CN114002336B - Method for analyzing and detecting thiophene ethylamine and related substances thereof - Google Patents
Method for analyzing and detecting thiophene ethylamine and related substances thereof Download PDFInfo
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Abstract
The invention relates to the technical field of chemical analysis, in particular to a method for analyzing and detecting thiophene ethylamine and related substances thereof. The invention provides a method for analyzing and detecting phenethylamine and related substances thereof, which utilizes a high performance liquid chromatography analysis method to effectively separate the phenethylamine and related substances thereof, and further analyzes the purity of the phenethylamine, thereby realizing the quality control of clopidogrel bulk drug and preparation thereof.
Description
Technical Field
The invention relates to the technical field of chemical analysis, in particular to a method for analyzing and detecting thiophene ethylamine and related substances thereof.
Background
Thiophene ethylamine is a key raw material for producing 4,5,6, 7-tetrahydrothiophene [3,2-c ] pyridine hydrochloride, and 4,5,6, 7-tetrahydrothiophene [3,2-c ] pyridine hydrochloride is an important medical intermediate, and is mainly used for synthesizing platelet coagulation inhibitors such as clopidogrel drugs. In order to improve the quality of clopidogrel drug production, the quality of the phenethylamine needs to be effectively analyzed and controlled according to drug production specifications and product standards, such as the rules of the United States Pharmacopeia (USP). The thiophene ethylamine process route is analyzed, and several impurity components which may be present are:
at present, a method for simply, conveniently, rapidly and low-cost analyzing and detecting the phenethylamine and related substances thereof is not reported.
Therefore, there is a need to develop a simple, rapid and convenient method for analyzing and detecting the phenethylamine and related substances, so as to realize the quality control of clopidogrel bulk drug and preparation thereof.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the related art to some extent. Therefore, an object of the present invention is to provide a method for analyzing and detecting phenethylamine and related substances, which uses a high performance liquid chromatography method to effectively separate the phenethylamine and related substances, and further analyze the purity of the phenethylamine, thereby realizing quality control of clopidogrel bulk drug and preparation thereof.
To this end, the first aspect of the present invention provides a method for the analytical detection of phenethylamine and related substances thereof. According to the embodiment of the invention, the method utilizes high performance liquid chromatography to detect the sample to be detected so as to obtain a chromatogram; and
based on the chromatogram, obtaining the content of the thiopheneethylamine in the sample to be detected;
wherein, the chromatographic conditions of the high performance liquid chromatography are as follows:
the chromatographic column adopts octadecylsilane chemically bonded silica gel column;
the Ghost peak trapping column is Welch Ghost-Buster;
the buffer solution is used as a mobile phase A, the organic solvent is used as a mobile phase B for gradient elution,
the gradient elution conditions are as follows:
| time (min) | A(％) | B(％) |
| 0 | 95 | 5 |
| 20 | 20 | 80 |
| 25 | 20 | 80 |
| 26 | 95 | 5 |
| 40 | 95 | 5 |
Wherein the mobile phase A is ammonium salt solution, preferably ammonium chloride aqueous solution, and the mobile phase B is acetonitrile.
The analysis and detection method can separate the thiophene ethylamine from the impurity A (2- (thiophene-3-yl) ethylamine), the impurity C (thiophene-2-formaldehyde) and the impurity D (thiophene) existing in the production process of the thiophene ethylamine, so that the purity of the thiophene ethylamine is analyzed and detected, the quality control of the thiophene ethylamine is realized, and the quality control of clopidogrel bulk drug and preparation thereof is realized.
The method for analyzing and detecting the phenethylamine and related substances thereof according to the embodiment of the invention can also have at least one of the following additional technical characteristics:
according to an embodiment of the invention, the mobile phase a has a concentration of 10mM.
According to an embodiment of the invention, the pH value of the mobile phase A is 9.10+/-0.05.
Preferably, the mobile phase A is an aqueous solution of ammonium chloride, and the preparation method comprises the following steps: dissolving a certain amount of ammonium chloride solid in water, ultrasonically dissolving, and regulating the pH value to 9.10+/-0.05 by using ammonia water.
The pH value of the mobile phase A can influence the separation result, and too high or too low pH value can lead the main component peak to generate tailing phenomenon, the peak shape is poor, and the separation effect with other impurity peaks is interfered.
According to an embodiment of the invention, the chromatographic column in the high performance liquid chromatography is Waters XB ridge C18.
According to a preferred embodiment of the present invention, the chromatographic column in the high performance liquid chromatography is Waters XB ridge C18 (150 mm. Times.4.6 mm,3.5 μm) and the Ghost peak trapping column is Welch Ghost-Buster (50 mm. Times.2.1 mm).
In chromatographic separation, particularly, subtle chromatographic peaks are easy to generate in the gradient elution process, commonly called ghost peaks mainly come from mobile phases and pipelines, and once ghost peaks appear in the development of the method, more time and effort are required for analyzing staff to eliminate the ghost peaks. In order to effectively trap the generated ghost peaks and eliminate the interference of the ghost peaks on the analysis of substances, the ghost peaks trapping column is arranged, so that impurities in solvents including organic solvents can be removed, and impurities in pipelines and mixers can be effectively trapped; the impurities are adsorbed, so that the peak appears more gentle, and the interference of the impurity peak and the solvent peak is reduced.
According to an embodiment of the invention, the column temperature of the chromatographic column is 38-42 ℃, preferably 40 ℃.
According to an embodiment of the invention, the flow rate of the mobile phase is 0.9 to 1.1ml/min, preferably 0.9 to 1.0ml/min.
The column temperature of the chromatographic column and the flow rate of the mobile phase affect the separation effect, and if the column temperature of the chromatographic column or the flow rate of the mobile phase is not within the above range, the separation effect is poor, the separation degree is less than 1.5, good separation between the main peak and the impurity peak is not achieved, and the peak shape is poor.
According to the embodiment of the invention, the detection wavelength in the high performance liquid chromatography is 208-212 nm, preferably 210nm.
According to an embodiment of the invention, the method further comprises the steps of:
(1) Taking a sample to be detected containing thiophene ethylamine, and dissolving the sample with acetonitrile to prepare a sample solution with the concentration of 2 mg/ml;
(2) And (3) 5 mu l of the sample solution in the step (1) is injected into a high performance liquid chromatograph, a chromatogram is recorded, and the separation and detection of the sample containing the thiophene ethylamine are completed.
In a second aspect, the invention provides an application of the method in the first aspect in analytical detection of clopidogrel or a salt thereof produced by using thiophene ethylamine as a raw material.
The method for analyzing and detecting the phenethylamine and related substances thereof provided by the invention can be realized according to the following method:
chromatographic conditions:
chromatographic column: waters XBiridge C18 (150 mm. Times.4.6 mm,3.5 μm);
ghost peak trapping column: welch Ghost-Buster 50mm 2.1mm;
mobile phase: phase A: 10mM ammonium chloride buffer (ph=9.10±0.05); and B phase: acetonitrile
Mobile phase flow rate: 0.9-1.1 ml/min;
column temperature of chromatographic column: 38-42 ℃;
detection wavelength: 208-212 nm
Sample injection amount: 5. Mu.L;
the gradient elution conditions are as follows:
| time (min) | A(％) | B(％) |
| 0 | 95 | 5 |
| 20 | 20 | 80 |
| 25 | 20 | 80 |
| 26 | 95 | 5 |
| 40 | 95 | 5 |
1) Taking a sample containing thiophene ethylamine, and dissolving the sample with acetonitrile of a mobile phase B to prepare a sample solution with the concentration of 2 mg/ml;
2) And 5. Mu.l of the sample solution obtained in the step 1) is injected into a high performance liquid chromatograph, a chromatogram is recorded, and the analysis and separation of the sample containing the thiophene ethylamine are completed.
When each component dissolved in the mobile phase passes through the stationary phase, the components are different in retention time in the stationary phase due to the difference in size and strength of the components which react with the stationary phase (adsorption, distribution, ion attraction, exclusion and affinity), so that the components flow out of the stationary phase successively. High performance liquid chromatography is based on this principle to achieve separation of substances.
The inventor creatively discovers that by adopting the method for analyzing and detecting the phenethylamine and related substances thereof, the purity of the phenethylamine can be analyzed, so that the quality control of the phenethylamine is realized, the quality control of clopidogrel bulk drug and preparation thereof is further realized, and if the content of the phenethylamine in a sample to be detected does not reach the standard, the sample to be detected cannot be used as a raw material for producing clopidogrel to produce clopidogrel.
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
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 shows a high performance liquid chromatogram of the resulting sample according to example 1 of the present invention;
FIG. 2 shows a high performance liquid chromatogram of the resulting sample according to example 2 of the present invention;
FIG. 3 shows a high performance liquid chromatogram of the resulting sample according to example 3 of the present invention;
FIG. 4 shows a high performance liquid chromatogram of the resulting sample according to example 4 of the present invention;
FIG. 5 shows a high performance liquid chromatogram of the resulting sample according to example 5 of the present invention;
FIG. 6 shows a high performance liquid chromatogram of the resulting sample according to example 6 of the present invention;
FIG. 7 shows a high performance liquid chromatogram of the resulting sample according to example 7 of the present invention;
FIG. 8 shows high performance liquid chromatograms (enlarged chromatogram merging around the main peak) of the samples of example 8 and comparative examples 1 to 7 of the present invention.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Samples containing thiophene ethylamine in the embodiment of the invention are purchased from mikrin, high performance liquid chromatograph: shimadzu LC-20A series high performance liquid chromatograph.
Example 1
1) Taking a sample containing thiophene ethylamine, and dissolving the sample with acetonitrile to prepare a sample solution with the concentration of 2 mg/ml;
2) And 5. Mu.l of the sample solution obtained in the step 1) is injected into a high performance liquid chromatograph, a chromatogram is recorded, and the analysis and separation of the sample containing the thiophene ethylamine are completed as shown in fig. 1.
Chromatographic conditions:
chromatographic column: waters XBiridge C18 (150 mm. Times.4.6 mm,3.5 μm);
ghost peak trapping column: welch Ghost-Buster 50mm 2.1mm;
mobile phase: phase A: 10mM ammonium chloride buffer (ph=9.10±0.05); and B phase: acetonitrile
Mobile phase flow rate: 1.0ml/min;
column temperature of chromatographic column: 40 ℃;
detection wavelength: 210nm;
sample injection amount: 5. Mu.L;
the gradient elution conditions are as follows:
| time (min) | A(％) | B(％) |
| 0 | 95 | 5 |
| 20 | 20 | 80 |
| 25 | 20 | 80 |
| 26 | 95 | 5 |
| 40 | 95 | 5 |
In fig. 1, the peak No. 1 is impurity a, the peak No. 2 is thiopheneethylamine, the peak No. 4 is impurity C, and the peak No. 11 is impurity D. Under the condition, the main peak of the thiophene ethylamine can be well separated from impurities, the separation degree of the main peak and the adjacent peaks is more than 1.5, and the main peak of the thiophene ethylamine is about 8.560 min.
Example 2
1) Taking a sample containing thiophene ethylamine, and dissolving the sample with acetonitrile to prepare a sample solution with the concentration of 2 mg/ml;
2) And 5. Mu.l of the sample solution obtained in the step 1) is injected into a high performance liquid chromatograph, a chromatogram is recorded, and analysis and separation of the sample containing the thiophene ethylamine are completed as shown in fig. 2.
Chromatographic conditions:
chromatographic column: waters XBiridge C18 (150 mm. Times.4.6 mm,3.5 μm);
ghost peak trapping column: welch Ghost-Buster 50mm 2.1mm;
mobile phase: phase A: 10mM ammonium chloride buffer (ph=9.10±0.05); and B phase: acetonitrile
Mobile phase flow rate: 1.0ml/min;
column temperature of chromatographic column: 40 ℃;
detection wavelength: 208nm;
sample injection amount: 5. Mu.L;
the gradient elution conditions are as follows:
| time (min) | A(％) | B(％) |
| 0 | 95 | 5 |
| 20 | 20 | 80 |
| 25 | 20 | 80 |
| 26 | 95 | 5 |
| 40 | 95 | 5 |
In fig. 2, the peak No. 1 is impurity a, the peak No. 2 is thiopheneethylamine, the peak No. 4 is impurity C, and the peak No. 12 is impurity D. Under the condition, the main peak of the thiophene ethylamine can be well separated from impurities, the separation degree of the main peak and the adjacent peaks is more than 1.5, and the main peak of the thiophene ethylamine is about 8.560 min.
Example 3
1) Taking a sample containing thiophene ethylamine, and dissolving the sample with acetonitrile to prepare a sample solution with the concentration of 2 mg/ml;
2) And 5. Mu.l of the sample solution obtained in the step 1) is injected into a high performance liquid chromatograph, a chromatogram is recorded, and the analysis and separation of the sample containing the thiophene ethylamine are completed as shown in fig. 3.
Chromatographic conditions:
chromatographic column: waters XBiridge C18 (150 mm. Times.4.6 mm,3.5 μm);
ghost peak trapping column: welch Ghost-Buster 50mm 2.1mm;
mobile phase: phase A: 10mM ammonium chloride buffer (ph=9.10±0.05); and B phase: acetonitrile
Mobile phase flow rate: 1.0ml/min;
column temperature of chromatographic column: 40 ℃;
detection wavelength: 212nm;
sample injection amount: 5. Mu.L;
the gradient elution conditions are as follows:
| time (min) | A(％) | B(％) |
| 0 | 95 | 5 |
| 20 | 20 | 80 |
| 25 | 20 | 80 |
| 26 | 95 | 5 |
| 40 | 95 | 5 |
In fig. 3, the peak No. 1 is impurity a, the peak No. 2 is thiopheneethylamine, the peak No. 4 is impurity C, and the peak No. 11 is impurity D. Under the condition, the main peak of the thiophene ethylamine can be well separated from impurities, the separation degree of the main peak and the adjacent peaks is more than 1.5, and the main peak of the thiophene ethylamine is about 8.560 min.
Example 4
1) Taking a sample containing thiophene ethylamine, and dissolving the sample with acetonitrile to prepare a sample solution with the concentration of 2 mg/ml;
2) And 5. Mu.l of the sample solution obtained in the step 1) is injected into a high performance liquid chromatograph, a chromatogram is recorded, and analysis and separation of the sample containing the thiophene ethylamine are completed as shown in fig. 4.
Chromatographic conditions:
chromatographic column: waters XBiridge C18 (150 mm. Times.4.6 mm,3.5 μm);
ghost peak trapping column: welch Ghost-Buster 50mm 2.1mm;
mobile phase: phase A: 10mM ammonium chloride buffer (ph=9.10±0.05); and B phase: acetonitrile
Mobile phase flow rate: 0.9ml/min;
column temperature of chromatographic column: 40 ℃;
detection wavelength: 210nm;
sample injection amount: 5. Mu.L;
the gradient elution conditions are as follows:
| time (min) | A(％) | B(％) |
| 0 | 95 | 5 |
| 20 | 20 | 80 |
| 25 | 20 | 80 |
| 26 | 95 | 5 |
| 40 | 95 | 5 |
In fig. 4, the peak No. 1 is impurity a, the peak No. 2 is thiopheneethylamine, the peak No. 5 is impurity C, and the peak No. 11 is impurity D. Under the condition, the main peak of the thiophene ethylamine can be well separated from impurities, the separation degree of the main peak and the adjacent peaks is more than 1.5, and the main peak of the thiophene ethylamine is about 9.309 min.
Example 5
1) Taking a sample containing thiophene ethylamine, and dissolving the sample with acetonitrile to prepare a sample solution with the concentration of 2 mg/ml;
2) And 5. Mu.l of the sample solution obtained in the step 1) is injected into a high performance liquid chromatograph, a chromatogram is recorded, and analysis and separation of the sample containing the thiophene ethylamine are completed as shown in fig. 5.
Chromatographic conditions:
chromatographic column: waters XBiridge C18 (150 mm. Times.4.6 mm,3.5 μm);
ghost peak trapping column: welch Ghost-Buster 50mm 2.1mm;
mobile phase: phase A: 10mM ammonium chloride buffer (ph=9.10±0.05); and B phase: acetonitrile
Mobile phase flow rate: 1.1ml/min;
column temperature of chromatographic column: 40 ℃;
detection wavelength: 210nm;
sample injection amount: 5. Mu.L;
the gradient elution conditions are as follows:
| time (min) | A(％) | B(％) |
| 0 | 95 | 5 |
| 20 | 20 | 80 |
| 25 | 20 | 80 |
| 26 | 95 | 5 |
| 40 | 95 | 5 |
In fig. 5, the peak No. 1 is impurity a, the peak No. 2 is thiopheneethylamine, the peak No. 4 is impurity C, and the peak No. 10 is impurity D. Under the condition, the main peak of the thiophene ethylamine can be well separated from impurities, the separation degree of the main peak and the adjacent peaks is more than 1.5, and the main peak of the thiophene ethylamine is about 7.944 min.
Example 6
1) Taking a sample containing thiophene ethylamine, and dissolving the sample with acetonitrile to prepare a sample solution with the concentration of 2 mg/ml;
2) And 5. Mu.l of the sample solution obtained in the step 1) is injected into a high performance liquid chromatograph, a chromatogram is recorded, and analysis and separation of the sample containing the thiophene ethylamine are completed as shown in fig. 6.
Chromatographic conditions:
chromatographic column: waters XBiridge C18 (150 mm. Times.4.6 mm,3.5 μm);
ghost peak trapping column: welch Ghost-Buster 50mm 2.1mm;
mobile phase: phase A: 10mM ammonium chloride buffer (ph=9.10±0.05); and B phase: acetonitrile
Mobile phase flow rate: 1.0ml/min;
column temperature of chromatographic column: 38 ℃;
detection wavelength: 210nm;
sample injection amount: 5. Mu.L;
the gradient elution conditions are as follows:
| time (min) | A(％) | B(％) |
| 0 | 95 | 5 |
| 20 | 20 | 80 |
| 25 | 20 | 80 |
| 26 | 95 | 5 |
| 40 | 95 | 5 |
In fig. 6, the peak No. 1 is impurity a, the peak No. 3 is thiopheneethylamine, the peak No. 5 is impurity C, and the peak No. 12 is impurity D. Under the condition, the main peak of the thiophene ethylamine can be well separated from impurities, the separation degree of the main peak and the adjacent peaks is more than 1.5, and the main peak of the thiophene ethylamine is about 8.197 min.
Example 7
1) Taking a sample containing thiophene ethylamine, and dissolving the sample with acetonitrile to prepare a sample solution with the concentration of 2 mg/ml;
2) And 5. Mu.l of the sample solution obtained in the step 1) is injected into a high performance liquid chromatograph, a chromatogram is recorded, and analysis and separation of the sample containing the thiophene ethylamine are completed as shown in fig. 7.
Chromatographic conditions:
chromatographic column: waters XBiridge C18 (150 mm. Times.4.6 mm,3.5 μm);
ghost peak trapping column: welch Ghost-Buster 50mm 2.1mm;
mobile phase: phase A: 10mM ammonium chloride buffer (ph=9.10±0.05); and B phase: acetonitrile
Mobile phase flow rate: 1.0ml/min;
column temperature of chromatographic column: 42 ℃;
detection wavelength: 210nm;
sample injection amount: 5. Mu.L;
the gradient elution conditions are as follows:
| time (min) | A(％) | B(％) |
| 0 | 95 | 5 |
| 20 | 20 | 80 |
| 25 | 20 | 80 |
| 26 | 95 | 5 |
| 40 | 95 | 5 |
In fig. 7, the peak No. 1 is impurity a, the peak No. 2 is thiopheneethylamine, the peak No. 4 is impurity C, and the peak No. 11 is impurity D. Under the condition, the main peak of the thiophene ethylamine can be well separated from impurities, the separation degree of the main peak and the adjacent peaks is more than 1.5, and the main peak of the thiophene ethylamine is about 8.035 min.
Example 8
The difference compared to example 1 is only that the column temperature in the chromatographic conditions is changed from 40℃to 41℃and the other is the same, the chromatogram is shown in FIG. 8 a. In FIG. 8a, the principal component has good peak symmetry, good peak shape, and good separation from adjacent peaks.
Comparative example 1
Compared with example 8, the difference is that the pH of the mobile phase A phase ammonium chloride buffer solution in chromatographic conditions is 8.07, and the other phases are the same, the obtained spectrum is shown in figure 8b, the main peak in figure 8b is triangular, the symmetry is poor, the peak outlet time is advanced, the main component peak has some tailing phenomenon, and whether the main component peak is separated from other peaks can not be judged.
Comparative example 2
The difference compared to example 8 is only that the pH of the mobile phase A ammonium chloride buffer in chromatographic conditions is 7.77, all other things being equal, the profile obtained is shown in FIG. 8 c. In fig. 8c, the main peak is triangular, the peak-out time is advanced, the symmetry is poor, the response is low, the main component peak has some tailing phenomenon, and whether the main component peak is separated from other peaks can not be judged.
Comparative example 3
1. Chromatographic conditions:
chromatographic column: CSH-C18 (150 mm. Times.4.6 mm,3.5 μm);
ghost peak trapping column: welch Ghost-Buster 50mm 2.1mm;
mobile phase: phase A: 10mM NH 4 FA buffer (ph=8.32); and B phase: acetonitrile
Mobile phase flow rate: 1.0ml/min;
column temperature of chromatographic column: 40 ℃;
detection wavelength: 210nm;
sample injection amount: 5. Mu.L;
the gradient elution conditions are as follows:
| time (min) | A(％) | B(％) |
| 0 | 95 | 5 |
| 20 | 20 | 80 |
| 25 | 20 | 80 |
| 26 | 95 | 5 |
| 40 | 95 | 5 |
1) Taking a sample containing thiophene ethylamine, and dissolving the sample with acetonitrile to prepare a sample solution with the concentration of 2 mg/ml;
2) And 5. Mu.l of the sample solution obtained in the step 1) is injected into a high performance liquid chromatograph, a chromatogram is recorded, and the analysis and separation of the sample containing the thiophene ethylamine are completed.
The separation results obtained are shown in fig. 8d, and small peaks appear before the main peak, which indicates that the main component peaks and other impurity peaks have poor separation effect.
Comparative example 4
Comparative example 4 differs from comparative example 3 only in the chromatographic column used: waters XBiridge C18 (150 mm. Times.4.6 mm,3.5 μm) gave the separation results shown in FIG. 8e with some tailing of the major component peaks.
Comparative example 5
1. Chromatographic conditions:
chromatographic column: waters XBiridge C18 (150 mm. Times.4.6 mm,3.5 μm);
ghost peak trapping column: welch Ghost-Buster 50mm 2.1mm;
mobile phase: phase A: 10mM NH 4 HCO 3 Buffer (ph=9.56); and B phase: acetonitrile
Mobile phase flow rate: 1.0ml/min;
column temperature of chromatographic column: 40 ℃;
detection wavelength: 210nm;
sample injection amount: 5. Mu.L;
the gradient elution conditions are as follows:
| time (min) | A(％) | B(％) |
| 0 | 95 | 5 |
| 20 | 20 | 80 |
| 25 | 20 | 80 |
| 26 | 95 | 5 |
| 40 | 95 | 5 |
1) Taking a sample containing thiophene ethylamine, and dissolving the sample with acetonitrile to prepare a sample solution with the concentration of 2 mg/ml;
2) And 5. Mu.l of the sample solution obtained in the step 1) is injected into a high performance liquid chromatograph, a chromatogram is recorded, and the analysis and separation of the sample containing the thiophene ethylamine are completed.
The separation results obtained are shown in FIG. 8f, in which the major component peaks have some tailing.
Comparative example 6
1. Chromatographic conditions:
chromatographic column: waters XBiridge C18 (150 mm. Times.4.6 mm,3.5 μm);
ghost peak trapping column: welch Ghost-Buster 50mm 2.1mm;
mobile phase: phase A: 10mMK 2 HPO 4 Buffer (ph=9.05); and B phase: acetonitrile
Mobile phase flow rate: 1.0ml/min;
column temperature of chromatographic column: 40 ℃;
detection wavelength: 210nm;
sample injection amount: 5. Mu.L;
the gradient elution conditions are as follows:
| time (min) | A(％) | B(％) |
| 0 | 95 | 5 |
| 20 | 20 | 80 |
| 25 | 20 | 80 |
| 26 | 95 | 5 |
| 40 | 95 | 5 |
1) Taking a sample containing thiophene ethylamine, and dissolving the sample with acetonitrile to prepare a sample solution with the concentration of 2 mg/ml;
2) And 5. Mu.l of the sample solution obtained in the step 1) is injected into a high performance liquid chromatograph, a chromatogram is recorded, and the analysis and separation of the sample containing the thiophene ethylamine are completed.
The separation result obtained is shown in FIG. 8g, the main component peak shape is triangle, the peak shape is asymmetric and sharp, and the peak shape is poor.
Comparative example 7
1. Chromatographic conditions:
chromatographic column: waters XBiridge C18 (150 mm. Times.4.6 mm,3.5 μm);
ghost peak trapping column: welch Ghost-Buster 50mm 2.1mm;
mobile phase: phase A: 0.01% NH 3 .H 2 O buffer (ph=9.17); and B phase: acetonitrile
Mobile phase flow rate: 1.0ml/min;
column temperature of chromatographic column: 40 ℃;
detection wavelength: 210nm;
sample injection amount: 5. Mu.L;
the gradient elution conditions are as follows:
| time (min) | A(％) | B(％) |
| 0 | 95 | 5 |
| 20 | 20 | 80 |
| 25 | 20 | 80 |
| 26 | 95 | 5 |
| 40 | 95 | 5 |
1) Taking a sample containing thiophene ethylamine, and dissolving the sample with acetonitrile to prepare a sample solution with the concentration of 2 mg/ml;
2) And 5. Mu.l of the sample solution obtained in the step 1) is injected into a high performance liquid chromatograph, a chromatogram is recorded, and the analysis and separation of the sample containing the thiophene ethylamine are completed.
The separation result obtained is shown in fig. 8h, the peak shape of the main component is triangular, the peak shape is asymmetric and sharp, and the peak shape is poor.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means 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 present invention. In this specification, schematic representations of the above terms are not necessarily directed 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, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (11)
1. A method for analyzing and detecting thiophene ethylamine and related substances thereof is characterized in that a sample to be detected is detected by utilizing a high performance liquid chromatography so as to obtain a chromatogram; and
based on the chromatogram, obtaining the content of the thiopheneethylamine in the sample to be detected;
wherein, the chromatographic conditions of the high performance liquid chromatography are as follows:
the chromatographic column adopts octadecylsilane chemically bonded silica gel column;
the Ghost peak trapping column is Welch Ghost-Buster;
the buffer solution is used as a mobile phase A, the organic solvent is used as a mobile phase B for gradient elution,
the gradient elution conditions are as follows:
Wherein the mobile phase A is an ammonium chloride aqueous solution, the mobile phase B is acetonitrile, and the pH value of the mobile phase A is 9.10+/-0.05.
2. The method of claim 1, wherein the mobile phase a is at a concentration of 10mM.
3. The method of claim 1, wherein the hplc column is Waters XBridge C18.
4. A method according to claim 3, wherein the column temperature of the chromatographic column is 38-42 ℃.
5. The method of claim 4, wherein the column temperature of the chromatographic column is 40 ℃.
6. The method according to claim 1, wherein the flow rate of the mobile phase is 0.9 to 1.1ml/min.
7. The method according to claim 6, wherein the flow rate of the mobile phase is 0.9 to 1.0ml/min.
8. The method according to claim 1, wherein the detection wavelength in the high performance liquid chromatography is 208 to 212nm.
9. The method of claim 8, wherein the detection wavelength in high performance liquid chromatography is 210nm.
10. The method according to any one of claims 1 to 9, characterized in that it further comprises the steps of:
(1) Taking a sample to be detected containing thiophene ethylamine, and dissolving the sample with acetonitrile to prepare a sample solution with the concentration of 2 mg/ml;
(2) And (3) 5 mu l of the sample solution in the step (1) is injected into a high performance liquid chromatograph, a chromatogram is recorded, and the separation and detection of the sample containing the thiophene ethylamine are completed.
11. Use of the method according to any one of claims 1 to 10 for the analytical detection of clopidogrel or a salt thereof produced from thiophene ethylamine.
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