Method for detecting three genotoxic impurities of candesartan cilexetil based on HPLC

Abstract

The invention provides a method for detecting three genotoxic impurities of candesartan cilexetil based on HPLC (High Performance Liquid Chromatography), and relates to the technical field of medicine detection. According to the present invention, the high performance liquid chromatography (HPLC) method is adopted to simultaneously determine the contents of the three genotoxic impurities such as 2-((t-butoxycarbonyl) amino)-3-nitrobenzoic acid ethyl ester, 2-cyano-4 '-bromomethyl biphenyl) and 2-[[(2-cyanobiphenyl-4-yl) methyl] amino]-3-nitrobenzoic acid ethyl ester in candesartan cilexetil, and the impurity control limit is researched; in the detection method, the separation degree of candesartan cilexetil and each impurity is greater than 1.5, the linear relationship with the chromatographic peak area in the corresponding range is good, and the correlation coefficients are greater than 0.9998. The average recovery rates (n is equal to 3) of the low-concentration level, the medium-concentration level and the high-concentration level of the impurities are 99.21%-101.79%, 97.89%-99.42% and 99.24%-100.05% respectively, and the RSD (Relative Standard Deviation) is 1.2%, 1.8% and 1.3% respectively. The detection method provided by the invention is strong in specificity, high in sensitivity, good in reproducibility and high in accuracy, an analysis method is simple, convenient and efficient, limit requirements can be met, and a new method is provided for quality control of candesartan cilexetil.

Description

Technical Field

[0001] The invention relates to the technical field of drug detection, and in particular to a method for detecting three genotoxic impurities of candesartan cilexetil based on HPLC. Background Art

[0002] Candesartan cilexetil, chemically known as (±)-1-[(cyclohexyloxy)carbonyloxy]ethyl 2-ethoxy-1[[2'-(1H-tetrazolyl-5-yl)biphenyl-4-yl]methyl]-1H-benzimidazole-7-carboxylate. It is often used as an antihypertensive drug in clinic and is an angiotensin II receptor antagonist. The mechanism of candesartan cilexetil's antihypertensive effect is that candesartan cilexetil, as a prodrug, is rapidly degraded into candesartan in the body. Candesartan can bind to the ATI receptors of vascular smooth muscles in the body, thereby antagonizing the vasoconstriction caused by angiotensin II and reducing peripheral vascular resistance throughout the body. The harm of hypertension to the human body is mainly manifested in damage to blood vessels throughout the body, sclerosis of brain blood vessels, and changes in heart structure and function. Candesartan cilexetil is mainly used in the treatment of primary hypertension, diabetic nephropathy, senile heart failure, improvement of calcium-phosphorus metabolism, and symptoms of cor pulmonale and respiratory failure.

[0003] Genotoxic impurities (GTIs) refer to substances that can directly or indirectly damage DNA, thereby causing gene mutations or cancer. Since candesartan cilexetil uses 2-cyano-4'-methylbiphenyl in its synthesis process, the intermediate impurity 2-((tert-butoxycarbonyl)amino)-3-nitrobenzoic acid ethyl ester and the impurity byproduct 2-[[(2-cyanobiphenyl-4-yl)methyl]amino]-3-nitrobenzoic acid ethyl ester are also produced during the synthesis process. The above three impurities are all genotoxic impurities in candesartan cilexetil.

[0004] The current quality standard of candesartan cilexetil does not include items for detecting ethyl 2-((tert-butoxycarbonyl)amino)-3-nitrobenzoate (impurity 1), 2-cyano-4'-bromomethylbiphenyl (impurity 2) and ethyl 2-[[(2-cyanobiphenyl-4-yl)methyl]amino]-3-nitrobenzoate (impurity 3). Publication Nos. CN117723694A, CN117725981A and CN117705980A disclose methods for detecting the above three impurities using UPLC-MS / MS liquid-mass spectrometry equipment, respectively. Although the above methods have the advantages of low detection limit and high sensitivity, in actual production detection, the detection cost of UPLC-MS / MS liquid-mass spectrometry equipment is higher, and the method uses acetonitrile as the mobile phase, which is highly toxic and expensive to use, and thus cannot be promoted and used in production enterprises. Therefore, the present invention aims to provide a detection method that is simpler and more popular, has a more convenient operation method, lower test cost, more environmentally friendly consumables, strong specificity, high sensitivity, and good accuracy, and can achieve the simultaneous determination of three genotoxic impurities in candesartan cilexetil. Summary of the invention

[0005] 1. Technical issues to be solved

[0006] In view of the shortcomings of the prior art, the present invention provides a method for detecting three genotoxic impurities of candesartan cilexetil based on HPLC. The present invention adopts a single wavelength gradient elution reversed phase high performance liquid chromatography method to simultaneously determine the three genotoxic impurities in candesartan cilexetil. The method has strong specificity, high sensitivity and good accuracy, and can provide a basis for the quality control of candesartan cilexetil.

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] The invention provides a method for detecting three genotoxic impurities of candesartan cilexetil based on HPLC, wherein the three genotoxic impurities are ethyl 2-((tert-butoxycarbonyl)amino)-3-nitrobenzoate (impurity 1), 2-cyano-4'-bromomethylbiphenyl (impurity 2) and ethyl 2-[[(2-cyanobiphenyl-4-yl)methyl]amino]-3-nitrobenzoate (impurity 3); the structural formula of impurity 1-2 is shown in formula 1-2:

[0010]

[0011] Specifically, the method comprises the following steps:

[0012] Step 1: Determine the liquid chromatography conditions

[0013] Chromatographic column: Agilent ZORBAX Eclipse Plus C18, 4.6 mm × 250 mm, 5 μm;

[0014] Column temperature: 40°C;

[0015] Mobile phase: Phase A is 0.05 mol·L-1 potassium dihydrogen phosphate solution with pH=3.5, and phase B is methanol;

[0016] Gradient elution: 0 min, 65% A; 0-10 min, 65% → 35% A; 10-20 min, 35% → 65% A; 20-21 min, 65% → 65% A; 21-30 min, 65% → 65% A;

[0017] The detection wavelength was 225 nm, the flow rate was 1.0 mL min-1, and the injection volume was 20 μL.

[0018] Step 2: Solution preparation

[0019] (1) Preparation of impurity reference solution: Take approximately 10 mg of each of impurity 1 reference, impurity 2 reference, and impurity 3 reference, accurately weigh them, place them in 100 mL volumetric flasks, dissolve them in mobile phase and dilute to the mark, shake well, and obtain the three impurity reference solutions;

[0020] (2) Preparation of mixed reference stock solution: Accurately weigh appropriate amounts of candesartan cilexetil and each impurity reference substance, dissolve them in mobile phase and quantitatively dilute them to make a mixed solution containing approximately 100 μg of each substance per 1 mL, which serves as the mixed reference stock solution of candesartan cilexetil and each impurity.

[0021] (3) Preparation of test solution: Take about 10 mg of candesartan cilexetil, accurately weigh it, place it in a 10 mL volumetric flask, dissolve it with mobile phase and dilute it to the mark, and shake well;

[0022] Step 3: respectively detect the reference solution, the mixed reference solution and the test solution under the above-mentioned liquid chromatography conditions.

[0023] (III) Beneficial effects

[0024] The present invention provides a method for simultaneously determining the contents of three kinds of genotoxic impurities in candesartan cilexetil based on HPLC, and discusses the limits of three kinds of impurities. In the detection method of the present invention, the separation degree of candesartan cilexetil and each impurity is greater than 1.5, and has a good linear relationship with the chromatographic peak area within the corresponding range, and the correlation coefficient is greater than 0.9998. The average recovery rate (n=3) of each impurity at low, medium and high concentration levels is 99.21% to 101.79%, 97.89% to 99.42%, 99.24% to 100.05%, respectively, and the RSD is 1.2%, 1.8%, and 1.3%, respectively. The detection method provided by the invention has strong specificity, high sensitivity, good reproducibility, high accuracy, and a simple and efficient analysis method, which can meet the limit requirements and is suitable for the simultaneous determination of the contents of 2-((tert-butoxycarbonyl)amino)-3-nitrobenzoic acid ethyl ester, 2-cyano-4'-bromomethylbiphenyl and 2-[[(2-cyanobiphenyl-4-yl)methyl]amino]-3-nitrobenzoic acid ethyl ester in candesartan cilexetil raw materials, thereby providing a new method for the quality control of candesartan cilexetil. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the system suitability chromatogram, 1 is impurity 1; 2 is impurity 2; 3 is candesartan cilexetil; 4 is impurity 3.

[0026] Figure 2 This is a specificity experimental chromatogram, A. blank solution; B. acid destruction; C. alkali destruction; D. oxidation destruction; E. high temperature destruction; F. light destruction; G. undestroyed. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are 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 creative work are within the scope of protection of the present invention.

[0028] Example

[0029] 1 Materials

[0030] 1.1 Instrument

[0031] Agilent 1260 high performance liquid chromatograph (Agilent, USA); XPE 205 electronic balance (accuracy 1 / 100,000, Mettler Toledo); CNC ultrasonic cleaner (KQ-250DV, Kunshan Ultrasonic Instrument Co., Ltd.); ultrapure water analyzer (Milli-Q Reference, Merck, Germany)

[0032] 1.2 Drug testing

[0033] Impurity 1 reference substance (Zhengzhou Alpha Chemical Co., Ltd., content mass fraction 98%, batch number A2856590912); impurity 2 reference substance (Shanghai McLean Biochemical Technology Co., Ltd., content mass fraction 98%, batch number C10094899); impurity 3 reference substance (Zhengzhou Alpha Chemical Co., Ltd., content mass fraction 98%, batch number A2856710908); candesartan cilexetil reference substance (batch number: 100685-201903, purity: 99.8%, China Food and Drug Inspection Institute); methanol was chromatographic grade (Merck, Germany); potassium dihydrogen phosphate and phosphoric acid were analytical grade, and water was ultrapure water (18.20 MΩ cm -1 ).

[0034] Candesartan cilexetil API (Batch numbers: 2304051, 2305072, 2306093, Company A).

[0035] 2 Methods and Results

[0036] 2.1 Liquid chromatography conditions

[0037] Using Agilent ZORBAX Eclipse Plus C 18 (4.6 mm × 250 mm, 5 μm) chromatographic column, column temperature was 40 °C, mobile phase was 0.05 mol·L -1 Potassium dihydrogen phosphate solution (adjusted to pH 3.5 with phosphoric acid) (A)-methanol (B), gradient elution, 0 min, 65% A; 0-10 min, 65%→35% A; 10-20 min, 35%→65% A; 20-21 min, 65%→65% A; 21-30 min, 65%→65% A. Detection wavelength was 225 nm, flow rate was 1.0 mL·min -1 , the injection volume was 20 μL.

[0038] 2.2 Solution preparation

[0039] 2.2.1 Preparation of blank solution: mobile phase.

[0040] 2.2.2 Preparation of impurity reference solution: Take approximately 10 mg of each of the impurity 1 reference substance, the impurity 2 reference substance and the impurity 3 reference substance, accurately weigh them, place them in 100 mL volumetric flasks, dissolve them with mobile phase and dilute to the scale, shake well, and obtain the three impurity reference solutions.

[0041] 2.2.3 Mixed reference substance stock solution: Accurately weigh appropriate amounts of candesartan cilexetil and each impurity reference substance, dissolve them in mobile phase and quantitatively dilute them to make a mixed solution containing approximately 100 μg of each substance per 1 mL, as the mixed reference substance stock solution of candesartan cilexetil and each impurity.

[0042] 2.2.4 Test solution: Take about 10 mg of this product (batch number: 2304051), accurately weigh it, put it into a 10 mL volumetric flask, dissolve it with mobile phase and dilute it to the scale, and shake it well.

[0043] 2.2.5 System suitability solution: Take about 10 mg of candesartan cilexetil reference substance, weigh it accurately, put it in a 10 mL volumetric flask, add 5 mL of mobile phase to dissolve it, accurately add 0.02 ml of each impurity reference substance solution under "2.2.2", dilute to the scale with mobile phase, shake well, and you will get 1 mg mL of candesartan cilexetil. -1 and each impurity 0.2μg·mL -1 of mixed solution.

[0044] 2.3 System suitability test

[0045] Take the system suitability solution under "2.2.5", inject and measure according to the chromatographic conditions under "2.1", and record the chromatogram. In the chromatogram of the system suitability solution, impurity 2, impurity 1, candesartan cilexetil and impurity 3 appear in sequence, the retention time of candesartan cilexetil is 15 minutes, and the separation between adjacent chromatographic peaks is greater than 1.5, indicating that the method has good system suitability. The results are as follows Figure 1 shown.

[0046] 2.4 Specificity test

[0047] Take 20μL of the blank solution for injection analysis, and the results show that the blank solution has no interference. Take about 10mg of this product (batch number: 2304051), accurately weigh it, put it in a 10mL volumetric flask, and perform forced degradation tests respectively. The experimental scheme is shown in Table 1. Take the above-mentioned forced degradation solutions and inject them according to the chromatographic conditions under "2.1" and record the chromatogram. The results of the forced degradation test show that the chromatographic conditions have good specificity, and the chromatographic peak of candesartan cilexetil and the chromatographic peak of impurities produced by destruction can be well separated. Candesartan cilexetil is relatively stable under high temperature and light conditions, and can be degraded to produce multiple impurities under acid, alkali and oxidative conditions. The typical chromatogram is shown in Figure 2 No genotoxic impurities 1 and 2 were detected in the test sample under acid, alkali, oxidation, high temperature and light conditions, and only genotoxic impurity 3 was detected.

[0048] Table 1 Forced degradation test plan

[0049]

[0050]

[0051] 2.5 Investigation of linear relationship

[0052] Accurately measure 1 ml of the mixed reference solution under item "2.2.3", place it in a 50 ml volumetric flask, dilute it to the mark with mobile phase, shake well, and use it as the linear reserve solution; accurately measure 0.2 ml, 0.8 ml, 1.0 ml, 1.2 ml, and 2.0 ml of the linear reserve solution respectively, place them in 10 ml volumetric flasks, dilute them to the mark with mobile phase, shake well, and obtain a series of linear solutions R1, R2, R3, R4, and R5. Inject and measure them respectively, record the chromatogram, draw the standard curve with the mass concentration of the reference substance as the abscissa (X) and the peak area as the ordinate (Y), and perform linear regression. The results are shown in Table 2.

[0053] 2.6 Limit of detection and limit of quantification

[0054] The linear stock solutions of candesartan cilexetil and each impurity under item "2.5" were gradually diluted, and the samples were analyzed according to the chromatographic conditions under item "2.1". The mass concentration corresponding to a signal-to-noise ratio (S / N) of 3 was the detection limit (LOD), and the mass concentration corresponding to a S / N of 10 was the quantification limit (LOQ). The measurement results are shown in Table 2.

[0055] Table 2 Regression equation, linear relationship, quantification limit and detection limit of candesartan cilexetil and three impurities

[0056]

[0057] 2.7 Injection precision test

[0058] Take the R3 solution under item "2.5" and inject it 6 times in succession. The retention time RSDs of ethyl 2-((tert-butoxycarbonyl)amino)-3-nitrobenzoate, 2-cyano-4'-bromomethylbiphenyl and 2-[[(2-cyanobiphenyl-4-yl)methyl]amino]-3-nitrobenzoate are 0.07%, 0.11% and 0.16%, respectively; the peak area RSDs are 1.2%, 1.4% and 1.0%, respectively, indicating that the instrument has good precision.

[0059] 2.8 Repeatability test

[0060] Take 6 portions of this product (batch number: 2304051), each portion is about 10 mg, accurately weighed, placed in a 10 mL volumetric flask, add 5 mL of mobile phase to dissolve, add 1 mL of R3 solution under the "2.5" item, dilute to the scale with mobile phase, shake well, prepare the test solution, inject and measure, record the chromatogram, calculate the RSD of the content of each component, which are 2.5%, 3.1%, and 1.8%, respectively. The results show that the method has good repeatability.

[0061] 2.9 Sample recovery test

[0062] Take about 10 mg of this product (batch number: 2304051), accurately weigh, prepare 9 portions in parallel, place them in 10 mL volumetric flasks, and then accurately add 3 portions of 0.8 ml, 1.0 ml and 1.2 ml of the linear reserve solution under "2.5", dissolve and dilute to the scale with mobile phase, shake well, and use them as low, medium and high concentration test solutions. Samples were injected and measured, and chromatograms were recorded. The results showed that the average recoveries of low, medium and high concentrations of impurity 1 were 99.2%, 100.1% and 101.8%, respectively, and the RSDs were 1.5%, 0.9% and 1.1%, respectively; the average recoveries of low, medium and high concentrations of impurity 2 were 97.9%, 99.4% and 98.7%, respectively, and the RSDs were 2.1%, 1.4% and 1.9%, respectively. The average recoveries of impurity 3 at low, medium and high concentrations were 99.2%, 99.9% and 100.0%, respectively, and the RSDs were 1.3%, 0.9% and 1.7%, respectively.

[0063] 2.10 Stability test

[0064] Take an appropriate amount of fine powder of this product (batch number: 2304051), place it in a 10mL volumetric flask, accurately add 1.0mL of the linear stock solution of candesartan cilexetil and each impurity under "2.5", and prepare a mixed solution containing about 0.2μg of candesartan cilexetil and each impurity in each 1mL according to the method under "2.2.4" as the test solution, and observe the changes in the impurity peak area at room temperature for 0, 2, 4, 8, 12, and 24 hours. As a result, the RSD of impurity 1 (n=6) is 1.1%; the RSD of impurity 2 (n=6) is 0.7%; the RSD of impurity 3 (n=6) is 1.4%, indicating that the test solution maintains good stability at room temperature for 24 hours.

[0065] 2.11 Sample determination

[0066] Three batches of candesartan cilexetil API (batch numbers: 2304051, 2305072, 2306093) were used to prepare test solutions according to the method under "2.2.4". The samples were injected for determination. The results showed that impurity 1 and impurity 2 were not detected in the samples, and the detection amount of impurity 3 was 0.010%.

[0067] 3 Discussions

[0068] 3.1 Study on the control limits of genotoxic impurities

[0069] The structures of the genotoxic impurities 2-((tert-butoxycarbonyl)amino)-3-nitrobenzoic acid ethyl ester and 2-[[(2-cyanobiphenyl-4-yl)methyl]amino]-3-nitrobenzoic acid ethyl ester both contain aromatic nitro compounds, and the structure of 2-cyano-4'-bromomethylbiphenyl contains a halogenated alkane warning structure. The three compounds belong to the third category of impurities with warning structures among genotoxic impurities, and their contents should be controlled. According to the threshold of toxicological concern (TTC), the acceptable limit of the carcinogenic risk of genotoxic impurities is 1.5μg·d -1 According to calculation, the maximum daily dose of candesartan cilexetil as the first-line drug for the clinical treatment of chronic hypertension is 8 mg, and the limits of the three genotoxic impurities are all 0.02% (1.5 μg·d -1 ÷8mg·d -1 × 100% = 0.02%).

[0070] 3.2 HPLC instrument selection

[0071] Publication Nos. CN117723694A, CN117725981A, and CN117705980A disclose methods for detecting the above three impurities using UPLC-MS / MS liquid-mass spectrometry equipment, respectively. Although the above methods have the advantages of low detection limit and high sensitivity, in actual production detection, the detection cost of UPLC-MS / MS liquid-mass spectrometry equipment is higher, and the method uses acetonitrile as the mobile phase, which is highly toxic and expensive to use, and cannot be promoted and used in production enterprises. Based on this, the present invention realizes the simultaneous detection of three genotoxic impurities by establishing a high-performance liquid chromatography method.

[0072] Using the detection method provided by the present invention, the detection limit of impurity 1 is: 1.1 ng·mL -1 ÷1mg·mL -1 ×100%=0.0001%, the detection limit of impurity 2 is: 0.7ng·mL -1 ÷1mg·mL -1 ×100%=0.00007%, the detection limit of impurity 3 is: 1.5ng·mL -1 ÷1mg·mL -1 ×100%=0.00015%, which is far below the limit requirement of 0.02% specified in item "3.1". High performance liquid chromatography equipment is more widely used among manufacturers, with simpler operation, lower drug quality control costs, and more convenient personnel training. Therefore, this study is more popular than UPLC-MS / MS method.

[0073] 3.3 Chromatographic conditions and test robustness

[0074] In this study, a diode array detector was used to scan at the full wavelength (200-400nm). Impurities 1-3 all had maximum absorption at (225±2)nm, with good peak shapes and tailing factors between 0.9 and 1.1, high sensitivity and low detection limits. Therefore, this experiment ultimately selected 225nm as the detection wavelength for the three genotoxic impurities.

[0075] The present invention explores the influence of detection conditions such as the composition of the mobile phase, gradient elution ratio, chromatographic column temperature, flow rate, chromatographic column brand and model on the detection results.

[0076] Determination of the organic phase of the mobile phase: We first compared the elution effects of methanol and acetonitrile on impurities as the organic phase. The results showed that when the sample was tested on the chromatographic column Agilent ZORBAX Eclipse Plus C18 (4.6mm×250mm, 5μm), when methanol was selected as the organic phase for liquid phase elution, the tailing factors of candesartan cilexetil, impurity 2 and impurity 3 were all 1.0, and the tailing factor of impurity 1 was 0.9; when acetonitrile was selected, the tailing factor of candesartan cilexetil was 1.1, the tailing factor of impurity 1 was 0.9, and the tailing factors of impurities 2 and impurity 3 were 1.0; the separation effect of methanol was significantly better than that of acetonitrile, and the methanol phase was less toxic than that of acetonitrile. In summary, the present invention uses methanol as the organic phase.

[0077] Determination of inorganic mobile phase: To ensure that the four components involved in the experiment (impurity 1, impurity 2, impurity 3 and candesartan cilexetil) can obtain the maximum separation degree while maintaining a good tailing factor, we investigated water, 0.1% glacial acetic acid aqueous solution and 0.05 mol·L -1 The results showed that when the mobile phase was methanol-water, the minimum separation between impurities was 2.1; when the mobile phase was methanol-0.1% glacial acetic acid solution, the minimum separation between impurities was 2.7, while when the mobile phase was methanol-0.05mol·L -1 When the potassium dihydrogen phosphate solution (the pH value is adjusted to 3.5 with phosphoric acid) is used, the minimum separation degree between impurities is 3.5. The larger the minimum separation degree between impurities, the better the separation effect of impurities and candesartan cilexetil. In summary, the present invention uses methanol-0.05mol·L -1 A potassium dihydrogen phosphate solution (the pH value was adjusted to 3.5 with phosphoric acid) was used as the mobile phase for gradient elution.

[0078] Determination of gradient elution program: Determine the mobile phase: 0.05mol·L -1Based on the potassium dihydrogen phosphate solution (adjusted to pH 3.5 with phosphoric acid) as phase A and methanol as phase B, the present invention explores the influence of different gradient elution degrees on the detection results. The results are shown in Table 3. When the gradient elution program is 0min, 65% A; 0-10min, 65%→35% A; 10-20min, 35%→65% A; 20-21min, 65%→65% A; 21-30min, 65%→65% A, the minimum separation between impurities is 4.7, and the minimum theoretical plate number of impurities is 35119, which well meets the system applicability requirements.

[0079] Table 3 Minimum separation degree and minimum theoretical pedal number of impurities under different gradient elution programs

[0080]

[0081]

[0082] The present invention also controls the column temperature (40±2°C), flow rate (1.0±0.2mL·min -1 ) and the influence of different brands and models of chromatographic columns such as Agilent ZORBAX Eclipse Plus C18 (4.6mm×250mm, 5μm) and Waters Symmetry C18 (4.6mm×250mm, 5μm) on the content determination results of the three impurities were investigated. The experimental results show that the above factors have little effect on the relative retention time and content of the three impurities. The recovery rates of the three impurities under different conditions are between 98.2% and 1102.1%, indicating that this method has good durability.

[0083] 3.4 Summary

[0084] The present invention provides a method for simultaneously determining the contents of three kinds of genotoxic impurities in candesartan cilexetil based on HPLC, and discusses the limits of the three impurities. The detection method provided by the present invention has strong specificity, high sensitivity, good reproducibility, high accuracy, can meet the limit requirements, and is suitable for the simultaneous determination of the contents of ethyl 2-((tert-butoxycarbonyl)amino)-3-nitrobenzoate, 2-cyano-4'-bromomethylbiphenyl and 2-[[(2-cyanobiphenyl-4-yl)methyl]amino]-3-nitrobenzoate in candesartan cilexetil raw materials.

[0085] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for detecting three genotoxic impurities of candesartan cilexetil based on HPLC, characterized in that: The method comprises the following steps: Step 1: Determine the liquid chromatography conditions Chromatographic column: Agilent ZORBAX Eclipse Plus C18, 4.6mm×250mm, 5μm; column temperature: 40℃; mobile phase: phase A is potassium dihydrogen phosphate solution, phase B is methanol; detection wavelength is 225nm, flow rate is 1.0mL / min, injection volume is 20μL; Step 2: Solution preparation (1) Preparation of impurity reference solution: Take approximately 10 mg of each of impurity 1 reference, impurity 2 reference, and impurity 3 reference, accurately weigh them, place them in 100 mL volumetric flasks, dissolve them in mobile phase and dilute to the mark, shake well, and obtain the three impurity reference solutions; (2) Preparation of mixed reference stock solution: Accurately weigh appropriate amounts of candesartan cilexetil and each impurity reference substance, dissolve them in mobile phase and quantitatively dilute them to make a mixed solution containing approximately 100 μg of each substance per 1 mL, which serves as the mixed reference stock solution of candesartan cilexetil and each impurity. (3) Preparation of test solution: Take about 10 mg of candesartan cilexetil, accurately weigh it, place it in a 10 mL volumetric flask, dissolve it with mobile phase and dilute it to the mark, and shake well; Step 3: respectively detect the reference solution, the mixed reference solution and the test solution under the above-mentioned liquid chromatography conditions.

2. A method for detecting three genotoxic impurities of candesartan cilexetil based on HPLC according to claim 1, characterized in that: Impurities 1-3 are ethyl 2-((tert-butoxycarbonyl)amino)-3-nitrobenzoate, 2-cyano-4'-bromomethylbiphenyl and ethyl 2-[[(2-cyanobiphenyl-4-yl)methyl]amino]-3-nitrobenzoate, respectively; the structural formula of impurity 1-2 is shown in Formula 1-2:

3. A method for detecting three genotoxic impurities of candesartan cilexetil based on HPLC according to claim 1, characterized in that: Phase A in the mobile phase is a 0.05 mol / L potassium dihydrogen phosphate solution with a pH of 3.

5.

4. A method for detecting three genotoxic impurities of candesartan cilexetil based on HPLC according to claim 1, characterized in that: The gradient elution program in the chromatographic conditions was: 0 min, 65% A; 0-10 min, 65%→35% A; 10-20 min, 35%→65% A; 20-21 min, 65%→65% A; 21-30 min, 65%→65% A.

5. A method for detecting three genotoxic impurities of candesartan cilexetil based on HPLC according to claim 1, characterized in that: The detection limit of impurity 1 of the method is 3.5 ng / mL, and the detection limit is 1.1 ng / mL; the detection limit of impurity 2 is 2.2 ng / mL, and the detection limit is 0.7 ng / mL; the detection limit of impurity 3 is 4.8 ng / mL, and the detection limit is 1.5 ng / mL.

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