Determination method for irbesartan and levoamlodipine compound tablet related substances

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for determining related substances of irbesartan and levoamlodipine compound tablets. According to the method, high performance liquid chromatography is adopted for detection, a chromatographic column is YMC-Triart C18, a 0.55% phosphoric acid aqueous solution with the pH value of 3.0-3.5 is used as a mobile phase A, methanol is used as a mobile phase B, and gradient elution is performed. According to the method, known impurities and various unknown impurities including amlodipine impurities A, B, D, E, F, G and H, irbesartan impurities I and the like can be measured under the same chromatographic condition, the separation degree of each chromatographic peak and a main peak or other impurity peaks meets the requirements, the peak shape is good, the method is high in specificity, good in reproducibility and high in accuracy, the sensitivity meets the analysis and detection requirements, and the method is suitable for industrial production. And a reliable detection method is provided for related substances of the irbesartan-levoamlodipine compound.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and particularly relates to a method for determining related substances in irbesartan-levamlodipine compound tablets. Background Art

[0002] The sources of related substances in compound preparations are relatively complex, mainly introduced by the main drugs, including raw materials / reactants that have not reacted completely in the synthesis of the main drugs, intermediates, side reaction products, and degradation products during storage, etc., which will affect the purity and efficacy of the product and pose certain safety risks.

[0003] This product is a combination tablet of irbesartan and levamlodipine, containing two antihypertensive active ingredients: irbesartan and levamlodipine besylate. Levoamlodipine besylate is a calcium channel blocker (CCB) drug that blocks extracellular calcium ions from entering myocardial and vascular smooth muscle cells through calcium ion channels in the cell membrane, directly relaxing vascular smooth muscle and producing an antihypertensive effect. Irbesartan is an angiotensin II receptor blocker (ARB) drug that selectively blocks the binding of Ang II to the AT1 receptor, inhibiting vasoconstriction and aldosterone release, resulting in a blood pressure-lowering effect. The two drugs have complementary mechanisms of action, synergistically enhancing efficacy and reducing toxicity. Their administration methods are consistent, and there are no drug interactions.

[0004] Compared to amlodipine, levamlodipine removes the nearly ineffective dextrorotatory component of amlodipine, retaining only the levorotatory isomer. Therefore, an equal amount of levamlodipine is twice as effective as amlodipine. That is, compared to irbesartan-levamlodipine combination tablets, the levamlodipine dosage in irbesartan-levamlodipine combination tablets is only half that of the latter. Therefore, compared to irbesartan-levamlodipine combination tablets, irbesartan-levamlodipine combination tablets contain a higher proportion of the raw material irbesartan, introducing more impurities. Levoamlodipine besylate contains a lower proportion of the raw material and is unstable, easily degrading to produce impurities, making its impurity detection difficult. These factors make the detection methods for irbesartan-levamlodipine combination tablets disclosed in the prior art difficult to apply to irbesartan-levamlodipine combination tablets.

[0005] At present, there is no determination method for the related substances in irbesartan levaquine combination tablets, and the quality control of the product is difficult to guarantee. Therefore, there is an urgent need to develop a determination method with strong specificity and high sensitivity that can effectively and accurately detect the related substances in irbesartan levaquine combination tablets. Summary of the Invention

[0006] In response to the problems existing in the prior art, the present invention provides a high-performance liquid chromatography method with strong specificity, good reproducibility and high accuracy, which can effectively identify the impurities of different main drugs in irbesartan and amlodipine compound tablets.

[0007] Specifically, to solve the above technical problems, the present invention provides the following technical solutions:

[0008] The present invention provides a method for determining related substances in irbesartan-levamlodipine compound tablets, characterized in that high performance liquid chromatography is used for detection, 0.55% phosphoric acid aqueous solution with a pH of 3.0 to 3.5 is used as mobile phase A, methanol is used as mobile phase B, and detection is performed according to the following gradient elution conditions:

[0009] Time (min) Mobile phase A (%) Mobile phase B (%) 0 50 50 50 20 80 60 10 90 70 10 90 71 50 50 85 50 50 .

[0010] In some specific embodiments, in the high performance liquid chromatography method, the flow rate is 0.6 to 1.0 ml / min; in some specific embodiments, the flow rate is 0.6 ml / min, 0.7 ml / min, 0.8 ml / min, 0.9 ml / min, 1.0 ml / min; preferably, in the high performance liquid chromatography method, the flow rate is 0.8 ml / min.

[0011] In some specific embodiments, in the high performance liquid chromatography method, the column temperature is 35-45°C. In some specific embodiments, the column temperature is 35°C, 40°C, or 45°C.

[0012] In some specific embodiments, in the high performance liquid chromatography method, the detection wavelength is 220 nm and 237 nm, and the injection volume is 10 to 20 μl.

[0013] Preferably, in the high performance liquid chromatography method, the chromatographic column is YMC-Triart C18.

[0014] In some specific embodiments, the analysis conditions of the high performance liquid chromatography are as follows: the chromatographic column is YMC-Triart C18, 0.55% phosphoric acid aqueous solution with a pH of 3.0 to 3.5 is used as mobile phase A, methanol is used as mobile phase B, the flow rate is 0.6 to 1.0 ml per minute, the column temperature is 35 to 45° C., the detection wavelengths are 220 nm and 237 nm, the injection volume is 10 to 20 μl, and the detection is performed under the gradient elution conditions shown in the table below:

[0015] Time (min) Mobile phase A (%) Mobile phase B (%) 0 50 50 50 20 80 60 10 90 70 10 90 71 50 50 85 50 50 .

[0016] In some specific embodiments, the high performance liquid chromatography method further includes a solution preparation step.

[0017] In some specific embodiments, the solution includes part or all of the reference substance stock solution, reference substance solution, positioning solution, system suitability solution, test solution, spiked test solution, sensitivity solution, and levamlodipine besylate alkali destruction solution; the preparation steps of the above solutions are not in particular order.

[0018] In some specific embodiments, the solvent used to prepare the solution is 0.55% phosphoric acid aqueous solution (pH 3.0-3.5)-methanol (20:80, v / v).

[0019] Furthermore, in the high performance liquid chromatography method, the system suitability solution is an alkali destruction solution of irbesartan, and the impurities introduced by different main drugs of irbesartan-amlodipine compound tablets are attributed.

[0020] In some specific embodiments, the related substances in the irbesartan-levoamlodipine compound tablets include, but are not limited to, amlodipine impurities A, B, D, E, F, G, H, irbesartan impurity I and other known impurities as well as various unknown impurities, wherein the specific structures of the known impurities are as follows:

[0021]

[0022]

[0023] Furthermore, in the irbesartan-levamlodipine compound tablets, at a wavelength of 237 nm, the amount of amlodipine impurity I shall not exceed 1.0% of the labeled amount of levamlodipine, and the amount of other individual impurities shall not exceed 0.5% of the labeled amount of levamlodipine; at a wavelength of 220 nm, if there is a chromatographic peak with a retention time consistent with that in the system suitability solution, it shall not exceed 0.2% of the labeled amount of irbesartan, and the total amount of impurities shall not exceed 0.5%; and the total amount of impurities at wavelengths of 220 nm and 237 nm shall not exceed 1.5%.

[0024] The present invention has the following beneficial effects:

[0025] The detection method of the present invention can realize the determination of known impurities including amlodipine impurities A, B, D, E, F, G, H and irbesartan impurity I and multiple unknown impurities under one chromatographic condition. The separation degree between each chromatographic peak and the main peak or other impurity peaks meets the requirements, the peak shape is good, the method has strong specificity, good reproducibility, high accuracy, and the sensitivity meets the analysis and detection requirements. The method provides a reliable detection method for related substances in irbesartan levamlodipine compound tablets. At the same time, the method can effectively and accurately attribute impurities introduced by each main drug in the irbesartan levamlodipine compound tablets, ensuring the scientificity and rationality of setting the limits of each impurity. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1AThis is a chromatogram of the spiked test solution in the specificity determination of Example 1 of the present invention.

[0027] Figure 1B 1 is a chromatogram of the blank solution in the specificity determination of Example 1 of the present invention.

[0028] Figure 2 This is a chromatogram of the system suitability in the specificity determination of Example 1 of the present invention.

[0029] Figure 3 This is the DAD scanning spectrum of the maximum absorption wavelength of each component contained in the spiked test solution in the specific determination of Example 1 of the present invention.

[0030] Figure 4 The chromatograms are of the system suitability solution and the test solution in Comparative Example 1 of the present invention.

[0031] Figure 5 The chromatograms are of the system suitability solution and the test solution in Comparative Example 2 of the present invention.

[0032] Figure 6 The chromatogram of the test solution in Comparative Example 3 of the present invention is shown.

[0033] Figure 7 This is a chromatogram of the spiked test solution in Comparative Example 4 of the present invention.

[0034] Figure 8 This is a chromatogram of the spiked test solution in Comparative Example 5 of the present invention.

[0035] Figure 9 1 is a chromatogram of the test solution in Example 1 of the present invention.

[0036] Figure 10 The chromatograms are of the system suitability solution and the alkali-destroyed solution of levamlodipine besylate in Screening Example 1 of the present invention.

[0037] Figure 11 The chromatogram of the test solution in Screening Example 2 of the present invention.

[0038] Figure 12 The chromatograms are of the amlodipine impurity H, amlodipine impurity B and spiked test solution in Screening Example 3 of the present invention.

[0039] Figure 13 The chromatograms are of the system suitability solution and the test solution in Screening Example 4 of the present invention.

[0040] Figure 14 The chromatogram of the test solution in Screening Example 5 of the present invention. DETAILED DESCRIPTION

[0041] In order to make the purpose and technical solution of the present invention more clear, the present invention is further described below in conjunction with the embodiments, but the scope of protection of the present invention is not limited to these embodiments, and the embodiments are only used to illustrate the present invention. It should be understood by those skilled in the art that any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the scope of protection of the present invention.

[0042] The time in the gradient elution conditions of the present invention may fluctuate within a certain range, such as within the range of ±5 min, ±4 min, ±3 min, or ±2 min.

[0043] Among them, phosphoric acid was chromatographically pure (Norsch), potassium dihydrogen phosphate was analytically pure (Norsch), methanol and acetonitrile were chromatographically pure (B&K), triethylamine was analytically pure (Chengdu Kelong), irbesartan (China Food and Drug Inspection Institutes), levamlodipine besylate (China Food and Drug Inspection Institutes), irbesartan levamlodipine tablets (specifications: irbesartan 150 mg and levamlodipine besylate (calculated as levamlodipine) 2.5 mg, batch number R12405041).

[0044] Amlodipine impurity A is 3-ethyl-5-methyl (4RS)-4-(2-chlorophenyl)-2-[[2-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethoxy]methyl]-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate, sourced from Shenzhen Botail Biotechnology;

[0045] Amlodipine impurity B is 3-ethyl-5-methyl (4RS)-4-(2-chlorophenyl)-6-methyl-2-[[2-[[2-(methylcarbamoyl)benzoyl]amino]-ethoxy]methyl-1,4-dihydropyridine-3,5-dicarboxylate, sourced from Shenzhen Botail Biotechnology;

[0046] Amlodipine impurity D is 3-ethyl-5-methyl-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methylpyridine-3,5-dicarboxylate, sourced from the China Food and Drug Inspection Institute;

[0047] Amlodipine impurity E is (4RS)-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylic acid diethyl ester, sourced from Shenzhen Botai Biotechnology;

[0048] Amlodipine impurity F is (4RS)-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylic acid dimethyl ester, sourced from Shenzhen Botai Biotechnology;

[0049] The source of amlodipine impurity G, 4-(2-chlorophenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate, is Shenzhen Botai Biotechnology.

[0050] The impurity H of amlodipine is 2-[[2-(4RS)-4-(2-chlorophenyl)-3-(ethoxycarbonyl)-5-(methoxycarbonyl)-6-methyl-1,4-dihydropyridin-2-yl]methoxy]ethyl]carbamoyl]benzoic acid, which is sourced from Shenzhen Botail Biotechnology.

[0051] Irbesartan impurity I is 1-(pentanoylamino)-N-[[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]cyclopentanecarboxamide, and its source is China Food and Drug Inspection Institute.

[0052] Example 1

[0053] The related substances of irbesartan and amlodipine compound tablets were detected by the following method:

[0054] Chromatographic conditions: YMC-Triart C18 column (4.6 mm × 250 mm, 3 μm) or equivalent column; 0.55% aqueous phosphoric acid (pH adjusted to 3.3 with triethylamine) as mobile phase A, methanol as mobile phase B, gradient elution according to Table 1; flow rate, 0.8 ml / min; column temperature, 40°C; detection wavelengths, 220 nm and 237 nm; injection volume, 20 μl.

[0055] Table 1 Linear gradient elution conditions

[0056] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 50 50 50 20 80 60 10 90 70 10 90 71 50 50 85 50 50

[0057]

Note

[0058] Solution preparation:

[0059] ①Solvent: 0.55% phosphoric acid aqueous solution (adjust pH to 3.3 with triethylamine)-methanol (20:80).

[0060] ② Stock solution of irbesartan impurity I reference substance: take about 12 mg of irbesartan impurity I reference substance, accurately weigh it, put it into a 10 ml volumetric flask, add an appropriate amount of solvent to dissolve and dilute to the scale, shake well, and obtain.

[0061] ③ Irbesartan impurity I positioning solution: Accurately measure 1 ml of ② Irbesartan impurity I reference substance stock solution, place it in a 100 ml volumetric flask, dilute to the scale with solvent, and shake well to obtain.

[0062] ④ Amlodipine impurity A, B, D, E, F, G, H reference substance stock solution: Take about 2.8 mg of each impurity reference substance, accurately weigh, and place it in a 10 ml volumetric flask respectively, add an appropriate amount of solvent to dissolve and dilute to the scale, shake well, and obtain the stock solution of each impurity reference substance.

[0063] ⑤ Amlodipine impurity A, B, D, E, F, G, H positioning solution: Accurately measure 1 ml each of ④ Amlodipine impurity A, B, D, E, F, G, H reference substance stock solution, place them in different 100 ml volumetric flasks, dilute to the scale with solvent, shake well, and obtain.

[0064] ⑥ Stock solution of irbesartan reference substance: Take about 12 mg of irbesartan reference substance, accurately weigh it, place it in a 10 ml volumetric flask, add an appropriate amount of solvent to dissolve and dilute to the scale, shake well, and obtain.

[0065] ⑦ Stock solution of levamlodipine besylate reference substance: Take about 13.9 mg of levamlodipine besylate reference substance, accurately weigh it, put it into a 100 ml volumetric flask, add an appropriate amount of solvent to dissolve it and dilute it to the scale with diluent, shake well, and it is ready.

[0066] ⑧ Reference solution: Accurately measure 1 ml each of irbesartan reference stock solution, irbesartan impurity I reference stock solution, levamlodipine besylate reference stock solution, and amlodipine impurity D reference stock solution, place them in the same 100 ml volumetric flask, dilute to the scale with solvent, and shake well to obtain.

[0067] ⑨ Sensitivity solution: Accurately measure 0.1 ml of the reference stock solution of levamlodipine besylate, place it in a 100 ml volumetric flask, dilute to the scale with solvent, and shake well.

[0068] ⑩ System suitability solution: After testing, it was found that levamlodipine besylate was unstable under light, alkali, oxidation and high temperature conditions, while irbesartan was unstable only under alkali destruction conditions and was relatively stable under other conditions; all impurities in the preparation could be well attributed. Because irbesartan is easily degraded only under alkali destruction conditions, its degradation impurities and unknown impurities can be well attributed in the finished preparation. Therefore, the present invention uses irbesartan in the preparation with the same concentration of alkali destruction test solution as the system suitability solution to attribute the impurities introduced by irbesartan in the finished preparation, and the corresponding impurities are calculated using the labeled amount of irbesartan.

[0069] Take about 120 mg of irbesartan sample, accurately weigh it, place it in a 10 ml volumetric flask, add 1 ml of 0.5 mol / L sodium hydroxide solution, let it stand for 0.5 h, add 1 ml of 0.5 mol / L hydrochloric acid solution to neutralize it, dissolve it with methanol and dilute it to the scale, and shake it well.

[0070] Test solution: Take 20 tablets of irbesartan and amlodipine compound tablets, accurately weigh them, grind them into powder, take about 2 g of fine powder, accurately weigh it, put it into a 100 ml volumetric flask, add 80 ml of methanol, shake for about 30 minutes to completely dissolve irbesartan and amlodipine besylate, dilute to the scale with mobile phase A, shake well, filter, and take the filtrate.

[0071] Spiked test solution: Take 20 tablets of irbesartan and amlodipine combination tablets, accurately weigh them, grind them into powder, take about 2 g of fine powder, accurately weigh it, place it in a 100 ml volumetric flask, add 80 ml of methanol, shake for about 30 minutes to completely dissolve irbesartan and amlodipine besylate, add 1 ml each of irbesartan impurity I reference substance stock solution and amlodipine impurity I reference substance stock solution, dilute to the scale with mobile phase A, and shake well.

[0072] Alkali destruction solution of levamlodipine besylate: Take about 13.9 mg of levamlodipine besylate reference substance, accurately weigh it, place it in a 100 ml volumetric flask, add 1 ml of 0.5 mol / L sodium hydroxide solution, let it stand for 0.5 h, add 1 ml of 0.5 mol / L hydrochloric acid solution to neutralize it, dissolve it with methanol and dilute it to the scale, and shake it well.

[0073] The specificity, solution stability, and quantification limit of the above chromatographic conditions were verified as follows:

[0074] (1) Specificity

[0075] Measurement and results:

[0076] Take blank solvent (i.e. ① solvent), ③ irbesartan impurity I positioning solution, ⑤ amlodipine impurity A, B, D, E, F, G, H positioning solution, ⑨ sensitivity solution, ⑩ system suitability solution, ⑧ reference solution, Test solution, 20 μl of each spiked test solution was injected into the liquid chromatograph and the chromatogram was recorded. The results are shown in Figures 1 (A, B) and Figure 2 .

[0077] The system suitability requirement is as follows: In the chromatogram of the reference solution (237 nm), amlodipine impurity I, levoamlodipine, and irbesartan should elute sequentially, with the theoretical plate number calculated based on the levoamlodipine peak being no less than 3000. In the chromatogram of the sensitivity solution (237 nm), the signal-to-noise ratio of the levoamlodipine peak height should be no less than 10.

[0078] From Figure 1 (A, B) and Figure 2 It can be seen that the blank solvent does not interfere with the detection; the separation between irbesartan, amlodipine besylate and various impurities is good, and the specificity meets the requirements; the system suitability also meets the requirements.

[0079] Pick Add 20 μl of the test solution to the liquid chromatograph and scan with a DAD detector at a wavelength of 200 nm to 400 nm. Record the chromatogram. Figure 3 And Table 2.

[0080] Table 2 Positioning results of spiked test solution

[0081]

[0082] As can be seen from the above table, irbesartan and irbesartan impurity I have a large absorption at a wavelength of 220nm, and amlodipine and amlodipine-related impurities have a large absorption at a wavelength of 237nm. Therefore, dual wavelengths of 220nm and 237nm are used to detect the main drug irbesartan and amlodipine besylate impurities respectively.

[0083] (2) Solution stability

[0084] Take the exclusive The spiked test solution was placed at room temperature. At 0, 11.5, and 34.5 hours, 20 μl was accurately measured and injected into the liquid chromatograph. The chromatogram was recorded and analyzed. The results are shown in Table 3.

[0085] Table 3 Stability results of spiked test solution

[0086]

[0087]

[0088] As shown in Table 3, compared with the initial 0 hour, the spiked test solution was placed at room temperature for 34.5 hours. The peak area ratio of the known impurities at each time point to the 0 hour peak area was in the range of 0.99 to 1.02. The number of impurities did not change within 34.5 hours. No other impurities greater than 0.05% were detected within 34.5 hours. The solution stability met the requirements.

[0089] (3) Limit of quantification

[0090] Appropriate amounts of specific ② irbesartan impurity I reference substance stock solution, ④ amlodipine impurity D reference substance stock solution, ⑥ irbesartan reference substance stock solution and ⑦ levamlodipine besylate reference substance stock solution were taken and diluted with solvent to an S / N ratio of approximately 10. The samples were used as quantitative limit solutions for determination. The specific results are shown in Table 4.

[0091] Table 4 Quantitation limit results

[0092]

[0093] As shown in Table 4, the method for determining related substances in irbesartan-levamlodipine compound tablets can accurately quantify other single impurities above 0.03%, and the sensitivity of the method meets the requirements.

[0094] Comparative Example 1

[0095] The difference between Comparative Example 1 and Example 1 is that the chromatographic column in Comparative Example 1 was changed to Phenomenex Luna C18 (4.6×250 mm, 5 μm); the mobile phase was changed to 0.55% phosphoric acid aqueous solution (adjusted to pH 3.0 with triethylamine)-methanol-acetonitrile (50:35:15) as the mobile phase, with isocratic elution and a flow rate of 1.0 ml per minute; the column temperature was 30°C; the detection wavelength was 237 nm; and the injection volume was 20 μl. Take the system suitability solution in the specificity ⑩, 20 μl of each test solution was injected into the liquid chromatograph and the chromatogram was recorded. The results were shown in Figure 4 . (This detection method refers to the determination method of related substances of amlodipine besylate included in the 2020 edition of the Chinese Pharmacopoeia).

[0096] Depend on Figure 4 It can be seen that the impurity peaks in the system suitability solution and the test solution were not effectively separated from the main peak and the impurity peaks, and the specificity did not meet the requirements.

[0097] Comparative Example 2

[0098] The difference between Comparative Example 2 and Example 1 is that the chromatographic column in Comparative Example 2 uses octadecylsilane bonded silica gel as the filler, and the mobile phase is changed to 0.7% triethylamine aqueous solution (adjusted to pH 3.2 with ammonium phosphate)-acetonitrile (62:38) as the mobile phase, with isocratic elution and a flow rate of 1.0 ml per minute; the column temperature is 30°C; the detection wavelength is 220 nm; and the injection volume is 10 μl. Take the system suitability solution in the specificity ⑩, Inject 10 μl of each test solution into the liquid chromatograph and record the chromatogram. Figure 5 . (This detection method refers to the determination method of related substances of irbesartan included in the 2020 edition of the Chinese Pharmacopoeia).

[0099] Depend on Figure 5 It can be seen that the impurity peaks in the system suitability solution and the test solution were not effectively separated from the main peak and the impurity peaks, and the specificity did not meet the requirements.

[0100] Comparative Example 3

[0101] The difference between Comparative Example 3 and Example 1 is that the mobile phase A of Comparative Example 3 is changed to 0.02 mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.0 with phosphoric acid)-methanol (50:50), the chromatographic column is GL Sciences Inertsil ODS-3 (equivalent to Wondasil C18 (150 mm × 4.6 mm, 3.5 μm): the chromatographic column GL Sciences Inertsil ODS-3 and Wondasil C18 have the same filler, carbon content of 15% and 9.5%, respectively, the L / dp value ratio is 1.17, and the column efficiency GL Sciences Inertsil ODS-3 is better than Wondasil C18) 4.6 × 250 mm 5 μm, the flow rate is 1.0 ml / min, and the linear gradient elution conditions are changed to those shown in Table 5. The remaining parameters of Comparative Example 3 refer to Example 1, and the specificity is taken as 20 μl of each test solution was injected into the liquid chromatograph and the chromatogram was recorded. The results were shown in Figure 6 、 Figure 9 . (For this detection method, please refer to patent document CN202111144844.X).

[0102] Table 5 Comparative Example 3 Linear Gradient Elution Conditions

[0103] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 100 0 5 100 0 20 60 40 30 60 40 30.1 100 0 40 100 0

[0104] As can be seen from the figure, compared with Example 1, the levamlodipine peak in the test solution was not effectively separated from the adjacent impurity peaks and the impurity peaks themselves, and many unknown impurities could not be detected after the main peak of irbesartan. The specificity and detection ability were both worse than those in Example 1.

[0105] Comparative Example 4

[0106] The difference between Comparative Example 4 and Example 1 is that the mobile phase A of Comparative Example 4 is changed to 0.7% triethylamine solution (pH adjusted to 3.0±0.1 with phosphoric acid), the mobile phase B is changed to methanol-acetonitrile (70:30), the chromatographic column is Ultimate LP-C184.6×250mm 5μm, the flow rate is 1.0ml / min, the column temperature is 25°C, the detection wavelength is 237nm, and the linear gradient elution conditions are as shown in Table 6. Add 20 μl of the test solution to the liquid chromatograph and record the chromatogram. Figure 7 . (For this detection method, please refer to patent document CN202311223357.1).

[0107] Table 6 Comparative Example 4 Linear Gradient Elution Conditions

[0108]

[0109]

[0110] As can be seen from the figure, compared with Example 1, the irbesartan peak and the adjacent impurity peaks, the levoamlodipine peak and the adjacent impurity peaks, and the impurity peaks in the spiked test solution were not effectively separated. There were many unknown impurities after the main peak of irbesartan that could not be detected. The specificity and detection ability were worse than those in Example 1.

[0111] Comparative Example 5

[0112] The difference between Comparative Example 5 and Example 1 is that the mobile phase A of Comparative Example 5 is changed to water-trifluoroacetic acid (100:0.02), the mobile phase B is changed to acetonitrile-trifluoroacetic acid (100:0.02), the chromatographic column is YMC Triart C18 (4.6×150mm 3μm), the flow rate is 1.0ml / min, the column temperature is 40°C, the detection wavelength is 237nm, and the linear gradient elution conditions are as shown in Table 7. Add 10 μl of the test solution to the liquid chromatograph and record the chromatogram. Figure 8 . (For this detection method, please refer to patent document CN202310947706.8).

[0113] Table 7 Comparative Example 5 Linear Gradient Elution Conditions

[0114] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 80 20 30 20 80 45 20 80 46 80 20 55 80 20

[0115] As can be seen from the figure, compared with Example 1, in the spiked test solution, the irbesartan peak and the levamlodipine peak overlap, and the known impurities and unknown impurities cannot be effectively separated. The specificity and detection ability are worse than those in Example 1.

[0116] Screening Example 1

[0117] Chromatographic conditions: The chromatographic column was GL Sciences Inertsil ODS-3 (4.6×250mm 5μm), the mobile phase A was 0.02 mol / L potassium dihydrogen phosphate solution (pH adjusted to 3.0 with phosphoric acid), the mobile phase B was methanol, the linear gradient elution conditions were changed to those shown in Table 8, the flow rate was 1.0 ml / min; the column temperature was 40°C, the detection wavelengths were 220 nm and 237 nm, the injection volume was 20 μl, and the specificity was determined by taking the solution ⑩ System suitability and 20 μl of each alkali-destroying solution of levamlodipine besylate was injected into the liquid chromatograph and the chromatogram was recorded. The results were shown in Figure 10 .

[0118] Table 8 Linear gradient elution conditions for screening example 1

[0119]

[0120]

[0121] As can be seen from the figure, the impurity peak of irbesartan overlaps with the impurity peak of levamlodipine, and the levamlodipine peak cannot be effectively separated from the adjacent impurity peaks and the impurity peak of irbesartan.

[0122] Screening Example 2

[0123] The difference between Screening Example 2 and Screening Example 1 is that the chromatographic column of Screening Example 2 is YMC-Triart C18 (4.6mm×250mm, 5μm), the linear gradient elution condition is changed to that shown in Table 9, and the other parameters of Screening Example 2 refer to Screening Example 1, and the specificity is taken as 20 μl of each test solution was injected into the liquid chromatograph and the chromatogram was recorded. The results were shown in Figure 11 .

[0124] Table 9 Screening Example 2 Linear Gradient Elution Conditions

[0125] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 50 50 5 50 50 30 30 70 40 15 85 50 15 85 51 50 50 60 50 50

[0126] As can be seen from the figure, by changing the chromatographic column and increasing the proportion of organic phase in the mobile phase, the separation between the impurities is improved, and unknown impurities are eluted after 45 minutes. The elution capacity of this method has increased, and further optimization is considered to improve the separation between the impurities.

[0127] Screening Example 3

[0128] The difference between Screening Example 3 and Screening Example 2 is that the chromatographic column of Screening Example 3 is YMC-Triart C18 (4.6 mm × 250 mm, 3 μm), the linear gradient elution conditions are changed to those shown in Table 10, and the other parameters refer to Screening Example 2, taking the specificity as ⑤ amlodipine impurities A, B, D, E, F, G, H positioning solution, Add 20 μl of each test solution, inject into the liquid chromatograph, record the chromatogram, and refer to the results. Figure 12 .

[0129] Table 10 Screening Example 3 Linear Gradient Elution Conditions

[0130]

[0131]

[0132] As can be seen from the figure, the amlodipine impurity H and the amlodipine impurity B merge into a peak, which is poorly separated. The amlodipine impurity E is wrapped with unknown impurities and cannot be separated. The specificity is difficult to meet the requirements.

[0133] Screening Example 4

[0134] The difference between Screening Example 4 and Screening Example 3 is that the mobile phase A of Screening Example 4 is changed to 0.55% phosphoric acid aqueous solution (adjusted to pH 3.3 with triethylamine). The other parameters of Screening Example 4 refer to Screening Example 3, and the system suitability solution and 20 μl of each test solution was injected into the liquid chromatograph and the chromatogram was recorded. The results were shown in Figure 13 .

[0135] As can be seen from the figure, the peak shapes of each chromatographic peak are better after changing the mobile phase system. However, some impurities are not eluted after 45 minutes, resulting in a decrease in the number of impurities. Therefore, the gradient elution conditions are further optimized to improve the elution capacity.

[0136] Screening Example 5

[0137] The difference between Screening Example 5 and Screening Example 4 is that the linear gradient elution conditions of Screening Example 5 are changed to those shown in Table 11. The other parameters of Screening Example 5 refer to those of Screening Example 4. Inject 20 μl of the test solution into the liquid chromatograph and record the chromatogram. Figure 14 .

[0138] Table 11 Screening Example 5 Linear Gradient Elution Conditions

[0139] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 50 50 50 20 80 60 10 90 70 10 90 71 50 50 85 50 50

[0140] As can be seen from the figure, further changing the gradient program can achieve good separation of the chromatographic peaks and more appropriate elution times of the impurities.

[0141] Unless otherwise defined, each technical and scientific term used herein has the same meaning as that generally understood by those of ordinary skill in the art to which the present disclosure belongs. For the purpose of description and disclosure, all patents, patent applications and other publications are expressly incorporated herein by reference. These publications are provided only because they are disclosed earlier than the filing date of the present application. All statements about the dates of these documents or the statements of the contents of these documents are based on the information available to the applicant and do not constitute any acknowledgment of the correctness of the dates of these documents or the contents of these documents. Moreover, in any country, any citation to these publications in this article does not constitute an acknowledgment that this publication becomes a part of the common general knowledge in this area.

[0142] Those skilled in the art will recognize that the scope of the present application is not limited to the various specific implementation modes and embodiments described above, and any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for determining related substances in irbesartan-levamlodipine compound tablets, characterized in that: Detection was performed by high performance liquid chromatography using a YMC-Triart C18 column, a 0.55% phosphoric acid aqueous solution with a pH of 3.0 to 3.5 as mobile phase A, and methanol as mobile phase B. Detection was performed under the following gradient elution conditions: 。 2. The method according to claim 1, characterized in that In the high performance liquid chromatography method, the flow rate is 0.6 to 1.0 ml / min.

3. The method according to claim 1, characterized in that In the high performance liquid chromatography method, the column temperature is 35-45°C.

4. The method according to claim 1, characterized in that In the high performance liquid chromatography method, the detection wavelengths are 220 nm and 237 nm.

5. The method according to claim 1, characterized in that: The high performance liquid chromatography method further comprises a solution preparation step.

6. The method according to claim 5, characterized in that The solution includes part or all of a reference substance stock solution, a reference substance solution, a positioning solution, a system suitability solution, a test substance solution, a spiked test substance solution, a sensitivity solution, and an alkali-destroying solution of levamlodipine besylate.

7. The method according to claim 5 or 6, characterized in that: The solvent used in preparing the solution is 0.55% phosphoric acid aqueous solution-methanol (20:80, v / v).

8. The method according to any one of claims 1 to 6, characterized in that: In the high performance liquid chromatography method, an alkali destruction solution of irbesartan is used as a system suitability solution.

9. The method according to claim 1, characterized in that: The known impurities in the related substances include amlodipine impurities A, B, D, E, F, G, H, and irbesartan impurity I.

10. The method according to claim 1, characterized in that: The analysis conditions of the HPLC method are as follows: a YMC-Triart C18 column, a 0.55% phosphoric acid aqueous solution at a pH of 3.0 to 3.5 as mobile phase A, methanol as mobile phase B, a flow rate of 0.6 to 1.0 ml per minute, a column temperature of 35 to 45° C., detection wavelengths of 220 nm and 237 nm, an injection volume of 10 to 20 μl, and detection under the gradient elution conditions shown in the table below:

Images