CN115343396A - Method for detecting related substances in diphenhydrasol hydrochloride - Google Patents
Method for detecting related substances in diphenhydrasol hydrochloride Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N2030/022—Column chromatography characterised by the kind of separation mechanism
- G01N2030/027—Liquid chromatography
Abstract
The invention relates to a method for detecting related substances in diphenhydrasol hydrochloride, which is characterized in that a proper chromatographic column and a proper column temperature are screened, a potassium dihydrogen phosphate aqueous solution and a phosphoric acid-acetonitrile solution are used as mixed mobile phases for gradient elution, and the elution time and the proportion of the mobile phases are optimized, so that the detected impurities are more, the main components and all the impurities have stronger retention capacity in the detection method, the response is higher, the separation among the components is good, the related substances in the diphenhydrasol hydrochloride can be rapidly and accurately monitored, and the method has important significance for the quality evaluation of the diphenhydrasol hydrochloride.
Description
Technical Field
The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a detection method of related substances in trihexyphenidyl hydrochloride.
Background
Trihexyphenidyl Hydrochloride (Trihexyphenidyl Hydrochloride) with chemical name of (+/-) -alpha-cyclohexyl-alpha-phenyl-1-piperidinepropanol Hydrochloride and molecular formula of C 20 H 31 NO & HCl, molecular weight 337.93, its structural formula is as follows:
the diphenhydrase hydrochloride is an anticholinergic drug, has the effects of directly inhibiting parasympathetic nervous system and relieving muscle spasm, is clinically used for treating various Parkinson's diseases, can improve symptoms such as rigidity, dyskinesia and tremor, and can also be used for the common extrapyramidal system reaction (EPS) of central nervous system drugs. In order to ensure the safety and effectiveness of the medicine, research, detection and monitoring are carried out on related substances of the medicine.
Disclosure of Invention
The invention aims to provide a method for detecting related substances in trihexyphenidyl hydrochloride on the basis of the prior art, which has the advantages of many detected impurities, high sensitivity, good specificity, good separation degree among impurity peaks, a main peak of trihexyphenidyl and adjacent impurity peaks, and capability of quickly, effectively and accurately monitoring the related substances in trihexyphenidyl hydrochloride.
The technical scheme of the invention is as follows:
a method for detecting related substances in diphenhydrasol hydrochloride is characterized in that the detection method adopts high performance liquid chromatography to carry out quantitative detection on the diphenhydrasol hydrochloride related substances, and the conditions of the high performance liquid chromatography comprise:
the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column; the column temperature is 30-40 ℃; gradient elution is carried out by taking a mobile phase A and a mobile phase B as a mixed mobile phase, wherein the mobile phase A is a potassium dihydrogen phosphate aqueous solution of 5-50 mmol/L, and the pH value of the solution is adjusted to 3.5-4.5 by phosphoric acid; the mobile phase B is 0.01 to 0.1 percent phosphoric acid-acetonitrile solution; the specific gradient elution procedure was as follows: (1) The volume ratio of mobile phase a to mobile phase B was kept constant at 85; (2) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 85 to 61; (3) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 61 to 30. The specific gradient elution procedure is shown in table 1 below.
TABLE 1 gradient elution procedure
| Time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 85 | 15 |
| 10 | 85 | 15 |
| 40 | 61 | 39 |
| 57 | 30 | 70 |
In a preferred embodiment, the present invention employs a chromatographic column Inertsil ODS-3 or YMC Pack ODS-AQ, wherein Inertsil ODS-3 is more effective. By adopting the chromatographic column and the specific elution process, under the coordination of other conditions, the detected impurities are more, the sensitivity is high, the specificity is good, the separation degree between each impurity peak, the main diphenhydrasol peak and the adjacent impurity peaks is good, and the related substances in the diphenhydrasol hydrochloride can be quickly, effectively and accurately monitored. Preferably, the column has a length of 150mm, a diameter of 4.6mm and a packing particle size of 3.5. Mu.m.
The invention takes a mobile phase A and a mobile phase B as a mixed mobile phase to carry out gradient elution, wherein the mobile phase A is 5-50 mmol/L potassium dihydrogen phosphate aqueous solution, and the pH value of the mobile phase A is adjusted to 3.5-4.5 by phosphoric acid. The mobile phase B is 0.01 to 0.1 percent phosphoric acid-acetonitrile solution. The invention uses the mobile phase A and the mobile phase B as the mixed mobile phase to carry out gradient elution, under the coordination of other conditions, the separation degree between each impurity peak, the main peak of the trihexyphenidyl and the adjacent impurity peaks is high, and the related substances in the trihexyphenidyl hydrochloride can be quickly, effectively and accurately monitored. Preferably, the mobile phase A is 10mmol/L potassium dihydrogen phosphate water solution, and the pH value is adjusted to 3.8-4.2 by phosphoric acid.
In a preferred embodiment, the preparation method of the mobile phase A is as follows: weighing 1.4g of anhydrous potassium dihydrogen phosphate, adding 1000mL of water for dissolving, and adjusting the pH value to 3.8-4.2 by using phosphoric acid to obtain the potassium dihydrogen phosphate.
In a more preferred embodiment, the mobile phase a is prepared as follows: weighing 1.4g of anhydrous potassium dihydrogen phosphate, adding 1000mL of water for dissolving, and adjusting the pH value to 4.0 by using phosphoric acid to obtain the potassium dihydrogen phosphate.
In a preferred embodiment, the mobile phase B is a 0.03 to 0.07% phosphoric acid-acetonitrile solution, and more preferably, the mobile phase B is a 0.05% phosphoric acid-acetonitrile solution.
The invention selects the solution of potassium dihydrogen phosphate water solution of 9-12 mmol/L-0.01-0.1 percent phosphoric acid-acetonitrile
In the process of gradient elution, the selection of the column temperature is important, the column temperature is 30-40 ℃, for example, 30 ℃, 35 ℃ or 40 ℃, under the coordination of other conditions, the detected impurities are more, the sensitivity is high, the specificity is good, and the separation degree between each impurity peak, the main peak of the trihexyphenidyl and the adjacent impurity peaks is good. When the column temperature is low, for example, 25 ℃, the separation effect of some impurities is poor, and it is difficult to accurately monitor the related substances in the diphenhydral hydrochloride.
According to the method for detecting the related substances in the diphenhydrasol hydrochloride, provided by the invention, in the gradient elution process, a proper mobile phase is screened out, and the elution time and the proportion of the mobile phase are optimized, so that the detected impurities are more, the main components and all the impurities have stronger retention capacity in the detection method, the response is higher, the separation among the components is good, the related substances in the diphenhydrasol hydrochloride can be rapidly and accurately monitored, and the method has important significance for the quality evaluation of the diphenhydrasol hydrochloride.
The detection method comprises the following high performance liquid chromatography conditions:
the detection wavelength is 200 to 220nm, preferably 210nm.
The column temperature is 30 to 40 ℃, preferably 30 to 35 ℃, more preferably 30 ℃.
Furthermore, the flow rate is 1.1-1.3 mL/min; preferably 1.2mL/min.
Furthermore, the sample injection amount is 10-50 mu L; preferably 20. Mu.L. For example: the sample volume was 10. Mu.l, 20. Mu.l or 50. Mu.l.
The invention provides a method for detecting related substances in diphenyol hydrochloride, wherein the related substances comprise the following substances:
the detection method provided by the invention comprises the following specific steps: respectively preparing impurity and main component positioning solution and sample solution, and calculating the content of each impurity. The solvent is a mixed solution of a mobile phase A and a mobile phase B with a volume ratio of 70, wherein the preparation method of the mobile phase A comprises the following steps: weighing 1.4g of anhydrous potassium dihydrogen phosphate, adding 1000mL of water for dissolving, and adjusting the pH value to 4.0 by using phosphoric acid to obtain the potassium dihydrogen phosphate; mobile phase B was 0.05% phosphoric acid-acetonitrile solution.
The impurity and main component (diphenhydrasol hydrochloride) positioning solution is as follows: taking samples of impurities 1, 2, 3,4, 5, 6, 7, 8 and 9 and the diphenhydrasol hydrochloride, adding a solvent (a mixed solution of a mobile phase A and a mobile phase B with a volume ratio of 70.
Test solution: the diphenhydrasol hydrochloride is taken and dissolved by a solvent (a mixed solution of mobile phase A and mobile phase B with the volume ratio of 70.
The invention determines the detection method of the invention by screening proper chromatographic conditions and carrying out chromatographic detection on the diphenhydrasol and the impurities, and the invention is specially verified by carrying out peak location test, interference test and degradation test on the diphenhydrasol and the impurities.
By adopting the technical scheme of the invention, the advantages are as follows:
the invention provides a method for detecting related substances in diphenhydrasol hydrochloride, which screens out proper chromatographic columns and column temperature, uses 9-50 mmol/L potassium dihydrogen phosphate aqueous solution and 0.01-0.1% phosphoric acid-acetonitrile solution as mixed mobile phase to carry out gradient elution, optimizes the elution time and the proportion of the mobile phase, ensures that the detected impurities are more, has stronger retention capacity of main components and all impurities in the detection method, has higher response and good separation among the components, can quickly and accurately monitor the related substances in the diphenhydrasol hydrochloride, and has important significance for the quality evaluation of the diphenhydrasol hydrochloride.
Drawings
FIG. 1 is a high performance liquid chromatogram of an impurity and principal component localization solution;
FIG. 2 is a high performance liquid chromatogram of the relevant substances in diphenhydrase hydrochloride of example 1;
FIG. 3 is a high performance liquid chromatogram of a mixed solution of diphenhydrase hydrochloride and impurities of example 2;
FIG. 4 is a high performance liquid chromatogram of a mixed solution of diphenhydrase hydrochloride and impurities of example 3;
FIG. 5 is a high performance liquid chromatogram of the related substances in the mixed solution of diphenhydramine hydrochloride and impurities of example 4;
FIG. 6 is a high performance liquid chromatogram of a mixed solution of diphenhydrase hydrochloride and impurities of example 5;
FIG. 7 is a high performance liquid chromatogram of the related substances in the mixed solution of diphenhydramine hydrochloride and impurities of example 6;
FIG. 8 is a high performance liquid chromatogram of a mixed solution of diphenhydrase hydrochloride and impurities of example 7;
FIG. 9 is a high performance liquid chromatogram of the relevant substances in the impurity mixed solution of comparative example 1;
FIG. 10 is a high performance liquid chromatogram of a related substance in a mixed solution of diphenhydrase hydrochloride and impurities of comparative example 2;
FIG. 11 is a high performance liquid chromatogram of a related substance in a mixed solution of diphenoxylate hydrochloride and impurities of comparative example 3;
Detailed Description
The detection method of the present invention is further illustrated by the following examples in conjunction with the drawings, but the present invention is not limited to these examples.
Example 1
High performance liquid chromatography conditions:
octadecylsilane chemically bonded silica is used as a filler, a chromatographic column is Inertsil ODS-3,4.6mm multiplied by 150mm,5 mu m; the preparation process of the mobile phase A with 10mmol/L potassium dihydrogen phosphate aqua includes the following steps: weighing 1.4g of anhydrous potassium dihydrogen phosphate, adding 1000mL of water for dissolving, and adjusting the pH value to 4.0 by using phosphoric acid; 0.05 percent phosphoric acid-acetonitrile solution is taken as a mobile phase B; carrying out gradient elution according to the following gradient elution process; the flow rate is 1.2mL/min per minute; the column temperature is 30 ℃; the detection wavelength is 210nm; the injection volume was 20. Mu.l.
The gradient elution process is as follows: (1) The volume ratio of mobile phase a to mobile phase B was kept constant at 85; (2) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 85 to 61; (3) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 61 to 30.
Sample preparation:
a sample of benzhexol hydrochloride was taken and dissolved in a solvent (a mixed solution of mobile phase A and mobile phase B at a volume ratio of 70.
And (3) test operation: sampling 20 μ l of the sample solution, and recording chromatogram
Preparing a test sample solution from a trihexyphenidyl hydrochloride sample, injecting a sample, recording a chromatogram, and calculating the content of impurities in the test sample by using a main component self-comparison method, wherein the result is shown in table 2 and figure 2.
TABLE 2 measurement results of the content of each impurity in diphenhydrasol hydrochloride
| | Impurity | 6 | Maximum single hetero | Total miscellaneous |
| Content (%) | 0.042 | 0.014 | 0.06 |
As can be seen from table 2 and fig. 2, the known impurity 6 was detected in the diphenhydrasol hydrochloride sample, the maximum single impurity was 0.014%, the total impurity was 0.06%, and the number of impurities was 2.
The detection method of the embodiment 1 of the invention is determined by screening a proper mobile phase and optimizing the proportion of each component in the mobile phase to perform chromatographic detection on the diphenhydrasol hydrochloride and 9 impurities.
The invention is specially verified through a peak location test, an interference test and a degradation test of each impurity and a main component (the diphenhydrasol hydrochloride).
The impurity and main component (dipheny hydrochloride) positioning solution is: taking samples of impurities 1, 2, 3,4, 5, 6, 7, 8 and 9 and the diphenhydrasol hydrochloride, adding a solvent (a mixed solution of a mobile phase A and a mobile phase B with a volume ratio of 70.
Each impurity and the localized solution of impurities and main component (diphenhydrasol hydrochloride) were collected and tested according to the HPLC conditions and method of example 1, and the results are shown in Table 3 and FIG. 1.
TABLE 3 results of specificity verification
| Order of appearance | t R (min) | Degree of separation |
| 1 | 5.175 | / |
| 8 | 6.419 | 3.68 |
| 7 | 11.670 | 11.55 |
| 2 | 25.434 | 25.85 |
| Unknown impurities | 28.073 | 5.99 |
| Benzhexol | 30.702 | 3.70 |
| 4 | 33.400 | 3.85 |
| 9 | 34.741 | 3.50 |
| 5 | 38.786 | 10.55 |
| 6 | 40.043 | 3.62 |
| 3 | 55.069 | 45.26 |
As can be seen from Table 3 and FIG. 1, the separation degree between each impurity peak, the main peak of trihexyphenidyl and the adjacent impurity peaks is greater than 2.0, the peak purity is good, and the specificity of the invention is good.
Respectively taking a diphenhydrasol hydrochloride reference substance and impurities 1, 2, 3,4, 5, 6, 7, 8 and 9 reference substances, precisely weighing, adding a solvent (a mixed solution of a mobile phase A and a mobile phase B with a volume ratio of 70; an appropriate amount of stock solution was precisely measured, diluted with a solvent (a mixed solution of mobile phase a and mobile phase B at a volume ratio of 70. Taking the concentration mu g/mL as a horizontal coordinate and the peak area as a vertical coordinate, performing linear regression and solving a regression equation, wherein the linear ranges and the linear equations of the diphenhydrasol hydrochloride and the impurities are shown in table 4.
The impurities and the main components are gradually diluted and then 20 mul of sample is injected, the S/N is more than or equal to 10 as the limit of quantification, and the S/N is more than or equal to 3 as the limit of detection, and the results are shown in table 4.
TABLE 4 detection limit, quantitation limit, and linearity results of diphenhydrasol hydrochloride and each impurity
As can be seen from Table 4, the detection sensitivity of the diphenhydrazole hydrochloride and each impurity in the detection method of the invention is higher, and the linear relation of each impurity in a lower concentration range is good.
Impurity control stock solution: an appropriate amount of each of the reference samples of impurities 1, 2, 3,4, 5, 6, 7, 8, and 9 was weighed precisely, dissolved in a solvent (a mixed solution of mobile phase a and mobile phase B in a volume ratio of 70.
Taking a proper amount of a test sample of the diphenhydrasol hydrochloride, adding a solvent (a mixed solution of a mobile phase A and a mobile phase B with a volume ratio of 70.
Taking about 20mg of a benzhexol hydrochloride sample, diluting 0.5mL, 1.0mL and 1.5mL of impurity reference stock solutions respectively to a scale by adding a solvent (a mixed solution of a mobile phase A and a mobile phase B with a volume ratio of 70.
The above background solution and recovery rate test sample solution were each injected into a liquid chromatograph at 20. Mu.l, and the results of the recovery rate test were shown in Table 5.
TABLE 5 results of recovery of trihexyphenidyl hydrochloride and impurities
As can be seen from Table 5, the detection method of the present invention has satisfactory recovery rate test and high recovery rate.
Example 2
High performance liquid chromatography conditions:
octadecylsilane chemically bonded silica is used as a filler, a chromatographic column is Inertsil ODS-3,4.6mm multiplied by 150mm,5 mu m; the preparation process of the mobile phase A with 10mmol/L potassium dihydrogen phosphate water solution is as follows: weighing 1.4g of anhydrous potassium dihydrogen phosphate, adding 1000mL of water for dissolving, and adjusting the pH value to 4.0 by using phosphoric acid; 0.05% phosphoric acid-acetonitrile solution as mobile phase B; gradient elution is carried out according to the following gradient elution process; the flow rate is 1.1mL/min per minute; the column temperature is 30 ℃; the detection wavelength is 210nm; the injection volume was 20. Mu.l.
The gradient elution process is as follows: (1) The volume ratio of mobile phase a to mobile phase B was kept constant at 85; (2) The volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 85 to 61; (3) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 61 to 30.
Sample preparation:
taking a sample of the benzhexol hydrochloride and impurities 1, 2, 3,4, 5, 6, 7, 8 and 9, adding a solvent (a mixed solution of a mobile phase A and a mobile phase B with a volume ratio of 70.
And (3) test operation: and (3) sampling 20 mu l of the mixed solution of the diphenhydrasol hydrochloride and the impurities, and recording a chromatogram, wherein the chromatogram is shown in figure 3.
As can be seen from fig. 3, the respective impurities were separated from the main component and the respective impurities at the baseline, and the degree of separation was good.
Example 3
High performance liquid chromatography conditions:
octadecylsilane chemically bonded silica is used as a filler, and a chromatographic column is Inertsil ODS-3,4.6mm multiplied by 150mm,5 mu m; the preparation process of the mobile phase A with 10mmol/L potassium dihydrogen phosphate aqua includes the following steps: weighing 1.4g of anhydrous potassium dihydrogen phosphate, adding 1000mL of water for dissolving, and adjusting the pH value to 4.0 by using phosphoric acid; 0.05% phosphoric acid-acetonitrile solution as mobile phase B; carrying out gradient elution according to the following gradient elution process; the flow rate is 1.3mL/min per minute; the column temperature is 30 ℃; the detection wavelength is 210nm; the injection volume was 20. Mu.l.
The gradient elution process is as follows: (1) The volume ratio of mobile phase a to mobile phase B was kept constant at 85; (2) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 85 to 61; (3) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 61.
Sample preparation:
taking a sample of the benzhexol hydrochloride and impurities 1, 2, 3,4, 5, 6, 7, 8 and 9, adding a solvent (a mixed solution of a mobile phase A and a mobile phase B with a volume ratio of 70.
And (3) test operation: and (3) sampling 20 mu l of the mixed solution of the diphenhydrasol hydrochloride and the impurities, and recording a chromatogram, wherein the chromatogram is shown in figure 4.
As can be seen from fig. 4, the respective impurities were separated from the main component and the respective impurities at the baseline, and the degree of separation was good.
Example 4
High performance liquid chromatography conditions:
octadecylsilane chemically bonded silica is used as a filler, a chromatographic column is Inertsil ODS-3,4.6mm multiplied by 150mm,5 mu m; the preparation process of the mobile phase A with 10mmol/L potassium dihydrogen phosphate water solution is as follows: weighing 1.4g of anhydrous potassium dihydrogen phosphate, adding 1000mL of water for dissolving, and adjusting the pH value to 3.8 by using phosphoric acid; 0.05% phosphoric acid-acetonitrile solution as mobile phase B; carrying out gradient elution according to the following gradient elution process; the flow rate is 1.2mL/min per minute; the column temperature is 30 ℃; the detection wavelength is 210nm; the injection volume was 20. Mu.l.
The gradient elution process is as follows: (1) The volume ratio of mobile phase a to mobile phase B was kept constant at 85; (2) The volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 85 to 61; (3) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 61 to 30.
Sample preparation:
taking a sample of the benzhexol hydrochloride and impurities 1, 2, 3,4, 5, 6, 7, 8 and 9, adding a solvent (a mixed solution of a mobile phase A and a mobile phase B with a volume ratio of 70.
And (3) test operation: and (3) sampling 20 mu l of the mixed solution of the diphenhydrasol hydrochloride and the impurities, and recording a chromatogram, wherein the chromatogram is shown in figure 5.
As can be seen from fig. 5, the respective impurities were separated from the main component and the respective impurities at the baseline, and the degree of separation was good.
Example 5
High performance liquid chromatography conditions:
octadecylsilane chemically bonded silica is used as a filler, a chromatographic column is Inertsil ODS-3,4.6mm multiplied by 150mm,5 mu m; the preparation process of the mobile phase A with 10mmol/L potassium dihydrogen phosphate aqua includes the following steps: weighing 1.4g of anhydrous potassium dihydrogen phosphate, adding 1000mL of water for dissolving, and adjusting the pH value to 4.2 by using phosphoric acid; 0.05 percent phosphoric acid-acetonitrile solution is taken as a mobile phase B; carrying out gradient elution according to the following gradient elution process; the flow rate is 1.2mL/min per minute; the column temperature is 30 ℃; the detection wavelength is 210nm; the injection volume was 20. Mu.l.
The gradient elution process is as follows: (1) The volume ratio of mobile phase a to mobile phase B was kept constant at 85; (2) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 85 to 61; (3) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 61 to 30.
Sample preparation:
taking a sample of the benzhexol hydrochloride and impurities 1, 2, 3,4, 5, 6, 7, 8 and 9, adding a solvent (a mixed solution of a mobile phase A and a mobile phase B with a volume ratio of 70.
And (3) test operation: and (3) sampling 20 mu l of the mixed solution of the diphenhydrasol hydrochloride and the impurities, and recording a chromatogram, wherein the chromatogram is shown in figure 6.
As can be seen from fig. 6, the respective impurities were separated from the main component and from each other by the baseline, and the degree of separation was good.
Example 6
High performance liquid chromatography conditions:
octadecylsilane chemically bonded silica is used as a filler, a chromatographic column is Inertsil ODS-3,4.6mm multiplied by 150mm,5 mu m; the preparation process of the mobile phase A with 10mmol/L potassium dihydrogen phosphate aqua includes the following steps: weighing 1.4g of anhydrous potassium dihydrogen phosphate, adding 1000mL of water for dissolving, and adjusting the pH value to 4.0 by using phosphoric acid; 0.05% phosphoric acid-acetonitrile solution as mobile phase B; gradient elution is carried out according to the following gradient elution process; the flow rate is 1.2mL/min per minute; the column temperature is 35 ℃; the detection wavelength is 210nm; the injection volume was 20. Mu.l.
The gradient elution process is as follows: (1) The volume ratio of mobile phase a to mobile phase B was kept constant at 85; (2) The volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 85 to 61; (3) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 61.
Sample preparation:
taking a diphenhydrasol hydrochloride sample and impurities 1, 2, 3,4, 5, 6, 7, 8 and 9, adding a solvent (a mixed solution of a mobile phase A and a mobile phase B with a volume ratio of 70.
And (3) test operation: and (3) sampling 20 mu l of the mixed solution of the diphenhydrasol hydrochloride and the impurities, recording a chromatogram, wherein the chromatogram is shown in figure 7.
As can be seen from fig. 7, the respective impurities were separated from the main component and from each other by the baseline, and the degree of separation was good.
Example 7
High performance liquid chromatography conditions:
octadecylsilane chemically bonded silica is used as a filler, and a chromatographic column is YMCPack ODS-AQ,4.6mm multiplied by 150mm,5 mu m; the preparation process of the mobile phase A with 10mmol/L potassium dihydrogen phosphate water solution is as follows: weighing 1.4g of anhydrous potassium dihydrogen phosphate, adding 1000mL of water for dissolving, and adjusting the pH value to 4.0 by using phosphoric acid; 0.05% phosphoric acid-acetonitrile solution as mobile phase B; carrying out gradient elution according to the following gradient elution process; the flow rate is 1.2mL/min per minute; the column temperature is 30 ℃; the detection wavelength is 210nm; the injection volume was 20. Mu.l.
The gradient elution process is as follows: (1) The volume ratio of mobile phase a to mobile phase B was kept constant at 85; (2) The volume ratio of the mobile phase A to the mobile phase B is uniformly graded from 85 to 61; (3) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 61 to 30.
Sample preparation:
taking a diphenhydrasol hydrochloride sample and impurities 1, 2, 3,4, 5, 6, 7, 8 and 9, adding a solvent (a mixed solution of a mobile phase A and a mobile phase B with a volume ratio of 70.
And (3) test operation: and (3) sampling 20 mu l of the mixed solution of the diphenhydrasol hydrochloride and the impurities, recording a chromatogram, wherein the chromatogram is shown in figure 8.
As can be seen from FIG. 8, the impurities were separated from the main component and the impurities by baseline separation, the degree of separation was good, and the retention time of the main component and the impurities was delayed from that of the Inertsil ODS-3 column.
Comparative example 1
High performance liquid chromatography conditions:
octadecylsilane chemically bonded silica is used as a filler, and a chromatographic column is Inertsil ODS-3,4.6mm multiplied by 150mm,5 mu m; the preparation process of the mobile phase A with 10mmol/L potassium dihydrogen phosphate aqua includes the following steps: weighing 1.4g of anhydrous potassium dihydrogen phosphate, adding 1000mL of water for dissolving, and adjusting the pH value to 4.0 by using phosphoric acid; 0.05% phosphoric acid-acetonitrile solution as mobile phase B; carrying out gradient elution according to the following gradient elution process; the flow rate is 1.2mL/min per minute; the column temperature is 30 ℃; the detection wavelength is 210nm; the injection volume was 20. Mu.l.
The gradient elution process is as follows: (1) The volume ratio of mobile phase a to mobile phase B was kept constant at 85; (2) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 85 to 40; (3) The volume ratio of mobile phase a to mobile phase B was kept constant at 40.
Sample preparation:
And (3) test operation: and (5) sampling 20 mu l of impurity mixed solution, recording a chromatogram, wherein the chromatogram is shown in figure 9.
As can be seen from fig. 9, the separation effect was poor because the peak-off times of impurity 8 and impurity 1 (6.395 min) overlapped.
Comparative example 2
High performance liquid chromatography conditions:
octadecylsilane chemically bonded silica is used as a filler, a chromatographic column is Inertsil ODS-3,4.6mm multiplied by 150mm,5 mu m; the preparation process of the mobile phase A with 10mmol/L potassium dihydrogen phosphate water solution is as follows: weighing 1.4g of anhydrous potassium dihydrogen phosphate, adding 1000mL of water for dissolving, and adjusting the pH value to 4.0 by using phosphoric acid; 0.05% phosphoric acid-acetonitrile solution as mobile phase B; gradient elution is carried out according to the following gradient elution process; the flow rate is 1.2mL/min per minute; the column temperature was 25 ℃; the detection wavelength is 210nm; the injection volume was 20. Mu.l.
The gradient elution process is as follows: (1) The volume ratio of mobile phase a to mobile phase B was kept constant at 85; (2) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 85 to 61; (3) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 61 to 30.
Sample preparation:
taking a diphenhydrasol hydrochloride sample and impurities 1, 2, 3,4, 5, 6, 7, 8 and 9, adding a solvent (a mixed solution of a mobile phase A and a mobile phase B with a volume ratio of 70.
And (3) test operation: and (3) sampling 20 mu l of the mixed solution of the diphenhydrasol hydrochloride and the impurities, and recording a chromatogram, wherein the chromatogram is shown in a figure 10.
As can be seen from fig. 10, impurity 4 (32.825 min) and impurity 9 (33.322 min) did not achieve baseline separation, and the separation effect was poor.
Comparative example 3
High performance liquid chromatography conditions:
octadecylsilane chemically bonded silica is used as a filler, and a chromatographic column is Inertsil ODS-3,4.6mm multiplied by 150mm,5 mu m; adjusting the pH value to 4.0 with 10mmol/L triethylamine water solution by using phosphoric acid to serve as a mobile phase A;0.05% phosphoric acid-acetonitrile solution as mobile phase B; carrying out gradient elution according to the following gradient elution process; the flow rate is 1.2mL/min per minute; the column temperature is 30 ℃; the detection wavelength is 210nm; the injection volume was 20. Mu.l.
The gradient elution process is as follows: (1) The volume ratio of mobile phase a to mobile phase B was kept constant at 85; (2) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 85 to 61; (3) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 61 to 30.
Sample preparation:
taking a sample of the benzhexol hydrochloride and impurities 1, 2, 3,4, 5, 6, 7, 8 and 9, adding a solvent (a mixed solution of a mobile phase A and a mobile phase B with a volume ratio of 70.
And (3) test operation: and (3) sampling 20 mu l of the mixed solution of the diphenhydrasol hydrochloride and the impurities, and recording a chromatogram, wherein the chromatogram is shown in figure 11.
As is clear from fig. 11, the main component peak retention time coincides with the peak appearance time of the impurities 2 and 7, and the impurity 3 cannot appear, resulting in poor separation effect.
Claims (10)
1. A detection method for related substances in trihexyphenidyl hydrochloride is characterized in that the detection method adopts high performance liquid chromatography to carry out quantitative detection on the trihexyphenidyl hydrochloride related substances, and the conditions of the high performance liquid chromatography comprise:
the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column; the column temperature is 30-40 ℃; taking a mobile phase A and a mobile phase B as a mixed mobile phase to carry out gradient elution, wherein the mobile phase A is a potassium dihydrogen phosphate aqueous solution of 5-50 mmol/L, and the pH value of the potassium dihydrogen phosphate aqueous solution is adjusted to 3.5-4.5 by phosphoric acid; the mobile phase B is 0.01 to 0.1 percent phosphoric acid-acetonitrile solution; the specific gradient elution procedure was as follows: (1) The volume ratio of mobile phase a to mobile phase B was kept constant at 85; (2) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 85 to 61; (3) The volume ratio of mobile phase a to mobile phase B was uniformly graded from 61 to 30.
2. The detection method according to claim 1, wherein the mobile phase A is 10mmol/L potassium dihydrogen phosphate water solution, and the pH value is adjusted to 3.8-4.2 by phosphoric acid; the mobile phase B is 0.03-0.07% phosphoric acid-acetonitrile solution, preferably 0.05% phosphoric acid-acetonitrile solution.
3. The detection method according to claim 2, wherein the mobile phase A is prepared by the following method: weighing 1.4g of anhydrous potassium dihydrogen phosphate, adding 1000mL of water for dissolving, and adjusting the pH value to 3.8-4.2 by using phosphoric acid to obtain the potassium dihydrogen phosphate; preferably, the pH is adjusted to 4.0 with phosphoric acid.
4. The detection method as claimed in claim 1, wherein the chromatographic column is Inertsil ODS-3 or YMCPack ODS-AQ.
5. The detection method according to claim 4, wherein the chromatographic column has a length of 150mm, a diameter of 4.6mm, and a packing particle size of 3.5 μm.
6. The detection method according to claim 1, wherein the high performance liquid chromatography conditions further comprise: the detection wavelength is 200 to 220nm, preferably 210nm.
7. The detection method according to claim 1, wherein the high performance liquid chromatography conditions further comprise: the column temperature is 30 to 35 ℃, preferably 30 ℃.
8. The detection method according to claim 1, wherein the high performance liquid chromatography conditions further comprise: the flow rate is 1.1-1.3 mL/min; preferably 1.2mL/min.
9. The detection method according to claim 1, wherein the high performance liquid chromatography conditions further comprise: the sample amount is 10-50 mu L; preferably 20. Mu.L.
10. The detection method according to claim 1, wherein the substances of interest are as follows:
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113552232A (en) * | 2020-04-24 | 2021-10-26 | 江苏天士力帝益药业有限公司 | Method for measuring content of impurity 1-phenyl-2-propenyl-1-ketone in trihexyphenidyl hydrochloride by high performance liquid phase method |
| CN113552240A (en) * | 2020-04-25 | 2021-10-26 | 江苏天士力帝益药业有限公司 | Method for measuring content of impurity 3-phenyl-1- (piperidine-1-yl) penta-3-alkoxide in diphenhydrasol hydrochloride |
| WO2021212535A1 (en) * | 2020-04-25 | 2021-10-28 | 江苏天士力帝益药业有限公司 | Method for refining benzhexol hydrochloride |
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| CN113552232A (en) * | 2020-04-24 | 2021-10-26 | 江苏天士力帝益药业有限公司 | Method for measuring content of impurity 1-phenyl-2-propenyl-1-ketone in trihexyphenidyl hydrochloride by high performance liquid phase method |
| CN113552240A (en) * | 2020-04-25 | 2021-10-26 | 江苏天士力帝益药业有限公司 | Method for measuring content of impurity 3-phenyl-1- (piperidine-1-yl) penta-3-alkoxide in diphenhydrasol hydrochloride |
| WO2021212535A1 (en) * | 2020-04-25 | 2021-10-28 | 江苏天士力帝益药业有限公司 | Method for refining benzhexol hydrochloride |
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