CN114088846A - Detection method of scopine and impurities thereof - Google Patents
Detection method of scopine and impurities thereof Download PDFInfo
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- CN114088846A CN114088846A CN202210057194.3A CN202210057194A CN114088846A CN 114088846 A CN114088846 A CN 114088846A CN 202210057194 A CN202210057194 A CN 202210057194A CN 114088846 A CN114088846 A CN 114088846A
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- 239000012535 impurity Substances 0.000 title claims abstract description 125
- 238000001514 detection method Methods 0.000 title claims abstract description 65
- FIMXSEMBHGTNKT-UHFFFAOYSA-N Scopine Natural products CN1C2CC(O)CC1C1C2O1 FIMXSEMBHGTNKT-UHFFFAOYSA-N 0.000 title claims abstract description 59
- FIMXSEMBHGTNKT-RZVDLVGDSA-N scopine Chemical compound C([C@@H]1N2C)[C@H](O)C[C@@H]2[C@@H]2[C@H]1O2 FIMXSEMBHGTNKT-RZVDLVGDSA-N 0.000 title claims abstract description 59
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- 238000000034 method Methods 0.000 claims abstract description 44
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- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 28
- 238000010828 elution Methods 0.000 claims abstract description 17
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims abstract description 12
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims abstract description 5
- 238000010829 isocratic elution Methods 0.000 claims description 25
- 239000002245 particle Substances 0.000 claims description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000000945 filler Substances 0.000 claims description 9
- 230000003247 decreasing effect Effects 0.000 claims description 8
- 239000000741 silica gel Substances 0.000 claims description 7
- 229910002027 silica gel Inorganic materials 0.000 claims description 7
- 229910000396 dipotassium phosphate Inorganic materials 0.000 claims description 4
- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 4
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 4
- 235000019800 disodium phosphate Nutrition 0.000 claims description 4
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 4
- 239000001488 sodium phosphate Substances 0.000 claims description 4
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
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- 238000012856 packing Methods 0.000 claims 1
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
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- LERNTVKEWCAPOY-VOGVJGKGSA-N C[N+]1(C)[C@H]2C[C@H](C[C@@H]1[C@H]1O[C@@H]21)OC(=O)C(O)(c1cccs1)c1cccs1 Chemical compound C[N+]1(C)[C@H]2C[C@H](C[C@@H]1[C@H]1O[C@@H]21)OC(=O)C(O)(c1cccs1)c1cccs1 LERNTVKEWCAPOY-VOGVJGKGSA-N 0.000 description 1
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- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- -1 scopine Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
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- 229960000257 tiotropium bromide Drugs 0.000 description 1
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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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
- G01N30/86—Signal analysis
- G01N30/8675—Evaluation, i.e. decoding of the signal into analytical information
- G01N30/8679—Target compound analysis, i.e. whereby a limited number of peaks is analysed
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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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/065—Preparation using different phases to separate parts of sample
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Abstract
The application belongs to the technical field of scopine, and particularly relates to a detection method of scopine and impurities thereof. The application provides a detection method of scopine and impurities thereof, which comprises the following steps: detecting scopine by high performance liquid chromatography; the mobile phase in the detection process of the high performance liquid chromatography is mobile phase A and mobile phase B; the mobile phase A is dipotassium hydrogen phosphate aqueous solution or disodium hydrogen phosphate aqueous solution, and the mobile phase B is acetonitrile aqueous solution; the pH of mobile phase A was 9.8; the high performance liquid detection process adopts a gradient elution mode to enable the mobile phase to pass through a chromatographic column. The application provides a detection method of scopine and impurities thereof, which can simultaneously and accurately detect 13 impurities in scopine.
Description
Technical Field
The application belongs to the technical field of scopine, and particularly relates to a detection method of scopine and impurities thereof.
Background
Both chemical raw material medicines and pharmaceutical preparations need to be continuously updated and developed on the basis of ensuring the stable and controllable quality of the medicines. The drug may bring or generate new impurities in the processes of starting materials, process synthesis, preparation and storage. Intermediate compounds such as scopine, which is a raw material for synthesizing tiotropium bromide, can be used in drug synthesis, but are easy to liquefy and unstable when exposed to air, and impurities of the compound can influence the synthesis of drugs and directly influence the clinical safety of the drugs. Therefore, the control of the scopine related substances (impurities) is a key project for product development and quality control. And no related technical method is available for simultaneously detecting scopine and related substances (impurities) thereof.
Disclosure of Invention
In view of this, the present application provides a method for detecting scopine and its impurities, which can simultaneously and accurately detect 13 impurities in scopine.
The application provides a detection method of scopine and impurities thereof, which comprises the following steps:
detecting scopine by high performance liquid chromatography;
the mobile phase in the detection process of the high performance liquid chromatography is a mobile phase A and a mobile phase B; the mobile phase A is a dipotassium phosphate aqueous solution or a disodium phosphate aqueous solution, and the mobile phase B is an acetonitrile aqueous solution; the pH value of the mobile phase A is 9.7-9.9;
in the detection process of the high performance liquid chromatography, a gradient elution mode is adopted to enable a mobile phase to pass through a chromatographic column, and the gradient elution method comprises the following steps:
performing isocratic elution on the mobile phase A and the mobile phase B within 0-5min, wherein the mass content of the mobile phase A in the mobile phase is 80% -95%, and the mass content of the mobile phase B in the mobile phase is 5% -20%;
in 5-15min, the mass content of the mobile phase A in the mobile phase is reduced from 80-95% to 15-30%, and the mass content of the mobile phase B in the mobile phase is increased from 5-20% to 70-85%;
performing isocratic elution on the mobile phase A and the mobile phase B within 15-20min, wherein the mass content of the mobile phase A in the mobile phase is 15-30%, and the mass content of the mobile phase B in the mobile phase is 70-85%;
in 20-21min, the mass content of the mobile phase A in the mobile phase is increased from 15% -30% to 80% -95%, and the mass content of the mobile phase B in the mobile phase is decreased from 70% -85% to 5% -20%;
and performing isocratic elution on the mobile phase A and the mobile phase B within 21-25min, wherein the mass content of the mobile phase A in the mobile phase is 80% -95%, and the mass content of the mobile phase B in the mobile phase is 5% -20%.
In another embodiment, the filler of the chromatographic column in the high performance liquid chromatography detection process is an octadecyl bonded silica gel or waters chromatographic column.
In another embodiment, the particle size of the chromatographic column filler is 3 μm to 5 μm.
In another embodiment, in the mobile phase A, the mass concentration of the dipotassium phosphate aqueous solution is 5-40 mmol/L, and the mass concentration of the disodium phosphate aqueous solution is 5-40 mmol/L.
In another embodiment, the volume fraction of the acetonitrile aqueous solution is 50% to 100%.
In another embodiment, the flow rate of the mobile phase in the high performance liquid chromatography detection process is 0.5-1.5 mL/min; the temperature of the chromatographic column is 15-25 ℃.
In another embodiment, the detection wavelength in the high performance liquid chromatography detection process is 200 nm-210 nm.
In another embodiment, the gradient elution method is:
performing isocratic elution on the mobile phase A and the mobile phase B within 0-5min, wherein the mass content of the mobile phase A in the mobile phase is 95%, and the mass content of the mobile phase B in the mobile phase is 5%;
in 5 min-15 min, the mass content of the mobile phase A in the mobile phase is reduced from 95% to 30%, and the mass content of the mobile phase B in the mobile phase is increased from 5% to 70%;
performing isocratic elution on the mobile phase A and the mobile phase B within 15-20min, wherein the mass content of the mobile phase A in the mobile phase is 30%, and the mass content of the mobile phase B in the mobile phase is 70%;
in 20-21min, the mass content of the mobile phase A in the mobile phase is increased to 95% from 30%, and the mass content of the mobile phase B in the mobile phase is decreased to 5% from 70%;
and (3) performing isocratic elution on the mobile phase A and the mobile phase B within 21-25min, wherein the mass content of the mobile phase A in the mobile phase is 95%, and the mass content of the mobile phase B in the mobile phase is 5%.
Specifically, the gradient elution method comprises the following steps:
performing isocratic elution on the mobile phase A and the mobile phase B within 0-5min, wherein the mass content of the mobile phase A in the mobile phase is 80%, and the mass content of the mobile phase B in the mobile phase is 20%;
in 5 min-15 min, the mass content of the mobile phase A in the mobile phase is reduced from 80% to 15%, and the mass content of the mobile phase B in the mobile phase is increased from 20% to 85%;
performing isocratic elution on the mobile phase A and the mobile phase B within 15-20min, wherein the mass content of the mobile phase A in the mobile phase is 15%, and the mass content of the mobile phase B in the mobile phase is 85%;
in 20-21min, the mass content of the mobile phase A in the mobile phase is increased from 15% to 80%, and the mass content of the mobile phase B in the mobile phase is decreased from 85% to 20%;
and (3) performing isocratic elution on the mobile phase A and the mobile phase B within 21-25min, wherein the mass content of the mobile phase A in the mobile phase is 80%, and the mass content of the mobile phase B in the mobile phase is 20%.
In another embodiment, the chromatographic column in the high performance liquid chromatography detection process is a C18 chromatographic column; the mobile phase A is a 10mmol/L disodium hydrogen phosphate aqueous solution, the mobile phase B is a 50% acetonitrile aqueous solution in volume fraction, and the pH value of the mobile phase A is 9.8; the flow rate of the mobile phase is 1 mL/min; the detection wavelength was 206 nm.
In another embodiment, the impurities in scopine include one or more of formula 1-formula 13;
Different from the conventional method for separating substances by a chromatographic column, the detection method disclosed by the application elutes impurities in scopine by using an alkaline mobile phase so as to separate spatial isomers, and separates configuration isomers (impurities of scopine) under a comparative limit condition, so that the problem of chromatographic column screening can be omitted. Performing gradient elution by using a mobile phase with a specific pH range, and performing high performance liquid chromatography detection by using high performance liquid chromatography at a proper flow rate and mobile phase content to analyze related substances in a sample solution. The method can effectively and simultaneously detect the impurities in 13 scopine, and the experimental data show that the method has high detection sensitivity and good specificity.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a high performance liquid chromatogram of an impurity mixed solution provided in example 1 of the present application;
FIG. 2 is a high performance liquid chromatogram of the impurity mixed solution provided in example 2 of the present application;
FIG. 3 is a high performance liquid chromatogram of the impurity mixed solution provided in example 3 of the present application;
FIG. 4 is a high performance liquid chromatogram of a sample spiking solution provided in example 4 of the present application;
FIG. 5 is a high performance liquid chromatogram of the impurity mixed solution provided in comparative example 1 of the present application;
fig. 6 is a high performance liquid chromatogram of the impurity mixed solution provided in comparative example 2 of the present application.
Detailed Description
The application provides a detection method of scopine and impurities thereof, which can simultaneously and accurately detect the scopine and 13 scopine impurities.
The technical solutions in the embodiments of the present application will be described clearly and completely below, and it should be understood that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
The raw materials and reagents used in the following examples are commercially available or self-made.
The structural formulas of the impurities 1 to 13 in the following examples are respectively represented by formulas 1 to 13, and the numbers correspond to one another, as shown in table 1 below.
TABLE 1
Example 1
The embodiment of the application provides a detection method of scopine and impurities thereof, which adopts a high performance liquid chromatography to detect scopine and comprises the following steps:
(1) an ultraviolet detector of a high performance liquid chromatograph is used, the wavelength of the ultraviolet detector is 206nm, and a chromatographic column with C18 bonded silica gel particles with the particle size of 3.5 mu m as a filler is selected. The mobile phase A is 10mmol/L disodium hydrogen phosphate aqueous solution (pH value is 9.8 by acid liquor or alkali liquor), and the mobile phase B is acetonitrile aqueous solution with volume fraction of 50%. In the high performance liquid detection process, a gradient elution mode is adopted to enable the mobile phase to pass through the chromatographic column, and the gradient elution program is set according to the following table 2, namely when 0-5min is carried out, isocratic elution is carried out on the mobile phase A80% and the mobile phase B20%, then the proportion of the mobile phase A is gradually reduced, and the proportion of the mobile phase B is increased. 5-15min, mobile phase A (80%) dropped to (15%) and mobile phase B (20%) increased to (85%); 15-20min for the mobile phase A to maintain 15% isocratic elution, and 85% for the mobile phase B to maintain isocratic elution; mobile phase a (15%) increased to (80%) and mobile phase B (85%) decreased to (20%) for 20-21 min; the mobile phase A keeps 80% isocratic elution and the mobile phase B keeps 20% isocratic elution for 21-25 min.
Table 2 gradient elution table for example 1
(2) Preparation of impurity mixed solution
The impurities were taken out in the corresponding volumetric flask according to the following table 3 and dissolved by adding methanol. The mixture was diluted to the scale with a blank solvent (50% acetonitrile in water) and shaken to prepare a mother liquor of each impurity. Precisely measuring 1mL of impurity 1 mother liquor and other impurity mother liquors (impurity 2-impurity 13 mother liquor) in volumetric flasks of 0.1mL to 10mL respectively; make up to 10mL with the blank solvent, shake well.
Table 3 table for preparation of mother liquor of each impurity of example 1
(3) The impurity mixed solution was sampled at 10. mu.L, the flow rate was 1.0mL/min, and the column temperature was 18 ℃. The detection pattern of the mixed solution of the impurities of scopine is shown in figure 1. In fig. 1, each peak was injected separately for localization, and as a result, it can be seen that one peak in fig. 1 is scopine, and 11 impurities (in the figure, the individual peak and other peaks are combined into the same peak) are detected at the same time.
Example 2
The embodiment of the application provides a detection method of scopine and impurities thereof, which adopts a high performance liquid chromatography to detect scopine and comprises the following steps:
(1) an ultraviolet detector of a high performance liquid chromatograph is used, the wavelength of the ultraviolet detector is 206nm, and a chromatographic column with C18 bonded silica gel particles with the particle size of 3.5 mu m as a filler is selected. The mobile phase A is 10mmol/L disodium hydrogen phosphate aqueous solution (pH value is 9.8 by acid liquor or alkali liquor), and the mobile phase B is acetonitrile aqueous solution with volume fraction of 50%. In the high performance liquid detection process, a gradient elution mode is adopted to enable the mobile phase to pass through the chromatographic column, and the gradient elution program is set according to the following table 4, namely when 0-5min is carried out, 95% of the mobile phase A and 5% of the mobile phase B are subjected to isocratic elution, then the proportion of the mobile phase A is gradually reduced, and the proportion of the mobile phase B is increased. 5-15min, mobile phase A (95%) decreased to (30%) and mobile phase B (5%) increased to (70%); keeping 30% isocratic elution of mobile phase A and 70% isocratic elution of mobile phase B for 15-20 min; mobile phase a (30%) increased to (95%) and mobile phase B (70%) decreased to (5%) for 20-21 min; the mobile phase A keeps 95% isocratic elution and the mobile phase B keeps 5% isocratic elution for 21-25 min.
Table 4 gradient elution table for example 1
(2) Preparing an impurity mixed solution: the same as in Table 3 of example 1.
(3) And taking the impurity mixed solution for testing. The sample introduction amount was 10. mu.L, the flow rate was 0.9mL/min, and the column temperature was 18 ℃. The detection pattern of the mixed solution of the impurities of scopine is shown in FIG. 2. In fig. 2, each peak was injected separately for localization, and as a result, it can be seen that one peak in fig. 2 is scopine, and 11 impurities (in the figure, the individual peak and other peaks are combined into the same peak) are detected at the same time.
Example 3
The embodiment of the application provides a detection method of scopine and impurities thereof, which adopts a high performance liquid chromatography to detect scopine and comprises the following steps:
(1) a high performance liquid chromatograph ultraviolet detector is used, the wavelength of the ultraviolet detector is set to be 206nm, and a chromatographic column with C18 bonded silica gel particles with the particle size of 3.5 mu m as a filler is selected. The mobile phase A is 10mmol/L disodium hydrogen phosphate aqueous solution (pH value is 9.8 by acid liquor or alkali liquor), and the mobile phase B is acetonitrile aqueous solution with volume fraction of 50%. The elution gradient pattern for mobile phase a and mobile phase B was set according to table 4 above.
(2) Preparing an impurity mixed solution: the same as in Table 3 of example 1.
(3) And taking the impurity mixed solution for testing. The sample introduction amount was 10. mu.L, the flow rate was 1.0mL/min, and the column temperature was 19 ℃. The detection pattern of the mixed solution of the impurities of scopine is shown in FIG. 3. In fig. 3, each peak was injected separately for localization, and as a result, it can be seen that one peak in fig. 3 is scopine and 10 impurities are detected simultaneously (in the figure, the individual peak and other peaks are combined into the same peak).
Example 4
The embodiment of the application provides a detection method of scopine and impurities thereof, which adopts a high performance liquid chromatography to detect scopine and comprises the following steps:
(1) a high performance liquid chromatograph ultraviolet detector is used, the wavelength of the ultraviolet detector is set to be 206nm, and a chromatographic column with C18 bonded silica gel particles with the particle size of 3.5 mu m as a filler is selected. The mobile phase A is 10mmol/L disodium hydrogen phosphate aqueous solution (pH value is 9.8 by acid liquor or alkali liquor), and the mobile phase B is acetonitrile aqueous solution with volume fraction of 50%. The elution gradient pattern for mobile phase a and mobile phase B was set according to table 4 above.
(2) Preparing a sample labeling solution:
taking about 40mg of a test sample (scopine), precisely weighing, placing in a 10mL volumetric flask, adding a blank solvent (50% acetonitrile aqueous solution) to dissolve the test sample, carrying out ultrasonic measurement if necessary, precisely weighing 1mL of impurity 1 mother liquor, adding 0.1mL of other impurity mother liquor (impurity 2-impurity 13 mother liquor) respectively, filling the blank solvent to a scale of 10mL, and shaking uniformly (preparing each impurity mother liquor as shown in the table 3 of the example 1) to prepare a test sample labeling solution.
(3) And adding a standard solution into the sample to be tested for testing. The sample introduction was 10. mu.L, the flow rate was 1.0mL/min, and the column temperature was 18 ℃. The detection spectrum of the sample solution is shown in FIG. 4. In fig. 4, each peak was injected separately for localization, and as a result, it can be seen that one peak in fig. 4 is scopine, and 10 impurities are detected simultaneously (in the figure, the individual peak and other peaks are combined into the same peak).
Example 5
The embodiment of the application provides a detection method of scopine and impurities thereof, which adopts a high performance liquid chromatography to detect scopine; the method is then validated methodologically, with validation items including system applicability, specificity, quantitation limit, detection limit, linearity, precision (including repeatability and intermediate precision), solution stability and durability, including:
(1) a high performance liquid chromatograph ultraviolet detector is used, the wavelength of the ultraviolet detector is set to be 206nm, and a chromatographic column with C18 bonded silica gel particles with the particle size of 3.5 mu m as a filler is selected. Mobile phase a was 10mmol/L aqueous disodium hydrogen phosphate solution and mobile phase B was 50% volume fraction aqueous acetonitrile. The elution gradient pattern for mobile phase a and mobile phase B was set according to table 4 above. The sample introduction was 10. mu.L, the flow rate was 1.0mL/min, and the column temperature was 18 ℃.
(2) Preparation of the solution
a. Preparing a system adaptive solution:
taking 20mg of scopine control and 2mg of impurity, placing the scopine control and the impurity into a 200mL volumetric flask, adding water to dissolve and dilute the scopine control to the scale, and shaking up the volume.
b. Preparing a test solution:
taking about 40mg of a test sample (scopine), precisely weighing, placing in a 10mL volumetric flask, adding a proper amount of a blank solvent (50% acetonitrile aqueous solution) for dissolving, diluting to a scale with the blank solvent, and shaking up.
c. Preparing a reference substance stock solution:
taking a scopine control product 20mg to 10mL in a volumetric flask, diluting the scopine control product to the scale with a blank solvent, and shaking up.
d. Preparing a reference substance solution:
precisely measuring the reference substance stock solution in a volumetric flask with the volume of 1-100 mL, diluting the reference substance stock solution to the scale with a blank solvent, and shaking up.
e. Preparing mother liquor of each impurity: the same as in Table 3 of example 1.
f. Preparing an impurity 1 positioning solution:
precisely measuring the impurity 1 mother liquor in a volumetric flask with 1-10 mL, diluting the volumetric flask with a blank solution to a scale, and shaking up.
g. Other impurity-locating solutions except impurity 1:
precisely measuring other impurities (2-13 impurities) mother liquor in a volumetric flask of 0.5mL to 50 mL. Dilute to the mark with the blank solution and shake up.
h. Preparing an impurity mixed solution:
precisely measuring 1mL of impurity 1 reference substance stock solution and other impurity mother solutions (impurity 2-impurity 13) in volumetric flasks of 0.1mL to 10mL respectively. Diluting with blank solvent to scale, and shaking up.
i. Preparing an impurity reference substance solution:
precisely measuring 10mL of impurity 1, and comparing with a stock solution and an impurity 2 mother solution. Respectively diluting the impurity 4 mother liquor and the impurity 5 mother liquor to the scale with a blank solvent in volumetric flasks of 1-100 mL, and shaking up.
j. Preparing a sample labeling solution:
taking about 40mg of a sample, precisely weighing, placing in a 10mL volumetric flask, adding a proper amount of blank solvent to dissolve the sample, and precisely weighing 1mL of impurity 1 by ultrasonic waves if necessary; adding 0.1mL of other impurity mother liquor into the mixture, diluting the mixture to a scale with a blank solvent, and shaking up.
k. Preparation of a linear mother solution:
precisely weighing about 60mg to 10mL of impurity 1 in a volumetric flask, adding 1mL of reference stock solution, impurity 2 mother solution, impurity 4 mother solution and impurity 5 mother solution, diluting to scale with a blank solvent, and shaking uniformly.
l, preparing quantitative limit and detection limit solutions of impurities 3 and 13:
5mL of each of the impurity 3 and the impurity 13 was precisely measured and added dropwise to a 10mL volumetric flask. Diluting the blank solution to a scale, and shaking up to be used as a quantitative limit solution of the impurity 3 and the impurity 8; 5mL of each of the impurity 3 and the impurity 13 was precisely measured and added dropwise to a 10mL volumetric flask. The blank solution was filled to 10mL scale and shaken up to be used as a limiting solution for the quantification of impurity 3 and impurity 8.
m, preparing a quantitative limit and detection limit solution for other impurities except for the impurity 3 and the impurity 13:
precisely measuring the impurity mixed solution in a volumetric flask of 3mL to 10mL, diluting the impurity mixed solution to a scale with a blank solvent, and shaking the solution uniformly to be used as a quantitative limiting solution. Precisely measuring 1mL of impurity mixed solution (impurity mixed solution in h), dropwise adding the impurity mixed solution into a 10mL volumetric flask, filling the impurity mixed solution with water to reach a 10mL scale mark, and shaking uniformly. As a detection limiting solution.
(3) And carrying out methodology verification on the established high performance liquid chromatography analysis method of the scopine and related substances thereof according to the chromatographic parameters. The verification items include system applicability, specificity, quantitation limit, detection limit, linearity, precision (including repeatability and intermediate precision), solution stability and durability.
(4) Methodological validation results
The methodology was verified by the above method, and the verification results are shown below:
the methodological validation of the embodiments of the present application resulted in system adaptability: the system adaptability solution separation degree is 5.9; the maximum value of the impurity reference substance solution peak area RSD is 1.6%.
The methodological validation of the embodiments of the present application shows specificity results: the blank solvent has no interference to the detection; the minimum value of the separation degree of each peak and adjacent peaks in the test solution is 3.5; the minimum value of the separation degree of each peak and the adjacent peak in the sample standard solution is 2.16; the purity of each peak in the sample solution meets the specification.
Methodological validation of the examples of the present application quantitative limit results: as shown in table 5.
TABLE 5
The methodological validation of the embodiments of the present application shows the detection limit results: as shown in table 6.
TABLE 6
The methodology of the examples of the present application verifies the linear results:
the linear equation of the result of the scopine linear solution is y =1506.6448x-0.2152, the r value is 0.9998, the F value RSD is 1.4%, and the ratio of the intercept to the 100% limit concentration result is 0.72%;
② the linear solution result linear equation of the impurity 1 is y =1178.4759x-1.8862, the r value is 1.0000, the F value RSD is 0.8 percent, and the ratio of the intercept to the 100 percent limit concentration result is 0.27 percent;
③ the linear solution result of the impurity 2 has the linear equation of y =21283.0698x-0.7735, the r value is 1.0000, the F value RSD is 0.7 percent, and the ratio of the intercept to the 100 percent limit concentration result is 0.55 percent;
fourthly, the linear equation of the result of the impurity 4 linear solution is y =22076.7516x +7.326, the r value is 1.0000, the F value RSD is 0.7%, and the ratio of the intercept to the 100% limit concentration result is 0.55%;
fifthly, the linear equation of the result of the impurity 5 linear solution is y =11800.533x +5.3217, the r value is 0.9999, the F value RSD is 3.0%, and the ratio of the intercept to the 100% limit concentration result is 2.21%.
Precision (repeatability and intermediate precision) results of the methodological validation of the examples of the present application: the reproducibility results are shown in Table 7, and the intermediate precision results are shown in Table 8.
TABLE 7
TABLE 8
Solution stability results verified by the methodology of the examples of the present application: the stability of the impurity 2 in the test sample exceeds the acceptable standard at the time point of 2h, so the stability of the test sample solution at normal temperature is 1 h, the change rate of the impurity reference solution in 9 h meets the acceptable standard, but the peak width of the impurity 1 peak gradually increases in the stability process, the retention time drifts forward, and the stability of the impurity reference solution is determined to be 5 h.
Durability results of methodology validation of the examples of the present application:
the resolution of all impurity peaks of instruments with different intermediate precision and chromatographic column conditions is more than 1.5, and the acceptable standard is met.
② the separation degree of scopine and impurity 12 is less than 1.5, and the separation degree of impurity 8 and impurity 9 is less than 1.5 under the condition of 0.9ml/min flow rate; the separation degree of the impurity 10 and the impurity 5 in the flow rate condition of 1.1ml/min is 1.4 and is less than the acceptable standard 1.5;
③ the impurity 12 peak and the impurity 2 peak in the conditions of 17 ℃ and 19 ℃ are superposed into a peak which can not be separated. The degree of separation does not meet the acceptance criteria because impurities 12 and 10 drift more than adjacent peaks in retention time when the flow rate or column temperature is changed.
Therefore, the method has good durability to different instruments and chromatographic columns, and does not have durability to flow rate and column temperature.
Comparative example 1
The comparative example of the application provides a control detection method, and the detection of scopine is carried out by adopting a high performance liquid chromatography, and the method comprises the following steps:
the comparative example method is similar to example 1 except that the mobile phase a has a pH of 9.6, the remaining steps are identical to example 1, the detection profile of the mixed solution of impurities of scopine is shown in fig. 5, and as can be seen from fig. 5, the method has requirements on the pH of the mobile phase, the impurities in the first 5 minutes are sensitive to pH changes of the mobile phase a, and other impurities cannot be separated at this pH.
Comparative example 2
The comparative example of the application provides a control detection method, and the detection of scopine is carried out by adopting a high performance liquid chromatography, and the method comprises the following steps:
the comparative example method is similar to example 2 except that the mobile phase A has a pH of 10.0, the remaining steps are identical to example 2, the detection profile of the mixed solution of impurities of scopine is shown in FIG. 5, and it can be seen from FIG. 6 that the method has requirements on the pH of the mobile phase, the impurities in the first 5 minutes are sensitive to pH changes of the mobile phase A, and other impurities cannot be separated at the pH.
In summary, the detection method (1) of the present application selects a chromatographic column using octadecylsilane chemically bonded silica as a filler; gradient elution is carried out by adopting a mobile phase, wherein the phase A of the mobile phase is a water phase prepared by phosphate, and the phase B of the mobile phase is acetonitrile; the detector adopted by the high performance liquid chromatography is an ultraviolet detector; (2) and (3) carrying out high performance liquid chromatography detection at a proper flow rate and column temperature, and analyzing related substances in the sample solution. The method can effectively and simultaneously detect the impurities in 13 scopine, and the detection method has high sensitivity and good specificity.
The foregoing is only a preferred embodiment of the present application and it should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present application, and these improvements and modifications should also be considered as the protection scope of the present application.
Claims (10)
1. A method for detecting scopine and impurities thereof comprises the following steps:
detecting scopine by high performance liquid chromatography;
the mobile phase in the detection process of the high performance liquid chromatography is a mobile phase A and a mobile phase B; the mobile phase A is a dipotassium phosphate aqueous solution or a disodium phosphate aqueous solution, and the mobile phase B is an acetonitrile aqueous solution; the pH value of the mobile phase A is 9.7-9.9;
in the detection process of the high performance liquid chromatography, a gradient elution mode is adopted to enable a mobile phase to pass through a chromatographic column, and the gradient elution method comprises the following steps:
performing isocratic elution on the mobile phase A and the mobile phase B within 0-5min, wherein the mass content of the mobile phase A in the mobile phase is 80% -95%, and the mass content of the mobile phase B in the mobile phase is 5% -20%;
in 5-15min, the mass content of the mobile phase A in the mobile phase is reduced from 80-95% to 15-30%, and the mass content of the mobile phase B in the mobile phase is increased from 5-20% to 70-85%;
performing isocratic elution on the mobile phase A and the mobile phase B within 15-20min, wherein the mass content of the mobile phase A in the mobile phase is 15-30%, and the mass content of the mobile phase B in the mobile phase is 70-85%;
in 20-21min, the mass content of the mobile phase A in the mobile phase is increased from 15% -30% to 80% -95%, and the mass content of the mobile phase B in the mobile phase is decreased from 70% -85% to 5% -20%;
and performing isocratic elution on the mobile phase A and the mobile phase B within 21-25min, wherein the mass content of the mobile phase A in the mobile phase is 80% -95%, and the mass content of the mobile phase B in the mobile phase is 5% -20%.
2. The detection method according to claim 1, wherein the chromatographic column filler in the high performance liquid chromatography detection process is an octadecyl bonded silica gel or waters chromatographic column.
3. The detection method according to claim 2, wherein the particle size of the column packing is 3 to 5 μm.
4. The detection method according to claim 1, wherein the mobile phase A has a mass concentration of the aqueous solution of dipotassium phosphate of 5 to 40mmol/L and a mass concentration of the aqueous solution of disodium phosphate of 5 to 40 mmol/L.
5. The detection method according to claim 1, wherein the volume fraction of the acetonitrile aqueous solution is 50% to 100%.
6. The detection method according to claim 1, wherein the flow rate of the mobile phase in the high performance liquid chromatography detection process is 0.5-1.5 mL/min; the temperature of the chromatographic column is 15-25 ℃.
7. The detection method according to claim 1, wherein the detection wavelength in the high performance liquid chromatography detection process is 200nm to 210 nm.
8. The detection method according to claim 1, wherein the gradient elution method comprises:
performing isocratic elution on the mobile phase A and the mobile phase B within 0-5min, wherein the mass content of the mobile phase A in the mobile phase is 95%, and the mass content of the mobile phase B in the mobile phase is 5%;
in 5 min-15 min, the mass content of the mobile phase A in the mobile phase is reduced from 95% to 30%, and the mass content of the mobile phase B in the mobile phase is increased from 5% to 70%;
performing isocratic elution on the mobile phase A and the mobile phase B within 15-20min, wherein the mass content of the mobile phase A in the mobile phase is 30%, and the mass content of the mobile phase B in the mobile phase is 70%;
in 20-21min, the mass content of the mobile phase A in the mobile phase is increased to 95% from 30%, and the mass content of the mobile phase B in the mobile phase is decreased to 5% from 70%;
and (3) performing isocratic elution on the mobile phase A and the mobile phase B within 21-25min, wherein the mass content of the mobile phase A in the mobile phase is 95%, and the mass content of the mobile phase B in the mobile phase is 5%.
9. The detection method according to claim 8, wherein the chromatographic column in the high performance liquid chromatography detection process is a C18 chromatographic column; the mobile phase A is a 10mmol/L disodium hydrogen phosphate aqueous solution, the mobile phase B is a 50% acetonitrile aqueous solution in volume fraction, and the pH value of the mobile phase A is 9.8; the flow rate of the mobile phase is 1 mL/min; the detection wavelength was 206 nm.
10. The detection method according to claim 1, wherein the impurities in scopine comprise one or more of formula 1-formula 13;
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