CN114184698A - High performance liquid chromatography for detecting content of beta-artemether and related substances - Google Patents
High performance liquid chromatography for detecting content of beta-artemether and related substances Download PDFInfo
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- 229960000981 artemether Drugs 0.000 title claims abstract description 113
- SXYIRMFQILZOAM-HVNFFKDJSA-N dihydroartemisinin methyl ether Chemical compound C1C[C@H]2[C@H](C)CC[C@H]3[C@@H](C)[C@@H](OC)O[C@H]4[C@]32OO[C@@]1(C)O4 SXYIRMFQILZOAM-HVNFFKDJSA-N 0.000 title claims abstract description 111
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 19
- 239000000126 substance Substances 0.000 title abstract description 7
- 238000000034 method Methods 0.000 claims abstract description 49
- 239000012535 impurity Substances 0.000 claims abstract description 41
- 238000001514 detection method Methods 0.000 claims abstract description 28
- 229960002521 artenimol Drugs 0.000 claims abstract description 17
- BJDCWCLMFKKGEE-ISOSDAIHSA-N artenimol Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2O[C@H](O)[C@@H]4C BJDCWCLMFKKGEE-ISOSDAIHSA-N 0.000 claims abstract description 17
- BJDCWCLMFKKGEE-KDTBHNEXSA-N Dihydroartemisinin (DHA) Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2O[C@@H](O)[C@@H]4C BJDCWCLMFKKGEE-KDTBHNEXSA-N 0.000 claims abstract description 15
- BLUAFEHZUWYNDE-NNWCWBAJSA-N artemisinin Chemical compound C([C@](OO1)(C)O2)C[C@H]3[C@H](C)CC[C@@H]4[C@@]31[C@@H]2OC(=O)[C@@H]4C BLUAFEHZUWYNDE-NNWCWBAJSA-N 0.000 claims abstract description 15
- 229960004191 artemisinin Drugs 0.000 claims abstract description 15
- 229930101531 artemisinin Natural products 0.000 claims abstract description 15
- SXYIRMFQILZOAM-CNNNLJIRSA-N α-artemether Chemical compound C1C[C@H]2[C@H](C)CC[C@H]3[C@@H](C)[C@H](OC)O[C@H]4[C@]32OO[C@@]1(C)O4 SXYIRMFQILZOAM-CNNNLJIRSA-N 0.000 claims abstract description 15
- 230000014759 maintenance of location Effects 0.000 claims abstract description 10
- 239000000203 mixture Substances 0.000 claims abstract description 6
- 238000004458 analytical method Methods 0.000 claims abstract description 4
- 239000013558 reference substance Substances 0.000 claims description 39
- 238000012360 testing method Methods 0.000 claims description 37
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- 239000000523 sample Substances 0.000 claims description 36
- 239000000243 solution Substances 0.000 claims description 23
- 239000012085 test solution Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000010828 elution Methods 0.000 claims description 11
- 239000002904 solvent Substances 0.000 claims description 10
- 239000012088 reference solution Substances 0.000 claims description 8
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 claims description 7
- 239000012488 sample solution Substances 0.000 claims description 7
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- 239000002245 particle Substances 0.000 claims description 2
- 239000008186 active pharmaceutical agent Substances 0.000 claims 2
- 229940088679 drug related substance Drugs 0.000 claims 2
- 239000008194 pharmaceutical composition Substances 0.000 claims 2
- 239000003814 drug Substances 0.000 abstract description 8
- 229940079593 drug Drugs 0.000 abstract description 7
- 239000000825 pharmaceutical preparation Substances 0.000 abstract description 4
- 238000003908 quality control method Methods 0.000 abstract description 2
- 239000002994 raw material Substances 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 description 8
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 6
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- 238000000926 separation method Methods 0.000 description 6
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 5
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- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 3
- 239000000377 silicon dioxide Substances 0.000 description 3
- 229930016266 dihydroartemisinin Natural products 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000003255 drug test Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 201000004792 malaria Diseases 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- WHTVZRBIWZFKQO-AWEZNQCLSA-N (S)-chloroquine Chemical compound ClC1=CC=C2C(N[C@@H](C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-AWEZNQCLSA-N 0.000 description 1
- -1 4.6 × 250mm 5 μm Substances 0.000 description 1
- 241000224016 Plasmodium Species 0.000 description 1
- 239000003139 biocide Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 229960003677 chloroquine Drugs 0.000 description 1
- WHTVZRBIWZFKQO-UHFFFAOYSA-N chloroquine Natural products ClC1=CC=C2C(NC(C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-UHFFFAOYSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- DYLGFOYVTXJFJP-MYYYXRDXSA-N lumefantrine Chemical compound C12=CC(Cl)=CC=C2C=2C(C(O)CN(CCCC)CCCC)=CC(Cl)=CC=2\C1=C/C1=CC=C(Cl)C=C1 DYLGFOYVTXJFJP-MYYYXRDXSA-N 0.000 description 1
- 229960004985 lumefantrine Drugs 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
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- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
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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
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a high performance liquid chromatography for detecting beta-artemether content and related substances, belonging to the technical field of drug analysis. Firstly, the invention provides a method for rapidly detecting whether a sample containing beta-artemether contains impurity A or not, and provides specific chromatographic conditions. According to the chromatographic conditions provided by the invention, the retention time of the impurity A is short, and the detection efficiency is high. Secondly, the invention also provides a method for synchronously detecting the contents of impurities of alpha-dihydroartemisinin, beta-dihydroartemisinin, artemisinin and alpha-artemether in the beta-artemether by using the chromatographic conditions provided by the invention. The method provided by the invention not only realizes the rapid detection of the sample containing the beta-artemether, but also realizes the quantitative detection of impurities in the sample, and provides scientific and efficient methodology basis for the quality control of the raw material medicine of the beta-artemether or certain compositions and pharmaceutical preparations containing the beta-artemether.
Description
Technical Field
The invention belongs to the technical field of drug analysis. In particular to a high-efficiency liquid chromatography method for detecting the content of beta-artemether and related substances.
Background
Beta-artemether (structural formula shown in figure 1) has the following molecular formula: c16H26O5White crystals or crystalline powder. The beta-artemether can be prepared into beta-artemether tablets, beta 0-artemether capsules, beta 1-artemether injection, or can be prepared into compound beta-artemether tablets with lumefantrine. The beta-artemether is a high-efficiency and quick-acting plasmodium erythrocytic internal phase killing agent. Can be used for treating chloroquine-resistant severe malaria and dangerous malaria with rapid and good therapeutic effect. In the aspect of the content of beta-artemether and related substance inspection methods, the Chinese pharmacopoeia and the international pharmacopoeia use a chromatographic column using octadecylsilane chemically bonded silica as a filler (the international pharmacopoeia stipulates that the specification is 4.6 multiplied by 250mm and 5 mu m); acetonitrile-water (62: 38) is used as a mobile phase; the detection wavelength is 216 nm; and (3) testing the injection volume of 20 mu L. In the process of testing beta-artemether by using a pharmacopoeia method, a chromatographic peak is found at about 6 relative retention time, and the chromatographic peak is found to be beta-artemether impurity by investigation. Meanwhile, the impurity is found in beta-artemether legal reference substances purchased by European drug quality management bureau (EDQM) and China food and drug testing research institute, and beta-artemether produced or purchased by other manufacturers.
In the process of testing the beta-artemether method in the Chinese pharmacopoeia and the international pharmacopoeia in use, if the impurities need to be tested, under the condition that the main peak of the beta-artemether is about 8 minutes, the retention time of the impurities is about 50 minutes, and the testing efficiency is low.
Therefore, it is necessary to establish a high-efficiency detection method for detecting the impurities in the beta-artemether. Also, there is a need to provide a method capable of analyzing the content of impurities in a sample containing β -artemether.
Disclosure of Invention
The invention aims to provide a method for rapidly detecting whether a sample containing beta-artemether contains impurity A.
Another object of the present invention is to provide a method capable of simultaneously quantifying impurities including alpha-dihydroartemisinin, beta-dihydroartemisinin, artemisinin and alpha-artemether in a sample containing beta-artemether.
In order to achieve the purpose, the invention provides the following technical scheme:
firstly, the invention provides a method for rapidly detecting whether a sample containing beta-artemether contains impurity A by using HPLC, which comprises the following steps:
dissolving a sample containing beta-artemether in a proper amount of solvent to prepare a test solution;
detecting the sample solution by HPLC, and observing that there is no chromatographic peak at the position with retention time of 26-27 min; wherein, the chromatographic conditions used by HPLC are as follows:
a chromatographic column: thermo HYPERSIL BDS C18;
sample introduction amount: 1-20 μ L;
column temperature: 30 ℃;
detection wavelength: 190nm-400 nm;
flow rate of mobile phase: 0.8-1.5 mL/min;
elution conditions: using acetonitrile as a mobile phase A and water as a mobile phase B, and carrying out gradient elution according to the following modes:
according to a specific embodiment of the present invention, the method further comprises, after the end of the above elution conditions, continuing to run with 68% of mobile phase a and 32% of mobile phase B for about 6min, and keeping the column in equilibrium.
According to a specific embodiment of the invention, the detection method provided by the invention comprises the steps of preparing a beta-artemether-containing sample, preparing a beta-artemether bulk drug, preparing a beta-artemether legal control, preparing a beta-artemether-containing composition or a pharmaceutical preparation, such as artemether tablets, compound artemether tablets or artemether injection.
According to the specific embodiment of the present invention, the sample amount in the detection method provided by the present invention may be 1. mu.L, 2. mu.L, 3. mu.L, 4. mu.L, 5. mu.L, 6. mu.L, 7. mu.L, 8. mu.L, 9. mu.L, 10. mu.L, 11. mu.L, 12. mu.L, 13. mu.L, 14. mu.L, 15. mu.L, 16. mu.L, 17. mu.L, 18. mu.L, 19. mu.L, 20. mu.L or a range thereof.
According to an embodiment of the present invention, in the detection method provided in the present invention, the flow rate of the mobile phase may be 0.8mL/min, 0.9mL/min, 1.0mL/min, 1.1mL/min, 1.2mL/min, 1.3mL/min, 1.4mL/min, 1.5mL/min or a range thereof.
According to a specific embodiment of the present invention, in the detection method provided in the present invention, the solvent is a mixed solvent of acetonitrile and water; preferably a mixed solvent of acetonitrile and water in a volume ratio of 1: 1.
According to the specific embodiment of the invention, in the detection method provided by the invention, the concentration of the test solution is 1mg/mL-10mg/mL calculated by beta-artemether. In some embodiments, the concentration of the test solution may be 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL or a range thereof, based on β -artemether.
According to a specific embodiment, the detection method provided by the invention is characterized in that the particle size of the chromatographic column Thermo HYPERSIL BDS C18 is 3-5 μm, and the length of the chromatographic column is 150mm or 250 mm.
According to a specific embodiment of the present invention, in the detection method provided by the present invention, the chromatographic conditions are further preferably:
a chromatographic column: thermo HYPERSIL BDS C18 with specification of 4.6 × 250mm 5 μm;
sample introduction amount: 5 mu L of the solution;
column temperature: 30 ℃;
detection wavelength: 216 nm;
flow rate of mobile phase: 1.0 mL/min;
elution conditions: using acetonitrile as a mobile phase A and water as a mobile phase B, and carrying out gradient elution according to the following modes:
secondly, the invention also provides a method for synchronously detecting the contents of alpha-dihydroartemisinin, beta-dihydroartemisinin, artemisinin, alpha-artemether and beta-artemether in a test sample by using HPLC, which comprises the following steps:
preparation of control solutions: taking a proper amount of beta-artemether reference substance, precisely weighing, placing in a volumetric flask, adding a solvent, diluting to a scale, and shaking up to obtain a reference substance solution;
preparation of a test solution: taking a proper amount of a test sample, precisely weighing, placing in a volumetric flask, adding a solvent, diluting to a scale, and shaking uniformly to obtain a test sample solution;
and (3) determination: taking a reference solution and a test solution respectively, carrying out high performance liquid chromatography analysis by adopting the chromatographic conditions provided by the invention, measuring the content of beta-artemether by an external standard method, and measuring the content of alpha-dihydroartemisinin, beta-dihydroartemisinin, artemisinin, alpha-artemether and other impurities in the test solution by an area normalization method.
According to the specific embodiment of the invention, in the quantitative detection method provided by the invention, the test sample comprises a beta-artemether bulk drug, a beta-artemether legal control, a composition or a pharmaceutical preparation containing beta-artemether, such as artemether tablets, compound artemether tablets or artemether injection.
According to a specific embodiment of the present invention, the solvent is a mixed solvent of acetonitrile and water at a volume ratio of 1: 1.
According to a specific embodiment of the present invention, there is provided the quantitative determination method wherein,
the linear equation of the beta-artemether is that y is 147.5x-13.143, and the linear range is 8mg/mL-12 mg/mL;
the linear equation of the alpha-dihydroartemisinin is that y is 914.82x-0.0608, and the linear range is 0.005mg/mL-0.1 mg/mL;
the linear equation of the beta-dihydroartemisinin is that y is 241.83x +0.0852, and the linear range is 0.005mg/mL-0.1 mg/mL;
the linear equation of the alpha-artemether is that y is 1503.2x +0.1483, and the linear range is 0.005mg/mL-0.1 mg/mL;
the linear equation for artemisinin is y-1535.4 x +0.1049, with a linear range of 0.005mg/mL to 0.1 mg/mL.
According to the specific embodiment of the present invention, in the quantitative determination method provided by the present invention, the calculation formula of the content of β -artemether in the test sample is: content (%) ═ AX/AR×mR/mXX ω, wherein AXIs the peak area of beta-artemether in the test solution; a. theRThe peak area of beta-artemether in the reference solution is shown; m isRIs the weight of the beta-artemether reference substance, and the unit is mg; m isXThe weight of the sample is mg; omega is the content of beta-artemether, and the unit is%.
In conclusion, compared with the existing method in the prior art, the method for rapidly detecting whether the sample containing the beta-artemether contains the impurity A by using the HPLC has the advantages of high separation degree of the impurity A from other components, fast peak appearance, shorter detection time, higher detection efficiency and lower detection cost.
Meanwhile, the chromatographic conditions in the method can be adopted to realize the separation of common impurities in the beta-artemether sample, such as alpha-dihydroartemisinin, beta-dihydroartemisinin, artemisinin and alpha-artemether, and further can carry out quantitative detection on the impurities. Namely, the method provided by the invention not only realizes the rapid detection of the sample containing the beta-artemether, but also realizes the quantitative detection of impurities in the sample, and provides scientific and efficient methodology basis for the quality control of the raw material drug of the beta-artemether or certain compositions and pharmaceutical preparations containing the beta-artemether.
Drawings
FIG. 1 is a structural diagram of beta-artemether.
FIGS. 2-6 are chromatograms of several control samples, respectively; wherein, FIG. 2 is a chromatogram of a reference substance beta-artemether, FIG. 3 is a chromatogram of a reference substance artemisinin, FIG. 4 is a chromatogram of beta-dihydroartemisinin, FIG. 5 is a chromatogram of alpha-artemether, and FIG. 6 is a chromatogram of alpha-dihydroartemisinin.
FIG. 7 is a graph showing the separation effect of the control mixed solution.
FIG. 8 is a chromatogram for detecting impurity A under the chromatographic conditions provided by the present invention.
Figures 9-14 are experimental chromatograms of results of six parallel experiments by the operator in the precision testing experiment of example 2.
Figures 15-20 are experimental chromatograms of results of 2 six parallel experiments by the operator in the precision testing experiment of example 2.
FIGS. 21A-23 are graphs showing the results of three replicates at a control concentration of 8mg/mL in the accuracy testing experiment of example 3.
FIGS. 24-26 are graphs showing the results of three replicates at a control concentration of 10mg/mL in the accuracy testing experiment of example 3.
FIGS. 27-29 are graphs showing the results of three replicates at a control concentration of 12mg/mL in the accuracy testing experiment of example 3.
FIG. 30 is a graph showing the results of the test for detectivity in example 4.
FIGS. 31-40 are line result validation chromatograms of β -artemether in example 5.
FIG. 41 is a chromatogram showing that the concentration of each impurity in example 6 is 0.05% of β -artemether.
FIG. 42 is a chromatogram showing that the concentration of each impurity in example 6 is 0.10% of β -artemether.
FIG. 43 is a chromatogram showing that the concentration of each impurity in example 6 is 0.20% of β -artemether.
FIG. 44 is a chromatogram showing that the concentration of each impurity in example 6 is 0.50% of that of β -artemether.
FIG. 45 is a chromatogram showing that the concentration of each impurity in example 6 is 1.0% of β -artemether.
Detailed Description
The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention.
[ interpretation of terms ]
In the invention, the "impurity A" refers to the chromatographic conditions for detecting beta-artemether according to the specification in the International pharmacopoeia: a chromatographic column using octadecylsilane chemically bonded silica as a filler, 4.6 × 250mm 5 μm, acetonitrile: the water volume ratio is 62: 38 is the mobile phase, the detection wavelength is 216nm, and the substance corresponds to the chromatographic peak at the relative retention time of about 6 when the sample volume is 20 μ L.
In the present invention, "HPLC" is an abbreviation for high performance liquid chromatography.
In the present invention, the "external standard method" is a method well known to those skilled in the art, and means a method of quantifying by comparing a response signal of a reference substance and a response signal of a component to be measured in a sample using a pure product of the component to be measured as the reference substance in chromatography.
The invention is further illustrated with reference to specific embodiments.
The chromatographic conditions adopted in the embodiment of the invention are as follows: adopting American Agilent high performance liquid chromatograph, and octadecylsilane chemically bonded silica as filler (Thermo HYPERSIL BDS C184.6 × 250mm 5 μm or equivalent chromatographic column); performing gradient elution according to the table 1 by using acetonitrile as a mobile phase A and water as a mobile phase B; the flow rate was 1.0mL per minute; the detection wavelength is 216 nm; the injection volume was 5. mu.L.
TABLE 1 gradient elution procedure
The implementation method comprises the following steps:
preparation of the solution:
taking about 100mg of a beta-artemether reference substance, putting the beta-artemether reference substance into a 10mL volumetric flask, adding acetonitrile-water (1:1) to dissolve and dilute the beta-artemether reference substance to a scale, and shaking the solution uniformly to serve as a reference substance solution.
About 100mg of a test sample is taken and placed in a 10mL volumetric flask, acetonitrile-water (1:1) is added for dissolving and diluting to the scale, and the solution is uniformly shaken to be used as a test sample solution.
The determination method comprises the following steps:
precisely measuring the test solution and the reference solution, respectively injecting into a liquid chromatograph, and recording the chromatogram. And calculating the content of the test sample according to an external standard method by using the peak area, and calculating the content of related substances in the test sample according to an area normalization method.
Example 1
Experimental materials: beta-artemether working reference substance, artemisinin working reference substance, dihydroartemisinin working reference substance (alpha-dihydroartemisinin, beta-dihydroartemisinin), and alpha-artemether working reference substance. The working reference substances are all synthesized and refined by research and development departments of the company and are obtained by calibrating legal reference substances purchased by QC departments and China food and drug testing research institutes.
The experimental steps are as follows:
taking acetonitrile-water (1:1) as a diluent, preparing a beta-artemether reference solution (10mg/mL), an artemisinin reference solution (0.1mg/mL), a dihydroartemisinin reference solution (alpha-dihydroartemisinin 0.1mg/mL, beta-dihydroartemisinin 0.1mg/mL), an alpha-artemether reference solution (0.1mg/mL) and a mixed solution (containing beta-artemether 10mg/mL and other impurities each 0.05 mg/mL). And sequentially injecting the solution for testing, and recording chromatograms (fig. 2 is a chromatogram of a reference substance beta-artemether, fig. 3 is a chromatogram of a reference substance artemisinin, fig. 4 is a chromatogram of beta-dihydroartemisinin, fig. 5 is a chromatogram of alpha-artemether, and fig. 6 is a chromatogram of alpha-dihydroartemisinin). Each control solution was taken to locate each component and the separation of each component was examined and the results are shown in Table 2. The separation degree effect of each component is ideal (fig. 7).
Table 2: relative retention time and separation of the components in the mixed solution
Taking a sample containing beta-artemether (beta-artemether bulk drug, produced by Zhang Jia hong Weisheng biological medicine Co., Ltd.) and using acetonitrile-water (1:1) as a diluent to prepare a test solution with the concentration of 10mg/mL, and carrying out sample injection test. The experimental results are shown in fig. 8 and table 2, and it can be seen from fig. 8 that the retention time of the impurity a is 26.669 by using the method provided by the present invention, compared with the prior art (the retention time is about 50min), the retention time is greatly shortened, and the detection efficiency is greatly improved.
Example 2
Experimental materials: beta-artemether working reference substance and beta-artemether produced by Zhang hong Kong Weisheng biomedicine GmbH.
The experimental steps are as follows:
taking a beta-artemether working reference substance and a beta-artemether test sample, preparing a reference substance solution and a test sample solution according to the method under the implementation method, and preparing six test sample solutions in parallel. The two operators independently check twice, sample introduction test and chromatogram recording. The contents of the test samples measured by the two operators (see table 3) were determined to be 0.62%, 0.37% and 0.00% respectively in precision from the results of the tests, and the results were accurate.
Table 3: precision test result of beta-artemether
Example 3
Experimental materials: beta-artemether working reference substance.
The experimental steps are as follows:
taking a beta-artemether working reference substance, preparing reference substance solutions with the concentrations of 8mg/mL, 10mg/mL and 12mg/mL according to the method under the implementation method, sequentially injecting samples for testing, and recording a chromatogram (wherein the first experiment is carried out five times when the concentration is 8mg/mL, and the result is averaged). The recovery rate of the beta-artemether reference substance at each concentration is calculated (see table 4), and the recovery rate at each concentration is good and the accuracy is high.
Table 4: accuracy test result of beta-artemether
Example 4
Experimental materials: beta-artemether working reference substance.
The experimental steps are as follows:
taking a beta-artemether working reference substance, preparing a reference substance solution according to the method under the implementation method item, diluting to 0.05% of the concentration, and performing sample injection test to obtain the solution with the beta-artemether peak signal-to-noise ratio of 11.04, high quantitative detection capacity and strong detection capacity (figure 30).
Example 5
Experimental materials: beta-artemether working reference substance.
The experimental steps are as follows:
taking a beta-artemether working reference substance, preparing two reference substance solutions with the concentrations of 8mg/mL, 9mg/mL, 10mg/mL, 11mg/mL and 12mg/mL according to the method under the implementation method item, sequentially injecting samples for testing, recording a chromatogram (see table 5), drawing a function of the measured beta-artemether peak area (y) and the concentration (x), observing whether the beta-artemether working reference substance is linear or not, and performing linear regression by using a least square method. The linear regression equation is that y is 147.5x-13.143, and the correlation coefficient r is 0.9999. The linear relation is good, which shows that the method has good linearity between 8mg/mL and 12mg/mL and accurate result.
Table 5: statistical table of peak area of beta-artemether in each concentration
| Weighing volume | Concentration of | |
|
Peak area mean |
| 80.12 | 8.012 | 1172.06 (fig. 31) | 1171.20 (fig. 32) | 1171.63 |
| 90.17 | 9.017 | 1312.46 (fig. 33) | 1310.83 (fig. 34) | 1311.64 |
| 100.02 | 10.002 | 1464.55 (fig. 35) | 1462.83 (fig. 36) | 1463.69 |
| 110.03 | 11.003 | 1612.21 (fig. 37) | 1608.67 (fig. 38) | 1610.44 |
| 120.14 | 12.014 | 1759.66 (fig. 39) | 1758.39 (Picture 40) | 1759.04 |
Example 6
Experimental materials: working reference substances of various impurities of beta-artemether.
The experimental steps are as follows:
preparing an impurity mixed solution with each impurity concentration of 0.2mg/mL by taking a working reference substance of each impurity of the beta-artemether, and preparing limit solutions by taking the working reference substance of the beta-artemether and the impurity mixed solution, wherein the concentration of the beta-artemether in the limit solutions is 10mg/mL, and the concentrations of the impurities are 0.05%, 0.10%, 0.20%, 0.50% and 1.0% of the concentration of the beta-artemether in sequence. And (3) taking the limiting solution, sequentially injecting samples, testing, recording a chromatogram (see table 5), drawing by using the functions of the peak area (y) and the concentration (x) of each measured impurity, observing whether the solution is linear, and performing linear regression by using a least square method. Wherein the regression equation of the alpha-dihydroartemisinin is that y is 914.82x-0.0608, and the correlation coefficient r is 0.9996; the regression equation of the beta-dihydroartemisinin is that y is 241.83x +0.0852, and the correlation coefficient r is 0.9988; the regression equation of artemisinin is that y is 1535.4x +0.1049, and the correlation coefficient r is 0.9999; the regression equation of the alpha-artemether is 1503.2x +0.1483, and the correlation coefficient r is 0.9997. The correlation coefficient r of each impurity for making a linear regression equation is not lower than 0.99, which shows that the method has good linearity and accurate determination when the concentration of each impurity is 0.005mg/mL-0.1 mg/mL.
Table 5: statistical table of peak areas of impurity limit solutions of various concentrations
| Concentration of impurities | Alpha-dihydroartemisinin | Beta-dihydroartemisinin | Artemisinin | Alpha-artemether | Beta-artemether |
| 0.05% (fig. 41) | 0.48 | 0.25 | 0.94 | 0.96 | 1549.22 |
| 0.10% (fig. 42) | 0.92 | 0.33 | 1.62 | 1.52 | 1496.91 |
| 0.20% (fig. 43) | 1.61 | 0.56 | 3.12 | 3.34 | 1488.23 |
| 0.50% (fig. 44) | 4.49 | 1.22 | 7.78 | 7.49 | 1497.75 |
| 1.00% (fig. 45) | 9.12 | 2.54 | 15.47 | 15.24 | 1493.21 |
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Various alternatives, modifications and combinations of the features of the invention can be made without departing from the spirit and nature of the invention as claimed, and such simple variations and combinations should also be considered as disclosed in the present application, all falling within the scope of the invention.
Claims (10)
1. A method for rapidly detecting whether a sample containing beta-artemether contains impurity A or not by using HPLC (high performance liquid chromatography), which comprises the following steps:
dissolving a sample containing beta-artemether in a proper amount of solvent to prepare a test solution;
detecting the sample solution by HPLC, and observing that there is no chromatographic peak at the position with retention time of 26-27 min; wherein, the chromatographic conditions used by HPLC are as follows:
a chromatographic column: thermo HYPERSIL BDS C18;
sample introduction amount: 1-20 μ L;
column temperature: 30 ℃;
detection wavelength: 190nm-400 nm;
flow rate of mobile phase: 0.8-1.5 mL/min;
elution conditions: using acetonitrile as a mobile phase A and water as a mobile phase B, and carrying out gradient elution according to the following modes:
2. the method of claim 1, wherein the beta-artemether-containing sample comprises a beta-artemether drug substance, a beta-artemether legal control, a beta-artemether-containing composition, or a pharmaceutical formulation.
3. The method according to claim 1, wherein the solvent is a mixed solvent of acetonitrile and water; preferably a mixed solvent of acetonitrile and water in a volume ratio of 1: 1.
4. The method of claim 1, wherein the concentration of the test solution is 1mg/mL to 10mg/mL, based on β -artemether.
5. The process as claimed in claim 1, wherein the Thermo HYPERSIL BDS C18 has a particle size of 3-5 μm and a column length of 150mm or 250 mm.
6. The method of any one of claims 1-5, wherein:
the chromatographic conditions used for HPLC were:
a chromatographic column: thermo HYPERSIL BDS C18 with specification of 4.6 × 250mm 5 μm;
sample introduction amount: 5 mu L of the solution;
column temperature: 30 ℃;
detection wavelength: 216 nm;
flow rate of mobile phase: 1.0 mL/min;
elution conditions: using acetonitrile as a mobile phase A and water as a mobile phase B, and carrying out gradient elution according to the following modes:
7. a method for synchronously detecting contents of alpha-dihydroartemisinin, beta-dihydroartemisinin, artemisinin, alpha-artemether and beta-artemether in a test sample by utilizing HPLC (high performance liquid chromatography), which comprises the following steps:
preparation of control solutions: taking a proper amount of beta-artemether reference substance, precisely weighing, placing in a volumetric flask, adding a solvent, diluting to a scale, and shaking up to obtain a reference substance solution;
preparation of a test solution: taking a proper amount of a test sample, precisely weighing, placing in a volumetric flask, adding a solvent, diluting to a scale, and shaking uniformly to obtain a test sample solution;
and (3) determination: taking a reference solution and a test solution respectively, carrying out high performance liquid chromatography analysis by adopting the chromatographic conditions of any one of claims 1-6, measuring the content of beta-artemether by an external standard method, and measuring the content of alpha-dihydroartemisinin, beta-dihydroartemisinin, artemisinin, alpha-artemether and other impurities in the test solution by an area normalization method.
8. The method of claim 7, wherein the test sample comprises a beta-artemether drug substance, a beta-artemether statutory control, a beta-artemether-containing composition, or a pharmaceutical formulation.
9. The method according to claim 8, wherein the solvent is a mixed solvent of acetonitrile and water in a volume ratio of 1: 1.
10. The method of claim 7, wherein,
the linear equation of the beta-artemether is that y is 147.5x-13.143, and the linear range is 8mg/mL-12 mg/mL;
the linear equation of the alpha-dihydroartemisinin is that y is 914.82x-0.0608, and the linear range is 0.005mg/mL-0.1 mg/mL;
the linear equation of the beta-dihydroartemisinin is that y is 241.83x +0.0852, and the linear range is 0.005mg/mL-0.1 mg/mL;
the linear equation of the alpha-artemether is that y is 1503.2x +0.1483, and the linear range is 0.005mg/mL-0.1 mg/mL;
the linear equation for artemisinin is y-1535.4 x +0.1049, with a linear range of 0.005mg/mL to 0.1 mg/mL.
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