CN111220715A - Method for separating and measuring safinamide mesylate and related substances thereof by liquid chromatography - Google Patents
Method for separating and measuring safinamide mesylate and related substances thereof by liquid chromatography Download PDFInfo
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- CN111220715A CN111220715A CN201811406645.XA CN201811406645A CN111220715A CN 111220715 A CN111220715 A CN 111220715A CN 201811406645 A CN201811406645 A CN 201811406645A CN 111220715 A CN111220715 A CN 111220715A
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- G—PHYSICS
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- 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
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- 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
- G01N30/38—Flow patterns
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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
- G01N30/38—Flow patterns
- G01N2030/388—Elution in two different directions on one stationary phase
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Abstract
The invention belongs to the field of analytical chemistry, and discloses a method for separating and determining safinamide mesylate and related substances thereof by using a liquid chromatography. The method has the advantages of strong specificity, high accuracy and simple and convenient operation.
Description
Technical Field
The invention belongs to the field of analytical chemistry, and particularly relates to a method for separating and determining safinamide mesylate and related substances thereof by liquid chromatography.
Background
The safinamide mesylate is a third-generation monoamine oxidase B (MAO-B) inhibitor, has the characteristics of high efficiency, reversibility, strong specificity and the like, and is commonly used for treating the Parkinson disease clinically. The title of the title is Safinamidesilate in English and 2(S) - [4- [ (3-Fluorobenzyloxy) benzamido]propionamide of the formula C17H19FN2O2•CH4O3And S. The structural formula of the safinamide mesylate is as follows:
in the process of synthesizing the safinamide mesylate, several important starting materials and intermediates can influence the purity of the drug due to incomplete removal, and the impurities which influence the purity of the drug are called process impurities; while the degradation impurities are generated during the production or storage of the drug. The related substances have no treatment effect, can influence the stability and curative effect of the medicament, and even harm human health, and the number of the related substances mainly controlling the synthesis of the safinamide mesylate is 3, wherein the number of the related substances is respectively as follows: the related substance 1, 4-Fluoro-benzaldehyde; the substance 2, 1-Fluoro-3- (2-iodo-ethyl) -benzene; the substance 3, 4- (3-Fluoro-benzoyl) -benzodiazepine; the structural formulas are respectively:
quality control is required for process impurities introduced in the synthesis of safinamide mesylate and degradation impurities generated during storage. Therefore, the method realizes the separation of the safinamide mesylate and the related substances thereof, and has important practical significance in the quality control of the safinamide mesylate raw material medicine and the preparation thereof.
Disclosure of Invention
The invention aims to provide a method for analyzing safinamide mesylate and related substances thereof, so that the separation and the determination of the safinamide mesylate and the related substances thereof are realized, the purity of the safinamide mesylate is ensured, and the quality control of the safinamide mesylate is realized.
The method for analyzing the safinamide mesylate and related substances thereof by using the liquid chromatography adopts a chromatographic column with phenyl silane bonded silica gel as a filler, and takes a buffer salt solution-organic phase with a certain proportion as a mobile phase.
The chromatographic column using phenyl silane bonded silica gel as a filler is selected from Agilent, YMC, Phenomene and other brands.
Said organic phase is selected from the group consisting of: acetonitrile, methanol, isopropanol, tetrahydrofuran, etc., with acetonitrile being preferred.
In the method, the mobile phase buffer salt solution-organic phase adopts gradient elution.
In the above-mentioned method, the buffer salt contained in the buffer salt solution is selected from the group consisting of phosphate, acetate, formate, perchlorate, and preferably phosphate.
Wherein the concentration of the buffered salt solution is 0.02 mol/L.
The separation and measurement method of the present invention can be realized by the following method:
1) a suitable amount of safinamide mesylate sample was taken, and the reaction solution was washed with a solvent (acetonitrile: water =1: 1) and the sample is dissolved to prepare a sample solution containing 0.1 to 1.5mg of safinamide mesylate per 1 mL.
2) Setting the flow rate of the mobile phase to be 0.5-1.5 mL/min, and preferably setting the flow rate of the mobile phase to be 1.0 mL/min; the detection wavelength is 200-230 nm, and the optimal detection wavelength is 215 nm; the temperature of the column oven is 20-40 ℃, and the temperature of the column oven is preferably 35 ℃.
3) And (2) injecting 10-50 mu L of the sample solution obtained in the step (1) into a liquid chromatograph to complete the separation and determination of the safinamide mesylate and related substances thereof. Wherein:
the type of the high performance liquid chromatograph has no special requirements, and the chromatograph adopted by the invention is Shimadzu:
LC-20AT pump, SPD-M20A detector, SIL-20AC autosampler, CBM-20A controller, CTO-10ASVP column oven, LC solution workstation
A chromatographic column: agilent (Phenyl, 250X 4.6 mm, 5 μm)
Mobile phase: a: 0.02mol/L potassium dihydrogen phosphate buffer solution (pH to 2.0), B: acetonitrile;
elution was performed with the following gradient:
| T(min) | 0 | 20 | 55 | 55.01 | 70 |
| B% | 15 | 30 | 70 | 15 | 15 |
flow rate: 1.0mL/min
Detection wavelength: 215nm
Sample introduction volume: 10 μ L
According to the invention, Agilent (Phenyl, 250 multiplied by 4.6 mm, 5 mu m) chromatographic column is adopted, so that the methanesulfonic acid safinamide and related substances thereof can be effectively separated. The invention solves the problem of separation and determination of the safinamide mesylate and related substances thereof, thereby ensuring the controllable quality of the safinamide mesylate raw material medicine and the preparation thereof.
Drawings
FIG. 1 is a HPLC chart of safinamide mesylate and related substances in example 1;
FIG. 2 is a HPLC chart of safinamide mesylate in example 1;
FIG. 3 is a blank solvent HPLC plot for example 1;
FIG. 4 is a HPLC chart of safinamide mesylate and related substances in example 2;
FIG. 5 is a HPLC chart of safinamide mesylate in example 2;
FIG. 6 is an HPLC chart of safinamide mesylate and related substances in example 3;
FIG. 7 is a HPLC chart of safinamide mesylate in example 3;
the specific implementation mode is as follows:
the present invention will be described in further detail with reference to the following examples, but it should not be construed that the scope of the above subject matter of the present invention is limited to the following examples, and that all the technologies realized based on the above contents of the present invention are within the scope of the present invention.
Example 1
Apparatus and conditions
High performance liquid chromatograph: shimadzu: LC-20AT pump, SPD-M20A detector, SIL-20AC autosampler, CBM-20A controller, CTO-10ASVP column oven, LC solution workstation
A chromatographic column: agilent (Phenyl, 250X 4.6 mm, 5 μm)
Mobile phase: a: 0.02mol/L potassium dihydrogen phosphate buffer solution (pH to 2.0), B: acetonitrile;
elution was performed with the following gradient:
| T(min) | 0 | 20 | 40 | 55 | 55.01 | 70 |
| B% | 15 | 30 | 60 | 70 | 15 | 15 |
flow rate: 1.0mL/min
Detection wavelength: 215nm
Sample introduction volume: 10 μ L
Experimental procedure
Taking a proper amount of safinamide mesylate and related substances thereof, dissolving a sample by using a solvent (acetonitrile: water =1: 1) to prepare a solution containing about 1.0mg/mL of safinamide mesylate; respectively taking a proper amount of impurity reference substances of the safinamide mesylate, dissolving the impurity reference substances by using an agent (acetonitrile: water =1: 1) to prepare an impurity mixed solution containing about 0.5mg/ml of each impurity; and taking an appropriate amount of the impurity mixed solution and the safinamide mesylate solution to prepare a system applicability solution. Performing high performance liquid chromatography analysis according to the conditions, and recording a chromatogram. The result is shown in attached figures 1-3, wherein figure 1 is a system applicability solution chromatogram, a chromatographic peak with retention time of 22.081min in the chromatogram is vortioxetine, and the rest is chromatographic peaks of various related substances; FIG. 2 is a chromatogram of safinamide mesylate, wherein the chromatographic peak with the retention time of 22.051min is safinamide mesylate; FIG. 3 is a solvent chromatogram.
Example 2
Apparatus and conditions
High performance liquid chromatograph: shimadzu: LC-20AT pump, SPD-M20A detector, SIL-20AC autosampler, CBM-20A controller, CTO-10ASVP column oven, LC solution workstation
A chromatographic column: YMC (Phenyl, 250X 4.6 mm, 5 μm)
Mobile phase: 0.02mol/L dipotassium hydrogenphosphate buffer solution (pH to 5.0), B: acetonitrile;
elution was carried out with the following gradient
| T(min) | 0 | 20 | 40 | 50 | 50.01 | 60 |
| B% | 15 | 30 | 55 | 70 | 15 | 15 |
Flow rate: 1.0mL/min
Detection wavelength: 215nm
Sample introduction volume: 10 μ L
Experimental procedure
Taking a proper amount of safinamide mesylate and related substances thereof, dissolving a sample by using a solvent (acetonitrile: water =1: 1) to prepare a solution containing about 1.0mg/mL of safinamide mesylate; respectively taking a proper amount of impurity reference substances of the safinamide mesylate, dissolving the impurity reference substances by using an agent (acetonitrile: water =1: 1) to prepare an impurity mixed solution containing about 0.5mg/ml of each impurity; and taking an appropriate amount of the impurity mixed solution and the safinamide mesylate solution to prepare a system applicability solution. Performing high performance liquid chromatography analysis according to the conditions, and recording a chromatogram. The result is shown in attached figures 4-5, and figure 4 is a system applicability solution chromatogram, in which the chromatographic peak with the retention time of 21.613min is vortioxetine, and the rest is the chromatographic peaks of various related substances; FIG. 5 is a chromatogram of safinamide mesylate, wherein the chromatographic peak with the retention time of 21.602min is safinamide mesylate;
example 3
Apparatus and conditions
High performance liquid chromatograph: shimadzu: LC-20AT pump, SPD-M20A detector, SIL-20AC autosampler, CBM-20A controller, CTO-10ASVP column oven, LC solution workstation
A chromatographic column: phenomenex (Phenyl, 250X 4.6 mm, 5 μm)
Mobile phase: 0.02mol/L ammonium acetate buffer solution (pH 4.0); phase B: acetonitrile
Elution was performed with the following gradient:
| T(min) | 0 | 30 | 40 | 48 | 48.01 | 60 |
| B% | 15 | 30 | 55 | 70 | 15 | 15 |
flow rate: 1.0mL/min
Detection wavelength: 215nm
Sample introduction volume: 10 μ L
Experimental procedure
Taking a proper amount of safinamide mesylate and related substances thereof, dissolving a sample by using a solvent (acetonitrile: water =1: 1) to prepare a solution containing about 1.0mg/mL of safinamide mesylate; respectively taking a proper amount of impurity reference substances of the safinamide mesylate, dissolving the impurity reference substances by using an agent (acetonitrile: water =1: 1) to prepare an impurity mixed solution containing about 0.5mg/ml of each impurity; and taking an appropriate amount of the impurity mixed solution and the safinamide mesylate solution to prepare a system applicability solution. Performing high performance liquid chromatography analysis according to the conditions, and recording a chromatogram. The result is shown in the attached figures 6-7, and figure 6 is a system applicability solution chromatogram, in which the chromatographic peak with the retention time of 29.458min is vortioxetine, and the rest is the chromatographic peaks of various related substances; FIG. 7 is a chromatogram of safinamide mesylate, wherein the chromatographic peak with the retention time of 29.420min is safinamide mesylate;
the following items of the analysis method of the substances related to the final product of the safinamide mesylate are verified:
system suitability test
Whether the chromatographic conditions were satisfactory or not was analyzed by using a mixed solution of safinamide mesylate and each related substance according to the chromatographic conditions determined in example 1. As can be seen from FIG. 1, under the condition, the separation degree between each related substance and the main peak meets the requirement, and both the peak purity and the single-point threshold value meet the requirement.
Stability of solution
The mixed solution of safinamide mesylate and the relevant substances thereof was injected at 0, 2, 4, 6, 8 and 12 hours according to the chromatographic conditions determined in example 1, and the stability of the solution was examined when the amount of the sample was determined, and it was found that the solution was stable within 12 hours.
Durability
Since the chromatographic conditions of the product are gradient elution and the corresponding chromatographic column model, column temperature, flow rate, pH value and the like are specified, the conditions are correspondingly finely adjusted to investigate the durability of the method. As a result, the method is found to have good durability under the conditions of chromatographic columns of different brands, column temperature variation of +/-5 ℃, flow rate variation of +/-0.2 mL/min, pH value variation of +/-0.2 and the like. Under the conditions of chromatographic columns of different brands, different column temperatures, different flow rates, different pH values and the like, the retention time of the safinamide mesylate and related substances thereof has no obvious change and can achieve the effect.
Claims (12)
1. A method for separating and measuring safinamide mesylate and related substances thereof by liquid chromatography is characterized by comprising the following steps: the chromatographic column takes phenyl silane bonded silica gel as a filler, and takes a buffer salt solution-organic phase with a certain proportion as a mobile phase.
2. The separation and measurement method according to claim 1, wherein the column is selected from columns of Agilent, YMC, Phenomene, and the like.
3. The separation assay of claim 1, wherein the organic phase is selected from one or more of the following compounds: acetonitrile, methanol, isopropanol, tetrahydrofuran, and the like.
4. The separation assay method of claim 3, wherein the organic phase is acetonitrile.
5. The isolation assay of claim 1, wherein said buffered salt solution is selected from the group consisting of: phosphates, acetates, formates, citrates, and the like.
6. The separation assay method according to claim 5, wherein the concentration of the buffer salt contained in the buffer salt solution is preferably 0.02 mol/L.
7. The isolation assay of claim 5, wherein said buffered salt solution is a buffer salt, preferably phosphate.
8. The separation assay method of claim 1, comprising the steps of:
1) a suitable amount of safinamide mesylate sample was taken, and the reaction solution was washed with a solvent (acetonitrile: water =1: 1) and dissolving the sample to prepare a sample solution containing 0.1-1.5 mg of safinamide mesylate and related substances thereof per 1 mL;
2) setting the flow rate of a mobile phase to be 1.0-2.0 mL/min, the detection wavelength to be 205-250 nm, and the temperature of a chromatographic column incubator to be 20-50 ℃;
3) and (2) injecting 10-50 mu L of the sample solution obtained in the step 1) into a liquid chromatograph to complete the separation and determination of the safinamide mesylate and related substances thereof.
9. The separation and analysis method according to claim 7, wherein the pH of the buffer is 2.0 to 5.0, preferably 3.0.
10. The separation and analysis method according to claim 8, wherein the flow rate of said mobile phase in step 2) is preferably 1.0 mL/min.
11. The separation analysis method according to claim 8, wherein said detection wavelength in step 2) is preferably 215 nm.
12. The separation and analysis method according to claim 8, wherein the temperature of said column oven of step 2) is preferably 35 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201811406645.XA CN111220715A (en) | 2018-11-23 | 2018-11-23 | Method for separating and measuring safinamide mesylate and related substances thereof by liquid chromatography |
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| CN201811406645.XA CN111220715A (en) | 2018-11-23 | 2018-11-23 | Method for separating and measuring safinamide mesylate and related substances thereof by liquid chromatography |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114965720A (en) * | 2021-02-20 | 2022-08-30 | 成都康弘药业集团股份有限公司 | Method for determining related substances of vortioxetine hydrobromide |
-
2018
- 2018-11-23 CN CN201811406645.XA patent/CN111220715A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114965720A (en) * | 2021-02-20 | 2022-08-30 | 成都康弘药业集团股份有限公司 | Method for determining related substances of vortioxetine hydrobromide |
| CN114965720B (en) * | 2021-02-20 | 2024-02-23 | 成都康弘药业集团股份有限公司 | Method for determining related substances of hydrobromic acid voltammetric acid |
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