Method for detecting N-nitroso amlodipine impurity in perindopril amlodipine tablet

Abstract

The invention relates to the technical field of medicine detection, in particular to a method for detecting N-nitroso amlodipine impurities in perindopril amlodipine tablets, which comprises the following steps: S1, preparing a test solution; s2, preparing a reference substance solution; the conditions of high performance liquid chromatography are as follows: Phenomenex Kinetex 2.6 [mu] m C18 100A is taken as a chromatographic column, a Phenomenex guard column is taken as a pre-column, an aqueous solution containing 0.1% (v / v) formic acid and 2mM ammonium acetate is taken as a mobile phase A, acetonitrile containing 0.1% (v / v) formic acid is taken as a mobile phase B, and the column temperature is 45 DEG C; and S4, performing chromatographic analysis. According to the present invention, the N-nitroso amlodipine impurity in the perindopril amlodipine tablet is determined through the high performance liquid chromatography, and the method has advantages of good specificity, good system applicability and good repeatability.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, in particular to a method for detecting N-nitrosoamlodipine impurities in perindopril amlodipine tablets. Background Art

[0002] Perindopril / Amlodipine Tablets are a combination antihypertensive drug consisting of perindopril (an ACE inhibitor) and amlodipine (a calcium channel blocker). They synergistically lower blood pressure through different mechanisms and are suitable for patients with essential hypertension whose condition is poorly controlled with a single medication. N-nitrosoamlodipine is a potential genotoxic impurity in perindopril / Amlodipine Tablets, with a control limit of 100 ppm.

[0003] Through literature research, no relevant reports on the detection of N-nitrosoamlodipine impurities in perindopril amlodipine tablets have been found at home or abroad. Summary of the Invention

[0004] Aiming at the current technical gap, the present invention proposes a method for detecting N-nitrosoamlodipine impurities in perindopril amlodipine tablets.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for detecting N-nitrosoamlodipine impurities in perindopril amlodipine tablets, comprising the following steps: S1. Prepare the test solution: take 5 perindopril amlodipine tablets, place them in a 25 mL volumetric flask and dissolve them in 20 mL of methanol. First, ultrasonically disintegrate for 5 minutes, then dilute to the mark with methanol, shake well, and then centrifuge at 4000 rpm for 10 minutes. After that, collect the supernatant to obtain the test solution, wherein the mass of perindopril amlodipine tablets is 15 mg, and each perindopril amlodipine tablet contains 5 mg of amlodipine besylate; S2. Prepare reference substance solution: accurately measure 2.5 mg of N-nitroso-amlodipine, place it in a 50 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to obtain reference substance dilution, accurately measure 1 mL of reference substance dilution, place it in a 100 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to obtain reference substance stock solution, accurately measure 10 mL of reference substance stock solution, place it in a 50 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to obtain reference substance solution; S3. HPLC: The test solution and the reference solution were analyzed by HPLC to obtain chromatograms. The HPLC conditions were as follows: a Phenomenex Kinetex 2.6 μm C18 100A column with a Phenomenex guard column as a pre-column, an aqueous solution containing 0.1% (v / v) formic acid and 2 mM ammonium acetate as mobile phase A, and acetonitrile containing 0.1% (v / v) formic acid as mobile phase B. The column temperature was 45°C. S4. Chromatographic analysis: According to the chromatogram, the external standard method is used for analysis to obtain the content of N-nitrosoamlodipine impurity in the perindopril amlodipine tablets.

[0006] Optionally, in S1, the ultrasonic frequency during ultrasonic disintegration is 35 kHz and the ultrasonic power is 100 W.

[0007] Optionally, in S3, mobile phase A and mobile phase B are ultrasonically degassed for 5 minutes before use.

[0008] Optionally, in S3, the elution mode of the high performance liquid chromatography analysis is gradient elution, and the gradient elution program is shown in Table 1: Table 1 Gradient elution program Time (min) Flow rate (mL / min) Mobile phase B (%) 2.00 0.600 55 2.10 0.600 95 4.00 0.600 95 4.10 0.600 55

[0009] Compared with the prior art, the present invention has the following beneficial effects: the present invention uses high performance liquid chromatography to determine N-nitrosoamlodipine impurities in perindopril amlodipine tablets, and has good specificity, system applicability and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 is the chromatogram of the blank solution; Figure 2 This is the chromatogram of the perindopril arginine bulk drug solution; Figure 3 This is a chromatogram of amlodipine besylate bulk drug solution; Figure 4 is the chromatogram of the excipient solution; Figure 5 is the chromatogram of the test solution; Figure 6 is the chromatogram of the reference solution; Figure 7 is the chromatogram of the spiked test solution; Figure 8 This is the relationship between actual concentration and peak area in the linear relationship. DETAILED DESCRIPTION

[0011] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0012] Embodiment: The present invention provides a method for detecting N-nitrosoamlodipine impurities in perindopril amlodipine tablets, comprising the following steps: S1. Prepare the test solution: take 5 perindopril amlodipine tablets, place them in a 25 mL volumetric flask and dissolve them in 20 mL of methanol. First, ultrasonically disintegrate for 5 minutes, then dilute to the mark with methanol, shake well, and then centrifuge at 4000 rpm for 10 minutes. After that, collect the supernatant to obtain the test solution, wherein the mass of perindopril amlodipine tablets is 15 mg, and each perindopril amlodipine tablet contains 5 mg of amlodipine besylate; S2. Prepare reference substance solution: accurately measure 2.5 mg of N-nitroso-amlodipine, place it in a 50 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to obtain reference substance dilution, accurately measure 1 mL of reference substance dilution, place it in a 100 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to obtain reference substance stock solution, accurately measure 10 mL of reference substance stock solution, place it in a 50 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to obtain reference substance solution; S3. HPLC: The test solution and the reference solution were analyzed by HPLC to obtain chromatograms. The HPLC conditions were as follows: a Phenomenex Kinetex 2.6 μm C18 100A column with a Phenomenex guard column as a pre-column, an aqueous solution containing 0.1% (v / v) formic acid and 2 mM ammonium acetate as mobile phase A, and acetonitrile containing 0.1% (v / v) formic acid as mobile phase B. The column temperature was 45°C. S4. Chromatographic analysis: According to the chromatogram, the external standard method is used for analysis to obtain the content of N-nitrosoamlodipine impurity in the perindopril amlodipine tablets.

[0013] In a specific embodiment of the present invention, in S1, the ultrasonic frequency during ultrasonic disintegration is 35 kHz, and the ultrasonic power is 100 W.

[0014] In a specific embodiment of the present invention, in S3, mobile phase A is prepared as follows: 2000 mL of ultrapure water is added with 2 mL of formic acid and 2 mL of 2M ammonium acetate solution, the mixture is shaken and then ultrasonically degassed for 5 minutes. Mobile phase B is prepared as follows: 2000 mL of acetonitrile is added with 2 mL of formic acid, and ultrasonically degassed for 5 minutes. Generally, the shelf life of mobile phase A is 7 days, and the shelf life of mobile phase B is 30 days.

[0015] In a specific embodiment of the present invention, in S3, the elution mode of the high performance liquid chromatography analysis is gradient elution, and the gradient elution program is shown in Table 1: Table 1 Gradient elution program Time (min) Flow rate (mL / min) Mobile phase B (%) 2.00 0.600 55 2.10 0.600 95 4.00 0.600 95 4.10 0.600 55

[0016] In a specific embodiment of the present invention, the formula for calculating the content of N-nitrosoamlodipine impurity in perindopril amlodipine tablets by external standard method is as follows: .

[0017] Where: A 供 is the peak area of N-nitroso-amlodipine in the test solution; A 对 is the peak area of N-nitroso-amlodipine in the reference solution; M 对 is the sample weight of the reference substance, (mg); P 对 is the content of the reference substance; V 对 is the dilution volume of the reference solution, (mL); V 供 is the dilution volume of the test solution, (mL); M 供 is the sample weight of the test sample, (mg); W is the average tablet weight of perindopril and amlodipine tablets (mg); Label strength: Based on 5 mg of amlodipine besylate per tablet.

[0018] The detection method disclosed in the present invention is described below in combination with methodology and specific examples.

[0019] 1. Analytical methods 1.1 Chromatographic conditions The chromatographic conditions disclosed in the present invention are adopted during the specific implementation.

[0020] 1.2 Solution preparation 1.2.1 Blank solution Methanol was used as the blank solution.

[0021] 1.2.2 Perindopril Arginine API Solution Take 50 mg of the raw material drug (batch number: PAEA-2-05221101), accurately weigh it, place it in a 50 mL volumetric flask, and dissolve it with diluent to 80% of the volume of the volumetric flask. Ultrasonicate for 5 minutes to disintegrate it, then dilute to the scale with diluent and shake well. Centrifuge the solution (4000 rpm / 10 min) and collect the supernatant to obtain the arginine paprika raw material drug solution.

[0022] 1.2.3 Amlodipine besylate API Solution Take 50 mg of the raw material drug (batch number: 07022530), accurately weigh it, place it in a 50 mL volumetric flask, dissolve it with diluent to 80% of the volume of the volumetric flask, sonicate for 5 minutes to dissolve it, then dilute to the scale with methanol and shake well; centrifuge the solution (4000 rpm / 10 min) and collect the supernatant to obtain the amlodipine besylate raw material drug solution.

[0023] 1.2.4 Excipient solution Take 183 mg of blank excipient (batch number: XR210525011601KF) (corresponding to 6.94 mg of amlodipine besylate raw material at the prescription ratio), accurately weigh, place in a 10 mL volumetric flask, dissolve with diluent to 80% of the volume of the volumetric flask, ultrasonicate for 5 minutes to disintegrate, then dilute to the scale with diluent and shake well; centrifuge the solution (4000 rpm / 10 min) and collect the supernatant to obtain the excipient solution.

[0024] 1.2.5 Test solution Take 5 tablets of the test sample (batch number: YZ20230601; specification: 15 mg, each tablet contains 5 mg of amlodipine besylate), accurately weigh, place in a 25 mL volumetric flask, dissolve about 80% of the volume of the flask with diluent, ultrasonicate for 5 minutes to disintegrate, then dilute to the scale with diluent, and shake well; centrifuge the solution (4000 rpm / 10 min) and collect the supernatant to obtain the test sample solution.

[0025] 1.2.6 Reference solution Accurately weigh 2.5 mg of N-nitroso-amlodipine, place it in a 50 mL volumetric flask, dissolve it with diluent, and dilute to the mark, shake well; accurately measure 1 mL, place it in a 100 mL volumetric flask, dissolve it with diluent, and dilute to the mark, shake well to obtain the reference substance stock solution; accurately measure 10 mL of the reference substance stock solution, place it in a 50 mL volumetric flask, dissolve it with diluent, and dilute to the mark, shake well to obtain the reference substance solution.

[0026] 1.2.7 Spiked test solution Take 5 tablets of the test sample (batch number: YZ20230601; specification: 15 mg, each tablet contains 5 mg of amlodipine besylate), accurately weigh them, place them in a 25 mL volumetric flask, accurately add 5 mL of the reference substance stock solution, dissolve it with diluent to 80% of the volume of the volumetric flask, sonicate for 5 minutes to disintegrate, then dilute to the scale with diluent and shake well; centrifuge the solution (4000 rpm / 10 min) and collect the supernatant to obtain the spiked test sample solution.

[0027] 1.2.8 Lin-200% solution Pipette 20 mL of the reference stock solution into a 50 mL volumetric flask, dissolve it with diluent, make up to the mark, and shake well.

[0028] 1.2.9 Lin-150% solution Pipette 15 mL of the reference stock solution into a 50 mL volumetric flask, dissolve it with diluent, make up to the mark, and shake well.

[0029] 1.2.10 Lin-100% solution Pipette 10 mL of the reference stock solution into a 50 mL volumetric flask, dissolve it with diluent, make up to the mark, and shake well.

[0030] 1.2.11 Lin-50% solution Pipette 5 mL of the reference stock solution into a 50 mL volumetric flask, dissolve it with diluent, make up to the mark, and shake well.

[0031] 1.2.12 Lin-10% solution Pipette 2 mL of the reference stock solution into a 100 mL volumetric flask, dissolve it with diluent and make up to the mark, then shake well.

[0032] 1.2.13 Repeatability of test solution Take 5 perindopril and amlodipine tablets (average tablet weight 196.53 mg), accurately weigh them, place them in a 25 mL volumetric flask, accurately add 5 mL of the reference substance stock solution, dissolve it with diluent to 80% of the volume of the volumetric flask, sonicate for 5 minutes to disintegrate, then dilute to the scale with diluent and shake well; centrifuge the solution (4000 rpm / 10 min) and collect the supernatant to obtain the reproducible test solution, which is prepared in parallel for 6 times.

[0033] 2. Validation of analytical methodology 2.1 Exclusivity Prepare blank solution, perindopril arginine bulk drug solution, amlodipine besylate bulk drug solution, excipient solution, test solution, reference solution, and spiked test solution according to 1.2 solution preparation. Take 5 μL of each solution, inject and analyze, and record the chromatogram. Figures 1 to 7 As shown, it can be seen that the blank solution, blank excipient, and arginine perindopril bulk drug solution have no mutual interference with N-nitroso-aminochloroquine. The specificity of the present invention is good.

[0034] 2.2 Linear relationship and range Prepare the solution according to 1.2, prepare Lin-200% solution, Lin-150% solution, Lin-100% solution, Lin-50% solution, and Lin-10% solution, measure 5uL of each, inject and analyze, and record the spectrum. Perform linear regression with the concentration of the analyte in each solution as the horizontal axis and the peak area of the analyte as the vertical axis, and list the regression equation, correlation coefficient, and linear relationship diagram, as shown in Table 2 and Figure 8 shown.

[0035] Table 2 Linear relationship test results From Table 2 and Figure 8 It can be seen that the correlation coefficient R 2 ≥0.9900, |intercept / A100%|≤10%. The linear relationship of the present invention is good.

[0036] 2.3 System Applicability Prepare the reference solution as the system suitability sample according to the solution preparation in 1.2. Calculate the mean and RSD of the peak area and retention time. Record the results in Table 3.

[0037] Table 3 System suitability results As shown in Table 3, the peak area RSD of the six measurements was 1.58%, less than 10%, and the retention time RSD was 0.17%, both less than 2%. The system of the present invention has good applicability.

[0038] 2.4 Repeatability test Prepare the blank solvent, reference solution, and six replicate test solutions (RTS-1 to RTS-6) according to the solution preparation procedure in 1.2. Aspirate 5 μL of each solution, inject, analyze, and record the chromatogram. Calculate the analyte content using the external standard method and record the results in Table 4.

[0039] Table 4 Repeatability test results As shown in Table 4, the relative standard deviation between the six measurements was 2.03%, which is less than 10%. The present invention has good repeatability.

[0040] 2.5 Solution stability Prepare the reference solution and spiked test solution according to 1.2. Store in a refrigerator at 4°C and at room temperature at 25°C, respectively. At different time points, sample 5 μL each time and analyze the solution. Record the chromatogram. Calculate the mean peak area and RSD value for each determination to investigate the stability of the solution under different conditions. The results are recorded in Tables 5 and 6, respectively.

[0041] Table 5 Solution stability test results (4°C) As shown in Table 5, the RSDs of the peak areas of the reference solution and the spiked test solution measured at different time points within 22 h at 4 °C were all less than 15%, indicating that the 4 °C solution had good stability.

[0042] Table 6 Solution stability test results (25°C) As shown in Table 6, the RSDs of the peak areas of the reference solution and the spiked test solution measured at different time points within 22 h at 25 °C were all less than 15%, indicating that the 25 °C solution had good stability.

[0043] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for detecting N-nitrosoamlodipine impurities in perindopril amlodipine tablets, characterized in that: The following steps are involved: S1. Prepare the test solution: take 5 perindopril amlodipine tablets, place them in a 25 mL volumetric flask and dissolve them in 20 mL of methanol. First, ultrasonically disintegrate for 5 minutes, then dilute to the mark with methanol, shake well, and then centrifuge at 4000 rpm for 10 minutes. After that, collect the supernatant to obtain the test solution, wherein the mass of perindopril amlodipine tablets is 15 mg, and each perindopril amlodipine tablet contains 5 mg of amlodipine besylate; S2. Prepare reference substance solution: accurately measure 2.5 mg of N-nitroso-amlodipine, place it in a 50 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to obtain reference substance dilution, accurately measure 1 mL of reference substance dilution, place it in a 100 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to obtain reference substance stock solution, accurately measure 10 mL of reference substance stock solution, place it in a 50 mL volumetric flask, dissolve it in methanol and dilute to the mark, shake well to obtain reference substance solution; S3. HPLC: The test solution and the reference solution were analyzed by HPLC to obtain chromatograms. The HPLC conditions were as follows: a Phenomenex Kinetex 2.6 μm C18 100A column with a Phenomenex guard column as a pre-column, an aqueous solution containing 0.1% (v / v) formic acid and 2 mM ammonium acetate as mobile phase A, and acetonitrile containing 0.1% (v / v) formic acid as mobile phase B. The column temperature was 45°C. S4. Chromatographic analysis: According to the chromatogram, the external standard method is used for analysis to obtain the content of N-nitrosoamlodipine impurity in the perindopril amlodipine tablets.

2. The method for detecting N-nitrosoamlodipine impurities in a perindopril amlodipine tablet according to claim 1, wherein: In S1, the ultrasonic frequency during ultrasonic disintegration was 35 kHz and the ultrasonic power was 100 W.

3. The method for detecting N-nitrosoamlodipine impurities in perindopril amlodipine tablets according to claim 1, characterized in that: In S3, mobile phase A and mobile phase B were ultrasonically degassed for 5 min before use.

4. The method for detecting N-nitrosoamlodipine impurities in perindopril amlodipine tablets according to claim 1, wherein: In S3, the elution mode of the high performance liquid chromatography analysis is gradient elution, and the gradient elution program is shown in Table 1: Table 1 Gradient elution program 。

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