Furthermore, the polymer impurities are nicorandil trimer and nicorandil tetramer, and their chemical structures are as follows: ; .

[0012] Further, the method for detecting the content of polymer impurities in Nicorandil tablets as described in any one of the above items, the specific detection steps are as follows: S1. Prepare the test solution, reference solution and system suitability solution separately and set aside; S2. Accurately measure equal amounts of the test solution, reference solution, and system suitability solution, and inject them into the HPLC respectively. Use mobile phase A and mobile phase B to separate and analyze by gradient elution, and record the chromatogram.

[0013] Furthermore, the specific preparation method of the test solution is: take the sample to be tested, add an appropriate amount of diluent, shake to dissolve, dilute with the diluent, shake well, filter, and take the filtrate; the concentration of the test solution is 1 mg / ml; The specific preparation method of the reference solution is: take Nicorandil reference, accurately weigh, add diluent to dissolve and quantify; the concentration of the reference solution is 10 μg / ml; The specific preparation method of the system suitability solution is: take a Nicorandil reference substance, place it at 70° C. for 2 hours, cool it, and then dissolve and dilute it with a diluent; the concentration of the system suitability solution is 0.8%.

[0014] On the one hand, the present invention adjusts the diluent in the method for detecting the polymer impurity content in Nicorandil tablets. The BP2021 standard uses a 7% methanol solution as a diluent, and the present invention uses a 0.1% trifluoroacetic acid solution as a diluent: in a 7% methanol solution, the excipient carboxymethylcellulose calcium in the homemade preparation and the reference preparation interferes with the determination of Nicorandil trimer and Nicorandil tetramer. Carboxymethylcellulose calcium and Nicorandil trimer and Nicorandil tetramer (both ionized organic matter) generate insoluble solids, resulting in a decrease in the content of Nicorandil trimer and Nicorandil tetramer in the solution and a decrease in the peak area; using a 0.1% trifluoroacetic acid solution (pH = 1.5-2.5) as a diluent can eliminate the interference of carboxymethylcellulose calcium with the determination of Nicorandil trimer and Nicorandil tetramer. On the other hand, the present invention optimizes the gradient elution time in the chromatographic conditions of the method for detecting the polymer impurity content in Nicorandil tablets, and the gradient elution parameters before and after optimization are as follows:

[0015] The retention times of the Nicorandil peak and each known impurity peak are all before 35 minutes. The present invention optimizes the gradient elution program and shortens the running time.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following specific beneficial effects: The present invention can prevent carboxymethylcellulose calcium and polymer impurities of nicorandil trimer and nicorandil tetramer from generating insoluble solids, thereby eliminating interference, accurately detecting polymer impurities, being easy to operate and having good accuracy; in addition, the present invention shortens the running time and makes detection faster by optimizing gradient elution. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 It is the chromatogram of the system suitability solution of related substance II; Figure 2 This is the linear graph of Nicorandil with a concentration between 0.1394 μg / ml and 21.4538 μg / ml; Figure 3It is the linear graph of Nicorandil trimer with a concentration between 0.2994 μg / ml and 9.0742 μg / ml; Figure 4 It is the linear graph of nicorandil tetramer with concentration between 0.4569 μg / ml and 9.1374 μg / ml; Figure 5 To validate the specific blank solvent spectrum for the detection method of polymer impurities in Nicorandil tablets; Figure 6 To validate the specific blank excipient spectrum for the detection method of polymer impurity content in Nicorandil tablets; Figure 7 Validation of the specific system suitability profile for the polymer impurity content test method in Nicorandil tablets; Figure 8 Spectrum of specific reference solution for verifying the detection method of polymer impurity content in Nicorandil tablets; Fig. 9 Spectrum of spiked solution of test sample for verification of polymer impurity content in Nicorandil tablets; Fig.10 Spectrum of test sample solution for verification of polymer impurity content detection method in Nicorandil tablets. DETAILED DESCRIPTION

[0017] The present invention is described in detail below in conjunction with the embodiments.

[0018] The instruments and chromatographic columns used in the present invention are shown in Table 1:

[0019] The reagents and reference substances used in the present invention are shown in Table 2:

[0020] The solution configuration method used in the present invention is as follows: 1. Diluent: trifluoroacetic acid-water mixture: trifluoroacetic acid and water are mixed in a volume ratio of 1:1000.

[0021] 2. Test solution: Take 20 tablets of this product (5 mg / tablet), place them in a 100 ml volumetric flask, add an appropriate amount of diluent, shake to dissolve, dilute to the scale with diluent, shake well, filter (0.45 μm filter membrane), take the filtrate to obtain a test solution with a concentration of 1 mg / ml.

[0022] 3. Reference solution: Take an appropriate amount of Nicorandil reference substance, weigh it accurately, add diluent to dissolve it and dilute it quantitatively to obtain a reference solution with a concentration of 10 μg / ml.

[0023] 4. System suitability solution: Take about 40 mg of Nicorandil reference substance, place it at 70℃ for 2 hours, let it cool, dissolve it in a diluent and dilute it to make a 0.8% Nicorandil solution (to produce Nicorandil trimer and Nicorandil tetramer).

[0024] The chromatographic conditions in the experiment of the present invention are as follows: octadecylsilane bonded silica gel is used as a filler (25 cm×4.6 mm, 5 μm); trifluoroacetic acid-water (1.5:965) is used as solution 1, triethylamine-tetrahydrofuran-methanol (2:10:25) is used as solution 2, solution 1 and solution 2 are mixed evenly as mobile phase A, methanol is used as mobile phase B, and gradient elution is performed according to Table 3; the flow rate is 1.0 ml per minute; the detection wavelength is 254 nm; the column temperature is 25° C.; the sample chamber temperature is 5° C.; and the injection volume is 10 μl.

[0025]

[0026] In the present invention, in the system suitability solution chromatogram, the nicorandil trimer and nicorandil tetramer peaks are referenced to the attached Figure 1 "Chromatogram of system usability solution of related substance II"; if there are nicorandil trimer and nicorandil tetramer peaks in the chromatogram of the test sample solution, the correction factors of each impurity are shown in Table 4. After multiplying the peak area of ​​each impurity by the correction factor, the peak area of ​​nicorandil is calculated using the external standard method and should comply with the corresponding limit provisions in Table 4.

[0027]

[0028] Implementation 1: Linear relationship: Nicorandil reference substance stock solution: Take an appropriate amount of Nicorandil reference substance, weigh accurately, add diluent to dissolve and quantitatively dilute to make a solution containing approximately 100 μg per 1 ml.

[0029] Nicorandil trimer reference substance stock solution: Take an appropriate amount of Nicorandil trimer reference substance, weigh accurately, add diluent to dissolve and quantitatively dilute to make a solution containing approximately 50 μg per 1 ml.

[0030] Nicorandil tetramer reference substance stock solution: Take an appropriate amount of Nicorandil tetramer reference substance, weigh accurately, add diluent to dissolve and quantitatively dilute to make a solution containing approximately 50 μg per 1 ml.

[0031] Accurately measure 0.2 ml, 0.5 ml, 0.8 ml, 1.0 ml, 1.2 ml, 1.5 ml and 2.0 ml of nicorandil reference substance stock solution, nicorandil trimer reference substance stock solution and nicorandil tetramer reference substance stock solution, respectively, and place them in 10 ml volumetric flasks, add diluent to dilute to the scale, shake well, and use them as linear 1, linear 2, linear 3, linear 4, linear 5, linear 6 and linear 7 solutions respectively.

[0032] Accurately measure the above solution and the quantitative limit solution, inject them into the liquid chromatograph respectively, record the chromatogram, and calculate the linear regression equation, such as Figure 2-4 shown.

[0033] The results showed that Nicorandil had a good linear relationship in the concentration range of 0.1394μg / ml~21.4538μg / ml, and the linear regression equation was: y = 13656x + 548.51, r = 0.9999.

[0034] Nicorandil trimer has a good linear relationship in the concentration range of 0.2994μg / ml~9.0742μg / ml, and the linear regression equation is: y = 10680x + 77.833, r = 0.9997.

[0035] Nicorandil tetramer has a good linear relationship in the concentration range of 0.4569μg / ml~9.1374μg / ml, and the linear regression equation is: y = 10251x - 932.63, r = 0.9999.

[0036] Actual sample testing (taking the testing of 2 batches of reference preparations and 5 batches of homemade preparations as an example): A trifluoroacetic acid-water mixture (1:1000) is used as a diluent, and a sample to be tested is dissolved in the diluent to prepare a sample solution with a concentration of 1 mg / ml. The polymer impurity content in the sample solution is detected by high performance liquid chromatography; wherein the chromatographic conditions are as follows: octadecylsilane bonded silica gel is used as a filler; a trifluoroacetic acid-water mixture is used as solution 1, a triethylamine-tetrahydrofuran-methanol mixture is used as solution 2, solution 1 and solution 2 are mixed evenly as mobile phase A, and methanol is used as mobile phase B; and gradient elution is performed.

[0037] The sample measurement results are shown in Table 5.

[0038]

[0039] The results showed that the determination results of polymer impurities in 2 batches of reference preparations and 5 batches of homemade Nicorandil tablets were in compliance with the regulations.

[0040] Implementation 2: Determination of correction factor and relative retention time: Linear 1 to linear 7 solutions of nicorandil trimer, nicorandil tetramer and nicorandil were prepared, and the correction factor and relative retention time of each impurity were determined on three instruments of different brands by different personnel on different dates.

[0041]

[0042] Calculation formula: Impurity correction factor = ( C 杂质 / A 杂质 ) / ( C 尼可地尔 / A 尼可地尔 ) Relative retention time of impurities = RT 杂质 / RT 尼可地尔 A 杂质 : Impurity peak area, A 尼可地尔 : Nicorandil peak area, C 杂质 : Impurity linear solution concentration (μg / ml), C 尼可地尔 : Nicorandil linear solution concentration (μg / ml), RT 杂质 : Impurity peak retention time (min), RT 尼可地尔 : Nicorandil peak retention time (min).

[0043] The results of the correction factors and relative retention times of various impurities are summarized as follows:

[0044] Exclusive properties: (1) Blank solvent: Accurately measure 10 μl of diluent (0.1% trifluoroacetic acid solution), inject it into the liquid chromatograph, and record the chromatogram. The results showed that the blank solvent did not interfere with the determination of nicorandil trimer and nicorandil tetramer.

[0045] (2) System suitability solution: Weigh about 40 mg of nicorandil reference substance, place it at 70°C for 2 hours, cool it down, and dilute it with a diluent to make a 0.8% nicorandil solution (to generate nicorandil trimer and nicorandil tetramer). Accurately measure 10 μl of system suitability solution, inject it into the liquid chromatograph, and record the chromatogram. The results showed that the relative retention times of the nicorandil trimer peak and the nicorandil tetramer peak were 1.30 and 1.63, respectively, and the resolution between the nicorandil trimer and the nicorandil tetramer was 6.31, and they could be effectively separated from adjacent chromatographic peaks.

[0046] (3) Nicotinic acid positioning solution: Take about 2 mg of nicotinic acid reference substance, weigh it accurately, put it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and use it as nicotinic acid positioning solution. Accurately measure 10 μl, inject it into the liquid chromatograph, and record the chromatogram.

[0047] (4) Nicotinic acid methyl ester positioning solution: Take about 2 mg of nicotinate methyl ester reference substance, weigh it accurately, put it in a 50 ml volumetric flask, dissolve it with diluent and dilute it to the scale, shake it well, and use it as the nicotinate methyl ester positioning solution. Accurately measure 10 μl, inject it into the liquid chromatograph, and record the chromatogram.

[0048] (5) Nicorandil EP Impurity A Location Solution: Take about 2 mg of Nicorandil EP Impurity A reference substance, accurately weigh it, place it in a 50 ml volumetric flask, dissolve it with a diluent and dilute it to the mark, shake it well, and use it as the Nicorandil EP Impurity A Location Solution. Accurately measure 10 μl, inject it into the liquid chromatograph, and record the chromatogram.

[0049] (6) Nicorandil EP Impurity B Location Solution: Take about 2 mg of Nicorandil EP Impurity B reference substance, accurately weigh it, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and use it as Nicorandil EP Impurity B Location Solution. Accurately measure 10 μl, inject it into the liquid chromatograph, and record the chromatogram.

[0050] (7) Nicorandil EP Impurity C Localization Solution: Take about 2 mg of Nicorandil EP Impurity C Reference Standard, weigh accurately, place in a 50 ml volumetric flask, add diluent to dissolve and dilute to scale, shake well, and use as Nicorandil EP Impurity C Localization Solution. Accurately measure 10 μl, inject into liquid chromatograph, and record the chromatogram.

[0051] (8) Location of Nicorandil EP Impurity D: Take about 2 mg of Nicorandil EP Impurity D reference substance, weigh it accurately, place it in a 50 ml volumetric flask, add 7% methanol solution to dissolve and dilute to the scale, shake well, and use it as the location of Nicorandil EP Impurity D. Accurately measure 10 μl, inject it into the liquid chromatograph, and record the chromatogram.

[0052] (9) Nicorandil hydroxy dimer localization solution: Take about 2 mg of Nicorandil hydroxy dimer reference substance, weigh it accurately, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and use it as Nicorandil hydroxy dimer localization solution. Accurately measure 10 μl, inject it into the liquid chromatograph, and record the chromatogram.

[0053] (10) Nicorandil dimer localization solution: Take about 2 mg of Nicorandil dimer reference substance, weigh it accurately, place it in a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and use it as Nicorandil dimer localization solution. Accurately measure 10 μl, inject it into the liquid chromatograph, and record the chromatogram.

[0054] (11) Nicorandil trimer localization solution: Take about 2.5 mg of Nicorandil trimer reference substance, weigh accurately, place in a 50 ml volumetric flask, dissolve with diluent and dilute to scale, shake well, and use as Nicorandil trimer localization solution. Accurately measure 10 μl, inject into liquid chromatograph, and record the chromatogram.

[0055] (12) Nicorandil tetramer localization solution: Take about 2.5 mg of Nicorandil tetramer reference substance, weigh accurately, place in a 50 ml volumetric flask, dissolve with diluent and dilute to scale, shake well, and use as Nicorandil tetramer localization solution. Accurately measure 10 μl, inject into liquid chromatograph, and record the chromatogram.

[0056] (13) Reference solution: Take an appropriate amount of Nicorandil reference substance, weigh it accurately, dissolve it in a diluent and quantitatively dilute it to make a solution containing about 10 μg per 1 ml as the reference solution. Accurately measure 10 μl and inject it into the liquid chromatograph to record the chromatogram.

[0057] (14) Test sample spike solution: Take 2 tablets of this product (batch number 20201209), place them in a 10 ml volumetric bottle, accurately add 0.5 ml each of nicotinic acid localization solution, nicotinic acid methyl ester localization solution, nicorandil EP impurity A localization solution, EP impurity B localization solution, EP impurity C localization solution, nicorandil EP impurity D localization solution, nicorandil hydroxy dimer localization solution, nicorandil dimer localization solution, and 1 ml each of nicorandil trimer localization solution and nicorandil tetramer localization solution, dissolve with diluent and dilute to scale, shake well, filter (0.45 μm filter membrane), and take the filtrate as the test sample spike solution. Accurately measure 10 μl of the test sample spike solution, inject it into the liquid chromatograph, and record the chromatogram. The results showed that the nicotinic acid peak, nicotinic acid methyl ester peak, nicorandil EP impurity A peak, nicorandil EP impurity B peak, nicorandil EP impurity C peak, nicorandil EP impurity D peak, nicorandil hydroxy dimer peak, nicorandil dimer peak, nicorandil trimer peak and nicorandil tetramer peak in the chromatogram of the test sample spiked solution could be effectively separated.

[0058] (15) Blank excipient solution: Take an appropriate amount of blank excipient of Nicorandil tablets (equivalent to approximately 25 mg of Nicorandil), accurately weigh, place in a 25 ml volumetric flask, add an appropriate amount of diluent, shake, dilute to scale with solvent diluent, shake well, filter (0.45 μm filter membrane), and take the filtrate as the blank excipient solution. Accurately measure 10 μl of the blank excipient solution, inject into the liquid chromatograph, and record the chromatogram. The results show that the blank excipient does not interfere with the determination of Nicorandil trimer and Nicorandil tetramer.

[0059] (16) Undamaged test sample solution: Take 5 tablets of this product (batch number: 20201209), place them in a 25 ml volumetric flask, add an appropriate amount of diluent, shake to dissolve, dilute to the scale with diluent, shake well, filter (0.45 μm filter membrane), and take the filtrate as the undamaged test sample solution; accurately measure 10 μl of the undamaged test sample solution, inject it into the liquid chromatograph, and record the chromatogram.

[0060] (17) Reference preparation test solution-1: Take 5 tablets of reference preparation (batch number: 18K050Z), place in a 25 ml volumetric flask, add an appropriate amount of diluent, shake to dissolve, dilute to the scale with diluent, shake well, filter (0.45 μm filter membrane), and take the filtrate as the reference preparation test solution-1; accurately measure 10 μl of the reference preparation test solution-1, inject into the liquid chromatograph, and record the chromatogram.

[0061] (18) Reference preparation test solution-2: Take 5 tablets of reference preparation (batch number: 20F060Z), place in a 25 ml volumetric flask, add an appropriate amount of diluent, shake to dissolve, dilute to the scale with diluent, shake well, filter (0.45 μm filter membrane), and take the filtrate as reference preparation test solution-2; accurately measure 10 μl of reference preparation test solution-2, inject into liquid chromatograph, and record the chromatogram.

[0062] (19) Photoirradiation-destroyed blank excipient: Take an appropriate amount of blank excipient of Nicorandil tablets (equivalent to approximately 25 mg of Nicorandil), weigh accurately, place in a 25 ml volumetric flask, add an appropriate amount of diluent, shake, dilute to scale with diluent, shake well, filter (0.45 μm filter membrane), and take the filtrate as the photoirradiation-destroyed blank excipient solution; accurately measure 10 μl of the photoirradiation-destroyed blank excipient solution, inject into the liquid chromatograph, and record the chromatogram. The results showed that the photoirradiation-destroyed blank excipient did not interfere with the determination of Nicorandil trimer and Nicorandil tetramer.

[0063] (20) Light-damaged test solution: Take 5 tablets of this product (batch number: 20201209), place them in a 25 ml volumetric bottle, place them in a drug strong light irradiation tester (5000 lx) for 48 hours, take them out, add an appropriate amount of diluent, shake to dissolve, dilute to the scale with diluent, shake well, filter (0.45 μm filter membrane), and take the filtrate as the light-damaged test solution; accurately measure 10 μl of the light-damaged test solution, inject it into the liquid chromatograph, and record the chromatogram. The results showed that Nicorandil tablets had good stability under light-damaged conditions.

[0064] (21) High humidity destruction blank excipient solution: Take an appropriate amount of blank excipient of Nicorandil tablets (equivalent to approximately 25 mg of Nicorandil), accurately weigh it, place it in a 25 ml volumetric flask, place it at 92.5% RH for 48 hours, take it out, add an appropriate amount of diluent, shake it, dilute it to the scale with diluent, shake it well, filter it (0.45 μm filter membrane), and take the filtrate as the high humidity destruction blank excipient solution; accurately measure 10 μl of the high humidity destruction blank excipient solution, inject it into the liquid chromatograph, and record the chromatogram. The results showed that the high humidity destruction blank excipient did not interfere with the determination of Nicorandil trimer and Nicorandil tetramer.

[0065] (22) High humidity damage test solution: Take 5 tablets of this product (batch number: 20201209), place them in a 25ml volumetric bottle, place them at 92.5% RH for 48 hours, take them out, add an appropriate amount of diluent, shake to dissolve, dilute to scale with diluent, shake well, filter (0.45μm filter membrane), and take the filtrate as the high humidity damage test solution; accurately measure 10μl of the high humidity damage test solution, inject it into the liquid chromatograph, and record the chromatogram. The results showed that Nicorandil tablets had good stability under high humidity damage conditions.

[0066] (23) Acid destruction blank solvent: accurately measure 2.5 ml of 1 mol / L hydrochloric acid solution, place it in a 25 ml volumetric flask, add diluent to dilute to the mark, shake well, filter (0.45 μm filter membrane), and take the filtrate as the acid destruction blank solvent; accurately measure 10 μl of the acid destruction blank solvent, inject it into the liquid chromatograph, and record the chromatogram. The results showed that the acid destruction blank solvent did not interfere with the determination of nicorandil trimer and nicorandil tetramer.

[0067] (24) Acid-destroyed blank excipient solution: Take an appropriate amount of blank excipient of Nicorandil tablets (equivalent to approximately 25 mg of Nicorandil), accurately weigh it, place it in a 25 ml volumetric flask, add 2.5 ml of 1 mol / L hydrochloric acid solution, shake for 10 minutes, add an appropriate amount of diluent, shake, dilute to scale with diluent, shake well, filter (0.45 μm filter membrane), and take the filtrate as the acid-destroyed blank excipient solution; accurately measure 10 μl of the acid-destroyed blank excipient solution, inject it into the liquid chromatograph, and record the chromatogram. The results showed that the acid-destroyed blank excipient did not interfere with the determination of Nicorandil trimer and Nicorandil tetramer.

[0068] (25) Acid-destroyed test solution: Take 5 tablets of this product (batch number: 20201209), place them in a 25 ml volumetric bottle, add 2.5 ml of 1 mol / L hydrochloric acid solution, shake for 10 minutes, add diluent to dissolve and dilute to scale, shake well, filter (0.45 μm filter membrane), and take the filtrate as the acid-destroyed test solution; accurately measure 10 μl of the acid-destroyed test solution, inject it into the liquid chromatograph, and record the chromatogram. The results showed that Nicorandil tablets had good stability under acid-destroyed conditions.

[0069] (26) Alkali destruction blank solvent: accurately measure 2 ml of 1 mol / L sodium hydroxide solution, place it in a 25 ml volumetric flask, add diluent to dilute to the mark, shake well, filter (0.45 μm filter membrane), and take the filtrate as the alkali destruction blank solvent; accurately measure 10 μl of alkali destruction blank solvent, inject it into the liquid chromatograph, and record the chromatogram. The results showed that the alkali destruction blank solvent did not interfere with the determination of nicorandil trimer and nicorandil tetramer.

[0070] (27) Alkali-destroyed blank excipient solution: Take an appropriate amount of blank excipient of Nicorandil tablets (equivalent to approximately 25 mg of Nicorandil), weigh accurately, place in a 25 ml volumetric flask, add 2 ml of 1 mol / L sodium hydroxide solution, shake for 10 minutes, add an appropriate amount of diluent, shake, dilute to scale with diluent, shake well, filter (0.45 μm filter membrane), and take the filtrate as the alkali-destroyed blank excipient solution; accurately measure 10 μl of the alkali-destroyed blank excipient solution and inject it into the liquid chromatograph, and record the chromatogram. The results showed that the alkali-destroyed blank excipient did not interfere with the determination of Nicorandil trimer and Nicorandil tetramer.

[0071] (28) Alkali-destroyed test solution: Take 5 tablets of this product (batch number: 20201209), place them in a 25 ml volumetric bottle, add 2 ml of 1 mol / L sodium hydroxide solution, shake for 10 minutes, add diluent to dissolve and dilute to scale, shake well, filter (0.45 μm filter membrane), and take the filtrate as the alkaline-destroyed test solution; accurately measure 10 μl of the alkaline-destroyed test solution, inject it into the liquid chromatograph, and record the chromatogram. The results showed that Nicorandil tablets were unstable under alkaline-destroyed conditions, and the impurity peaks produced could be effectively separated from the Nicorandil trimer peak and Nicorandil tetramer peak.

[0072] (29) Oxidative destruction blank solvent: Accurately measure 2.5 ml of hydrogen peroxide solution (30%), place it in a 25 ml volumetric flask, add diluent to dilute to the mark, shake well, filter (0.45 μm filter membrane), and take the filtrate as the oxidative destruction blank solvent; accurately measure 10 μl of oxidative destruction blank solvent, inject it into the liquid chromatograph, and record the chromatogram. The results showed that the oxidative destruction blank solvent did not interfere with the determination of nicorandil trimer and nicorandil tetramer.

[0073] (30) Oxidative destruction blank excipient solution: Take an appropriate amount of blank excipient of Nicorandil tablets (equivalent to approximately 25 mg of Nicorandil), accurately weigh it, place it in a 25 ml volumetric flask, add 2.5 ml of hydrogen peroxide solution (30%), shake for 10 minutes, add an appropriate amount of diluent, shake, dilute to scale with diluent, shake well, filter (0.45 μm filter membrane), and take the filtrate as the oxidative destruction blank excipient solution; accurately measure 10 μl of the oxidative destruction blank excipient solution, inject it into the liquid chromatograph, and record the chromatogram. The results showed that the oxidative destruction blank excipient did not interfere with the determination of Nicorandil trimer and Nicorandil tetramer.

[0074] (31) Oxidative damage test solution: Take 5 tablets of this product (batch number: 20201209), place them in a 25 ml volumetric bottle, add 2.5 ml of hydrogen peroxide solution (30%), shake for 10 minutes, add diluent to dissolve and dilute to scale, shake well, filter (0.45 μm filter membrane), and take the filtrate as the oxidative damage test solution; accurately measure 10 μl of the oxidative damage test solution, inject it into the liquid chromatograph, and record the chromatogram. The results showed that Nicorandil tablets had good stability under oxidative damage conditions.

[0075] (32) High temperature destruction (60℃ water bath) blank solvent: Take 2.5ml of diluent, place in a 25ml volumetric flask, heat in a 60℃ water bath for 1 hour, take out, cool, add diluent to dilute to scale, shake well, filter (0.45μm filter membrane), and take the filtrate as the high temperature destruction (60℃ water bath) blank solvent; accurately measure 10μl of high temperature destruction blank solvent, inject into liquid chromatograph, and record the chromatogram. The results showed that the high temperature destruction blank solvent did not interfere with the determination of nicorandil trimer and nicorandil tetramer.

[0076] (33) High temperature destruction (60℃ water bath) blank excipient solution: Take an appropriate amount of blank excipient of Nicorandil tablets (equivalent to 25mg of Nicorandil), weigh accurately, place in a 25ml volumetric flask, add 2.5ml of mobile phase, heat in a 60℃ water bath for 1 hour, take out, cool, dilute to scale with diluent, shake well, filter (0.45μm filter membrane), and take the filtrate as the blank excipient solution of high temperature destruction (60℃ water bath); accurately measure 10μl of the blank excipient solution of high temperature destruction (60℃ water bath), inject into the liquid chromatograph, and record the chromatogram. The results showed that the blank excipient of high temperature destruction (60℃ water bath) did not interfere with the determination of Nicorandil trimer and Nicorandil tetramer.

[0077] (34) High temperature destruction (60℃ water bath) test solution: Take 5 Nicorandil tablets (batch number: 20201209), place them in a 25ml volumetric flask, add 2.5ml of mobile phase, heat in a 60℃ water bath for 1 hour, take out, cool, add diluent to dissolve and dilute to scale, shake well, filter (0.45μm filter membrane), and take the filtrate as the high temperature destruction (60℃ water bath) test solution; accurately measure 10μl of the high temperature destruction (60℃ water bath) test solution, inject it into the liquid chromatograph, and record the chromatogram. The results showed that Nicorandil tablets were unstable under high temperature (60℃ water bath) destruction conditions, and the impurity peaks produced could be effectively separated from the Nicorandil trimer peak and Nicorandil tetramer peak.

[0078] (35) High temperature destruction (50℃ solid) blank excipient solution: Take an appropriate amount of blank excipient of Nicorandil tablets (equivalent to 25mg of Nicorandil), weigh accurately, place in a 25ml volumetric flask, place at 50℃ for 48h, take out, cool, add an appropriate amount of diluent, shake, dilute to scale with diluent, shake well, filter (0.45μm filter membrane), and take the filtrate as the high temperature destruction (50℃ solid) blank excipient solution; accurately measure 10μl of the high temperature destruction (50℃ solid) blank excipient solution, inject into the liquid chromatograph, and record the chromatogram. The results showed that the high temperature destruction (50℃ solid) blank excipient did not interfere with the determination of Nicorandil trimer and Nicorandil tetramer.

[0079] (36) High temperature destruction (50℃ solid) test solution: Take 5 tablets of this product (batch number: 20201209), place them in a 25ml volumetric bottle, place them at 50℃ for 48h, take them out, cool them, add diluent to dissolve and dilute to the scale, shake them well, filter them (0.45μm filter membrane), and take the filtrate as the high temperature destruction (50℃ solid) test solution; accurately measure 10μl of the high temperature destruction (50℃ solid) test solution, inject it into the liquid chromatograph, and record the chromatogram. The results showed that Nicorandil tablets were unstable under high temperature (50℃ solid) destruction conditions, and the impurity peaks produced could be effectively separated from the Nicorandil trimer peak and Nicorandil tetramer peak.

[0080] The results showed that blank solvent and blank excipients did not interfere with the determination of nicorandil trimer and nicorandil tetramer; the nicorandil trimer peak and nicorandil tetramer peak could be effectively separated from the impurity peaks; Nicorandil tablets were stable under light, high humidity, acid and oxidation conditions, but unstable under alkali destruction, 50°C (solid) high temperature and 60°C (water bath) high temperature conditions, and the nicorandil trimer peak, tetramer peak and the impurity peaks produced by degradation could be effectively separated; the analysis method of polymer impurities in nicorandil tablets had good specificity.

[0081]

[0082] Repeatability: Test sample spiked solution: Take 2 tablets of this product (batch number: 20201209), place in a 10 ml volumetric flask, accurately add 1 ml each of nicorandil trimer reference stock solution and nicorandil tetramer reference stock solution, add appropriate amount of diluent, shake to dissolve, dilute to the scale with solvent, shake well, filter (0.45 μm filter membrane), and take the filtrate (prepare 6 portions in parallel).

[0083] Accurately measure the reference solution, test solution, and test spike solution, inject them into the liquid chromatograph, and record the chromatogram. Investigate the average value and RSD of each impurity content in the test spike solution.

[0084]

[0085] The results showed that the analytical method for polymer impurities in Nicorandil tablets had good repeatability.

[0086] Detection limit, quantification limit: The detection limit and quantification limit were determined by the signal-to-noise ratio method. The detection limit was determined by the corresponding concentration when the signal-to-noise ratio was approximately 3:1, and the quantification limit was determined by the corresponding concentration when the signal-to-noise ratio was approximately 10:1.

[0087] (1) Detection limit of Nicorandil Accurately measure 5 ml of Nicorandil quantitative limit solution, place it in a 15 ml volumetric flask, dilute to the scale with diluent, shake well, and use this as Nicorandil detection limit solution with a concentration of 0.04648 mg / ml.

[0088] (2) Detection limit of Nicorandil trimer Accurately measure 5 ml of Nicorandil trimer quantitative limit solution, place it in a 15 ml volumetric flask, dilute to the scale with diluent, shake well, and use this as the Nicorandil trimer detection limit solution with a concentration of 0.09982 mg / ml, which is 2.00% of the limit.

[0089] (3) Nicorandil tetramer detection limit Accurately measure 5 ml of the nicorandil tetramer quantitative limit solution, place it in a 15 ml volumetric flask, dilute to the scale with diluent, shake well, and use it as the nicorandil tetramer detection limit solution with a concentration of 0.1523 mg / ml, which is 3.05% of the limit.

[0090] (4) Nicorandil limit of quantification Accurately measure 0.13 ml of the Nicorandil reference stock solution under the linear item, place it in a 100 ml volumetric flask, dilute it to the mark with a diluent, shake it well, and use it as the Nicorandil quantitative limit solution with a concentration of 0.1394 mg / ml. Take the Nicorandil quantitative limit solution and repeat the injection 6 times to examine the peak area RSD. The results are shown in Table 8.

[0091]

[0092] (5) Limit of quantification of Nicorandil trimer Accurately measure 0.66 ml of the Nicorandil trimer reference stock solution under the linear item, place it in a 100 ml volumetric flask, dilute it to the mark with a diluent, shake it well, and use it as the Nicorandil trimer quantitative limit solution, with a concentration of 0.2994 mg / ml, which is 5.99% of the limit. Take the Nicorandil trimer quantitative limit solution, repeat the injection 6 times, and examine the peak area RSD. The results are shown in Table 9.

[0093]

[0094] (6) Limit of quantification of Nicorandil tetramer Accurately measure 1 ml of the stock solution of Nicorandil tetramer reference substance under the linear item, place it in a 100 ml volumetric flask, dilute it to the mark with diluent, shake it well, and use it as the quantification limit solution of Nicorandil tetramer, with a concentration of 0.4569 mg / ml, which is 9.14% of the limit. Take the quantification limit solution of Nicorandil tetramer, repeat the injection 6 times, and examine the peak area RSD. The results are shown in Table 10.

[0095]

[0096] The results showed that the quantification limit solutions of nicorandil, nicorandil trimer and nicorandil tetramer had good precision.

[0097] Accuracy: 10% spiked recovery solution: Take 2 tablets of this product (Batch No.: 20201209), place in a 10 ml volumetric flask, accurately add 0.1 ml each of nicorandil trimer reference stock solution and nicorandil tetramer reference stock solution, add appropriate amount of diluent, shake to dissolve, dilute to the scale with diluent, shake well, filter (0.45 μm filter membrane), and take the filtrate (prepare 3 portions in parallel).

[0098] 80% spiked recovery solution: Take 2 tablets of this product (Batch No.: 20201209), place in a 10 ml volumetric flask, accurately add 0.8 ml each of nicorandil trimer reference stock solution and nicorandil tetramer reference stock solution, add appropriate amount of diluent, shake to dissolve, dilute to the scale with diluent, shake well, filter (0.45 μm filter membrane), and take the filtrate (prepare 3 portions in parallel).

[0099] 100% spiked recovery solution: Take 2 tablets of this product (Batch No.: 20201209), place in a 10 ml volumetric flask, accurately add 1 ml each of nicorandil trimer reference stock solution and nicorandil tetramer reference stock solution, add appropriate amount of diluent, shake to dissolve, dilute to the scale with diluent, shake well, filter (0.45 μm filter membrane), and take the filtrate (prepare 3 portions in parallel).

[0100] 120% spiked recovery solution: Take 2 tablets of this product (Batch No.: 20201209), place in a 10 ml volumetric flask, accurately add 1.2 ml each of nicorandil trimer reference stock solution and nicorandil tetramer reference stock solution, add appropriate amount of diluent, shake to dissolve, dilute to the scale with diluent, shake well, filter (0.45 μm filter membrane), and take the filtrate (prepare 3 portions in parallel).

[0101] Equal amounts of nicorandil trimer reference stock solution, nicorandil tetramer reference stock solution, reference solution, test solution, 10% spiked recovery solution, 80% spiked recovery solution, 100% spiked recovery solution and 10% spiked recovery solution were accurately measured and injected into liquid chromatography respectively to investigate the average values ​​and RSDs of the spiked recoveries of nicorandil trimer and nicorandil tetramer.

[0102]

[0103]

[0104] The results showed that the analytical method for polymer impurities in Nicorandil tablets had good accuracy.

[0105] Durability: Prepare 1 portion of test sample solution and 1 portion of test sample spiked solution (preparation method is the same as under repeatability). According to the analytical method for related substance II, the RSD of the contents of nicorandil trimer and nicorandil tetramer were investigated under different chromatographic columns, different flow rates (1.0±0.1ml / min) and different column temperatures (25±2℃). The results are shown in Table 13 below.

[0106]

[0107] The results showed that the analytical method for polymer impurities in Nicorandil tablets had good robustness.

[0108] In summary, the method for detecting the content of polymer impurities in Nicorandil tablets provided by the present invention can avoid the formation of insoluble solids between carboxymethylcellulose calcium and the polymer impurities of Nicorandil trimer and Nicorandil tetramer by selecting 0.1% trifluoroacetic acid solution (pH about 2.0) as a diluent, thereby eliminating interference, accurately detecting polymer impurities, being easy to operate, and having good accuracy. In addition, the optimization of gradient elution shortens the running time and makes the detection faster.

[0109] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for detecting the content of polymer impurities in Nicorandil tablets, characterized in that: The detection method comprises: dissolving a sample to be tested with a diluent to obtain a sample solution, and using high performance liquid chromatography to detect the polymer impurity content in the sample solution; wherein the diluent is a trifluoroacetic acid-water mixture; the chromatographic conditions are: using octadecylsilane bonded silica gel as a filler; using a trifluoroacetic acid-water mixture as solution 1, using a triethylamine-tetrahydrofuran-methanol mixture as solution 2, mixing solution 1 and solution 2 as mobile phase A, and using methanol as mobile phase B; and performing gradient elution.

2. The method for detecting the content of polymer impurities in Nicorandil tablets according to claim 1, characterized in that: In the diluent, the pH of the mixture of trifluoroacetic acid and water is 1.5-2.

5.

3. The method for detecting the content of polymer impurities in Nicorandil tablets according to claim 1, characterized in that: The mobile phase is subjected to linear gradient elution as shown in the following table: 。 4. The method for detecting the content of polymer impurities in Nicorandil tablets according to claim 1, characterized in that: In the solution 1, the pH of the mixture of trifluoroacetic acid and water is 1.5-2.5; In the solution 2, the volume ratio of triethylamine, tetrahydrofuran and methanol is 2:10:25; In the mobile phase A, the volume ratio of solution 1 to solution 2 is 1:

1.

5. The method for detecting the content of polymer impurities in Nicorandil tablets according to claim 1, characterized in that: Chromatographic conditions also include: Flow rate: 1.0 ml / min; Detection wavelength: 254 nm; Column temperature: 25 °C; Sample chamber temperature: 5 °C; Injection volume: 10 μl.

6. The method for detecting the content of polymer impurities in Nicorandil tablets according to claim 1, characterized in that: The polymer impurities are nicorandil trimer and nicorandil tetramer, and their chemical structures are as follows: ； 。 7. The method for detecting the content of polymer impurities in Nicorandil tablets according to any one of claims 1 to 6, characterized in that: The specific detection steps are as follows: S1. Prepare the test solution, reference solution and system suitability solution separately and set aside; S2. Accurately measure equal amounts of the test solution, reference solution, and system suitability solution, and inject them into the HPLC respectively. Use mobile phase A and mobile phase B to separate and analyze by gradient elution, and record the chromatogram.

8. The method for detecting the content of polymer impurities in Nicorandil tablets according to claim 7, characterized in that: The specific preparation method of the test solution is: take the sample to be tested, add an appropriate amount of diluent, shake to dissolve, dilute with solvent, shake well, filter, and take the filtrate; the concentration of the test solution is 1 mg / ml; The specific preparation method of the reference solution is: take Nicorandil reference, accurately weigh, add diluent to dissolve and quantify; the concentration of the reference solution is 10 μg / ml; The specific preparation method of the system suitability solution is: take a Nicorandil reference substance, place it at 70° C. for 2 hours, cool it, and then dissolve and dilute it with a diluent; the concentration of the system suitability solution is 0.8%.

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