CN114397375A - Method for detecting related substances of arbidol hydrochloride intermediate - Google Patents
Method for detecting related substances of arbidol hydrochloride intermediate Download PDFInfo
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- CN114397375A CN114397375A CN202111502474.2A CN202111502474A CN114397375A CN 114397375 A CN114397375 A CN 114397375A CN 202111502474 A CN202111502474 A CN 202111502474A CN 114397375 A CN114397375 A CN 114397375A
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Abstract
The invention relates to the technical field of pharmaceutical analysis, and particularly discloses a method for detecting substances related to an arbidol hydrochloride intermediate. The detection method comprises the following steps: preparing a test solution and a reference solution; detecting the test solution and the reference solution by adopting liquid chromatography, wherein the chromatographic conditions of the liquid chromatography are as follows: performing gradient elution by using a C18 chromatographic column and acetonitrile-water as a mobile phase A in a volume ratio of 63-67: 33-37 and acetonitrile-methanol-water as a mobile phase B in a volume ratio of 38-42: 18-22. The detection method provided by the invention realizes quantitative and qualitative analysis of impurities INT1-B, INT1-C, INT1-D, INT1-E and INT1-F in the Sr-5 bulk drug, has strong specificity and high sensitivity, and meets the detection requirement of the Sr-5 bulk drug.
Description
Technical Field
The invention relates to the technical field of pharmaceutical analysis, in particular to a method for detecting substances related to an arbidol hydrochloride intermediate.
Background
Abidol Hydrochloride (Arbidol Hydrochloride) is a non-nucleoside broad-spectrum antiviral drug with immune enhancement effect, has a chemical name of 1-methyl-2-thiophenylmethyl-4-dimethylaminomethyl-5-hydroxy-6-bromo-1H-indole-3-ethyl formate Hydrochloride, and is used for treating upper respiratory tract infection caused by influenza A virus and influenza B virus. Arbidol is a non-nucleoside antiviral drug developed by the soviet union chemical research center, first marketed in russia in 1993, where it has been used for many years to treat influenza. Currently, the therapeutic effect of arbidol is recognized and favored by international experts and is marketed in some countries in japan and europe, with a permission to market in china in 2006.
The Sr-5 chemical name is 6-bromo-5-hydroxy-1-methyl-2-phenylthiomethyl indole-3-carboxylic acid ethyl ester, which is an important intermediate for synthesizing the arbidol hydrochloride, and is mainly synthesized by taking ethyl acetate and methylamine aqueous solution as raw materials through condensation methylaminoylation, Nenissiki cyclization, acylation, bromination and phenylthionation reactions. Due to the long synthesis time and the possibility of introducing byproducts and incompletely reacted raw materials into the product, Sr-5 is used as an important intermediate for synthesizing the arbidol hydrochloride, and the quality control of the Sr-5 has great influence on the synthesis of the arbidol hydrochloride.
Both the by-products introduced during the synthesis of Sr-5 and the unreacted raw materials may become impurities during the synthesis of arbidol hydrochloride, and no relevant literature reports a detection method for the impurities at present, so that a detection method capable of effectively controlling the quality of Sr-5 is urgently needed to be developed, so as to ensure the effective control of the quality of arbidol hydrochloride and improve the industrial standard.
Disclosure of Invention
In view of the above, the method for detecting substances related to the arbidol hydrochloride intermediate provided by the invention has excellent sensitivity, precision and linear relationship, and can meet the detection requirements of INT1-B, INT1-C, INT1-D, INT1-E and INT1-F in Sr-5 bulk drugs, wherein the structures and names of INT1-B, INT1-C, INT1-D, INT1-E and INT1-F are shown in the following table.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
the invention provides a detection method of related substances of an arbidol hydrochloride intermediate, wherein the arbidol hydrochloride intermediate is 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester (Sr-5), and the detection method comprises the following steps:
step one, preparing a test solution and a reference solution;
preparing a reference substance solution: taking 5-acetoxyl-6-bromo-2-bromomethyl-1-methylindole-3-carboxylic acid ethyl ester, 1, 2-dimethyl-6-bromo-5-hydroxyindole-3-carboxylic acid ethyl ester, 6, 7-dibromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester, 6-bromo-5-acetoxyl-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester, 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester and 5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester as reference substances, preparing a reference substance solution by using a solvent;
preparing a test solution: preparing a sample of 6-bromo-5-hydroxy-1-methyl-2-phenylthiomethyl indole-3-carboxylic acid ethyl ester into a test solution by using a solvent;
step two, detecting the test solution and the reference solution by adopting liquid chromatography, wherein the chromatographic conditions of the liquid chromatography are as follows:
performing gradient elution by using a C18 chromatographic column and acetonitrile-water as a mobile phase A in a volume ratio of 63-67: 33-37 and acetonitrile-methanol-water as a mobile phase B in a volume ratio of 38-42: 18-22, wherein the detection wavelength is 249-259 nm.
Compared with the prior art, the detection method for related substances of the arbidol hydrochloride intermediate has the following advantages:
the method for detecting substances related to the arbidol hydrochloride intermediate realizes quantitative and qualitative analysis of impurities INT1-B, INT1-C, INT1-D, INT1-E and INT1-F in 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester (Sr-5), has strong specificity and high sensitivity, meets the detection requirements of INT1-B, INT1-C, INT1-D, INT1-E and INT1-F in Sr-5 bulk drugs, and has good linear relation, high repeatability and correlation coefficient in the linear range of a standard curve larger than 0.99; in addition, the detection method has the advantages of fast analysis time, capability of quickly and accurately detecting impurities INT1-B, INT1-C, INT1-D, INT1-E and INT1-F in the Sr-5 bulk drug, reliable and controllable whole operation process, suitability for practical application and popularization and wide application prospect.
Alternatively, the procedure for the gradient elution is as follows:
0min → 14min, 100% mobile phase a, 0% mobile phase B;
14min → 14.1min, 0% mobile phase a, 100% mobile phase B;
14.1min → 25min, 0% mobile phase a, 100% mobile phase B;
25min → 25.1min, 100% mobile phase a, 0% mobile phase B;
25.1min → 35min, 100% mobile phase A, 0% mobile phase B.
The gradient elution has a remarkable influence on the separation effect and the peak shape of a detection target, so the preferable gradient elution sequence in the application can separate INT1-B, INT1-C, INT1-D, INT1-E, INT1-F from 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester (Sr-5) or other interferents, and the separation effect is excellent.
Optionally, the mobile phase a is acetonitrile-water with a volume ratio of 65:35, and the mobile phase B is acetonitrile-methanol-water with a volume ratio of 40:40: 20.
Further optionally, the detection wavelength is 254 nm.
Optionally, the flow rate is 0.9 ml/min-1.1 ml/min, and the column temperature is 25-35 ℃.
Further alternatively, the flow rate was 1ml/min and the column temperature was 30 ℃.
The optimal flow rate and the optimal column temperature can ensure that the impurity INT1-B, INT1-C, INT1-D, INT1-E, INT1-F can generate peaks in a short time, reduce the analysis time and have excellent separation effect on chromatographic peaks.
Optionally, the chromatography column has a specification of 4.6mm x 250mm x 5 μm.
Optionally, the chromatography column is model number CAPCELL PAK C18.
Different chromatographic columns have larger difference on the retention performance of the compound, so that CAPCELL PAK C18 columns with the specification of 4.6mm multiplied by 250mm multiplied by 5 mu mm are adopted in the method, the target can be quickly and effectively separated, and the peak shape is better.
Optionally, the concentration of the test solution is 0.28mg/ml to 0.32 mg/ml.
Further optionally, the concentration of the test solution is 0.3 mg/ml.
Optionally, the concentrations of ethyl 5-acetoxy-6-bromo-2-bromomethyl-1-methylindole-3-carboxylate, ethyl 1, 2-dimethyl-6-bromo-5-hydroxyindole-3-carboxylate, ethyl 6, 7-dibromo-5-hydroxy-1-methyl-2-phenylthiomethyl indole-3-carboxylate, ethyl 6-bromo-5-acetoxy-1-methyl-2-phenylthiomethyl indole-3-carboxylate and ethyl 5-hydroxy-1-methyl-2-phenylthiomethyl indole-3-carboxylate in the control solution are all 3 μ g/ml.
Optionally, the solvent is acetonitrile.
Acetonitrile is preferably used as a solvent, so that substances except INT1-B, INT1-C, INT1-D, INT1-E and INT1-F can be extracted as little as possible, INT1-B, INT1-C, INT1-D, INT1-E and INT1-F in Sr-5 bulk drugs can be extracted as much as possible, detection of liquid chromatography is basically free of interference, and the detection result of the liquid chromatography provided by the invention is more accurate.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a high performance liquid chromatogram of a control solution provided in example 1 of the present invention;
FIG. 2 is a high performance liquid chromatogram of a test solution provided in example 1 of the present invention;
FIG. 3 is a high performance liquid chromatogram of a blank solution provided in example 1 of the present invention;
FIG. 4 is a linear relationship diagram of INT1 provided by the present invention;
FIG. 5 is a linear relationship diagram of INT1-B provided by the present invention;
FIG. 6 is a linear relationship diagram of INT1-C provided by the present invention;
FIG. 7 is a linear relationship diagram of INT1-D provided by the present invention;
FIG. 8 is a linear relationship diagram of INT1-E provided by the present invention;
FIG. 9 is a linear relationship diagram of INT1-F provided by the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
The embodiment of the invention provides a method for detecting related substances of an arbidol hydrochloride intermediate, which comprises the following steps:
preparing a test solution, a reference solution and a blank solution;
preparing a reference substance solution: taking 5-acetoxyl-6-bromo-2-bromomethyl-1-methylindole-3-carboxylic acid ethyl ester, 1, 2-dimethyl-6-bromo-5-hydroxyindole-3-carboxylic acid ethyl ester, 6, 7-dibromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester, 6-bromo-5-acetoxyl-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester, 5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester and 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester as reference substances, preparing a reference substance solution with the concentration of each component being 3 mug/ml by using acetonitrile;
preparing a test solution: taking a 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester sample, and preparing a test solution with the concentration of 0.3mg/ml by using acetonitrile;
the blank solution is acetonitrile solvent;
and step two, detecting the reference solution, the test solution and the blank solution by adopting liquid chromatography, and recording spectrograms, wherein the chromatograms are respectively shown in figure 1, figure 2 and figure 3.
The chromatographic conditions of the liquid chromatogram are as follows:
adopting CAPCELL PAK C18 chromatographic column with specification of 4.6mm × 250mm × 5 μm, acetonitrile-water with volume ratio of 65:35 as mobile phase A, acetonitrile-methanol-water with volume ratio of 40:40:20 as mobile phase B, gradient elution with flow rate of 1ml/min, column temperature of 30 ℃, detection wavelength of 254nm, and sample injection volume of 10 μ L, wherein the procedure of gradient elution is as follows:
0min → 14min, 100% mobile phase a, 0% mobile phase B;
14min → 14.1min, 0% mobile phase a, 100% mobile phase B;
14.1min → 25min, 0% mobile phase a, 100% mobile phase B;
25min → 25.1min, 100% mobile phase a, 0% mobile phase B;
25.1min → 35min, 100% mobile phase A, 0% mobile phase B.
As can be seen from FIG. 1, the chromatographic peak separation degree of ethyl 6-bromo-5-hydroxy-1-methyl-2-phenylthiomethyl indole-3-carboxylate (main component INT1) from other impurities INT1-B, INT1-C, INT1-D, INT1-E, INT1-F is good, wherein a chromatographic peak of the impurity INT1-B appears at a retention time of 12.941min, a chromatographic peak of the impurity INT1-C appears at a retention time of 5.562min, a chromatographic peak of impurity INT1-D appeared at retention time 19.733min, a chromatographic peak of impurity INT1-E appeared at retention time 19.171, a chromatographic peak of impurity INT1-F appeared at retention time 7.374min, a chromatographic peak of ethyl 6-bromo-5-hydroxy-1-methyl-2-phenylthiomethyl indole-3-carboxylate appeared at a retention time of 11.75 min.
As can be seen from FIG. 2, the chromatographic peak separation was good for INT1-C, INT1-D, INT1-E and INT1 (ethyl 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylate), and neither INT1-B nor INT1-F was detected.
As can be seen from figures 1-3, the acetonitrile solvent and the Sr-5 bulk drug have no interference to the detection of the impurity INT1-B, INT1-C, INT1-D, INT1-E, INT1-FD, which indicates that the liquid chromatography provided by the invention has good specificity.
Example 2
The embodiment of the invention provides a method for detecting related substances of an arbidol hydrochloride intermediate, which comprises the following steps:
step one, preparing a test solution and a reference solution;
preparing a reference substance solution: taking 5-acetoxyl-6-bromo-2-bromomethyl-1-methylindole-3-carboxylic acid ethyl ester, 1, 2-dimethyl-6-bromo-5-hydroxyindole-3-carboxylic acid ethyl ester, 6, 7-dibromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester, 6-bromo-5-acetoxyl-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester, 5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester and 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester as reference substances, preparing a reference substance solution with the concentration of each component being 3 mug/ml by using acetonitrile;
preparing a test solution: taking a 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester sample, and preparing a test solution with the concentration of 0.4mg/ml by using acetonitrile;
step two, detecting the reference substance solution and the test substance solution by adopting liquid chromatography, wherein the chromatographic conditions of the liquid chromatography are as follows:
adopting CAPCELL PAK C18 chromatographic column with specification of 4.6mm × 250mm × 5 μm, acetonitrile-water with volume ratio of 63:37 as mobile phase A, acetonitrile-methanol-water with volume ratio of 38:42:20 as mobile phase B, gradient elution with flow rate of 1ml/min, column temperature of 30 ℃, detection wavelength of 254nm, and sample injection volume of 10 μ L, wherein the procedure of gradient elution is as follows:
0min → 14min, 100% mobile phase a, 0% mobile phase B;
14min → 14.1min, 0% mobile phase a, 100% mobile phase B;
14.1min → 25min, 0% mobile phase a, 100% mobile phase B;
25min → 25.1min, 100% mobile phase a, 0% mobile phase B;
25.1min → 35min, 100% mobile phase A, 0% mobile phase B.
Example 3
The embodiment of the invention provides a method for detecting related substances of an arbidol hydrochloride intermediate, which comprises the following steps:
step one, preparing a test solution and a reference solution;
preparing a reference substance solution: taking 5-acetoxyl-6-bromo-2-bromomethyl-1-methylindole-3-carboxylic acid ethyl ester, 1, 2-dimethyl-6-bromo-5-hydroxyindole-3-carboxylic acid ethyl ester, 6, 7-dibromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester, 6-bromo-5-acetoxyl-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester, 5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester and 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester as reference substances, preparing a reference substance solution with the concentration of each component being 3 mug/ml by using acetonitrile;
preparing a test solution: taking a 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester sample, and preparing a test solution with the concentration of 0.2mg/ml by using acetonitrile;
step two, detecting the reference substance solution and the test substance solution by adopting liquid chromatography, wherein the chromatographic conditions of the liquid chromatography are as follows:
adopting CAPCELL PAK C18 chromatographic column with specification of 4.6mm × 250mm × 5 μm, acetonitrile-water with volume ratio of 67:33 as mobile phase A, acetonitrile-methanol-water with volume ratio of 42:40:18 as mobile phase B, gradient elution with flow rate of 1ml/min, column temperature of 30 ℃, detection wavelength of 254nm, and sample injection volume of 10 μ L, wherein the procedure of gradient elution is as follows:
0min → 14min, 100% mobile phase a, 0% mobile phase B;
14min → 14.1min, 0% mobile phase a, 100% mobile phase B;
14.1min → 25min, 0% mobile phase a, 100% mobile phase B;
25min → 25.1min, 100% mobile phase a, 0% mobile phase B;
25.1min → 35min, 100% mobile phase A, 0% mobile phase B.
Example 4
The embodiment of the invention provides a method for detecting related substances of an arbidol hydrochloride intermediate, which comprises the following steps:
step one, preparing a test solution and a reference solution;
preparing a reference substance solution: taking 5-acetoxyl-6-bromo-2-bromomethyl-1-methylindole-3-carboxylic acid ethyl ester, 1, 2-dimethyl-6-bromo-5-hydroxyindole-3-carboxylic acid ethyl ester, 6, 7-dibromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester, 6-bromo-5-acetoxyl-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester, 5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester and 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester as reference substances, preparing a reference substance solution with the concentration of each component being 3 mug/ml by using acetonitrile;
preparing a test solution: taking a 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester sample, and preparing a test solution with the concentration of 0.3mg/ml by using acetonitrile;
step two, detecting the reference substance solution and the test substance solution by adopting liquid chromatography, wherein the chromatographic conditions of the liquid chromatography are as follows:
adopting CAPCELL PAK C18 chromatographic column with specification of 4.6mm × 250mm × 5 μm, acetonitrile-water with volume ratio of 65:35 as mobile phase A, acetonitrile-methanol-water with volume ratio of 40:40:20 as mobile phase B, gradient elution with flow rate of 1.1ml/min, column temperature of 35 deg.C, detection wavelength of 259nm, and sample injection volume of 10 μ L, wherein the procedure of gradient elution is as follows:
0min → 14min, 100% mobile phase a, 0% mobile phase B;
14min → 14.1min, 0% mobile phase a, 100% mobile phase B;
14.1min → 25min, 0% mobile phase a, 100% mobile phase B;
25min → 25.1min, 100% mobile phase a, 0% mobile phase B;
25.1min → 35min, 100% mobile phase A, 0% mobile phase B.
Example 5
The embodiment of the invention provides a method for detecting related substances of an arbidol hydrochloride intermediate, which comprises the following steps:
step one, preparing a test solution and a reference solution;
preparing a reference substance solution: taking 5-acetoxyl-6-bromo-2-bromomethyl-1-methylindole-3-carboxylic acid ethyl ester, 1, 2-dimethyl-6-bromo-5-hydroxyindole-3-carboxylic acid ethyl ester, 6, 7-dibromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester, 6-bromo-5-acetoxyl-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester, 5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester and 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester as reference substances, preparing a reference substance solution with the concentration of each component being 3 mug/ml by using acetonitrile;
preparing a test solution: taking a 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester sample, and preparing a test solution with the concentration of 0.3mg/ml by using acetonitrile;
the blank solution is acetonitrile solvent;
step two, detecting the reference substance solution and the test substance solution by adopting a liquid chromatogram, wherein the chromatographic conditions of the liquid chromatogram are as follows:
adopting CAPCELL PAK C18 chromatographic column with specification of 4.6mm × 250mm × 5 μm, acetonitrile-water with volume ratio of 65:35 as mobile phase A, acetonitrile-methanol-water with volume ratio of 40:40:20 as mobile phase B, gradient elution with flow rate of 0.9ml/min, column temperature of 25 deg.C, detection wavelength of 249nm, and sample injection volume of 10 μ L, wherein the procedure of gradient elution is as follows:
0min → 14min, 100% mobile phase a, 0% mobile phase B;
14min → 14.1min, 0% mobile phase a, 100% mobile phase B;
14.1min → 25min, 0% mobile phase a, 100% mobile phase B;
25min → 25.1min, 100% mobile phase a, 0% mobile phase B;
25.1min → 35min, 100% mobile phase A, 0% mobile phase B.
Examples 2 to 5 each achieved good separation of ethyl 6-bromo-5-hydroxy-1-methyl-2-phenylthiomethyl indole-3-carboxylate (main component INT1) from other impurities INT1-B, INT1-C, INT1-D, INT1-E, INT1-F, and good resolution of chromatographic peaks.
Example 6 detection and quantitation limits
Detection limit: the control solution with the concentration of 3 mug/ml of each component prepared in example 1 is diluted quantitatively by acetonitrile step by step, and then detection is performed by liquid chromatography, the specific conditions of the liquid chromatography are as described in example 1, a spectrogram is recorded, the detection limit is obtained according to the signal-to-noise ratio of not less than 3:1, and the result is shown in table 1.
And (4) quantitative limit: the control solution prepared in example 1 with the concentration of each component of 3 mug/ml is diluted quantitatively by acetonitrile step by step, and then detected by liquid chromatography, wherein the specific conditions of the liquid chromatography are as described in example 1, a spectrogram is recorded, the signal-to-noise ratio is not less than 10:1 as a quantitative limit, and the signal-to-noise ratio is not less than 10:1 as a detection limit, and the results are shown in table 1.
TABLE 1 detection Limit and quantitation Limit test results
Example 7 repeatability test
Taking the same batch of samples, preparing 6 parts of test solution according to the test preparation method of the detection method of the related substances of the arbidol hydrochloride intermediate in the example 1, and detecting by adopting liquid chromatography, wherein the specific conditions of the liquid chromatography are as described in the example 1, and the results are shown in table 2. As can be seen from Table 2, the RSD% of the results of 6 times of INT1-C, INT1-D, INT1-E and INT1 (ethyl 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylate) is less than 5%, and both INT1-B and INT1-F are not detected, which indicates that the detection method provided by the application has good repeatability.
TABLE 2 results of the repeatability tests
Example 8 intermediate precision test
The test sample solution prepared in example 1 was taken and tested by liquid chromatography, the specific conditions of which were as described in example 1, and different testers were used to sample 6 times at different times, 10 μ L each time, and the test results are shown in table 3. As can be seen from Table 3, the RSD% of 12 times of results of INT1-C, INT1-D, INT1-E and INT1 (ethyl 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylate) is less than 5%, and both INT1-B and INT1-F are not detected, which indicates that the intermediate precision of the detection method provided by the application is good.
TABLE 3 results of intermediate precision test
Example 9 stability
The test solution prepared in example 1 was allowed to stand for 0h, 2h, 4h, 6h, and 8h, respectively, and then detected by liquid chromatography, wherein the specific conditions of the liquid chromatography are as described in example 1, 10. mu.l of the sample was injected each time, a spectrum was recorded, the content thereof was examined, and the RSD (%) thereof was calculated, and the test results are shown in Table 4 below. As can be seen from Table 4, the RSD% of each of INT1-C, INT1-D, INT1-E and INT1 (ethyl 6-bromo-5-hydroxy-1-methyl-2-phenylthiomethyl indole-3-carboxylate) was less than 5% and neither of INT1-B and INT1-F was detected when the test solutions were left at room temperature for 8 hours, indicating that the stability of the above test solutions was good.
TABLE 4 stability test results
Example 10 Linear relationship
Taking a proper amount of 6-bromo-5-hydroxy-1-methyl-2-phenylthiomethyl indole-3-carboxylic acid ethyl ester, dissolving with acetonitrile, quantitatively diluting to prepare a series of concentration solutions, detecting by liquid chromatography, wherein the conditions of the liquid chromatography are specifically shown in example 1, recording a spectrogram, drawing a standard curve by taking the concentration (mu g/ml) as a horizontal coordinate and taking a peak area as a vertical coordinate, and calculating a regression equation, and the result is shown in table 5 and fig. 4. From the results, it can be seen that INT1 (ethyl 6-bromo-5-hydroxy-1-methyl-2-phenylthiomethyl indole-3-carboxylate) has a good linear relationship in the concentration range of 0.1259. mu.g/ml to 6.0446. mu.g/ml.
TABLE 5INT1 Linear test results
Dissolving 5-acetoxyl-6-bromo-2-bromomethyl-1-methylindole-3-carboxylic acid ethyl ester (INT1-B) with acetonitrile, quantitatively diluting to prepare solutions with a series of concentrations, detecting by liquid chromatography, wherein the liquid chromatography conditions are specifically shown in example 1, recording a spectrogram, drawing a standard curve by taking the concentration (mu g/ml) as a horizontal coordinate and taking a peak area as a vertical coordinate, and calculating a regression equation, wherein the results are shown in Table 6 and figure 5. From the results, it was found that INT1-B was linearly related in the concentration range of 0.09623. mu.g/ml to 6.3720. mu.g/ml.
TABLE 6 INT1-B Linear test results
Dissolving 1, 2-dimethyl-6-bromo-5-hydroxyindole-3-carboxylic acid ethyl ester (INT1-C) in acetonitrile, quantitatively diluting to prepare solutions with a series of concentrations, detecting by liquid chromatography, wherein the conditions of the liquid chromatography are specifically shown in example 1, recording a spectrogram, drawing a standard curve by taking the concentration (mu g/ml) as a horizontal coordinate and taking a peak area as a vertical coordinate, and calculating a regression equation, wherein the results are shown in Table 7 and FIG. 6. From the results, it can be seen that INT1-C was linearly related well in the concentration range of 0.04153. mu.g/ml to 6.2291. mu.g/ml.
TABLE 7 INT1-C Linear test results
Dissolving 6, 7-dibromo-5-hydroxy-1-methyl-2-phenylthiomethyl indole-3-carboxylic acid ethyl ester (INT1-D) with acetonitrile, quantitatively diluting to prepare solutions with a series of concentrations, detecting by liquid chromatography, wherein the liquid chromatography conditions are specifically shown in example 1, recording a spectrogram, drawing a standard curve by taking the concentration (mu g/ml) as a horizontal coordinate and taking a peak area as a vertical coordinate, and calculating a regression equation, wherein the results are shown in Table 8 and figure 7. From the results, it was found that INT1-D was linearly related in the concentration range of 0.18528. mu.g/ml to 5.8508. mu.g/ml.
TABLE 8 INT1-D Linear test results
6-bromo-5-acetoxy-1-methyl-2-phenylthiomethyl indole-3-carboxylic acid ethyl ester (INT1-E) is dissolved in acetonitrile and quantitatively diluted to prepare solutions with a series of concentrations, liquid chromatography is adopted for detection, the conditions of the liquid chromatography are specifically shown in example 1, a spectrogram is recorded, a standard curve is drawn by taking the concentration (mu g/ml) as a horizontal coordinate and a peak area as a vertical coordinate, a regression equation is calculated, and the results are shown in Table 9 and figure 8. From the results, it can be seen that INT1-E was linearly related well in the concentration range of 0.12274. mu.g/ml to 5.8913. mu.g/ml.
TABLE 9 INT1-E Linear test results
Dissolving 5-hydroxy-1-methyl-2-phenylthiomethyl indole-3-carboxylic acid ethyl ester (INT1-F) in acetonitrile, quantitatively diluting to obtain a series of concentration solutions, detecting by liquid chromatography, wherein the liquid chromatography conditions are specifically shown in example 1, recording a spectrogram, drawing a standard curve by taking the concentration (mu g/ml) as a horizontal coordinate and taking a peak area as a vertical coordinate, and calculating a regression equation, and the results are shown in Table 10 and FIG. 8. From the results, it was found that INT1-F was linearly related in the concentration range of 0.05314. mu.g/ml to 17.7119. mu.g/ml.
TABLE 10 INT1-F Linear test results
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A detection method of related substances of an Arbidol hydrochloride intermediate is disclosed, wherein the Arbidol hydrochloride intermediate is 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester, and is characterized in that: the detection method comprises the following steps:
step one, preparing a test solution and a reference solution;
preparing a reference substance solution: taking 5-acetoxyl-6-bromo-2-bromomethyl-1-methylindole-3-carboxylic acid ethyl ester, 1, 2-dimethyl-6-bromo-5-hydroxyindole-3-carboxylic acid ethyl ester, 6, 7-dibromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester, 6-bromo-5-acetoxyl-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester, 6-bromo-5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester and 5-hydroxy-1-methyl-2-thiophenylmethylindole-3-carboxylic acid ethyl ester as reference substances, preparing a reference substance solution by using a solvent;
preparing a test solution: preparing a sample of 6-bromo-5-hydroxy-1-methyl-2-phenylthiomethyl indole-3-carboxylic acid ethyl ester into a test solution by using a solvent;
step two, detecting the test solution and the reference solution by adopting liquid chromatography, wherein the chromatographic conditions of the liquid chromatography are as follows:
performing gradient elution by using a C18 chromatographic column and acetonitrile-water as a mobile phase A in a volume ratio of 63-67: 33-37 and acetonitrile-methanol-water as a mobile phase B in a volume ratio of 38-42: 18-22, wherein the detection wavelength is 249-259 nm.
2. The method for detecting substances related to the arbidol hydrochloride intermediate as claimed in claim 1, wherein: the procedure for the gradient elution was as follows:
0min → 14min, 100% mobile phase a, 0% mobile phase B;
14min → 14.1min, 0% mobile phase a, 100% mobile phase B;
14.1min → 25min, 0% mobile phase a, 100% mobile phase B;
25min → 25.1min, 100% mobile phase a, 0% mobile phase B;
25.1min → 35min, 100% mobile phase A, 0% mobile phase B.
3. The method for detecting substances related to the arbidol hydrochloride intermediate as claimed in claim 1, wherein: the mobile phase A is acetonitrile-water with a volume ratio of 65:35, and the mobile phase B is acetonitrile-methanol-water with a volume ratio of 40:40: 20.
4. The method for detecting substances related to the arbidol hydrochloride intermediate as claimed in claim 1, wherein: the detection wavelength is 254 nm.
5. The method for detecting substances related to the arbidol hydrochloride intermediate as claimed in claim 1, wherein: the flow rate is 0.9 ml/min-1.1 ml/min, and the column temperature is 25 ℃ to 35 ℃.
6. The method for detecting substances related to the arbidol hydrochloride intermediate as claimed in claim 5, wherein: the flow rate was 1ml/min and the column temperature was 30 ℃.
7. The method for detecting substances related to the arbidol hydrochloride intermediate as claimed in claim 1, wherein: the specification of the chromatographic column is 4.6mm × 250mm × 5 μm.
8. The method for detecting substances related to the arbidol hydrochloride intermediate as claimed in claim 7, wherein: the column was model number CAPCELL PAK C18.
9. The method for detecting substances related to the arbidol hydrochloride intermediate as claimed in claim 1, wherein: the concentration of the test solution is 0.25 mg/ml-0.35 mg/ml.
10. The method for detecting substances related to the arbidol hydrochloride intermediate as claimed in claim 1, wherein: the concentrations of ethyl 5-acetoxy-6-bromo-2-bromomethyl-1-methylindole-3-carboxylate, ethyl 1, 2-dimethyl-6-bromo-5-hydroxyindole-3-carboxylate, ethyl 6, 7-dibromo-5-hydroxy-1-methyl-2-phenylthiomethyl indole-3-carboxylate, ethyl 6-bromo-5-acetoxy-1-methyl-2-phenylthiomethyl indole-3-carboxylate, ethyl 6-bromo-5-hydroxy-1-methyl-2-phenylthiomethyl indole-3-carboxylate and ethyl 5-hydroxy-1-methyl-2-phenylthiomethyl indole-3-carboxylate in the control solution were adjusted to obtain the same All are 3 mug/ml; and/or
The solvent is acetonitrile.
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