CN106918672B - Method for measuring related substances in efinaconazole raw material medicine and preparation thereof by using HPLC - Google Patents
Method for measuring related substances in efinaconazole raw material medicine and preparation thereof by using HPLC Download PDFInfo
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Abstract
The invention discloses a method for determining related substances in an efinaconazole raw material medicine and a preparation thereof by using HPLC, wherein the analysis conditions of the method are as follows: the chromatographic column adopts an octadecylsilane chemically bonded silica gel column; and (3) gradient elution is carried out by using a mixed solvent of a formate buffer solution and a first organic solvent as a mobile phase. By adopting the method, the related substances in the efinaconazole raw material medicine and the preparation thereof can be quickly and efficiently separated under the same chromatographic condition, and the quality of the raw material medicine and the preparation can be effectively controlled. The detection method has the advantages of strong specificity, high precision, strong accuracy and convenient operation, and can effectively control the product quality.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for determining related substances in an efinaconazole raw material medicine and a preparation thereof by utilizing High Performance Liquid Chromatography (HPLC).
Background
Onychomycosis is a commonly known infectious disease occurring on the nails of human fingers (toes) and is caused by the infection of a large group of microorganisms called pathogenic fungi, pathogenic bacteria invade the distal subungual bed first, the cutis under the subungual bed is hyperplastic and thickened, the nail plate can turn white, brown to grey brown, the surface is uneven, the surface is glossy, and the nail plate is absent and crisp. The existing treatment method has certain limitations. Currently, over-the-counter or over-the-counter medications provide limited efficacy and often require frequent debridement, or scraping, cutting or removal of the nails while the medication is being administered. Prescribed oral drugs are of limited use due to drug interactions and serious safety concerns.
In the prior art, the best therapy for treating onychomycosis is external therapy, wherein the local application is the simplest and most convenient. The external antifungal agent has the most direct curative effect, wherein the liniment has excellent penetrability to the nail plate, can permeate into the inner side of the nail plate after being applied on the outer side of the nail plate, and can directly exert bacteriostatic or bactericidal activity.
Efinaconazole (Efinaconazole) is a new triazole antifungal drug developed by DOW pharmaceutical company, canada, and obtained FDA approval for marketing at 6.6.2014 under the trade name jubilia (topical solution containing 10% by mass of Efinaconazole). Jubilia is the first triazole antifungal drug for external use, can be applied topically, is applied through a unique built-in dosage control type nail brush, can dry quickly, does not need to remove excessive drug, is mainly used for treating tinea manuum and tinea pedis (namely onychomycosis) caused by trichophyton rubrum and trichophyton gypseum, does not need to consider systemic side effects such as drug interaction or acute liver injury, and has little side effect.
However, the current methods for analyzing related substances in the raw drug and preparation of efinaconazole still need to be improved.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, one object of the present invention is to provide a high performance liquid chromatography method for rapidly analyzing and separating the degradation products, process side reaction impurities and intermediates of the efinaconazole, thereby realizing the separation and determination of related substances in the efinaconazole bulk drug and the preparation thereof under the same chromatographic condition.
The chemical name of efinaconazole is (2R,3R) -2- (2, 4-difluorophenyl) -3- (4-methylenepiperidin-1-yl) -1- (1H-1,2, 4-triazol-1-yl) -2-butanol, the structural formula of which is:
the synthetic route of the efinaconazole is as follows:
the raw material drug of the efinaconazole and related substances of the preparation thereof have the following three sources: the first is raw material, intermediate and degradation product brought in the synthesis process of the product; the second is a byproduct generated in the synthesis reaction; and the third is a degradation product generated due to the influence of the environment in the storage process, and mainly comprises the following components:
impurity 3 is a side reaction product: e- (3R) -2- (2, 4-difluorophenyl) -3- (4-methylenepiperidin-1-yl) -1- (1H-1,2, 4-triazol-1-yl) -1-butene;
impurity 4 is a side reaction product: (2R,3R) -2- (2, 4-difluorophenyl) -3- (4-methylenepiperidin-1-yl) -1- (4H-1,2, 4-triazol-4-yl) -2-butanol;
impurity 5 is a side reaction product: (R, E) -3- (2, 4-difluorophenyl) -4- (1H-1,2, 4-triazol-1-yl)) -2-hydroxy-3-butene;
impurity 6 is a side reaction product: 1- ((6-fluoro-2-methylbenzofuran-3-yl) methyl) -1H-1,2, 4-triazole;
impurity 7 is a side reaction product, a degradation product: (2R,3R) -2- (2, 4-difluorophenyl) -3 (4-methyl-3, 6-dihydropyridin-1 (2H) -yl) -1- (1H-1,2, 4-triazol-1-yl) -2-butanol.
The impurities which are possibly generated in the processes of synthesizing, producing and storing the raw material drug of the efinaconazole and preparing and storing the preparation of the efinaconazole all need to be strictly controlled so as to ensure the product quality. Therefore, the realization of the rapid separation and analysis of the related substances in the efinaconazole and the preparation thereof has important practical significance in the aspects of synthesis and quality control of the preparation process.
The detection method of related substances of the efinaconazole is not reported, the difficulty of the detection method is that various process by-product impurities, diastereoisomer impurities, degradation products and the like can be generated in the synthesis process, and the impurities which can not be removed after refining are similar to the chromatographic behavior of the main drug and are not easy to be completely separated.
In one aspect of the invention, the invention provides a method for measuring related substances in an efinaconazole raw material medicine and a preparation thereof by using HPLC. The chromatographic conditions of the method are as follows: the chromatographic column is an octadecylsilane chemically bonded silica gel column; the mobile phase is a mixed solvent of formate buffer and a first organic solvent.
Therefore, the related substances in the efinaconazole raw material medicine and the preparation thereof can be quickly and efficiently separated under the same chromatographic condition, and the quality of the raw material medicine and the preparation can be effectively controlled. The detection method has the advantages of high sensitivity, strong specificity, high precision, strong accuracy and convenient operation, and can effectively control the quality of products.
The method for determining related substances in the efinaconazole bulk drug and the preparation thereof by using HPLC according to some embodiments of the invention can also have the following additional technical characteristics:
according to some embodiments of the invention, the conditions of the gradient elution are: the volume ratio of the formate buffer to the first organic solvent is (65-55)%, at 0-3 min: (35-45)%; at 6-25 minutes, the volume ratio of the formate buffer solution to the first organic solvent is 45% to 55%; at 30-35 minutes, the volume ratio of the formate buffer solution to the first organic solvent is 20% to 80%; the volume ratio of the formate buffer to the first organic solvent is (20-65)%: (80-35)%. The separation obtained is the best and the peak shape is the best.
According to one embodiment of the invention, the conditions of the gradient elution are:
| time/min | Formate buffer/%) | First organic solvent/%) |
| 0 | 65 | 35 |
| 3 | 65 | 35 |
| 6 | 45 | 55 |
| 25 | 45 | 55 |
| 30 | 20 | 80 |
| 35 | 20 | 80 |
| 36 | 65 | 35 |
| 42 | 65 | 35 |
According to some embodiments of the invention, the particle size of the chromatography column is between 3 μm and 5 μm, preferably 3 μm. This can further improve the degree of separation.
According to some embodiments of the invention, the formate buffer is an ammonium formate buffer. Thereby, the peak shape can be further improved.
According to some embodiments of the invention, the formate buffer is adjusted to a pH of 5.6-6.0, preferably 5.8, with formic acid or ammonia water. Thereby, the degree of separation between adjacent chromatographic peaks can be further improved.
According to an embodiment of the present invention, the concentration of formate in the formate buffer is 0.001mol/L to 0.10mol/L, preferably 0.005mol/L to 0.05mol/L, and most preferably 0.01 mol/L. Thereby, damage to the instrument and the column can be minimized while ensuring the separation effect.
According to an embodiment of the present invention, the first organic solvent is methanol and/or acetonitrile, preferably acetonitrile. This can further improve the separation efficiency.
According to an embodiment of the present invention, the analysis method of the present invention further comprises: and preparing a sample to be detected into a sample solution by adopting a mixed solution of a second organic solvent and water, and injecting the sample solution into a high performance liquid chromatograph to complete the determination of related substances in the efinaconazole raw material medicine and the preparation thereof.
According to an embodiment of the invention, the second organic solvent is methanol and/or acetonitrile, preferably acetonitrile.
According to an embodiment of the present invention, the volume ratio of water and the second organic solvent in the mixed solution of the second organic solvent and water is (0%: 100%) to (60%: 40%), and preferably 50%: 50%.
According to some embodiments of the present invention, the volume of the sample solution injected into the high performance liquid chromatograph is 2 μ L to 20 μ L, preferably 10 μ L.
According to some embodiments of the invention, the detection wavelength of the HPLC is 254nm to 262nm, preferably 260 nm. This can significantly improve the detection sensitivity.
According to an embodiment of the invention, the flow rate of the mobile phase is between 0.8mL/min and 1.2mL/min, preferably 1.0 mL/min. This can further improve the degree of separation.
According to some embodiments of the invention, the column temperature of the chromatography column is between 25 ℃ and 40 ℃, preferably 35 ℃. Thereby, the degree of separation between adjacent chromatographic peaks can be significantly improved.
According to the embodiment of the invention, the method for determining the related substances in the itraconazole and the preparation thereof can be realized according to the following steps:
(1) taking a proper amount of a sample to be detected, and adding acetonitrile: the mixture of water (50: 50 by volume) was dissolved to prepare a sample solution containing about 10mg/ml per 1 ml.
(2) The temperature of the chromatographic column is set to be 25-40 ℃, and preferably 35 ℃. The flow rate of the mobile phase is 0.8-1.2 mL/min, preferably 1.0mL/min, and the detection wavelength is 254-262 nm, preferably 260 nm.
(3) And (2) taking the sample solution of (1) into a sample volume of 2-20 mu L, preferably 10 mu L, and injecting the sample solution into a high performance liquid chromatograph to complete the determination of the related substances in the itraconazole and the preparation thereof.
The method can thoroughly separate all components of related substances in the efinaconazole raw material medicine and the preparation thereof, has good separation degree of impurity peaks and main peaks before and after the main peak, strong operability, strong specificity of an analysis method, high precision, strong accuracy and convenient operation, and can effectively control the quality of the raw material medicine and the preparation thereof.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1: high performance liquid chromatogram of the obtained efinaconazole and seven impurities thereof according to the conditions of the example 1;
FIG. 2: a high performance liquid chromatogram of the efinaconazole and seven impurities thereof obtained according to the conditions of the example 2;
FIG. 3: high performance liquid chromatogram of the obtained efinaconazole and seven impurities thereof according to the conditions of the example 3;
FIG. 4: high performance liquid chromatogram of the obtained efinaconazole and seven impurities thereof according to the conditions of the example 4;
FIG. 5: a high performance liquid chromatogram of the efinaconazole bulk drug obtained according to the conditions of the example 4;
FIG. 6: high performance liquid chromatogram of the obtained efinaconazole and seven impurities thereof according to the conditions of the example 5;
FIG. 7: high performance liquid chromatogram of the blank auxiliary material obtained according to the conditions of the example 5;
FIG. 8: a high performance liquid chromatogram of the efinaconazole preparation obtained according to the conditions of the example 5;
FIG. 9: a high performance liquid chromatogram of the efinaconazole impurity 3 obtained under the conditions of the comparative example 1;
FIG. 10: the high performance liquid chromatogram of the efinaconazole and seven impurities thereof obtained according to the conditions of the comparative example 1;
FIG. 11: a high performance liquid chromatogram of the efinaconazole impurity 3 obtained under the conditions of the comparative example 2;
FIG. 12: high performance liquid chromatogram of the efinaconazole and seven impurities thereof obtained according to the conditions of the comparative example 2.
Detailed Description
The following examples are provided to further illustrate the invention and its embodiments. The specific details given in the examples are for illustrative purposes only and should not be construed as limiting the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
The efinaconazole raw material medicine used in the embodiment of the invention is self-prepared by the applicant according to the synthesis route. The used efinaconazole preparation is self-made by the unit of the applicant.
Example 1
The instrument comprises the following steps: agilent 1260 high performance liquid chromatograph, 1260Infinity diode array detector
A chromatographic column: CAPCELL PAK MG IIC 18 (150X 4.6mm,3 μm)
Mobile phase A: 0.005mol/L ammonium formate buffer (pH adjusted to 5.6 with formic acid or ammonia)
Mobile phase B: acetonitrile
Gradient elution is shown in the following table:
| time/min | Formate buffer/%) | First organic solvent/%) |
| 0 | 65 | 35 |
| 3 | 65 | 35 |
| 6 | 45 | 55 |
| 25 | 45 | 55 |
| 30 | 20 | 80 |
| 35 | 20 | 80 |
| 36 | 65 | 35 |
| 42 | 65 | 35 |
Flow rate: 1.0mL/min
Detection wavelength: 260nm
Column temperature: 30 deg.C
Sample introduction volume: 10 μ L
Diluent agent: acetonitrile: water 50:50(v/v)
Operating time: 42 minutes
The experimental steps are as follows:
preparing an impurity positioning solution: precisely weighing about 20mg, 10mg, 20mg and 20mg of the impurities 1,2, 3, 4, 5, 6 and 7 respectively, placing the weighed materials into a 20mL volumetric flask, adding a diluent to dilute the materials to a scale, and shaking the materials uniformly to obtain a solution a. Accurately weighing 100mg of the itraconazole, placing the itraconazole in a 10mL volumetric flask, transferring 0.2mL of the solution a into the volumetric flask, adding a diluent to dilute the solution a to a scale, and shaking up to obtain the itraconazole.
Taking the impurity positioning solution, performing high performance liquid chromatography under the above chromatographic conditions, and recording chromatogram, the result is shown in figure 1.
In fig. 1, the chromatographic peaks with retention time of 4.250min, 6.380min, 7.922min, 11.384min, 17.446min, 18.170min, 18.637min and 19.724min are the chromatographic peaks of impurity 5, impurity 1, impurity 6, impurity 4, impurity 2, impurity 7, impurity 3 and itraconazole, wherein the separation degree of impurity 2 and impurity 7 is 2.09, the separation degree of impurity 7 and impurity 3 is 1.25, and the separation degree of impurity 3 and itraconazole is 2.45.
The result shows that the efinaconazole and related substances in the preparation thereof can be well separated under the same chromatographic condition.
Example 2
The instrument comprises the following steps: agilent 1260 high performance liquid chromatograph, 1260Infinity diode array detector
A chromatographic column: CAPCELL PAK MG IIC 18 (150X 4.6mm,3 μm)
Mobile phase A: 0.01mol/L ammonium formate buffer (pH adjusted to 5.8 with formic acid or ammonia)
Mobile phase B: acetonitrile
Gradient elution is shown in the following table:
| time/min | Formate buffer/%) | First organic solvent/%) |
| 0 | 65 | 35 |
| 3 | 65 | 35 |
| 6 | 45 | 55 |
| 25 | 45 | 55 |
| 30 | 20 | 80 |
| 35 | 20 | 80 |
| 36 | 65 | 35 |
| 42 | 65 | 35 |
Flow rate: 1.0mL/min
Detection wavelength: 260nm
Column temperature: 35 deg.C
Sample introduction volume: 10 μ L
Diluent agent: acetonitrile: water 50:50(v/v)
Operating time: 42 minutes
The experimental steps are as follows:
preparing an impurity positioning solution: precisely weighing about 20mg, 10mg, 20mg and 20mg of the impurities 1,2, 3, 4, 5, 6 and 7 respectively, placing the weighed materials into a 20mL volumetric flask, adding a diluent to dilute the materials to a scale, and shaking the materials uniformly to obtain a solution a. Accurately weighing 100mg of the itraconazole, placing the itraconazole in a 10mL volumetric flask, transferring 0.2mL of the solution a into the volumetric flask, adding a diluent to dilute the solution a to a scale, and shaking up to obtain the itraconazole.
Taking the impurity positioning solution, performing high performance liquid chromatography under the above chromatographic conditions, and recording chromatogram, the result is shown in figure 2.
In fig. 2, the chromatographic peaks with retention times of 4.247min, 6.343min, 7.882min, 11.355min, 17.240min, 17.935min, 18.451min and 19.412min are chromatographic peaks of impurity 5, impurity 1, impurity 6, impurity 4, impurity 2, impurity 7, impurity 3 and itraconazole respectively, the separation degree of impurity 2 and impurity 7 is 2.08, the separation degree of impurity 7 and impurity 3 is 1.45, and the separation degree of impurity 3 and itraconazole is 2.27.
The chromatogram shows that the related substances in the itraconazole and the preparation thereof can be well separated under the same chromatographic condition.
Example 3
The instrument comprises the following steps: agilent 1260 high performance liquid chromatograph, 1260Infinity diode array detector
A chromatographic column: CAPCELL PAK MG IIC 18 (150X 4.6mm,3 μm)
Mobile phase A: 0.05mol/L ammonium formate buffer (pH adjusted to 6.0 with formic acid or ammonia)
Mobile phase B: acetonitrile
Gradient elution is shown in the following table:
| time/min | Formate buffer/%) | First organic solvent/%) |
| 0 | 65 | 35 |
| 3 | 65 | 35 |
| 6 | 45 | 55 |
| 25 | 45 | 55 |
| 30 | 20 | 80 |
| 35 | 20 | 80 |
| 36 | 65 | 35 |
| 42 | 65 | 35 |
Flow rate: 1.0mL/min
Detection wavelength: 260nm
Column temperature: 40 deg.C
Sample introduction volume: 10 μ L
Diluent agent: methanol: water 80:20(v/v)
Operating time: 42 minutes
The experimental steps are as follows:
preparing an impurity positioning solution: precisely weighing about 20mg, 10mg, 20mg and 20mg of the impurities 1,2, 3, 4, 5, 6 and 7 respectively, placing the weighed materials into a 20mL volumetric flask, adding a diluent to dilute the materials to a scale, and shaking the materials uniformly to obtain a solution a. Accurately weighing 100mg of the itraconazole, placing the itraconazole in a 10mL volumetric flask, transferring 0.2mL of the solution a into the volumetric flask, adding a diluent to dilute the solution a to a scale, and shaking up to obtain the itraconazole.
Taking the impurity positioning solution, performing high performance liquid chromatography under the above chromatographic conditions, and recording chromatogram, the result is shown in figure 3.
In fig. 3, the chromatographic peaks with retention times of 4.171min, 6.197min, 7.761min, 11.206min, 16.832min, 17.559min, 18.144min and 18.835min are chromatographic peaks of impurity 5, impurity 1, impurity 6, impurity 4, impurity 2, impurity 7, impurity 3 and itraconazole respectively, the separation degree of impurity 2 from impurity 7 is 2.21, the separation degree of impurity 7 from impurity 3 is 1.67, and the separation degree of impurity 3 from itraconazole is 1.68.
The chromatogram shows that the related substances in the itraconazole and the preparation thereof can be well separated under the same chromatographic condition.
Example 4
The instrument comprises the following steps: agilent 1260 high performance liquid chromatograph, 1260Infinity diode array detector
A chromatographic column: CAPCELL PAK MG IIC 18 (150X 4.6mm,3 μm)
Mobile phase A: 0.01mol/L ammonium formate buffer (pH adjusted to 5.8 with formic acid or ammonia)
Mobile phase B: acetonitrile
Gradient elution is shown in the following table:
| time/min | Formate buffer/%) | First organic solvent/%) |
| 0 | 65 | 35 |
| 3 | 65 | 35 |
| 6 | 45 | 55 |
| 25 | 45 | 55 |
| 30 | 20 | 80 |
| 35 | 20 | 80 |
| 36 | 65 | 35 |
| 42 | 65 | 35 |
Flow rate: 1.0mL/min
Detection wavelength: 260nm
Column temperature: 35 deg.C
Sample introduction volume: 10 μ L
Diluent agent: acetonitrile: water 50:50(v/v)
Operating time: 42 minutes
The experimental steps are as follows:
preparing an impurity positioning solution: precisely weighing about 20mg, 10mg, 20mg and 20mg of the impurities 1,2, 3, 4, 5, 6 and 7 respectively, placing the weighed materials into a 20mL volumetric flask, adding a diluent to dilute the materials to a scale, and shaking the materials uniformly to obtain a solution a. Accurately weighing 100mg of the itraconazole, placing the itraconazole in a 10mL volumetric flask, transferring 0.2mL of the solution a into the volumetric flask, adding a diluent to dilute the solution a to a scale, and shaking up to obtain the itraconazole.
Preparing a test solution: precisely weighing about 100mg of a self-made efinaconazole raw material medicine, placing the self-made efinaconazole raw material medicine into a 10mL volumetric flask, adding a diluent to dilute the raw material medicine to a scale, and shaking up the raw material medicine to obtain the final product.
And (3) taking the impurity positioning solution and the sample solution, respectively carrying out high performance liquid chromatography analysis under the chromatographic conditions, and recording a chromatogram, wherein the result is shown in the attached figures 4-5.
The chromatographic peaks of the impurity positioning solution with retention time of 4.247min, 6.343min, 7.882min, 11.355min, 17.240min, 17.935min, 18.451min and 19.412min are chromatographic peaks of impurity 5, impurity 1, impurity 6, impurity 4, impurity 2, impurity 7, impurity 3 and itraconazole respectively, the separation degree of impurity 2 and impurity 7 is 2.08, the separation degree of impurity 7 and impurity 3 is 1.45, and the separation degree of impurity 3 and itraconazole is 2.27, specifically shown in fig. 4.
The chromatographic peaks of the test solution with retention time of 6.255min, 17.934min and 19.260min are respectively the chromatographic peaks of impurity 1, impurity 7 and itraconazole, the area normalized content thereof is respectively 0.01%, 0.02% and 99.97%, and the other known impurities are not detected, as shown in fig. 5. The single impurity of related substances of the efinaconazole bulk drug is less than 0.1 percent, the total impurity is less than 0.5 percent, and the result shows that the efinaconazole bulk drug meets the quality requirement, and the method can be used for quality detection of the efinaconazole bulk drug.
The chromatogram shows that the related substances of the efinaconazole bulk drug can be well separated under the same chromatographic condition.
Example 5
The instrument comprises the following steps: agilent 1260 high performance liquid chromatograph, 1260Infinity diode array detector
A chromatographic column: CAPCELL PAK MG IIC 18 (150X 4.6mm,3 μm)
Mobile phase A: 0.01mol/L ammonium formate buffer (pH adjusted to 5.8 with formic acid or ammonia)
Mobile phase B: acetonitrile
Gradient elution is shown in the following table:
flow rate: 1.0mL/min
Detection wavelength: 260nm
Column temperature: 35 deg.C
Sample introduction volume: 10 μ L
Diluent agent: acetonitrile: water 50:50(v/v)
Operating time: 42 minutes
The experimental steps are as follows:
preparing an impurity positioning solution: precisely weighing about 20mg, 10mg, 20mg and 20mg of the impurities 1,2, 3, 4, 5, 6 and 7 respectively, placing the weighed materials into a 20mL volumetric flask, adding a diluent to dilute the materials to a scale, and shaking the materials uniformly to obtain a solution a. Accurately weighing 100mg of the itraconazole, placing the itraconazole in a 10mL volumetric flask, transferring 0.2mL of the solution a into the volumetric flask, adding a diluent to dilute the solution a to a scale, and shaking up to obtain the itraconazole.
Blank adjuvant solution: precisely weighing about 0.9g (equivalent to 100mg of the efinaconazole-containing adjuvant) of the efinaconazole (anhydrous ethanol: water: diisopropyl adipate with a weight ratio of 38: 2: 5) into a 10ml measuring flask, dissolving with acetonitrile, fixing the volume to the scale, shaking up, and filtering.
Test solution: precisely weighing an efinaconazole preparation (the weight ratio of the efinaconazole raw material drug to absolute ethyl alcohol to water to diisopropyl adipate is 5: 38: 2: 5) about 1g to 10ml in a measuring flask, dissolving the mixture by using acetonitrile, fixing the volume to a scale, shaking up and filtering.
And (3) respectively carrying out high performance liquid chromatography analysis on the impurity positioning solution, the blank auxiliary material solution and the sample solution under the chromatographic conditions, and recording a chromatogram, wherein the result is shown in the attached figures 6-8.
The chromatographic peaks of the impurity positioning solution with retention time of 4.247min, 6.343min, 7.882min, 11.355min, 17.240min, 17.935min, 18.451min and 19.412min are chromatographic peaks of impurity 5, impurity 1, impurity 6, impurity 4, impurity 2, impurity 7, impurity 3 and itraconazole respectively, the separation degree of impurity 2 and impurity 7 is 2.08, the separation degree of impurity 7 and impurity 3 is 1.45, and the separation degree of impurity 3 and itraconazole is 2.27, specifically shown in fig. 6.
A peak of the adjuvant with a retention time of 35.199min was found in the blank adjuvant solution chromatogram, in particular in FIG. 7.
The chromatographic peaks of the sample solution with retention time of 6.282min, 17.933min and 19.248min are chromatographic peaks of impurity 1, impurity 7 and itraconazole, the area normalized content of which is 0.01%, 0.02% and 99.96%, respectively, an unknown impurity peak with retention time of 10.131min is provided, the area normalized content of which is 0.01%, and the rest known impurities are not detected, which is shown in fig. 8 specifically. The related substances of the efinaconazole preparation are less than 0.1 percent of single impurity and less than 0.5 percent of total impurity, the result shows that the efinaconazole preparation meets the quality requirement, and the method can be used for the quality detection of the efinaconazole preparation.
The chromatogram shows that the related substances of the efinaconazole preparation can be well separated under the same chromatographic condition.
Comparative example 1
The instrument comprises the following steps: agilent 1260 high performance liquid chromatograph, 1260Infinity diode array detector
A chromatographic column: CAPCELL PAK MG IIC 18 (150X 4.6mm,3 μm)
Mobile phase A: 0.01mol/L ammonium formate buffer (pH adjusted to 5.8 with formic acid or ammonia)
Mobile phase B: acetonitrile
Gradient elution is shown in the following table:
| time/min | Formate buffer/%) | First organic solvent/%) |
| 0 | 65 | 35 |
| 3 | 65 | 35 |
| 8 | 42 | 58 |
| 25 | 42 | 58 |
| 30 | 20 | 80 |
| 35 | 20 | 80 |
| 36 | 65 | 35 |
| 42 | 65 | 35 |
Flow rate: 1.0mL/min
Detection wavelength: 260nm
Column temperature: 35 deg.C
Sample introduction volume: 10 μ L
Diluent agent: acetonitrile: water 50:50(v/v)
Operating time: 42 minutes
The experimental steps are as follows:
preparing an impurity positioning solution: precisely weighing about 20mg, 10mg, 20mg and 20mg of the impurities 1,2, 3, 4, 5, 6 and 7 respectively, placing the weighed materials into a 20mL volumetric flask, adding a diluent to dilute the materials to a scale, and shaking the materials uniformly to obtain a solution a. Accurately weighing 100mg of the itraconazole, placing the itraconazole in a 10mL volumetric flask, transferring 0.2mL of the solution a into the volumetric flask, adding a diluent to dilute the solution a to a scale, and shaking up to obtain the itraconazole.
Preparing an impurity 3 positioning solution: precisely weighing about 20mg of impurity 3, placing the impurity in a 20mL volumetric flask, adding a diluent to dilute the impurity to a scale, and shaking up. And putting another 1.0mL of the solution into a 100mL measuring flask, adding a diluent to dilute the solution to a scale, and shaking up the solution to obtain the composition.
Taking the impurity positioning solution and the impurity 3 positioning solution, respectively performing high performance liquid chromatography under the above chromatographic conditions, and recording chromatogram, the results are shown in figures 9 and 10.
Impurity 3 the peak of the chromatogram with retention time of 16.526min in the solution chromatogram is located as impurity 3, see in particular fig. 9.
The chromatographic peaks of the impurity positioning solution with retention time of 4.002min, 6.078min, 8.048min, 11.045min, 15.084min, 15.997min and 16.520min are chromatographic peaks of impurity 5, impurity 1, impurity 6, impurity 4, impurity 2, impurity 7 and itraconazole (containing impurity 3), respectively, the separation degree of impurity 2 and impurity 7 is 3.85, the separation degree of impurity 7 and itraconazole is 1.82, and impurity 3 and itraconazole are not separated, specifically shown in fig. 10.
The chromatogram shows that when the gradient elution is changed to 8 min-25 min based on example 2, the ratio of formate buffer to acetonitrile is kept to be 42 percent to 58 percent, and the related substances in the itraconazole and the preparation thereof can not be well separated under the same chromatographic condition.
Comparative example 2
The instrument comprises the following steps: agilent 1260 high performance liquid chromatograph, 1260Infinity diode array detector
A chromatographic column: CAPCELL PAK MG IIC 18 (150X 4.6mm,3 μm)
Mobile phase A: 0.01mol/L ammonium acetate buffer (pH adjusted to 5.8 with acetic acid or ammonia)
Mobile phase B: acetonitrile
Gradient elution is shown in the following table:
flow rate: 1.0mL/min
Detection wavelength: 260nm
Column temperature: 35 deg.C
Sample introduction volume: 10 μ L
Diluent agent: acetonitrile: water 50:50(v/v)
Operating time: 42 minutes
The experimental steps are as follows:
preparing an impurity positioning solution: precisely weighing about 20mg, 10mg, 20mg and 20mg of impurities 1,2, 3, 4, 5, 6 and 7 respectively, placing in a 20mL volumetric flask, adding a diluent to dilute to a scale, and shaking uniformly to obtain a solution a. Accurately weighing 100mg of the itraconazole, placing the itraconazole in a 10mL volumetric flask, transferring 0.2mL of the solution a into the volumetric flask, adding a diluent to dilute the solution a to a scale, and shaking up to obtain the itraconazole.
Preparing an impurity 3 positioning solution: precisely weighing about 20mg of impurity 3, placing the impurity in a 20mL volumetric flask, adding a diluent to dilute the impurity to a scale, and shaking up. And putting another 1.0mL of the solution into a 100mL measuring flask, adding a diluent to dilute the solution to a scale, and shaking up the solution to obtain the composition.
Taking the impurity locating solution and the impurity 3 locating solution, respectively performing high performance liquid chromatography under the above chromatographic conditions, and recording chromatogram, the results are shown in figures 11 and 12.
Impurity 3 the peak of the chromatogram with retention time of 19.474min in the solution map was located as impurity 3, see in particular fig. 11.
The chromatographic peaks with retention time of 4.206min, 6.282min, 7.842min, 11.362min, 17.408min, 18.739min and 19.479min in the impurity positioning solution chromatogram are chromatographic peaks of impurity 5, impurity 1, impurity 6, impurity 4, impurity 2, impurity 7 and itraconazole (containing impurity 3), the separation degree of impurity 2 and impurity 7 is 3.92, the separation degree of impurity 7 and itraconazole is 1.71, and impurity 3 and itraconazole are not separated, specifically shown in fig. 12.
The chromatogram shows that when the type of the buffer salt is changed into ammonium acetate on the basis of example 2, the efinaconazole and related substances in the preparation thereof cannot be well separated under the same chromatographic condition.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (5)
1. A method for measuring related substances in an efinaconazole raw material medicament and a preparation thereof by utilizing HPLC is characterized in that the chromatographic conditions are as follows:
the chromatographic column is an octadecylsilane chemically bonded silica gel column; the mobile phase is a mixed solvent of formate buffer solution and a first organic solvent;
the conditions for gradient elution were:
The particle size of the chromatographic column is 3-5 μm,
the formate buffer solution is ammonium formate buffer solution, the pH value of the formate buffer solution is adjusted to 5.6-6.0 by adopting formic acid or ammonia water, the concentration of the formate buffer solution is 0.005 mol/L-0.05 mol/L,
the first organic solvent is acetonitrile;
the method further comprises:
preparing a sample to be detected into a sample solution by adopting a mixed solution of a second organic solvent and water;
injecting the sample solution into a high performance liquid chromatograph to complete the determination of related substances in the efinaconazole raw material medicine and the preparation thereof;
the second organic solvent is methanol and/or acetonitrile;
the volume ratio of water to the second organic solvent in the mixed solution of the second organic solvent and water is (0% to 100%) -60% to 40%;
the volume of the sample solution injected into the high performance liquid chromatograph is 2-20 mu L;
the detection wavelength of the high performance liquid chromatograph is 254 nm-262 nm;
the flow rate of the mobile phase is 0.8mL/min to 1.2 mL/min;
the column temperature of the chromatographic column is 25-40 ℃;
wherein, the related substances in the raw material drug of the itraconazole and the preparation thereof comprise:
impurity 1 is the raw material: 1- (((2R,3S) -2- (2, 4-difluorophenyl) -3-methyloxiranyl-2-yl) methyl) -1H-1,2, 4-triazole;
impurity 2 is a side reaction product, a diastereoisomeric impurity: (2S,3R) -2- (2, 4-difluorophenyl) -3- (4-methylenepiperidin-1-yl) -1- (1H-1,2, 4-triazol-1-yl) -2-butanol;
impurity 3 is a side reaction product: e- (3R) -2- (2, 4-difluorophenyl) -3- (4-methylenepiperidin-1-yl) -1- (1H-1,2, 4-triazol-1-yl) -1-butene;
impurity 4 is a side reaction product: (2R,3R) -2- (2, 4-difluorophenyl) -3- (4-methylenepiperidin-1-yl) -1- (4H-1,2, 4-triazol-4-yl) -2-butanol;
impurity 5 is a side reaction product: (R, E) -3- (2, 4-difluorophenyl) -4- (1H-1,2, 4-triazol-1-yl)) -2-hydroxy-3-butene;
impurity 6 is a side reaction product: 1- ((6-fluoro-2-methylbenzofuran-3-yl) methyl) -1H-1,2, 4-triazole;
impurity 7 is a side reaction product, a degradation product: (2R,3R) -2- (2, 4-difluorophenyl) -3 (4-methyl-3, 6-dihydropyridin-1 (2H) -yl) -1- (1H-1,2, 4-triazol-1-yl) -2-butanol.
2. The method of claim 1, wherein the chromatographic column has a particle size of 3 μm.
3. The method according to claim 1, wherein the formate buffer is adjusted to a pH of 5.8 with formic acid or ammonia.
4. The method of claim 1, wherein the second organic solvent is acetonitrile;
the volume ratio of water to the second organic solvent in the mixed solution of the second organic solvent and water is 50% to 50%;
the volume of the sample solution injected into the high performance liquid chromatograph is 10 mu L;
the detection wavelength of the high performance liquid chromatograph is 260 nm.
5. The method of claim 1, wherein the flow rate of the mobile phase is 1.0 mL/min;
the column temperature of the chromatographic column was 35 ℃.
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