Method for detecting N-nitroso benazepril impurity in levamlodipine benazepril

Abstract

The invention discloses a method for detecting N-nitroso benazepril impurities in levamlodipine benazepril, which comprises the following steps: grinding a benazepril hydrochloride tablet to be detected, mixing and dissolving the benazepril hydrochloride tablet with 1-methyl-2-pyrrolidone and a solvent according to the mass ratio of the effective amount of benazepril hydrochloride to the mass of 1-methyl-2-pyrrolidone of (10-50): 1, filtering, and concentrating to obtain the N-nitroso benazepril impurities in levamlodipine benazepril. Obtaining a test solution; and detecting the impurities of the N-nitroso benazepril in the test solution by adopting liquid chromatography-tandem mass spectrometry. The detection method provided by the invention is a liquid chromatography-mass spectrometry method, has the characteristics of strong specificity, high sensitivity, high accuracy and the like, and is suitable for trace detection of N-nitroso benazepril in the levamlodipine benazepril capsule preparation.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and detection, and particularly relates to a method for detecting N-nitrosobenazepril impurities in levamlodipine benazepril. Background Art

[0002] The statements herein merely provide background information related to the present invention and do not necessarily constitute prior art.

[0003] In recent years, a variety of pril drugs have been recalled from the market due to the detection of nitrosamine impurity levels higher than ADI, such as enalapril, quinapril and other related preparations.

[0004] Levoamlodipine benazepril capsule is a complex high-end hard capsule preparation, which contains a film-coated tablet of benazepril hydrochloride and a white powder prepared from an appropriate amount of excipients and levamlodipine besylate. Since the raw material of benazepril hydrochloride is used in the formulation, the primary amino group contained in the raw material may react with the residual nitrosamines in the excipients to produce the nitrosamine impurity N-nitrosobenazepril, which increases the cancer risk of the product. The N-nitrosobenazepril impurity has high toxicity and low limit (ADI is 1500ng / day), and the maximum daily dose of benazepril hydrochloride is 40mg, so the quality control limit of N-nitrosobenazepril is 37.5ppm. However, there is no detection method for controlling N-nitrosobenazepril at present, so it is very necessary to establish a quantitative detection method for nitrosamine impurities N-nitrosobenazepril in levovamlodipine benazepril capsules. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for detecting N-nitrosobenazepril impurities in levoamlodipine benazepril. The method provided by the present invention has the characteristics of strong specificity, high sensitivity, high accuracy, etc., is universal and practical, and is simple and efficient in operation.

[0006] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0007] A method for detecting N-nitrosobenazepril impurities in levamlodipine benazepril comprises the following steps:

[0008] The benazepril hydrochloride tablets in the contents of the levamlodipine benazepril capsule to be tested are ground, mixed and dissolved with 1-methyl-2-pyrrolidone and a solvent, the mass ratio of the effective amount of benazepril hydrochloride to 1-methyl-2-pyrrolidone being 10-50:1, and filtered to obtain a test solution;

[0009] The test solution was tested for N-nitrosobenazepril impurities by liquid chromatography-tandem mass spectrometry.

[0010] The effective amount of benazepril hydrochloride refers to the mass of benazepril hydrochloride in the levamlodipine benazepril capsule to be tested.

[0011] The choice of solvent for nitrosamine impurity detection is usually to use acetonitrile-water / or methanol-water for dissolution detection or to add a small amount of formic acid, methylamine and other solutions to the above mobile phase. There are several problems with the existing solvent selection:

[0012] ① For solvents such as acetonitrile-water / or methanol-water, as the proportion of water increases, the recovery rate of N-nitrosobenazepril impurity test results becomes higher. The reason is that as the amount of water increases, the degree of benazepril hydrochloride in the free state becomes higher. The free benazepril will react with the residual nitrite in the excipients to produce N-nitrosobenazepril, resulting in a false positive in the sample test result, causing the N-nitrosobenazepril test result of the product to be high, which cannot truly reflect the impurity level of N-nitrosobenazepril in the product.

[0013] ②By adding an acidic solvent such as 0.1% formic acid or an alkaline solvent such as methylamine to the mobile phase, benazepril hydrochloride can always be kept in a molecular state or a pure ionic state, which to a certain extent avoids the re-production of N-nitrosobenazepril in the preparation and detection process of the test solution, thereby avoiding the phenomenon of false positives. However, in the detection process of N-nitrosobenazepril impurities in the levamlodipine benazepril capsule of this product, only formic acid or methylamine is added, and the recovery rate RSD% of the product detection is still high, generally exceeding 10% (RSD should be ≤5%), and the accuracy of the analytical method is low.

[0014] In order to solve the above problems, the core of the present invention is to add 1-methyl-2-pyrrolidone to the solvent when preparing the test solution. The addition of 1-methyl-2-pyrrolidone makes the extraction of N-nitrosobenazepril more complete, and at the same time inhibits the test sample from continuing to produce N-nitrosobenazepril in the solution state, thereby avoiding the false positive phenomenon. In short, the addition of 1-methyl-2-pyrrolidone improves the sensitivity, specificity and accuracy of the analytical method.

[0015] In some embodiments, the mass ratio of the effective amount of benazepril hydrochloride to 1-methyl-2-pyrrolidone is 10-30:1; preferably 15-25:1; and more preferably 20:1.

[0016] In some embodiments, the solvent is 1-methyl-2-pyrrolidone, formic acid or methylamine.

[0017] Preferably, the concentration of 1-methyl-2-pyrrolidone in the test solution is 0.03-0.1 mg / ml; preferably 0.03-0.7 mg / ml; more preferably 0.05 mg / ml.

[0018] In some embodiments, ultrasound is applied to the mixed system during the dissolution to assist the dissolution and promote the extraction of the target.

[0019] In some embodiments, when the test solution is detected, the liquid chromatography conditions are: the filler of the chromatographic column is octadecylsilane bonded silica gel; the mobile phase A is a mixture of formic acid and water, and the volume percentage of formic acid is 0.08-0.12%; the mobile phase B is a mixture of formic acid, water and acetonitrile, and the volume percentage of formic acid is 0.08-0.12%.

[0020] Preferably, the column temperature during liquid chromatography detection is 28-32°C.

[0021] Preferably, the gradient elution program of the liquid chromatography is: 0-3min, mobile phase A is 60%, the purpose is to elute the unretained blank excipient peak; 3-10min, mobile phase A changes from 60% to 0, the purpose is to elute the N-nitrosobenazepril peak; 10-12min, mobile phase A is 0, the purpose is to elute the chromatographic peak with small polarity; 12-12.5min, mobile phase A changes from 0 to 60%, the purpose is to return to the initial state of the mobile phase; 12.5-15min, mobile phase A is 60%, the purpose is to balance the chromatographic column to facilitate the next injection of the sample; % is the volume percentage.

[0022] In some embodiments, the detector for mass spectrometry detection is a triple quadrupole mass spectrometry detector.

[0023] Preferably, the ion source for mass spectrometry detection is an electrospray ionization source; and the mode is a positive ion scanning mode.

[0024] The beneficial effects achieved by one or more embodiments of the present invention are as follows:

[0025] The detection method of the invention is a liquid chromatography-mass spectrometry method, which has the characteristics of strong specificity, high sensitivity, high accuracy and the like, and is suitable for the trace detection of N-nitrosobenazepril in levoamlodipine-benazepril capsule preparations. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 This is a typical chromatogram of the N-nitrosobenazepril reference substance solution in the embodiment of the present invention;

[0028] Figure 2 This is a typical chromatogram of a blank solvent solution in an embodiment of the present invention;

[0029] Figure 3It is a typical chromatogram of the blank auxiliary material solution in the embodiment of the present invention. DETAILED DESCRIPTION

[0030] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0031] Structural formula:

[0032]

[0033] The present invention will be further described below in conjunction with the embodiments.

[0034] Example 1

[0035] Chromatographic column: Octadecylsilane bonded silica gel as filler (Agilent SB-C18, 2.1 mm × 100 mm, 1.8 μm or a chromatographic column with equivalent performance);

[0036] Mobile phase A: formic acid-water (0.1:100);

[0037] Mobile phase B: formic acid-water-acetonitrile (0.1:5:95);

[0038] Column temperature: 30°C;

[0039] Flow rate: 0.3ml / min;

[0040] Solvent: acetonitrile-water (80:20);

[0041] 1-Methyl-2-pyrrolidone solution: Take an appropriate amount of 1-methyl-2-pyrrolidone (CAS No.: 872-50-4) and dilute it with methanol to make a solution containing about 10 mg per 1 ml;

[0042] Injection volume: 10 μl;

[0043] The gradient elution program is shown in Table 1.

[0044] Table 1

[0045]

[0046] Detector: Triple quadrupole mass spectrometer (AB Sciex Triple Quad5500 or mass spectrometer with equivalent performance)

[0047] Ion source: electrospray ionization (ESI);

[0048] Mode: positive ion scanning mode;

[0049] Mass spectrometry detection mode: Select reaction detection (SIM) and scan mode as SRM.

[0050] The quantitative ions and the qualitative ions are shown in Table 2.

[0051] Table 2

[0052]

[0053]

[0054] Ion source temperature: 350°C;

[0055] Atomization temperature: 350℃;

[0056] Ionization current: 4A;

[0057] Sheath gas: 45arb; Auxiliary gas: 15arb.

[0058] (3) Sample preparation

[0059] Test solution: Take 10 benazepril hydrochloride tablets, accurately weigh, grind, accurately weigh an appropriate amount of the ground powder (equivalent to approximately 10 mg of benazepril hydrochloride), place it in a 10 ml volumetric flask, add 50 μl of 1-methyl-2-pyrrolidone solution and 3 ml of acetonitrile-water (80:20), ultrasonically dissolve the benazepril hydrochloride and impurities for 10 minutes, dilute to the scale with acetonitrile-water (80:20), shake well, filter, and take the filtrate.

[0060] Reference solution: Take about 9.5 mg of N-nitrosobenazepril reference substance, accurately weigh, place in a 250 ml volumetric flask, add 10 ml of acetonitrile-water (80:20), sonicate to dissolve, dilute to scale with acetonitrile-water (80:20), shake well, accurately measure 10 ml, place in a 100 ml volumetric flask, dilute to scale with acetonitrile-water (80:20), shake well, accurately measure 1 ml, place in a 100 ml volumetric flask, add 500 μl of 1-methyl-2-pyrrolidone solution, dilute to scale with acetonitrile-water (80:20), shake well.

[0061] Determination method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatography-mass spectrometer, record the mass spectrum, and calculate by peak area according to the external standard method.

[0062] The calculation formula is as follows:

[0063]

[0064] Where: Sample A: is the mass spectrum peak area of ​​N-nitrosobenazepril in the test solution;

[0065] A pair: the mass spectrum peak area of ​​N-nitrosobenazepril in the reference solution;

[0066] m: the weight of N-nitrosobenazepril reference substance, mg;

[0067] msample: the sample weight of N-nitrosobenazepril test sample, g;

[0068] V: dilution multiple of N-nitrosobenazepril reference solution;

[0069] V sample: dilution multiple of the test solution;

[0070] The standard stipulates that N-nitrosobenazepril shall not exceed 37.5ppm.

[0071] The methodological study of the above methods is as follows:

[0072] 1. Specificity test

[0073] Blank solution: Take 50 μl of 1-methyl-2-pyrrolidone solution and an appropriate amount of solvent [acetonitrile-water (80:20)].

[0074] Blank excipient solution: Take about 70 mg of blank excipient, accurately weigh, place in a 10 ml volumetric flask, add 50 μl of 1-methyl-2-pyrrolidone solution and 3 ml of acetonitrile-water (80:20), sonicate for 10 minutes to dissolve, dilute to scale with acetonitrile-water (80:20), shake well, filter, and take the filtrate.

[0075] Test solution: Take 10 benazepril hydrochloride tablets, accurately weigh, grind, accurately weigh an appropriate amount of the ground powder (approximately equivalent to 10 mg of benazepril hydrochloride), put it in a 10 ml volumetric flask, add 50 μl of 1-methyl-2-pyrrolidone solution and 3 ml of acetonitrile-water (80:20), ultrasonically dissolve the benazepril hydrochloride for 10 minutes, dilute to the scale with acetonitrile-water (80:20), shake well, filter, and take the filtrate.

[0076] Reference solution: Take about 9.5 mg of N-nitrosobenazepril reference substance, accurately weigh, place in a 250 ml volumetric flask, add 10 ml of acetonitrile-water (80:20), sonicate to dissolve, dilute to scale with acetonitrile-water (80:20), shake well, accurately measure 10 ml, place in a 100 ml volumetric flask, dilute to scale with acetonitrile-water (80:20), shake well, accurately measure 1 ml, place in a 100 ml volumetric flask, add 500 μl of 1-methyl-2-pyrrolidone solution, dilute to scale with acetonitrile-water (80:20), shake well.

[0077] Accurately measure 10 μl of each of the above solutions, inject them into liquid chromatography-mass spectrometry, record the chromatogram, and the test results are shown in Table 3.

[0078] Table 3 N-nitrosobenazepril detection specificity test results

[0079]

[0080] Conclusion: Blank solvent and blank excipients do not interfere with the detection of N-nitrosobenazepril. The method has good specificity. Figure 1 , Figure 2 and Figure 3 shown.

[0081] 2. Linearity and range tests

[0082] Reference substance stock solution: Take about 9.5 mg of N-nitrosobenazepril reference substance, accurately weigh, place in a 250 ml volumetric flask, add 10 ml of acetonitrile-water (80:20), sonicate to dissolve, dilute to the mark with acetonitrile-water (80:20), shake well, accurately measure 10 ml, place in a 100 ml volumetric flask, dilute to the mark with acetonitrile-water (80:20), shake well, accurately measure 2 ml, place in a 100 ml volumetric flask, dilute to the mark with acetonitrile-water (80:20), shake well.

[0083] Linear series solution: Accurately measure 1 ml, 2.5 ml, 5 ml, 6 ml, and 7.5 ml of the linear stock solution and place them in different 10 ml volumetric flasks, dilute to the scale with 50 μl of 1-methyl-2-pyrrolidone solution and acetonitrile-water (80:20), and shake well.

[0084] Accurately measure 10 μl of each of the above linear series solutions, inject them into the liquid chromatography-mass spectrometer, and record the chromatogram. Perform linear regression on the peak area with concentration (including the quantitative limit concentration solution). The results are shown in Table 4 below.

[0085] Table 4 N-nitrosobenazepril linearity and range test results

[0086]

[0087] Conclusion: From the test results, we can see that the linear regression equation of N-nitrosobenazepril is: A=12.04C-4.37, and the linear correlation coefficient r=0.9991, which meets the verification requirements. The results show that N-nitrosobenazepril has a good linear relationship in the range of 1.50ng / ml-56.14ng / ml.

[0088] 3. Limit of detection and limit of quantification

[0089] Under the experimental conditions, take the linear series solutions under the linearity and range test items, dilute them step by step, and inject them separately to determine the detection limit and quantification limit of this product and each impurity. Accurately measure 20μl of the quantification limit solution, inject it into the liquid chromatograph, record the chromatogram, repeat the injection for 6 times, and examine the repeatability of the quantification limit.

[0090] Table 5 Results of the quantification limit test of N-nitrosobenazepril

[0091]

[0092] Table 6 N-nitrosobenazepril detection limit test results

[0093]

[0094]

[0095] Table 7 N-nitrosobenazepril quantitative limit repeatability test results - peak area

[0096]

[0097] Table 8 Results of repeatability test of limit of quantitation of N-nitrosobenazepril - retention time (min)

[0098] name 1 2 3 4 5 6 average value RSD% Peak 1 6.928 6.942 6.942 6.955 6.942 6.942 6.9418 0.13 Peak 2 7.302 7.288 7.275 7.302 7.275 7.302 7.2907 0.19

[0099] Conclusion: The test results show that when the quantitative limit solution was injected 6 times continuously, the RSD of the retention time was less than 1.0%, and the RSD of the sum of peak areas was less than 15%, which met the verification requirements.

[0100] 4. System suitability test

[0101] Take 10 μl of the "reference solution" under the specificity item, inject it into the liquid chromatography-mass spectrometer, repeat the injection five times, and record the chromatogram. The test results are shown in Tables 9 and 10 below.

[0102] Table 9 System suitability test results - peak area

[0103]

[0104] Table 10 System suitability test results - retention time (min)

[0105] name 1 2 3 4 5 average value RSD(%) Peak 1 6.942 6.928 6.928 6.942 6.942 6.936 0.12 Peak 2 7.289 7.289 7.288 7.289 7.289 7.289 0.01

[0106] Conclusion: From the test results, we can see that the reference solution was measured 5 times, the RSD of the sum of peak areas was less than 10%, and the RSD of retention time was less than 1%, which met the validation requirements and confirmed that the system precision was good.

[0107] 5. Repeatability test

[0108] Preparation of test solution: Take 10 tablets of Benazepril Hydrochloride, weigh accurately, grind finely, weigh an appropriate amount of fine powder (equivalent to 10 mg of Benazepril Hydrochloride), put it in a 10 ml volumetric flask, add 50 μl of 1-methyl-2-pyrrolidone solution and 3 ml of acetonitrile-water (80:20), sonicate for 10 minutes to dissolve the Benazepril Hydrochloride, dilute to scale with acetonitrile-water (80:20), shake well, filter, and take the filtrate. (Prepare 6 portions in parallel)

[0109] Preparation of reference solution: Take about 9.5 mg of N-nitrosobenazepril reference substance, accurately weigh, place in a 250 ml volumetric flask, add 10 ml of acetonitrile-water (80:20), sonicate to dissolve, dilute to scale with acetonitrile-water (80:20), shake well, accurately measure 10 ml, place in a 100 ml volumetric flask, dilute to scale with acetonitrile-water (80:20), shake well, accurately measure 1 ml, place in a 100 ml volumetric flask, add 500 μl of 1-methyl-2-pyrrolidone solution, dilute to scale with acetonitrile-water (80:20), shake well.

[0110] Accurately measure the test solution and the reference solution, inject them into the liquid chromatography-mass spectrometer, and record the chromatogram. Calculate the peak area according to the external standard method, and calculate the RSD of the 6 content determination results at the same time. The results are shown in Table 11.

[0111] Table 11 N-nitrosobenazepril repeatability test results

[0112]

[0113] Conclusion: The test results show that the same batch of samples were measured 6 times in parallel, and the average detection amount of N-nitrosobenazepril in 6 test solutions was 10.45ppm, with a range of less than 3ppm, which met the validation requirements and confirmed that the method had good repeatability.

[0114] 6. Accuracy test

[0115] Test solution: Take 10 benazepril hydrochloride tablets, accurately weigh, grind, accurately weigh an appropriate amount of the ground powder (approximately equivalent to 10 mg of benazepril hydrochloride), put it in a 10 ml volumetric flask, add 50 μl of 1-methyl-2-pyrrolidone solution and 3 ml of acetonitrile-water (80:20), ultrasonically dissolve the benazepril hydrochloride for 10 minutes, dilute to the scale with acetonitrile-water (80:20), shake well, filter, and take the filtrate.

[0116] Reference solution: Take about 9.5 mg of N-nitrosobenazepril reference substance, accurately weigh, place in a 250 ml volumetric flask, add 10 ml of acetonitrile-water (80:20), sonicate to dissolve, dilute to scale with acetonitrile-water (80:20), shake well, accurately measure 10 ml, place in a 100 ml volumetric flask, dilute to scale with acetonitrile-water (80:20), shake well, accurately measure 1 ml, place in a 100 ml volumetric flask, add 500 μl of 1-methyl-2-pyrrolidone solution, dilute to scale with acetonitrile-water (80:20), shake well.

[0117] Stock solution of reference substance: take about 9.5 mg of N-nitrosobenazepril reference substance, accurately weigh, place in a 250 ml volumetric flask, add 10 ml of acetonitrile-water (80:20), sonicate to dissolve, dilute to scale with acetonitrile-water (80:20), shake well, accurately measure 10 ml, place in a 100 ml volumetric flask, dilute to scale with acetonitrile-water (80:20), shake well, accurately measure 2 ml, place in a 100 ml volumetric flask, dilute to scale with acetonitrile-water (80:20), shake well.

[0118] Recovery solution: Take 10 benazepril hydrochloride tablets, accurately weigh, grind, accurately weigh an appropriate amount of the ground powder (equivalent to approximately 10 mg of benazepril hydrochloride), place in a 10 ml volumetric bottle, accurately measure 2.5 ml, 5 ml and 7.5 ml of the reference stock solution, ultrasonicate for 10 minutes to dissolve the benazepril hydrochloride, dilute to scale with 50 μl of 1-methyl-2-pyrrolidone solution and acetonitrile-water (80:20), shake well, filter, and take the filtrate. Use them as the recovery solutions of N-nitrosobenazepril (18.75 ppm, 37.5 ppm, 56.25 ppm) respectively. (Prepare 3 copies for each concentration in the same way).

[0119] Accurately measure 10 μl of each of the above solutions and inject them into the liquid chromatography-mass spectrometer respectively, record the chromatogram, measure the peak area, and calculate the recovery rate. The results are shown in Table 12.

[0120]

[0121] Table 12 N-nitrosobenazepril recovery test results

[0122]

[0123] Conclusion: From the test results, we can see that three levels of added recovery, low, medium and high, were carried out respectively. Three recovery reference solutions were prepared for each concentration for determination, with a total of 9 groups of results. The recovery range was between 80% and 120%, which all met the validation requirements, proving that this method has good accuracy.

[0124] 7. Sample inspection

[0125] N-nitrosobenazepril was determined by HPLC-MS / MS (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0512) and mass spectrometry (Chinese Pharmacopoeia 2020 Edition, Part IV, General Rules 0431) with low temperature injection.

[0126] The solvent was acetonitrile-water (80:20).

[0127] Preparation of 1-methyl-2-pyrrolidone solution: Take an appropriate amount of 1-methyl-2-pyrrolidone and dilute it with methanol to make a solution containing about 10 mg per 1 ml.

[0128] Preparation of test solution: Take 10 benazepril hydrochloride tablets, accurately weigh, grind, accurately weigh an appropriate amount of the ground powder (approximately equivalent to 10 mg of benazepril hydrochloride), put it in a 10 ml volumetric flask, add 50 μl of 1-methyl-2-pyrrolidone solution and 3 ml of acetonitrile-water (80:20), ultrasonically dissolve the benazepril hydrochloride for 10 minutes, dilute to the scale with acetonitrile-water (80:20), shake well, filter, and take the filtrate.

[0129] Preparation of reference solution: Take about 9.5 mg of N-nitrosobenazepril reference substance, accurately weigh, place in a 250 ml volumetric flask, add 10 ml of acetonitrile-water (80:20), sonicate to dissolve, dilute to scale with acetonitrile-water (80:20), shake well, accurately measure 10 ml, place in a 100 ml volumetric flask, dilute to scale with acetonitrile-water (80:20), shake well, accurately measure 1 ml, place in a 100 ml volumetric flask, add 500 μl of 1-methyl-2-pyrrolidone solution, dilute to scale with acetonitrile-water (80:20), shake well.

[0130] Chromatographic conditions

[0131] Octadecylsilane bonded silica gel was used as filler (Agilent SB-C18, 2.1 mm × 150 mm, 1.8 μm or a chromatographic column with equivalent column energy); 0.1% formic acid solution was used as mobile phase A, acetonitrile-water-formic acid (95:5:0.1) was used as mobile phase B, and linear gradient elution was performed according to Table 13; the flow rate was 0.3 ml per minute; the column temperature was 30°C, and the injection volume was 10 μl.

[0132] Table 13 Gradient elution program

[0133]

[0134] Table 14 Mass spectrometry conditions

[0135]

[0136] Table 15 Quantitative ions and qualitative ions

[0137]

[0138] Determination method: Accurately measure the test solution and the reference solution, inject them into the liquid chromatography-mass spectrometer, and record the chromatogram.

[0139] Limit: If the above peaks are present in the chromatogram of the test solution, the peak area calculated by the external standard method shall not exceed 37.5 ppm.

[0140] Table 16 Test results of process validation batches and reference preparations N-nitrosobenazepril

[0141]

[0142] Conclusion: The N-nitrosobenazepril test results of process validation batch samples and reference preparations were less than 30% of PDE, and the quality of the two was equivalent.

[0143] In summary, the method provided in Example 1 can be used as a method for detecting nitrosamine impurities N-nitrosamine benazepril in amlodipine benazepril capsules. This method has the characteristics of strong specificity, high sensitivity, high accuracy, etc., has the advantages of universality and practicality, and is easy to operate and efficient.

[0144] 8. Screening of solvents for preparing test samples

[0145] The test results of N-nitrosobenazepril method determination are shown in Table 17.

[0146] Table 17

[0147]

[0148]

[0149] In Table 17, solvent refers to the solvent used to prepare the test solution. In the comparative example, the difference from the example is that 1-methyl-2-pyrrolidone is not added to the test solution, and the test samples are prepared using the various solvents in Table 17. The rest is the same as the accuracy test in Example 1.

[0150] The liquid chromatography conditions and mass spectrometry detection conditions are the same as those in Example 1.

[0151] As shown in Table 17, when formic acid or methylamine was not added to the solvent, the recovery rate of N-nitrosobenazepril was much higher than 100%, and an obvious false positive occurred; after formic acid or methylamine was added to the solvent, the recovery rate of N-nitrosobenazepril decreased to a certain extent, but the RSD increased significantly, resulting in a decrease in the accuracy of the detection. After 1-methyl-2-pyrrolidone was added to the solvent, the recovery rate of N-nitrosobenazepril was more appropriate, and the RSD was much lower than 5%, which met the requirements and had good detection accuracy.

[0152] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for detecting N-nitrosobenazepril impurities in levamlodipine benazepril, characterized in that: The steps include: The benazepril hydrochloride tablets to be tested are ground, mixed and dissolved with 1-methyl-2-pyrrolidone and a solvent, the mass ratio of the effective amount of benazepril hydrochloride to 1-methyl-2-pyrrolidone being 10-50:1, and filtered to obtain a test solution; The test solution was tested for N-nitrosobenazepril impurities by liquid chromatography-tandem mass spectrometry.

2. The method for detecting N-nitrosobenazepril impurities in levamlodipine benazepril according to claim 1, characterized in that: The mass ratio of the effective amount of benazepril hydrochloride to 1-methyl-2-pyrrolidone is 10-30:1; preferably 15-25:1; more preferably 20:1; Preferably, the solvent is 1-methyl-2-pyrrolidone, formic acid or methylamine.

3. The method for detecting N-nitrosobenazepril impurities in levamlodipine benazepril according to claim 1, characterized in that: The concentration of 1-methyl-2-pyrrolidone in the test solution is 0.03-0.1 mg / ml; preferably 0.03-0.7 mg / ml; preferably 0.05 mg / ml.

4. The method for detecting N-nitrosobenazepril impurities in levamlodipine benazepril according to claim 1, characterized in that: During the dissolution, ultrasound is applied to the mixed system.

5. The method for detecting N-nitrosobenazepril impurities in levamlodipine benazepril according to claim 1, characterized in that: When the test solution is tested, the liquid chromatography conditions are as follows: the filler of the chromatographic column is octadecylsilane bonded silica gel; the mobile phase A is a mixture of formic acid and water, and the volume percentage of formic acid is 0.08-0.12%.

6. The method for detecting N-nitrosobenazepril impurities in levamlodipine benazepril according to claim 5, characterized in that: Mobile phase B is a mixture of formic acid, water and acetonitrile, and the volume percentage of formic acid is 0.08-0.12%.

7. The method for detecting N-nitrosobenazepril impurities in levamlodipine benazepril according to claim 6, characterized in that: The column temperature during liquid chromatography detection was 28-32°C.

8. The method for detecting N-nitrosobenazepril impurities in levamlodipine benazepril according to claim 7, characterized in that: The gradient elution program of the liquid chromatography was as follows: 0-3 min, mobile phase A was 60%; 3-10 min, mobile phase A changed from 60% to 0; 10-12 min, mobile phase A was 0; 12-12.5 min, mobile phase A changed from 0 to 60%; 12.5-15min, mobile phase A is 60%; % is volume percentage.

9. The method for detecting N-nitrosobenazepril impurities in levamlodipine benazepril according to claim 1, characterized in that: The detector for mass spectrometry detection is a triple quadrupole mass spectrometer.

10. The method for detecting N-nitrosobenazepril impurities in levamlodipine benazepril according to claim 9, characterized in that: The ion source of mass spectrometry detection was an electrospray ionization source; the mode was positive ion scanning.

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