CN110514759B - Method for detecting azide in candesartan cilexetil - Google Patents
Method for detecting azide in candesartan cilexetil Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 29
- GHOSNRCGJFBJIB-UHFFFAOYSA-N Candesartan cilexetil Chemical compound C=12N(CC=3C=CC(=CC=3)C=3C(=CC=CC=3)C3=NNN=N3)C(OCC)=NC2=CC=CC=1C(=O)OC(C)OC(=O)OC1CCCCC1 GHOSNRCGJFBJIB-UHFFFAOYSA-N 0.000 title claims abstract description 27
- 229960004349 candesartan cilexetil Drugs 0.000 title claims abstract description 24
- 150000001540 azides Chemical class 0.000 title description 29
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- 238000001514 detection method Methods 0.000 claims abstract description 35
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- 239000012085 test solution Substances 0.000 claims abstract description 7
- FWFGVMYFCODZRD-UHFFFAOYSA-N oxidanium;hydrogen sulfate Chemical compound O.OS(O)(=O)=O FWFGVMYFCODZRD-UHFFFAOYSA-N 0.000 claims abstract description 6
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- 238000001914 filtration Methods 0.000 claims abstract description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims abstract description 4
- IJCMDGBKHKZSJL-UHFFFAOYSA-N acetonitrile sulfuric acid Chemical compound S(=O)(=O)(O)O.S(O)(O)(=O)=O.C(C)#N IJCMDGBKHKZSJL-UHFFFAOYSA-N 0.000 claims abstract description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 claims abstract description 3
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- 238000010268 HPLC based assay Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 claims 1
- 239000002904 solvent Substances 0.000 claims 1
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 abstract description 28
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- NNOHXABAQAGKRZ-UHFFFAOYSA-N 3,5-dinitrobenzoyl chloride Chemical compound [O-][N+](=O)C1=CC(C(Cl)=O)=CC([N+]([O-])=O)=C1 NNOHXABAQAGKRZ-UHFFFAOYSA-N 0.000 description 1
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- OESFSXYRSCBAQJ-UHFFFAOYSA-M sodium;3-carboxy-3,5-dihydroxy-5-oxopentanoate;2-hydroxypropane-1,2,3-tricarboxylic acid Chemical compound [Na+].OC(=O)CC(O)(C(O)=O)CC(O)=O.OC(=O)CC(O)(C(O)=O)CC([O-])=O OESFSXYRSCBAQJ-UHFFFAOYSA-M 0.000 description 1
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Images
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
Abstract
The invention relates to the technical field of chemical analysis, in particular to a method for detecting an azide compound in candesartan cilexetil. The detection method comprises the steps of taking a candesartan cilexetil raw material to be detected, adding acetonitrile, ultrasonically dissolving, and diluting by adopting a sulfuric acid aqueous solution; the volume ratio of the acetonitrile to the sulfuric acid water is 40: 60-60: 40; fully shaking and filtering, and taking filtrate as a test solution for later use; and the chromatographic column takes octadecylsilane chemically bonded silica as a stationary phase, acetonitrile-sulfuric acid aqueous solution as a mobile phase A and acetonitrile-water as a mobile phase B for gradient elution, HPLC detection is carried out, and the chromatogram is recorded. By adopting the detection method, the sodium azide in the candesartan cilexetil can be quickly, conveniently and quantitatively detected, so that the quality of the candesartan cilexetil product is effectively controlled.
Description
Technical Field
The invention relates to the technical field of chemical analysis, in particular to a method for detecting an azide compound in candesartan cilexetil.
Technical Field
Candesartan cilexetil is a prodrug of candesartan, which is rapidly and completely hydrolyzed to candesartan in the gastrointestinal tract. Candesartan is an angiotensin II AT1 receptor antagonist, antagonizes angiotensin II vasoconstriction by binding to vascular smooth muscle AT1 receptor, thereby reducing peripheral vascular resistance, and is a long-acting receptor antagonist.
The azide compound is one of key intermediates in the synthesis process of candesartan cilexetil medicaments, and can inhibit the activity of cytochrome oxidase and various enzymes, cause phosphorylation and abnormal cell respiration, and has the main acute toxicity effect of causing extreme reduction of vascular tension, which is one of common genotoxic impurities, so that the content of the azide in the medicaments and the intermediates thereof must be strictly controlled in the medicament production process.
Kingomong et al (HPLC for residual sodium azide in azido binders, analytical instrumentation, 1999, 1 st, 38-40) reported extraction of binder samples with citrate sodium citrate buffer, concentration of the extract with 3, 5-dinitrobenzoyl chloride (DNBC) under weakly acidic conditions for UV derivatization.
Chinese patent CN201810852355.1 discloses a method for determining azide compounds in drugs or intermediates thereof by a derivatization HPLC method, and particularly discloses that the azide compounds are subjected to derivatization reaction for 5-60 min by using biphenyl chloride derivatization reagents, and reaction liquid is used as a sample for detection; and taking the generated derivatization reaction solution as a sample, and determining a derivatization product between 220 and 300nm by using an HPLC-DAD method, thereby realizing the quantitative detection of the azide compound in the drug or the synthetic intermediate thereof. However, the method needs derivatization operation, and has complicated steps and long time consumption.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for detecting an azide compound in candesartan cilexetil, which can rapidly and conveniently separate and quantitatively determine sodium azide in candesartan cilexetil, thereby effectively controlling the quality of the candesartan cilexetil product, and which completely meets the standards in the aspects of system applicability, specificity, detectability and quantitativity, linear range, recovery rate, repeatability, etc., and has a novel detection method with high durability.
The invention adopts the following technical scheme:
a method for detecting azide compounds in candesartan cilexetil comprises the steps of taking a to-be-detected product of a candesartan cilexetil raw material drug, adding acetonitrile, ultrasonically dissolving, and diluting by adopting a sulfuric acid water solution, wherein the volume ratio of the acetonitrile to the sulfuric acid water is 40: 60-60: 40; fully shaking and filtering, and taking filtrate as a test solution for later use; and the chromatographic column takes octadecylsilane chemically bonded silica as a stationary phase, acetonitrile-sulfuric acid aqueous solution as a mobile phase A and acetonitrile-water as a mobile phase B for gradient elution, HPLC detection is carried out, and the chromatogram is recorded.
Preferably, the volume ratio of acetonitrile to aqueous sulfuric acid in the mobile phase a is 40: 60.
preferably, the volume ratio of acetonitrile to water in the mobile phase B is 90: 10.
preferably, the sulfuric acid aqueous solution is a 0.0009mol/L sulfuric acid solution.
Preferably, the temperature of the stationary phase is 28-32 ℃, and the flow rate of the mobile phase is 0.6-0.8 mL/min.
Preferably, the detection wavelength of the HPLC detection is 205 nm.
Preferably, the sample volume for HPLC detection is 50. mu.l.
Preferably, in the gradient elution, the elution procedure of the mobile phase a and the mobile phase B is as follows:
0-15min, the volume ratio of the mobile phase A to the mobile phase B is 100: 0, performing isocratic elution;
and (3) 15-16min, wherein the volume ratio of the mobile phase A to the mobile phase B is linearly changed to 10: 90, performing linear gradient elution;
16-30min, wherein the volume ratio of the mobile phase A to the mobile phase B is 10: 90, performing isocratic elution;
30-31min, the volume ratio of the mobile phase A to the mobile phase B is linearly changed into 100: 10, performing linear gradient elution;
31-40min, wherein the volume ratio of the mobile phase A to the mobile phase B is 100: and 0, performing isocratic elution.
Compared with the prior art, the method can quickly, conveniently and quantitatively determine the sodium azide in the candesartan cilexetil, so that the quality of the candesartan cilexetil product is effectively controlled, and the method completely meets the standard in the aspects of system applicability, specificity, detectability, quantitativity, linear range, recovery rate, repeatability and the like and has high durability.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a diagram of the ultraviolet spectrum of sodium azide;
FIG. 2 is a liquid chromatogram of a blank solution detected by the method of the present invention;
FIG. 3 is a liquid chromatogram of a solution of a reference substance detected by the method of the present invention;
FIG. 4 is a system-adapted solution phase chromatogram using the method of the present invention;
FIG. 5 is a linear relationship diagram of sodium azide.
Detailed Description
In order to better illustrate the content of the invention, the invention is further verified by the following specific examples. It should be noted that the examples are given for the purpose of describing the invention more directly and are only a part of the present invention, which should not be construed as limiting the invention in any way.
The detection method according to the present invention, wherein the ratio of the sulfuric acid aqueous solution: the sulfuric acid aqueous solution is prepared by measuring 18ml of sulfuric acid solution with the concentration of 0.05mol/L and adding the sulfuric acid solution into 1000ml of purified water to prepare 0.0009mol/L sulfuric acid aqueous solution. Diluent agent: the volume ratio of acetonitrile to sulfuric acid water is 40: 60 as a diluent.
Example 1: HPLC detection of candesartan cilexetil bulk drug
The detection method provided by the invention has the following specific implementation mode:
the instrument comprises the following steps: waters e2695-2489 high performance liquid chromatograph
A chromatographic column: octadecylsilane chemically bonded silica gel as filler (Waters Atlantis T3C 184.6X 250mm, 5 μm)
Mobile phase A: the volume ratio of acetonitrile to sulfuric acid aqueous solution is 40: 60 as mobile phase a.
And (3) mobile phase B: the volume ratio of acetonitrile to water is 90: 10 as mobile phase B.
Gradient elution was performed as in table 1 below:
TABLE 1 elution procedure
Column temperature: 30 deg.C
Flow rate: 0.7ml/min
Sample introduction amount: 50 μ l
Detection wavelength: 205nm
A workstation: empower 3
Test solution: precisely weighing about 80mg of a candesartan cilexetil raw material drug to be tested, putting the candesartan cilexetil raw material drug into a 10ml measuring flask, adding 4ml of acetonitrile, ultrasonically dissolving, then adding a sulfuric acid water solution to scale, shaking up, filtering, and taking a subsequent filtrate as a test sample solution.
Control stock solutions: taking about 30mg of sodium azide reference substance, placing the sodium azide reference substance into a 250ml measuring flask, adding a diluent to dissolve and dilute the sodium azide reference substance to a scale, and shaking up; precisely measuring 1.0ml, placing into a 100ml measuring flask, adding diluent to scale, and shaking to obtain reference stock solution.
Control solution: precisely measuring 1.0ml of the control stock solution, placing into a 10ml measuring flask, adding diluent to scale, shaking to obtain control solution (corresponding to about N-azido in each 1 ml)3－0.08. mu.g).
System applicability solution: precisely weighing about 80mg of a sample to be tested, placing the sample in a 10ml measuring flask, adding 4ml of acetonitrile for ultrasonic dissolution, precisely weighing 1.0ml of a reference substance stock solution into the measuring flask, adding a sulfuric acid aqueous solution to a scale, shaking up, filtering, and taking a subsequent filtrate as a system applicability solution.
Precisely measuring the reference solution, the system applicability solution and the test solution, respectively 50 μ l, injecting into a liquid chromatograph, recording the chromatogram in the first 16 minutes (the main elution component after 16 minutes and no collection is needed) for inspecting the signal-to-noise ratio of the azide peak; the degree of separation of the azide peak from adjacent peaks; if an impurity peak which is consistent with the retention time of the azide peak exists in the chromatogram obtained by the test solution, the content of the azide is calculated according to an external standard method.
Example 2: determination of detection wavelength
The instrument comprises the following steps: ultraviolet-visible spectrophotometer
Detection wavelength: 200-400nm full wavelength scanning
Precisely weighing about 30mg of sodium azide reference substance, placing the sodium azide reference substance into a 250ml measuring flask, adding a diluent to dissolve and dilute the sodium azide reference substance to a scale, and shaking up to be used as reference substance mother liquor. Precisely measuring 5.0ml of the reference mother solution, placing the reference mother solution into a 10ml measuring flask, adding a diluent to the scale, and shaking up to be used as a wavelength detection reference solution.
Taking a proper amount of sodium azide wavelength detection reference solution, measuring according to 'ultraviolet-visible spectrophotometry operating procedure', recording a spectrum scanning curve graph, wherein an ultraviolet scanning result is shown in attached figure 1.
Referring to fig. 1, the results from the uv scan can be obtained: sodium azide has a large absorption at about the wavelength 205nm, so 205nm is chosen as the optimum detection wavelength for the detector.
Example 3: system suitability and specificity testing
The system applicability of the embodiment simultaneously inspects the system applicability of the instrument during continuous sample introduction.
Blank solution: a diluent;
control solution, system applicability solution: the preparation method is the same as that of example 1;
the relative standard deviations of retention time and peak area were calculated for 1 pin of blank solution, 6 pins of control solution, and 1 pin of system applicability solution in the order of chromatographic conditions in example 1, and the results are shown in Table 2.
TABLE 2 System suitability results
And (4) conclusion: referring to fig. 2, the blank solution is undisturbed. Referring to the attached figure 3, in the chromatogram obtained from the reference solution, the signal-to-noise ratio of the azide peak is over 100 and is far higher than the signal-to-noise ratio of the general limit by 20-30, the retention time RSD is less than 1.0 percent and as low as 0.007 percent, and the peak area RSD is less than 5 percent;
referring to FIG. 4, the azide peak retention time in the system suitability solution was 6.114, consistent with the azide peak retention time in the control solution, and the separation of the azide peak from the adjacent peak was 3.5, which is already greater than 1.5 of the standard requirement. The analysis method is good in system applicability and specificity.
Example 4: detection limit and quantification limit
According to the detection signal-to-noise ratio limiting method, the signal-to-noise ratio is generally 3: 1 or 2: the detection limit is determined by the corresponding concentration or the amount of the human injection instrument at the time 1, and the quantitative signal-to-noise ratio limiting method generally adopts the signal-to-noise ratio of 10: the corresponding concentration at1 or the amount injected into the instrument determines the limit of quantitation.
Limit of quantitation (LOQ) solution: precisely measuring 5.0ml of the control solution, placing the control solution in a 20ml measuring flask, adding a diluent to the scale, and shaking up to obtain a limit of quantitation (LOQ) solution. 6 parts are prepared in parallel.
Limit of detection (LOD) solution: precisely measuring 3.0ml of the quantitative limiting solution, putting the quantitative limiting solution into a 10ml measuring flask, adding a diluent to the scale, and shaking up to be used as a limit of detection (LOD) solution.
6 parts of quantitative limiting solution were introduced into the column under the conditions of chromatography in example 1, and the results are shown in Table 3, wherein 1 pin of each of the quantitative limiting solutions was used and 3 pins of each of the quantitative limiting solutions were used for detection.
TABLE 3 Azide quantitation Limit and detection Limit results
And (4) conclusion: the signal-to-noise ratio of an azide peak in a chromatogram obtained from 6 parts of LOQ solution is 10-13, so that quantitative detection of azide with the concentration of more than 2.38ppm in a sample can be ensured; the signal-to-noise ratio of the azide peak in the chromatogram obtained by using the 3-needle LOD solution is 3, so that the azide with the concentration of more than 0.71ppm in the sample can be detected, and the good sensitivity of the method is proved.
Example 5: linearity and range
The apparatus and reagents were the same as in example 1.
Taking the concentration (in N)3 －Calculated) 0.0190. mu.g/ml, 0.0380. mu.g/ml, 0.0609. mu.g/ml, 0.0761. mu.g/ml, 0.1141. mu.g/ml, 0.1521. mu.g/ml, respectively as azide controlsReference solutions to the LOQ, 50%, 80%, 100%, 150%, 200% limit; the results of the linear regression plots of peak area as ordinate and concentration as abscissa, determined under the chromatographic conditions of example 1, are shown in Table 4 and FIG. 5:
TABLE 4 Azide Linear and Range results
And (4) conclusion: azides meet the criteria of at least in the range of the LOQ value to the 200% limit at concentrations ranging from 0.0190 to 0.1521 μ g/mL and a correlation coefficient of 0.9994 with a Y-axis intercept of 7% of the 100% response value, indicating that the assay detects azides with a good linearity at concentrations ranging from 0.0190 to 0.1521 μ g/mL.
Example 6: repeatability test
The precision of the method is judged by measuring the closeness between the obtained results through multiple sampling.
Control solution: the preparation method is the same as that of example 1.
Repetitive solution: the preparation method is the same as that of the system applicable solution in example 1. 6 parts are prepared in parallel.
The sample was injected with 2 samples of control solution and 1 sample of 6 replicates under the chromatographic conditions of example 1, and the azide content was calculated to determine the relative standard deviation, the results of which are shown in Table 5.
TABLE 5 repeatability results
And (4) conclusion: the RSD of the azide content in 6 replicate solutions was 5%, indicating that the assay is well reproducible.
Example 7: recovery test
The accuracy of the process was ensured by determining the recovery between the theoretical addition of azide and the actual detection.
Respectively weighing proper amount of candesartan cilexetil and placing into a proper volumetric flaskThe candesartan cilexetil was given a concentration of 8mg/ml and divided into 3 groups of 3 portions each. Add appropriate amounts of azide control to 3 sets of concentrations (in N)3 －Calculated) were 0.0192. mu.g/ml, 0.0767. mu.g/ml and 0.1150. mu.g/ml, which correspond to LOQ, 100% and 150% sample recovery solutions, and the prepared solutions were measured under the chromatographic conditions of example 1 to calculate the recovery rates according to the following formulas, and the results are shown in Table 6.
Since no azide was detected in the test solution, the amount of azide was 0 ng/ml.
TABLE 6 recovery of Azide from sample recovery solution at varying limiting concentrations
And (4) conclusion: the method has the advantages that azide with the limit concentration of LOQ-150% is added into a test sample, the recovery rate is 83.83-105.62%, and the RSD is less than 10%, so that the method is good in accuracy.
Example 8: durability test
The durability means a degree of tolerance to which the measurement result is not affected when the measurement conditions are slightly changed. Typical variables in liquid chromatography are: column temperature, flow rate, chromatography column, etc. The flow rates for this study were 0.6ml/min, 0.7ml/min, and 0.8 ml/min; the column temperature was 28 ℃, 30 ℃ and 32 ℃; chromatographic columns 1 and 2 (two chromatographic columns are of the same type, with different batch numbers, and the specific information is shown in table 7), and the chromatographic conditions except for flow rate, column temperature and chromatographic column are the same as in example 1.
TABLE 7 chromatographic column information Table
The blank solution and the system applicability solution are respectively taken and measured under different flow rates, column temperatures and chromatographic column conditions, and the peak results are shown in the following table 8:
TABLE 8 durability test results
Note: "\" indicates that the comparison with the predetermined value is unchanged.
And (3) detection results: when the flow velocity and the column temperature of the mobile phase in the chromatographic parameter conditions are slightly changed or different batch number chromatographic columns of the same type are replaced, the blank is not interfered, the separation degree of the azide and the adjacent peak in the solution with the system applicability is more than 1.5, and the method is proved to have good durability.
Claims (5)
1. A method for detecting azide compounds in candesartan cilexetil is characterized by comprising the steps of taking a to-be-detected candesartan cilexetil raw material drug, adding acetonitrile to the to-be-detected candesartan cilexetil raw material drug, ultrasonically dissolving the drug, and diluting the drug by using a sulfuric acid aqueous solution; the volume ratio of the acetonitrile to the sulfuric acid water is 40: 60-60: 40; fully shaking and filtering, and taking filtrate as a test solution for later use; performing gradient elution on a chromatographic column by using octadecylsilane chemically bonded silica as a stationary phase, acetonitrile-sulfuric acid aqueous solution as a mobile phase A and acetonitrile-water as a mobile phase B, performing HPLC detection, and recording a map;
the volume ratio of acetonitrile to sulfuric acid aqueous solution in the mobile phase A is 40: 60, adding a solvent to the mixture;
the volume ratio of acetonitrile to water in the mobile phase B is 90: 10;
in the gradient elution, the elution procedure of the mobile phase A and the mobile phase B is as follows:
0-15min, the volume ratio of the mobile phase A to the mobile phase B is 100: 0, performing isocratic elution;
and (3) 15-16min, wherein the volume ratio of the mobile phase A to the mobile phase B is linearly changed into 10: 90, performing linear gradient elution;
16-30min, wherein the volume ratio of the mobile phase A to the mobile phase B is 10: 90, performing isocratic elution;
30-31min, the volume ratio of the mobile phase A to the mobile phase B is linearly changed into 100: 10, performing linear gradient elution;
31-40min, wherein the volume ratio of the mobile phase A to the mobile phase B is 100: and 0, performing isocratic elution.
2. The detection method according to claim 1, wherein the aqueous sulfuric acid solution is a 0.0009mol/L sulfuric acid solution.
3. The detection method according to claim 1, wherein the temperature of the stationary phase is 28 to 32 ℃ and the flow rate of the mobile phase is 0.6 to 0.8 mL/min.
4. The detection method according to claim 1, wherein the detection wavelength of the HPLC detection is 205 nm.
5. The method of claim 1, wherein the HPLC assay is carried out in a sample volume of 50. mu.l.
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| CN101936961A (en) * | 2009-07-01 | 2011-01-05 | 浙江华海药业股份有限公司 | High performance liquid chromatography for analyzing candesartan cilexetil |
| CN102670604A (en) * | 2012-05-18 | 2012-09-19 | 四川升和药业股份有限公司 | Composition containing candesartan and amlodipine, preparation process, testing process and application thereof |
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| CN101936961A (en) * | 2009-07-01 | 2011-01-05 | 浙江华海药业股份有限公司 | High performance liquid chromatography for analyzing candesartan cilexetil |
| CN102670604A (en) * | 2012-05-18 | 2012-09-19 | 四川升和药业股份有限公司 | Composition containing candesartan and amlodipine, preparation process, testing process and application thereof |
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Denomination of invention: A detection method for azide compounds in candesartan ester Granted publication date: 20220520 Pledgee: Bank of Changsha Limited by Share Ltd. Liuyang branch Pledgor: TIANDI HENGYI PHARMACEUTICAL Co.,Ltd. Registration number: Y2024980011973 |