CN117607308A - Liquid chromatography detection method for trace azide impurities in irbesartan - Google Patents
Liquid chromatography detection method for trace azide impurities in irbesartan Download PDFInfo
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- CN117607308A CN117607308A CN202311695393.8A CN202311695393A CN117607308A CN 117607308 A CN117607308 A CN 117607308A CN 202311695393 A CN202311695393 A CN 202311695393A CN 117607308 A CN117607308 A CN 117607308A
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- 239000012535 impurity Substances 0.000 title claims abstract description 134
- 150000001540 azides Chemical class 0.000 title claims abstract description 86
- 239000002947 C09CA04 - Irbesartan Substances 0.000 title claims abstract description 46
- 238000001514 detection method Methods 0.000 title claims abstract description 46
- YCPOHTHPUREGFM-UHFFFAOYSA-N irbesartan Chemical compound O=C1N(CC=2C=CC(=CC=2)C=2C(=CC=CC=2)C=2[N]N=NN=2)C(CCCC)=NC21CCCC2 YCPOHTHPUREGFM-UHFFFAOYSA-N 0.000 title claims abstract description 46
- 229960002198 irbesartan Drugs 0.000 title claims abstract description 46
- 238000004811 liquid chromatography Methods 0.000 title claims abstract description 23
- 239000000243 solution Substances 0.000 claims abstract description 59
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims abstract description 51
- 239000013558 reference substance Substances 0.000 claims abstract description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000002994 raw material Substances 0.000 claims abstract description 10
- 239000006228 supernatant Substances 0.000 claims abstract description 9
- 238000010790 dilution Methods 0.000 claims abstract description 8
- 239000012895 dilution Substances 0.000 claims abstract description 8
- 238000001816 cooling Methods 0.000 claims abstract description 6
- 238000010438 heat treatment Methods 0.000 claims abstract description 6
- LANSUNWWSUCCJT-UHFFFAOYSA-N 2-[4-(azidomethyl)phenyl]benzonitrile Chemical compound C1=CC(CN=[N+]=[N-])=CC=C1C1=CC=CC=C1C#N LANSUNWWSUCCJT-UHFFFAOYSA-N 0.000 claims abstract description 4
- HIWODOJPZXUTRT-UHFFFAOYSA-N 5-[2-[4-(azidomethyl)phenyl]phenyl]-2h-tetrazole Chemical compound C1=CC(CN=[N+]=[N-])=CC=C1C1=CC=CC=C1C1=NNN=N1 HIWODOJPZXUTRT-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000002156 mixing Methods 0.000 claims abstract description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 32
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 18
- 238000000034 method Methods 0.000 claims description 18
- 239000007788 liquid Substances 0.000 claims description 13
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 11
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 11
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 11
- 238000010828 elution Methods 0.000 claims description 9
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 9
- 239000000377 silicon dioxide Substances 0.000 claims description 9
- 239000000945 filler Substances 0.000 claims description 8
- 230000035945 sensitivity Effects 0.000 abstract description 8
- 231100000024 genotoxic Toxicity 0.000 abstract description 3
- 230000001738 genotoxic effect Effects 0.000 abstract description 3
- 239000002904 solvent Substances 0.000 description 26
- 239000000523 sample Substances 0.000 description 21
- 239000011550 stock solution Substances 0.000 description 20
- 238000012360 testing method Methods 0.000 description 20
- 238000004128 high performance liquid chromatography Methods 0.000 description 16
- 239000012488 sample solution Substances 0.000 description 15
- 230000000052 comparative effect Effects 0.000 description 13
- 230000014759 maintenance of location Effects 0.000 description 8
- 238000011084 recovery Methods 0.000 description 8
- 238000005303 weighing Methods 0.000 description 8
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 6
- 238000004090 dissolution Methods 0.000 description 6
- 230000004807 localization Effects 0.000 description 5
- 239000007858 starting material Substances 0.000 description 5
- 239000012085 test solution Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 101100449813 Schizosaccharomyces pombe (strain 972 / ATCC 24843) gti1 gene Proteins 0.000 description 3
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000002425 crystallisation Methods 0.000 description 3
- 230000008025 crystallization Effects 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000008186 active pharmaceutical agent Substances 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 229940088679 drug related substance Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000010812 external standard method Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 238000002211 ultraviolet spectrum Methods 0.000 description 2
- PAKGYCNZUGIDHV-UHFFFAOYSA-N SR-49498 Chemical compound C=1C=C(C=2C(=CC=CC=2)C=2NN=NN=2)C=CC=1CNC(=O)C1(NC(=O)CCCC)CCCC1 PAKGYCNZUGIDHV-UHFFFAOYSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000007674 genetic toxicity Effects 0.000 description 1
- 231100000025 genetic toxicology Toxicity 0.000 description 1
- 231100000086 high toxicity Toxicity 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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
-
- 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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
Abstract
The invention discloses a liquid chromatography detection method for trace azide impurities in irbesartan, which comprises the following steps: adding irbesartan raw material into acetonitrile, heating and dissolving at 65-75 ℃, cooling, adding water for dilution, mixing, standing at room temperature for 0.5-1.5h, centrifuging, and taking supernatant; respectively preparing reference substance solutions of azide impurity 1 and azide impurity 2; detecting the supernatant and the reference substance solution by adopting liquid chromatography; wherein, the impurity 1 is 5- (4 '-azidomethyl- [1,1' -biphenyl ] -2-yl) -1H-tetrazole; impurity 2 is 4 '-azidomethyl- [1,1' -biphenyl ] -2-carbonitrile. The detection method provided by the invention has the characteristics of high sensitivity, strong specificity, good accuracy and high sensitivity, and the sensitivity can reach 1ppm, so that the genotoxic impurities in the irbesartan can be efficiently, quickly and conveniently detected.
Description
Technical Field
The invention belongs to the technical field of impurity detection, and particularly relates to a liquid chromatography detection method for trace azide impurities in irbesartan.
Background
The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.
Irbesartan drug substance is at risk of generating and remaining 5- (4 '- (azidomethyl) - [1,1' -biphenyl ] -2-yl) -1H-tetrazole (GTI 2) and parent 4'- (azidomethyl) - [1,1' -biphenyl ] -2-nitrile (GTI 1), and the two impurities have genetic toxicity and high toxicity, so that the two impurities need to be controlled and detected for controlling the quality of irbesartan drug substance. These two azide impurities are low in limit and need to be controlled to be not more than five parts per million (5 ppm), and the methods disclosed at present are all liquid methods, expensive in equipment and poor in popularity of various companies.
In addition, the concentration of irbesartan in the sample solution is large, various unknown impurities can be detected, the peak is continuous, and the measurement interference of irbesartan and various unknown impurities on the impurities 1 and 2 is difficult to be simultaneously considered. There are reports that the detection of these two impurities separately requires two methods to detect, resulting in large detection workload and long detection time.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide a liquid chromatography detection method for trace azide impurities in irbesartan.
In order to achieve the above object, the present invention is realized by the following technical scheme:
a liquid chromatography detection method for trace azide impurities in irbesartan comprises the following steps:
adding irbesartan raw material into acetonitrile, heating and dissolving at 65-75 ℃, cooling, adding water for dilution, mixing, standing at room temperature for 0.5-1.5h, centrifuging, and taking supernatant;
respectively preparing reference substance solutions of azide impurity 1 and azide impurity 2;
detecting the supernatant and the reference substance solution by adopting liquid chromatography;
wherein, the impurity 1 is 5- (4 '-azidomethyl- [1,1' -biphenyl ] -2-yl) -1H-tetrazole; impurity 2 is 4 '-azidomethyl- [1,1' -biphenyl ] -2-carbonitrile.
The irbesartan is heated and dissolved at 65-75 ℃, so that the irbesartan raw material and impurities can be fully dissolved in acetonitrile, the accuracy of impurity detection is improved, and the waste of acetonitrile can be avoided.
In some embodiments, the volume ratio of acetonitrile to water is 1:0.8-1.5 when the irbesartan starting material is extracted. The solubility of the irbesartan sample can be increased by acetonitrile, and according to experiments, the solubility of impurities can be increased by acetonitrile, so that the inclusion of irbesartan precipitation on the impurities 1 and 2 is reduced in the sample preparation process, and the accuracy of the determination of the impurities 1 and 2 is ensured. In the extraction process, the addition amount of acetonitrile is too small, the sample cannot be dissolved, the addition amount of acetonitrile is too large, the elution time of the sample can be prolonged, and the peak shapes of the impurity 1 and the impurity 2 in the chromatogram are poor.
Preferably, the volume ratio of acetonitrile to water is 1:1.
In some embodiments, the temperature of the room temperature stationary extraction is 10-30 ℃.
In some embodiments, the chromatographic column is octadecylsilane chemically bonded silica as a packing material, XB-C18,4.6mm by 250mm,5 μm, for liquid chromatography detection.
Preferably, the mobile phase a detected by liquid chromatography is: 0.005mol/L potassium dihydrogen phosphate solution with pH value of 2.7; mobile phase B was methanol.
Preferably, the detection wavelength of the liquid chromatograph is 224nm. The ultraviolet spectrogram of the impurity 1 (figure 8) and the ultraviolet spectrogram of the impurity 2 (figure 9) are comprehensively compared, the impurity 2 is weak in absorption, but has maximum absorption at the wavelength of 224.1nm, the detection capability and the detection noise are comprehensively considered, and 224nm is selected as the detection wavelength of the liquid chromatograph.
Preferably, the gradient elution procedure is: 0-45min, mobile phase B55%; 45-50min, 55% -70% of mobile phase B; 50-75min, mobile phase B70%; 75-76min, 70% -55% of mobile phase B; 76-85min, 55% of mobile phase B and percentage by volume.
Experiments show that when the mobile phase B is 55%, each impurity peak in each irbesartan crude drug does not interfere with the measurement of GTI1 and GTI2, and then the eluting capacity of the mobile phase B is increased by adding the mobile phase B (methanol), so that each impurity in the chromatographic column is flushed faster, and finally the chromatographic column is balanced by returning to the proportion of 55% of the mobile phase B, so that the purpose of balancing the chromatographic column is achieved, and preparation is made for next sample injection.
In some embodiments, the column temperature is 30 ℃.
In some embodiments, the sample volume is 50 μl and the mobile phase flow rate is 1ml/min.
The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:
the detection method provided by the invention has the characteristics of high sensitivity, strong specificity, good accuracy and high sensitivity, and the sensitivity can reach 1ppm, so that the genotoxic impurities in the irbesartan can be efficiently, quickly and conveniently detected. The quality control of the genotoxic impurities is enhanced in the detection of the irbesartan finished product, so that the quality control of the irbesartan bulk drug is improved. The detection method can be carried out by adopting a high-performance liquid chromatograph, and a higher-precision analysis instrument is not needed, so that the detection cost is greatly reduced.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.
FIG. 1 is an HPLC chart of a solution of a control (A) and a test substance (B) in comparative example 1 of the present invention;
FIG. 2 is an HPLC profile of an azide impurity 1 peak localization solution in an embodiment of the present invention;
FIG. 3 is an HPLC profile of an azide impurity 2 peak localization solution in an embodiment of the present invention;
fig. 4 is an HPLC profile of a mixed solution of irbesartan, azido impurity 1, azido impurity 2 in the embodiment of the present invention, wherein a is a full view, and B is a partial enlarged view;
FIG. 5 is a quantitative limit of detection test HPLC chart of an azide impurity study in the embodiment of the invention, wherein A is a full chart, B is a partial enlarged chart of 0-1.5min, and C is a partial enlarged chart of 10-30 min;
FIG. 6 is a standard curve of impurity 1 in an embodiment of the present invention;
FIG. 7 is a standard curve of impurity 2 in an embodiment of the present invention;
FIG. 8 is an ultraviolet spectrum of impurity 1 according to an embodiment of the present invention;
FIG. 9 is an ultraviolet spectrum of impurity 2 according to an embodiment of the present invention;
FIG. 10 is an HPLC chart of a control solution (1) in comparative example 2 of the present invention;
FIG. 11 is an HPLC chart of a sample solution (1) in comparative example 2 of the present invention;
FIG. 12 is an HPLC chart of a control solution (1) in comparative example 3 of the present invention;
FIG. 13 is an HPLC chart of a sample solution (1) in comparative example 3 of the present invention;
FIG. 14 is an HPLC chart of a control solution (2) in comparative example 4 of the present invention;
FIG. 15 is an HPLC chart of a control solution (1) in comparative example 5 of the present invention;
FIG. 16 is an HPLC chart of a sample solution (1) in comparative example 5 of the present invention.
Detailed Description
It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Example 1
Solvent: acetonitrile-water (50:50).
Preparing a test solution:
taking about 100mg of irbesartan raw material, precisely weighing, placing into a 10ml measuring flask, adding 5ml of acetonitrile, heating in a water bath at 70 ℃ to dissolve, taking out, cooling to room temperature, adding water to dilute to a scale, shaking uniformly, standing at room temperature for 1 hour, centrifuging at 4000 rpm for 20 minutes, and taking supernatant.
Preparing a reference substance solution:
taking a proper amount of each of the azide impurity 1 reference substance and the azide impurity 2 reference substance, precisely weighing, adding a solvent for dissolution and dilution to prepare a solution containing about 0.05 mug of each of the azide impurity 1 reference substance and the nitrogen impurity 2 reference substance in each 1 ml.
Wherein the structural formulas of the impurity 1, the impurity 2 and the impurity A are shown in the table 1.
TABLE 1
Chromatographic conditions: octadecylsilane chemically bonded silica as filler (XB-C18, 4.6mm x 250mm,5 μm or column with comparable performance); gradient elution was carried out according to Table 2 with 0.005mol/L potassium dihydrogen phosphate solution (pH adjusted to 2.7 with phosphoric acid) as mobile phase A and methanol as mobile phase B; the column temperature is 30 ℃; the flow rate is 1.0ml per minute; the detection wavelength is 224nm; the sample volume was 50. Mu.l.
TABLE 2
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 45 | 55 |
| 45 | 45 | 55 |
| 50 | 30 | 70 |
| 75 | 30 | 70 |
| 76 | 45 | 55 |
| 85 | 45 | 55 |
The measuring method precisely measures the solution of the sample and the solution of the reference substance, respectively injects the solution into a liquid chromatograph, and records the chromatograms.
The limiting test solution chromatograms include azide impurity 1 peak and azide impurity 2 peak, and the peak areas are calculated according to an external standard method, and the peak areas are not more than 5ppm.
The above examination method was used while changing the solvent, and the recovery rate of the corresponding azide impurity 2 is shown in table 3.
TABLE 3 Table 3
The mixing of different solvents (0.005 mol/L potassium dihydrogen phosphate solution (pH 2.8), water, methanol and acetonitrile), the dissolution mode of the sample and the precipitation mode of the sample are compared, and the difference of recovery rates of GTI1 and GTI2 is measured due to the difference of the dissolution degree of the sample and the influence of the crystallization temperature on the crystallization speed caused by different solvents.
Because the temperature of placement can affect the rate of crystallization of the sample, too low a temperature, too rapid sample precipitation can result in encapsulation of GTI1 and GTI2 as the sample precipitates, thereby affecting the accuracy of the assay.
Comparative example 1
Octadecylsilane chemically bonded silica (XB-C18, 4.6 mm. Times.250 mm,5 μm, manufactured by Yuehu materials Co., ltd.) was used as a filler; gradient elution was carried out according to Table 4 with 0.005mol/L potassium dihydrogen phosphate solution as mobile phase A and methanol as mobile phase B; the detection wavelength is 210nm; the column temperature is 30 ℃; the flow rate is 1.0ml per minute; the sample volume was 50. Mu.l.
TABLE 4 Table 4
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 55 | 45 |
| 10 | 45 | 55 |
| 50 | 30 | 70 |
| 55 | 30 | 70 |
| 56 | 55 | 45 |
| 65 | 55 | 45 |
Precisely measuring 50 μl of each of the control solution and the sample solution, injecting into a liquid chromatograph, and recording the chromatogram. The chromatogram is shown in FIG. 1, the retention time of azide impurity 1 is 17.933min, the retention time of azide impurity 2 is 38.633min, and the test sample solution interferes with the determination of azide impurity 1.
Example 2
Specificity test:
irbesartan impurity A about 1.8mg is taken, precisely weighed, placed in a 200ml measuring flask, diluted to a scale by adding a solvent, and uniformly shaken to be used as an interference test solution.
Taking about 2mg of azide impurity 1 reference substance, precisely weighing, placing into a 200ml measuring flask, adding a solvent for dissolution and dilution to a scale, shaking uniformly, and positioning a stock solution as an azide impurity 1 peak; precisely measuring 1ml of the stock solution, placing the stock solution into a 200ml measuring flask, adding a solvent for dilution to a scale, and shaking the stock solution to be uniform to obtain the azide impurity 1 peak positioning solution.
Taking about 2mg of azide impurity 2 reference substance, precisely weighing, placing into a 200ml measuring flask, adding a solvent for dissolution and dilution to a scale, shaking uniformly, and positioning a stock solution as an azide impurity 2 peak; 1ml of the stock solution is precisely measured, placed in a 200ml measuring flask, diluted to a scale by adding a solvent, and shaken uniformly to serve as an azide impurity 2 peak positioning solution.
Taking 1ml of each of the azide impurity 1 peak positioning stock solution and the azide impurity 2 peak positioning stock solution, placing the stock solutions into a 200ml measuring flask, adding a solvent to dilute to a scale, and shaking uniformly to obtain a reference substance solution.
Taking about 100mg of irbesartan raw material, precisely weighing, placing into a 10ml measuring flask, adding 5ml of acetonitrile, heating in a 70 ℃ water bath to dissolve, taking out, cooling to room temperature, adding water to dilute to a scale, shaking uniformly, standing at room temperature for 1 hour, centrifuging at 4000 revolutions per minute for 20 minutes, and taking the supernatant as a test sample solution.
Taking 1ml of each of the azide impurity 1 peak positioning stock solution and the azide impurity 2 peak positioning stock solution, placing the stock solutions into a 10ml measuring flask, adding a solvent to dilute the stock solutions to a scale, and shaking the stock solutions uniformly to serve as mixed impurity stock solutions; taking about 100mg of irbesartan raw material, precisely weighing, placing into a 10ml measuring flask, adding 5ml of acetonitrile, heating in a water bath at 70 ℃ to dissolve, taking out, cooling to room temperature, adding 0.5ml of mixed impurity stock solution, adding water to dilute to a scale, shaking uniformly, standing at room temperature for 1 hour, centrifuging at 4000 rpm for 20 minutes, and taking supernatant as a mixed solution.
Chromatographic conditions: octadecylsilane chemically bonded silica as filler (XB-C18, 4.6mm x 250mm,5 μm or column with comparable performance); gradient elution was carried out according to Table 2 with 0.005mol/L potassium dihydrogen phosphate solution (pH adjusted to 2.7 with phosphoric acid) as mobile phase A and methanol as mobile phase B; the column temperature is 30 ℃; the flow rate is 1.0ml per minute; the detection wavelength is 224nm; the sample volume was 50. Mu.l and the chromatogram was recorded.
Azide impurity 1 peak localization solution HPLC profile as shown in fig. 2; azide impurity 2 peak localization solution HPLC profile as shown in fig. 3; the HPLC chart of the mixed solution of irbesartan, azido impurity 1 and azido impurity 2 is shown in figure 4.
Table 5 results of the azide impurity check peak localization test in irbesartan starting material
The test result shows that the impurity A (retention time is 16.410) does not interfere with the determination of azide impurity, and the azide impurity 1 and the azide impurity 2 are well separated and have good specificity.
Example 3
Sensitivity test.
And (3) taking a proper amount of each of the azide impurity 1 reference substance and the azide impurity 2 reference substance, adding a solvent to dissolve and dilute the mixture to prepare a solution with a series of concentration, wherein the solution takes the S/N (signal to noise ratio) of about 10 as a quantitative limit and takes the S/N (signal to noise ratio) of about 3 as a detection limit. The azide impurity study limit of detection assay HPLC profile is shown in fig. 5.
Table 6 test results of quantitative limit and detection limit of azide impurity examination in irbesartan raw material
| Component name | Quantitative limit (ng) | Detection limit (ng) | Limit of quantification (ppm) | Limit (ppm) | Limit/quantitative limit |
| Azido impurity 1 | 0.477 | 0.143 | 1 | 5 | 5 |
| Azido impurity 2 | 0.501 | 0.150 | 1 | 5 | 5 |
The test result shows that the limit of each impurity/quantitative limit is more than 5, and the detection sensitivity of the method is good.
Example 4
Linearity test
Taking a proper amount of each of the azide impurity 1 reference substance and the azide impurity 2 reference substance, precisely weighing, adding a solvent for dissolving and diluting to prepare a solution containing about 1 mug of each of the azide impurity 1 reference substance and the nitrogen impurity 2 reference substance in each 1ml, and shaking uniformly to serve as a linear test stock solution; 1ml, 2.5ml, 4ml, 5ml and 7.5ml of linear test stock solutions are respectively measured precisely, placed in different 100ml measuring flasks, diluted to scale by adding solvent, and uniformly shaken to be respectively used as a series of linear test concentration solutions of 20% group, 50% group, 80% group, 100% group and 150% group.
The solvent and 50. Mu.l of each of the above solutions were precisely measured, injected into a liquid chromatograph, and the chromatograms were recorded to perform linear regression of peak areas with concentrations.
Table 7 azide impurity examination Linear test results in irbesartan starting Material
The test results show that the linear relationship between the azide impurity 1 and the azide impurity 2 is good in the range of 0.01-0.075 mug/ml (0.0001-0.00075%), as shown in fig. 6 and 7.
Example 5
Accuracy test
Sample solutions with recovery rates of 1ppm, 2.5ppm, 5ppm and 7.5ppm were prepared respectively,
precisely measuring 50 μl of each sample solution, respectively injecting into a liquid chromatograph, and recording the chromatogram. And taking a proper amount of each of the azide impurity 1 reference substance and the azide impurity 2 reference substance, adding a solvent to dissolve and dilute the mixture to prepare a solution containing about 0.05 mug of each impurity in each 1ml, and taking the solution as a reference substance solution. The chromatogram was recorded by the same method. Recovery was calculated as peak area by the external standard method.
Table 8 results of accuracy test for azide impurity examination in irbesartan starting material
The results of the multiple sample assays are shown in Table 9.
Table 9 results of azide impurity examination test in irbesartan starting material
| Lot number | Azide impurity 1 (ppm) | Azide impurity 2 (ppm) |
| 80321016501 | 1.04 | Not detected |
| 80320112523 | 0.92 | Not detected |
| 80320116501 | 0.39 | Not detected |
| 80320102511 | 1.17 | Not detected |
| 80320122501 | 1.34 | 0.13 |
Comparative example 2
Octadecylsilane chemically bonded silica (XB-C18, 4.6mm.times.150mm, 5 μm, manufactured by Yuehu materials Co., ltd.) was used as a filler; performing gradient elution by taking 0.005mol/L potassium dihydrogen phosphate solution as a mobile phase A and methanol as a mobile phase B according to the following table; the detection wavelength is 210nm; the column temperature is 30 ℃; the flow rate is 1.0ml per minute; the sample volume was 50. Mu.l.
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 55 | 45 |
| 20 | 30 | 70 |
| 25 | 30 | 70 |
| 26 | 55 | 45 |
| 40 | 55 | 45 |
Control solution (1): taking 1.8mg of each of the azide impurity 1 reference substance and the azide impurity 2 reference substance, precisely weighing, placing into a 200ml measuring flask, adding a solvent [ acetonitrile-water (50:50) ] to dilute to scale, and shaking uniformly to serve as a reference substance stock solution. Precisely measuring 1ml of reference stock solution, placing into a 100ml measuring flask, diluting to scale with solvent, shaking, and taking as reference solution (1).
Test solution (1): about 218mg of the product is taken, precisely weighed, placed in a 50ml measuring flask, added with a proper amount of solvent, dissolved by ultrasonic, diluted to a scale by the solvent, shaken uniformly, filtered, and the subsequent filtrate is taken as the sample solution (1).
Precisely measuring 50 μl of each of the control solution (1) and the sample solution (1), injecting into a liquid chromatograph, and recording the chromatograms. As shown by the chromatogram, the retention time of the azide impurity 1 is 12.658min, the retention time of the azide impurity 2 is 20.878min, and the sample solution interferes with the peak of the azide impurity.
Comparative example 3
Octadecylsilane chemically bonded silica (XB-C18, 4.6 mm. Times.250 mm,5 μm, manufactured by Yuehu materials Co., ltd.) was used as a filler; performing gradient elution by taking 0.005mol/L potassium dihydrogen phosphate solution as a mobile phase A and methanol as a mobile phase B according to the following table; the detection wavelength is 210nm; the column temperature is 30 ℃; the flow rate is 1.0ml per minute; the sample volume was 50. Mu.l.
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 55 | 45 |
| 10 | 45 | 55 |
| 50 | 30 | 70 |
| 55 | 30 | 70 |
| 56 | 55 | 45 |
| 65 | 55 | 45 |
Control solution (2): taking a proper amount of each of the azide impurity 1 reference substance and the azide impurity 2 reference substance, adding a solvent [ methanol-water (50:50) ] to quantitatively dilute the mixture to prepare a solution containing about 0.09 mug of each of the azide impurity 1 and the azide impurity 2 in each 1ml, and shaking the solution uniformly to obtain a reference substance solution (2).
Test solution (2): taking a proper amount of the product, adding a proper amount of a solvent, carrying out ultrasonic dissolution, quantitatively diluting with the solvent to prepare a solution containing about 20mg of irbesartan per 1ml, shaking uniformly, filtering, and taking a subsequent filtrate as a sample solution (2).
Precisely measuring 50 μl of each of the control solution (2) and the sample solution (2), injecting into a liquid chromatograph, and recording the chromatograms. As can be seen from the chromatogram, the retention time of the azide impurity 1 is 17.933min, the retention time of the azide impurity 2 is 38.633min, and the test sample solution interferes with the determination of the azide impurity 1.
Comparative example 4
Octadecylsilane chemically bonded silica (XB-C18, 4.6 mm. Times.250 mm,5 μm, manufactured by Yuehu materials Co., ltd.) was used as a filler; performing gradient elution by taking 0.005mol/L potassium dihydrogen phosphate solution as a mobile phase A and methanol as a mobile phase B according to the following table; the detection wavelength is 210nm; the column temperature is 30 ℃; the flow rate is 1.0ml per minute; the sample volume was 50. Mu.l.
| Time (minutes) | Mobile phase a (%) | Mobile phase B (%) |
| 0 | 45 | 55 |
| 30 | 45 | 55 |
| 31 | 30 | 70 |
| 45 | 30 | 70 |
| 46 | 55 | 45 |
| 60 | 55 | 45 |
The control solution (2. Mu.l) was precisely measured and injected into a liquid chromatograph, and the chromatogram was recorded. As can be seen from the chromatogram, the retention time of the azide impurity 2 is 33.358min, which is too close to the gradient peak.
Comparative example 5
Octadecylsilane chemically bonded silica (XB-C18, 4.6 mm. Times.250 mm,5 μm, manufactured by Yuehu materials Co., ltd.) was used as a filler; 0.005mol/L potassium dihydrogen phosphate solution (pH 2.8) -methanol (45:55) is taken as a mobile phase; the detection wavelength is 210nm; the column temperature is 30 ℃; the flow rate is 1.0ml per minute; the sample volume was 50. Mu.l.
Recovery rate solution: taking a proper amount of each of the azide impurity 1 reference substance, the azide impurity 2 reference substance and the irbesartan raw material, and adding a solvent for quantitative dilution to prepare about 0.05 mug of each of the azide impurity 1 and the azide impurity 2 and about 10mg of each of the azide impurity 2 in 1ml, wherein the solution is used as a recovery rate solution.
The control solution and the recovery solution were prepared using 0.005mol/L potassium dihydrogen phosphate solution (pH 2.8) -methanol (50:50), water-methanol (50:50) water-acetonitrile (50:50) as solvents, respectively.
And precisely measuring 50 mu l of each of the control solution and the recovery solution, injecting into a liquid chromatograph, and recording a chromatogram. From the chromatogram, the main component interferes with the measurement of azide impurity 2.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A liquid chromatography detection method for trace azide impurities in irbesartan is characterized by comprising the following steps of: the method comprises the following steps:
adding irbesartan raw material into acetonitrile, heating and dissolving at 65-75 ℃, cooling, adding water for dilution, mixing, standing at room temperature for 0.5-1.5h, centrifuging, and taking supernatant;
respectively preparing reference substance solutions of azide impurity 1 and azide impurity 2;
detecting the supernatant and the reference substance solution by adopting liquid chromatography;
wherein, the impurity 1 is 5- (4 '-azidomethyl- [1,1' -biphenyl ] -2-yl) -1H-tetrazole; impurity 2 is 4 '-azidomethyl- [1,1' -biphenyl ] -2-carbonitrile.
2. The liquid chromatography detection method of trace azide impurities in irbesartan according to claim 1, wherein the method comprises the following steps: when the irbesartan raw material is extracted, the volume ratio of acetonitrile to water is 1:0.8-1.5.
3. The liquid chromatography detection method of trace azide impurities in irbesartan according to claim 2, wherein: the volume ratio of acetonitrile to water is 1:1.
4. The liquid chromatography detection method of trace azide impurities in irbesartan according to claim 1, wherein the method comprises the following steps: the temperature of standing and extracting at room temperature is 10-30 ℃.
5. The liquid chromatography detection method of trace azide impurities in irbesartan according to claim 1, wherein the method comprises the following steps: in liquid chromatography detection, the chromatographic column is octadecylsilane chemically bonded silica as filler, XB-C18,4.6mm by 250mm,5 μm.
6. The liquid chromatography detection method of trace azide impurities in irbesartan according to claim 5, wherein the method comprises the following steps: the mobile phase A detected by liquid chromatography is as follows: 0.005mol/L potassium dihydrogen phosphate solution with pH value of 2.7; mobile phase B was methanol.
7. The liquid chromatography detection method of trace azide impurities in irbesartan according to claim 5, wherein the method comprises the following steps: the detection wavelength of the liquid chromatograph was 224nm.
8. The liquid chromatography detection method of trace azide impurities in irbesartan according to claim 5, wherein the method comprises the following steps: the gradient elution procedure was: 0-45min, mobile phase B55%; 45-50min, 55% -70% of mobile phase B; 50-75min, mobile phase B70%; 75-76min, 70% -55% of mobile phase B; 76-85min, 55% of mobile phase B and percentage by volume.
9. The liquid chromatography detection method of trace azide impurities in irbesartan according to claim 5, wherein the method comprises the following steps: the column temperature was 30 ℃.
10. The liquid chromatography detection method of trace azide impurities in irbesartan according to claim 5, wherein the method comprises the following steps: the sample volume was 50. Mu.l and the mobile phase flow rate was 1ml/min.
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