CN111693626A - Method for detecting dehydroepiandrosterone acetate and related substances thereof and application - Google Patents
Method for detecting dehydroepiandrosterone acetate and related substances thereof and application Download PDFInfo
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Abstract
The invention relates to the technical field of chemical drug analysis methods, in particular to a detection method and application of dehydroepiandrosterone acetate and related substances thereof. The detection method of dehydroepiandrosterone acetate and related substances thereof comprises the following steps: detecting the test solution by high performance liquid chromatography; the detection conditions of the chromatogram comprise: the detection wavelength is 201-220 nm; mobile phase A: adjusting the pH of a mixture of acetonitrile, water and triethylamine to 6.4-6.7; mobile phase B: acetonitrile; in the mobile phase A, before the pH of the mixture is adjusted, the volume ratio of acetonitrile to water to triethylamine is (100-200): 897-799): 3-1; elution was carried out with a gradient using mobile phase a and mobile phase B. The detection conditions of the invention can realize one-time and high-efficiency separation of dehydroepiandrosterone acetate and related substances thereof, have strong specificity and good durability, and can be used for research and quality control of dehydroepiandrosterone acetate.
Description
Technical Field
The invention relates to the technical field of chemical drug analysis methods, in particular to a detection method and application of dehydroepiandrosterone acetate and related substances thereof.
Background
Dehydroepiandrosterone acetate, a compound 5-androstene-3 beta-ol-17-one-3-acetate, is a key material for synthetic drugs, such as abiraterone acetate, mifepristone, norethindrone and ethinylestradiol, and has the following structure:
dehydroepiandrosterone acetate is an important component of many bulk drugs, and if the dehydroepiandrosterone acetate contains impurities, the impurities or converted substances of the impurities can enter subsequent reactions during the production of the bulk drugs, so that the production quality of the bulk drugs is affected. Moreover, certain quality attributes of dehydroepiandrosterone acetate are changed under the influence of impurities, and the quality of the raw material medicines is finally influenced. Therefore, it is important for impurity control of dehydroepiandrosterone acetate.
In the prior art, some reports about a detection method of dehydroepiandrosterone acetate related substances, but due to different synthesis processes, the generated impurities are different, and the existing detection method cannot completely separate the dehydroepiandrosterone acetate from the two impurities. Therefore, it is highly desirable to provide a novel analysis method for dehydroepiandrosterone acetate, which has good specificity, repeatability and accuracy on the basis of ensuring the efficient separation of various related substances and effective components, thereby better realizing the quality control of dehydroepiandrosterone acetate.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a detection method of dehydroepiandrosterone acetate and related substances thereof, which can separate two related substances of dehydroepiandrosterone acetate at one time with high efficiency, and has strong specificity and good durability.
The second purpose of the invention is to provide the application of the detection method of dehydroepiandrosterone acetate and related substances thereof in the quality control of the raw material or preparation of the dehydroepiandrosterone acetate.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the detection method of dehydroepiandrosterone acetate and related substances thereof comprises the following steps:
detecting the test solution by high performance liquid chromatography;
the detection conditions of the high performance liquid chromatography comprise:
the detection wavelength is 201-220 nm;
mobile phase A: adjusting the pH of a mixture of acetonitrile, water and triethylamine to 6.4-6.7; mobile phase B: acetonitrile;
in the mobile phase A, before the pH of the mixture is adjusted, the volume ratio of acetonitrile to water to triethylamine is (100-200): 897-799): 3-1;
performing gradient elution by using the mobile phase A and the mobile phase B;
the process of gradient elution includes: the volume ratio of mobile phase a to mobile phase B was changed from 95: 5 to 5: 95.
In a preferred embodiment of the present invention, the process of gradient elution comprises: the volume ratio of the mobile phase A to the mobile phase B is changed from 50: 50 to 15: 85 within 0-12 min; and the volume ratio of the mobile phase A to the mobile phase B is 15: 85 within 12-40 min.
Specifically, the volume ratio of mobile phase A to mobile phase B is preferably changed from 50: 50 to 15: 85 at 0-12 min.
By adopting the detection conditions, the dehydroepiandrosterone acetate and related substances can be separated at one time and efficiently, the specificity is strong, the durability is good, and the method can be used for research and quality control of the dehydroepiandrosterone acetate.
In a specific embodiment of the present invention, the related substances include I and II, which have the following structural formulas:
wherein the related substance has a molecular formula of C19H28O2Molecular weight is 288; the related substance has a molecular formula of C19H26O3The molecular weight is 302. The polarity difference of related substances I and II is very small, and the prior art has no detection standard for related substances I and II including dehydroepiandrosterone acetate.
According to the raw material synthesis process of dehydroepiandrosterone acetate, related substances I and II contained in the dehydroepiandrosterone acetate are controlled, and the two related substances can be efficiently separated at one time by the detection condition of the high performance liquid chromatography, so that the research method of the dehydroepiandrosterone acetate related substances is established.
As in the different embodiments, the detection wavelength may be 201nm, 202nm, 203nm, 204nm, 205nm, 206nm, 207nm, 208nm, 209nm, 210nm, 211nm, 212nm, 213nm, 214nm, 215nm, 216nm, 217nm, 218nm, 219nm, 220nm, etc., preferably 205 to 215nm, more preferably 210 nm.
In a specific embodiment of the present invention, the preparation method of the mobile phase a comprises: uniformly mixing water, triethylamine and acetonitrile in proportion, and adjusting the pH to 6.4-6.7 by using a pH regulator; specifically, after water and triethylamine are mixed uniformly, acetonitrile is added, and then a pH regulator is adopted to regulate the pH value to 6.4-6.7. Wherein the pH regulator can be glacial acetic acid or sodium hydroxide aqueous solution (the concentration is 0.1-1 mol/L). Preferably, the pH of the mixture is adjusted to 6.5-6.6, preferably 6.5. In actual operation, glacial acetic acid pH regulator is adopted for regulation, and after regulation, sodium hydroxide aqueous solution pH regulator is adopted for regulation back.
As in the different embodiments, the amount of triethylamine in the mobile phase a may be 1mL, 2mL, 3mL, etc., in 1000mL before adjusting the pH of the mixture; the amount of water used can be 100mL, 105mL, 110mL, 115mL, 120mL, 125mL, 130mL, 135mL, 140mL, 145mL, 150mL, 155mL, 160mL, 165mL, 170mL, 175mL, 180mL, 185mL, 190mL, 195mL, 200mL, and the like; the amount of acetonitrile used may be 897mL, 894mL, 893mL, 892mL, 889mL, 888mL, 887mL, 884mL, 883mL, 882mL, 879mL, 878mL, 877mL, 874mL, 873mL, 872mL, 869mL, 868mL, 867mL, 864mL, 863mL, 862mL, 859mL, 858mL, 857mL, 854mL, 853mL, 852mL, 849mL, 848mL, 847mL, 845mL, 844mL, 843mL, 842mL, 839mL, 838mL, 837mL, 834mL, 833mL, 832mL, 829mL, 828mL, 827mL, 824mL, 823mL, 822mL, 819mL, 818mL, 817mL, 814mL, 813mL, 812mL, 809mL, 808mL, 807mL, 804mL, 803mL, 802, 799mL, and the like.
In a preferred embodiment of the invention, the mobile phase a has a volume ratio of acetonitrile, water and triethylamine of (150 to 160): 849 to 839: 1, preferably 155: 844: 1, before adjusting the pH of the mixture.
By adopting the mobile phase A and matching with the mobile phase B, under a certain gradient elution condition, the separation degree between each related substance and the main component can be ensured, and the detection accuracy is improved.
In a specific embodiment of the present invention, the flow rate of the gradient elution is 0.8 to 1.2mL/min, preferably 0.9 to 1.1mL/min, and more preferably 1.0 mL/min.
As in various embodiments, the flow rate of the gradient elution can be 0.8mL/min, 0.85mL/min, 0.9mL/min, 0.95mL/min, 1.0mL/min, 1.05mL/min, 1.1mL/min, 1.15mL/min, 1.2mL/min, and the like.
In a specific embodiment of the present invention, the column of the high performance liquid chromatography is any one of an octadecylsilane-bonded silica column, an octaalkylsilane-bonded silica column and a cyano column, and is preferably an octadecylsilane-bonded silica column. In particular, an Agilent SB-C18 (150X 4.6mm, 5 μm) column, or a column of comparable performance, may be used.
In a specific embodiment of the invention, the column temperature of the chromatographic column is 25-40 ℃, and preferably 30 ℃.
As in the different embodiments, the column temperature of the chromatographic column can be 25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃ and so on.
In the embodiment of the present invention, the sample amount of the sample solution may be 10 to 20 μ L, such as 10 μ L, 12 μ L, 14 μ L, 15 μ L, 16 μ L, 18 μ L, 20 μ L, etc., and preferably 20 μ L.
In a preferred embodiment of the present invention, the detection conditions of the high performance liquid chromatography include:
the detection wavelength is 210 nm;
mobile phase A: a mixture of acetonitrile, water and triethylamine, the pH of the mixture being adjusted to 6.5; mobile phase B: acetonitrile;
in the mobile phase A, before the pH of the mixture is adjusted, the volume ratio of acetonitrile to water to triethylamine is 155: 844: 1; performing gradient elution by using the mobile phase A and the mobile phase B;
the flow rate of the gradient elution is 1.0 mL/min;
the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column, and the column temperature of the chromatographic column is 30 ℃.
In a specific embodiment of the present invention, the method for preparing the test solution comprises: and dissolving the sample by using the mobile phase B, and diluting by using a diluent. The diluent is mobile phase A and/or mobile phase B. Preferably, the volume ratio of the mobile phase a to the mobile phase B in the dilution is between 100: 0 and 0: 100; the diluent is a mixed solvent of the mobile phase A and the mobile phase B with the volume ratio of 50: 50. Wherein, the specific dilution factor can be adjusted according to the actual demand. In a specific embodiment of the invention, the concentration of dehydroepiandrosterone acetate in the test solution is 0.5-5 mg/mL, such as 2 mg/mL. In the specific operation, the concentration of dehydroepiandrosterone acetate in the sample solution is not limited to this, as long as the concentration x the sample injection volume is larger than the quantitative limit of each impurity.
In a specific embodiment of the present invention, the test sample comprises a material or a preparation containing dehydroepiandrosterone acetate.
In a preferred embodiment of the present invention, the content of dehydroepiandrosterone acetate and/or related substances in the test solution is calculated by an external standard method.
In a specific embodiment, the method for calculating the content of dehydroepiandrosterone acetate comprises the following steps: respectively injecting the standard series working solutions of dehydroepiandrosterone acetate into a high performance liquid chromatograph, measuring corresponding chromatographic peak areas under the detection condition of the high performance liquid chromatograph, and drawing a dehydroepiandrosterone acetate standard curve by taking the concentration of the standard working solutions as a horizontal coordinate and the peak areas as a vertical coordinate; substituting the chromatographic peak area of dehydroepiandrosterone acetate in the chromatographic detection result of the test solution into the standard curve of dehydroepiandrosterone acetate, and calculating to obtain the content of dehydroepiandrosterone acetate in the test solution. The concentration range of the dehydroepiandrosterone acetate standard series working solution is 4-500 mu g/mL, and for example, the working solutions with the concentrations of about 4.65 mu g/mL, 9.29 mu g/mL, 46.45 mu g/mL, 92.90 mu g/mL, 232.25 mu g/mL, 464.50 mu g/mL and the like can be specifically adopted.
In a specific embodiment, the method for calculating the content of the related substance comprises: respectively injecting related substance standard series working solutions into a high performance liquid chromatograph, measuring corresponding chromatographic peak areas under the detection condition of the high performance liquid chromatograph, and drawing a related substance standard curve by taking the concentration of the standard working solutions as a horizontal coordinate and the peak areas as a vertical coordinate; substituting the chromatographic peak area of the related substances in the chromatographic detection result of the test solution into the dehydroepiandrosterone acetate standard curve, and calculating to obtain the concentration of the related substances in the test solution. Wherein the concentration range of the relevant substance standard series working solution is 4-500 mug/mL.
Specifically, for the substance I concerned, working solutions having concentrations of about 4.092. mu.g/mL, 8.184. mu.g/mL, 40.92. mu.g/mL, 81.84. mu.g/mL, 204.6. mu.g/mL, 409.2. mu.g/mL, etc., respectively, can be specifically used. As the substance II concerned, specifically, working solutions having concentrations of about 5. mu.g/mL, 10. mu.g/mL, 50. mu.g/mL, 100. mu.g/mL, 250. mu.g/mL, 500. mu.g/mL, etc., respectively, can be used.
Dissolving dehydroepiandrosterone acetate, a related substance I and a related substance II in the standard series working solution by using a mobile phase B to respectively obtain a dehydroepiandrosterone acetate mother solution, a related substance I mother solution and a related substance II mother solution, and diluting the solutions to preset concentrations by using a diluent; wherein the diluent is a mixed solvent of mobile phase A and mobile phase B at a volume ratio of 50: 50.
In another embodiment of the present invention, the same volume of the test solution and the same volume of the reference solution are respectively detected by the high performance liquid chromatography, and dehydroepiandrosterone acetate and/or related substances are analyzed according to the detection result of the high performance liquid chromatography. Knowing the content of the dehydroepiandrosterone acetate and/or related substances in the reference solution, calculating the content of the dehydroepiandrosterone acetate and/or related substances in the test solution by a conventional calculation method.
The invention also provides application of the detection method of any dehydroepiandrosterone acetate and related substances thereof in quality control of dehydroepiandrosterone acetate raw materials or preparations.
Compared with the prior art, the invention has the beneficial effects that:
(1) the detection method can realize one-time high-efficiency separation of two related substances with small polarity difference of dehydroepiandrosterone acetate, and has the advantages of strong specificity, good durability and high sensitivity;
(2) the detection method can be used for quality control of the raw material or preparation of dehydroepiandrosterone acetate.
Drawings
In order to more clearly illustrate the embodiments of the present invention 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, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a linear relationship chart of dehydroepiandrosterone acetate according to example 7 of the present invention;
FIG. 2 is a linear relationship diagram of a related substance I provided in example 8 of the present invention;
FIG. 3 is a linear relationship diagram of related substance II provided in example 9 of the present invention;
FIG. 4 is a chromatogram corresponding to mixed sample injection in the specificity test of Experimental example 1 of the present invention;
FIG. 5 is a chromatogram corresponding to the single injection of dehydroepiandrosterone acetate in the specificity test of Experimental example 1 of the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and the detailed description, but those skilled in the art will understand that the following described embodiments are some, not all, of the embodiments of the present invention, and are only used for illustrating the present invention, and should not be construed as limiting the scope of the present invention. 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 invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In the specific implementation mode, the verification of the items such as specificity, detection limit and quantification limit, precision, linearity and range, accuracy, solution stability and the like is performed according to the technical guide principle of verification of chemical drug quality control analysis method, the technical guide principle of standardized process established by chemical drug quality standard, the technical guide principle of chemical drug impurity research, the technical guide principle of chemical drug residual solvent research and the related guide principle in the appendix of the current edition of the pharmacopoeia of the people's republic of China.
The test solution in the following embodiment is mainly prepared from dehydroepiandrosterone acetate and related substances I and II, and in actual detection, the test solution is prepared by dissolving and diluting a raw material or preparation containing dehydroepiandrosterone acetate to be detected.
Example 1
The embodiment provides a method for detecting dehydroepiandrosterone acetate and related substances thereof, which comprises the following steps:
(1) test solution (containing substances I and II)
Preparation of a test solution: precisely measuring 0.5mL of solution of a related substance I and 0.5mL of solution of a related substance II respectively, placing the solutions in a 100mL volumetric flask, precisely measuring about 50mg of dehydroepiandrosterone acetate sample, placing the sample in the volumetric flask, adding 20mL of acetonitrile for dissolution, then using diluent for constant volume to scale, and preparing to obtain solutions containing 10 mu g of the related substances I and II respectively and 0.5mg of dehydroepiandrosterone acetate per 1 mL;
the preparation method of the solution of the related substance I comprises the following steps: weighing about 20mg of a related substance I sample, placing the sample in a 10mL volumetric flask, adding 5mL of acetonitrile for dissolution, then using a diluent for constant volume to scale, shaking up, and preparing a related substance I solution containing 2.0mg in 1mL of the solution; the preparation method of the solution of the related substance II comprises the following steps: weighing about 20mg of a related substance II sample, placing the sample in a 10mL volumetric flask, adding 5mL of acetonitrile for dissolution, then fixing the volume to a scale with a diluent, shaking up, and preparing a related substance II solution containing 2.0mg in 1mL of the solution;
the diluent used above is a mixed solvent of mobile phase a and mobile phase B at a volume ratio of 50: 50.
(2) High performance liquid chromatography detection conditions
The instrument comprises the following steps: agilent 1260 high performance liquid chromatography system and workstation, ultraviolet detector, detection wavelength: 210 nm;
a chromatographic column: agilent SB-C18(150 mm. times.4.6 mm, 5 μm) column;
mobile phase: mobile phase a and mobile phase B; wherein, the preparation of the mobile phase A comprises the following steps: mixing 844mL of water and 1mL of triethylamine uniformly, adding 155mL of acetonitrile, and adjusting the pH value to 6.5 by using a sodium hydroxide aqueous solution or glacial acetic acid; the mobile phase B is acetonitrile; carrying out gradient elution according to a gradient table 1;
flow rate: 1.0 mL/min;
column temperature: 30 ℃;
sample introduction volume: 20 μ L.
TABLE 1 gradient elution schedule (vol.)
| Time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 50 | 50 |
| 12 | 15 | 85 |
| 40 | 15 | 85 |
(3) Detection step
And (3) precisely measuring 20 mu L of the test solution, injecting into a liquid chromatograph, detecting according to the conditions in the step (2), and recording a chromatogram. In the chromatogram, each peak is related substance II, related substance I and dehydroepiandrosterone acetate in turn, the retention time is 2.216min, 4.639min and 11.035min respectively, and the separation degree among the related substances and between the related substances and the dehydroepiandrosterone acetate is good, which is shown in table 2.
TABLE 2 separation data between the components
Example 2
This example refers to the detection method of example 1, with the only difference that: and (3) in the detection condition of the high performance liquid chromatography in the step (2), the column temperature is 25 ℃.
Example 3
This example refers to the detection method of example 1, with the only difference that: and (3) in the detection condition of the high performance liquid chromatography in the step (2), the column temperature is 40 ℃.
The degrees of separation between the substances of examples 1 to 3 and between the substances and dehydroepiandrosterone acetate are shown in Table 3.
TABLE 3 separation data between the components
Example 4
This example refers to the detection method of example 1, with the only difference that: in the detection condition of the high performance liquid chromatography in the step (2), the preparation of the mobile phase A comprises the following steps: 897mL of water and 3mL of triethylamine were mixed, 100mL of acetonitrile was added, and the pH was adjusted to 6.5 with aqueous sodium hydroxide or glacial acetic acid.
Example 5
This example refers to the detection method of example 1, with the only difference that: in the detection condition of the high performance liquid chromatography in the step (2), the preparation of the mobile phase A comprises the following steps: water 799mL and 1mL triethylamine were mixed well, acetonitrile 200mL was added, and pH was adjusted to 6.5 with aqueous sodium hydroxide or glacial acetic acid.
The degrees of separation between the related substances and dehydroepiandrosterone acetate of examples 1, 4 and 5 are shown in Table 4.
TABLE 4 separation data between the components
Example 6
This example refers to the detection method of example 1, with the only difference that:
in the step (1), the preparation of the test solution comprises: dissolving a sample to be detected containing dehydroepiandrosterone acetate in acetonitrile to prepare a mother solution containing about 5mg/mL of dehydroepiandrosterone acetate, and then diluting the mother solution to a test solution containing about 0.5mg/mL of dehydroepiandrosterone acetate by adopting a diluent for detection.
Example 7
The embodiment provides a method for detecting the content of dehydroepiandrosterone acetate, which comprises the following steps:
(1) precisely weighing about 20mg of dehydroepiandrosterone acetate, placing into a 10mL volumetric flask, adding 5mL of acetonitrile for dissolving, diluting with diluent to a constant volume of 10mL, and shaking up to obtain stock solution; wherein the diluent was the same as that in example 1;
preparation of standard series working solution:
placing 5mL of stock solution into a 20mL volumetric flask, dissolving with diluent, fixing the volume, and shaking up to obtain a linear 6 solution;
placing 5mL of linear 6 solution into a 10mL volumetric flask, dissolving with diluent, fixing the volume, and shaking up to obtain linear 5 solution;
placing 2mL of linear 6 solution into a 10mL volumetric flask, dissolving with diluent, fixing the volume, and shaking up to obtain a linear 4 solution;
putting 1mL of linear 6 solution into a 10mL volumetric flask, dissolving with diluent, fixing the volume, and shaking up to obtain a linear 3 solution;
placing 2mL of the linear 3 solution into a 10mL volumetric flask, dissolving the linear 3 solution by using a diluent, fixing the volume, and shaking up to obtain a linear 2 solution;
the linear 3 solution 1mL is placed in a 10mL volumetric flask, dissolved with the diluent and added to the constant volume, and shaken up to obtain the linear 1 solution.
(2) Precisely measuring 20 μ L of each of the standard series working solutions, injecting into a liquid chromatograph, detecting according to the detection conditions in the embodiment 1, and recording the chromatogram; the test results are shown in Table 5, and a linear regression equation is obtained by using the concentration C (μ g/mL) as the abscissa and the corresponding peak area as the ordinate, and the linear relationship is shown in FIG. 1.
The linear relation between the concentration of dehydroepiandrosterone acetate and the peak area is good, and the verification requirement is met.
TABLE 5 Linear test results for dehydroepiandrosterone acetate
Example 8
This example provides a method for detecting the content of a substance i, comprising the steps of:
precisely weighing about 20mg of related substance I, placing the related substance I in a 10mL volumetric flask, adding 5mL of acetonitrile for dissolving, diluting with a diluent to a constant volume of 10mL, and shaking up to be used as a stock solution; wherein the diluent was the same as that in example 1;
preparation of standard series working solution:
placing 5mL of stock solution into a 20mL volumetric flask, dissolving with diluent, fixing the volume, and shaking up to obtain a linear 6 solution;
placing 5mL of linear 6 solution into a 10mL volumetric flask, dissolving with diluent, fixing the volume, and shaking up to obtain linear 5 solution;
placing 2mL of linear 6 solution into a 10mL volumetric flask, dissolving with diluent, fixing the volume, and shaking up to obtain a linear 4 solution;
putting 1mL of linear 6 solution into a 10mL volumetric flask, dissolving with diluent, fixing the volume, and shaking up to obtain a linear 3 solution;
placing 2mL of the linear 3 solution into a 10mL volumetric flask, dissolving the linear 3 solution by using a diluent, fixing the volume, and shaking up to obtain a linear 2 solution;
the linear 3 solution 1mL is placed in a 10mL volumetric flask, dissolved with the diluent and added to the constant volume, and shaken up to obtain the linear 1 solution.
(2) Precisely measuring 20 μ L of each of the standard series working solutions, injecting into a liquid chromatograph, detecting according to the detection conditions in the embodiment 1, and recording the chromatogram; the test results are shown in Table 6, and a linear regression equation is obtained by using the concentration C (μ g/mL) as the abscissa and the corresponding peak area as the ordinate, and the linear relationship is shown in FIG. 2.
The linear relation between the concentration of the related substance I of dehydroepiandrosterone acetate and the peak area is good, and the verification requirement is met.
TABLE 6 results of the linearity test on substance I
Example 9
The embodiment provides a method for detecting the content of a related substance II, which comprises the following steps:
precisely weighing about 20mg of related substance II, placing the related substance II in a 10mL volumetric flask, adding 5mL of acetonitrile for dissolving, diluting with a diluent to a constant volume of 10mL, and shaking up to be used as a stock solution; wherein the diluent was the same as that in example 1;
preparation of standard series working solution:
placing 5mL of stock solution into a 20mL volumetric flask, dissolving with diluent, fixing the volume, and shaking up to obtain a linear 6 solution;
placing 5mL of linear 6 solution into a 10mL volumetric flask, dissolving with diluent, fixing the volume, and shaking up to obtain linear 5 solution;
placing 2mL of linear 6 solution into a 10mL volumetric flask, dissolving with diluent, fixing the volume, and shaking up to obtain a linear 4 solution;
putting 1mL of linear 6 solution into a 10mL volumetric flask, dissolving with diluent, fixing the volume, and shaking up to obtain a linear 3 solution;
placing 2mL of the linear 3 solution into a 10mL volumetric flask, dissolving the linear 3 solution by using a diluent, fixing the volume, and shaking up to obtain a linear 2 solution;
the linear 3 solution 1mL is placed in a 10mL volumetric flask, dissolved with the diluent and added to the constant volume, and shaken up to obtain the linear 1 solution.
(2) Precisely measuring 20 μ L of each of the standard series working solutions, injecting into a liquid chromatograph, detecting according to the detection conditions in the embodiment 1, and recording the chromatogram; the test results are shown in Table 7, and a linear regression equation is obtained by using the concentration C (μ g/mL) as the abscissa and the corresponding peak area as the ordinate, and the linear relationship is shown in FIG. 3.
The linear relation between the concentration of the related substance II of dehydroepiandrosterone acetate and the peak area is good, and the verification requirement is met.
TABLE 7 results of the linearity test on substance II
Experimental example 1
Specificity
Resolution analysis process
(1) Solution preparation
Dehydroepiandrosterone acetate test solution: weighing about 20mg of dehydroepiandrosterone acetate sample, placing the sample in a 10mL volumetric flask, adding 5mL of acetonitrile for dissolution, then using the diluent in the embodiment 1 to fix the volume to a scale, shaking up, and preparing a test solution containing about 2.0mg of dehydroepiandrosterone acetate in 1mL of solution.
Localization solution of related substance I: weighing 20mg of the related substance I sample, placing the sample in a 10mL volumetric flask, adding 5mL of acetonitrile for dissolution, then using the diluent in the example 1 to fix the volume to a scale, shaking up, and preparing a sample solution containing about 2.0mg of the related substance I in 1mL of solution.
Substance II localization solution: weighing 20mg of a related substance II sample, placing the sample in a 10mL volumetric flask, adding 5mL of acetonitrile for dissolution, then fixing the volume to the scale by using the diluent in the example 1, shaking up, and preparing a test solution containing about 2.0mg of the related substance II in 1mL of solution.
Mixing the solution: test solution added with related substances
Precisely measuring 0.5mL of the positioning solution of the related substance I and 0.5mL of the positioning solution of the related substance II respectively, placing the positioning solution of the related substance I and the positioning solution of the related substance II in a 100mL volumetric flask, precisely measuring about 50mg of dehydroepiandrosterone acetate in the volumetric flask, adding 20mL of acetonitrile for dissolving, adding the diluent in the embodiment 1 for constant volume, and preparing a mixed solution containing 10 mu g of the related substances I and II respectively and 0.5mg of dehydroepiandrosterone acetate in each 1mL of the mixed solution.
(2) Detection of
Samples were injected in the order of Table 8 below, and detection was carried out under the detection conditions in example 1, and the chromatogram was recorded. Wherein, the blank solution is: mixed solution of mobile phase a and mobile phase B at a volume ratio of 50: 50.
TABLE 8 specificity sample introduction sequence
| Sample name | Number of samples introduced |
| Blank solution | 1 |
| Dehydroepiandrosterone acetate test solution | 1 |
| Positioning solution of related substance I | 1 |
| Related substance II positioning solution | 1 |
| Mixed solution | 1 |
(3) Analysis results
The specificity results are shown in table 9.
TABLE 9 specificity results
From the above specificity results, it can be known that the main peak and known impurities in the sample can be completely separated from dehydroepiandrosterone acetate, and the inspection of related substances is not interfered. The relevant spectra are shown in FIGS. 4 and 5. The separation degrees of dehydroepiandrosterone acetate, the related substance I and the related substance II meet the requirements, so the detection method can stably and effectively detect the related substances in the dehydroepiandrosterone acetate.
Experimental example 2
Detection limit and quantification limit
And (4) quantitative limit: and (3) taking the solution of dehydroepiandrosterone acetate and each related substance to dilute in sequence (the diluent is the diluent in the embodiment 1), and when the S/N is approximately equal to 10, the solution is the quantification limit of the dehydroepiandrosterone acetate and each related substance. And (5) taking 6 continuous sample introduction needles of the quantitative limit concentration solution, and counting the retention time and S/N.
Detection limit: and (3) sequentially diluting the dehydroepiandrosterone acetate and the solutions of the related substances (the diluent is the diluent in the example 1), and determining the detection limits of the dehydroepiandrosterone acetate and the related substances when the S/N is approximately equal to 3. And (5) sampling samples in sequence, and counting the retention time and S/N.
The detection limit and quantification limit test results of dehydroepiandrosterone acetate and related substances I and II are shown in tables 10 and 11, respectively.
TABLE 10 detection and quantitation limits for dehydroepiandrosterone acetate and related substances
| Name (R) | Dehydroepiandrosterone acetate | Related substance I | Related substance II |
| Detection limit (ng/mL) | 69.986 | 30.69 | 3.00 |
| Limit of quantitation (ng/mL) | 199.96 | 102.30 | 10.00 |
TABLE 11 detection and quantitation Limit statistics for dehydroepiandrosterone acetate and related substances
Experimental example 3
Precision degree
(1) Method of producing a composite material
Samples of the same lot were sampled, and 6 parts of the mixed solution were prepared in parallel as a repetitive test solution with reference to the method for preparing the mixed solution in the specificity test of Experimental example 1.
Each of 6 test solutions (10. mu.L) was measured out precisely, and the results are shown in Table 12, where the results were obtained by measuring 10. mu.L each of the test solutions, injecting the sample solution into a liquid chromatograph under the chromatographic conditions in example 1, recording the chromatogram, calculating the RSD (%) of the main peak content in the test solution, and calculating the RSD (%) of the content of the substance concerned.
Table 12 repeatability data
From the results in Table 12, it is understood that RSD (%) of the content of repetitive impurities is less than 2% and meets the standard.
Experimental example 4
Recovery rate
(1) Method of producing a composite material
Mixed stock solution of related substances I and II: accurately weighing 10.37mg of related substance I and 10.33mg of related substance II, placing in a 10mL measuring flask, adding acetonitrile to dissolve and dilute to scale, and shaking up to obtain mixed stock solution of related substances I and II.
80% recovery sample solution: precisely measuring 0.4mL of mixed stock solution of related substances I and II, placing the mixed stock solution into a 100mL measuring flask, adding the diluent to a constant volume to scale, and shaking up to obtain the final product.
100% recovery sample solution: precisely measuring 0.5mL of mixed stock solution of related substances I and II, placing the mixed stock solution into a 100mL measuring flask, adding the diluent to a constant volume to reach a scale, and shaking up to obtain the final product.
120% recovery sample solution: precisely measuring 0.6mL of mixed stock solution of related substances I and II, placing the mixed stock solution into a 100mL measuring flask, adding the diluent to a constant volume to reach a scale, and shaking up to obtain the final product.
3 parts of solution is prepared in parallel for each concentration, and the sample injection is repeated. The value of the weight/the value of the theoretical weight was calculated, and the recovery (%) was calculated.
Each 10. mu.L of the above-mentioned concentration solution was precisely measured, and the solution was injected into a liquid chromatograph under the chromatographic detection conditions in example 1 to measure, and the chromatogram was recorded. The contents of the related substances I and II and the recovery rates were calculated by peak areas according to the external standard method, and the results are shown in tables 13 and 14.
(2) Results
TABLE 13 results on recovery of substance I
TABLE 14 results on recovery of substance II
The result shows that the average recovery rate of the related substance I and the related substance II under each concentration term is within the range of 98.0-102.0 percent, and the accuracy is good.
Experimental example 5
Stability of solution
(1) Method of producing a composite material
50mg of dehydroepiandrosterone acetate is precisely weighed, placed in a 100mL volumetric flask, dissolved in 20mL of acetonitrile, diluted to a constant volume to a scale (the same diluent as that in example 1), and shaken up to serve as a test solution.
After standing at room temperature for 12 hours, 10. mu.L of the mixture was measured precisely at the end of 0 th, 2 nd, 4 th, 6 th, 8 th, 10 th and 12 th hours, and the mixture was poured into a liquid chromatograph under the conditions for chromatography in example 1 to measure the mixture, and a chromatogram was recorded.
(2) Results
TABLE 15 solution stability results
The test result shows that the RSD value of the peak area is less than 2.0% within 12h, which indicates that the test solution is stable after being placed at room temperature for 12 h.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. The method for detecting dehydroepiandrosterone acetate and related substances thereof is characterized by comprising the following steps of:
detecting the test solution by high performance liquid chromatography;
the detection conditions of the high performance liquid chromatography comprise:
the detection wavelength is 201-220 nm;
mobile phase A: adjusting the pH of a mixture of acetonitrile, water and triethylamine to 6.4-6.7; mobile phase B: acetonitrile;
in the mobile phase A, before the pH of the mixture is adjusted, the volume ratio of acetonitrile to water to triethylamine is (100-200): 897-799): 3-1;
performing gradient elution by using the mobile phase A and the mobile phase B;
the process of gradient elution includes: the volume ratio of mobile phase a to mobile phase B was changed from 95: 5 to 5: 95.
2. The detection method according to claim 1, wherein the gradient elution process comprises: the volume ratio of the mobile phase A to the mobile phase B is changed from 50: 50 to 15: 85 within 0-12 min; the volume ratio of the mobile phase A to the mobile phase B is 15: 85 within 12-40 min;
preferably, the related substances include I and II, and the structural formulas are respectively as follows:
3. the detection method according to claim 1 or 2, wherein the detection wavelength is 205 to 215 nm;
preferably, the detection wavelength is 210 nm.
4. The detection method according to claim 1 or 2, wherein the volume ratio of acetonitrile, water and triethylamine in the mobile phase A is (150-160): 849-839): 1;
preferably, the volume ratio of acetonitrile to water to triethylamine is 155: 844: 1.
5. The detection method according to claim 1 or 2, wherein the flow rate of the gradient elution is 0.8 to 1.2 mL/min;
preferably, the flow rate of the gradient elution is 0.9-1.1 mL/min;
more preferably, the flow rate of the gradient elution is 1.0 mL/min.
6. The detection method according to claim 1 or 2, wherein the high performance liquid chromatography column is any one of an octadecylsilane bonded silica gel column, an octaalkylsilane bonded silica gel column, and a cyano column;
and/or the column temperature of the high performance liquid chromatography is 25-40 ℃;
preferably, the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column;
preferably, the column temperature is 30 ℃.
7. The detection method according to claim 1 or 2, wherein the content of dehydroepiandrosterone acetate and/or related substances in the test solution is calculated by an external standard method;
preferably, the method for calculating the content of dehydroepiandrosterone acetate and/or related substances comprises the following steps: respectively injecting standard series working solutions of dehydroepiandrosterone acetate and/or related substances into a high performance liquid chromatograph, measuring corresponding chromatographic peak areas under the detection conditions of the high performance liquid chromatograph, and drawing a standard curve of the dehydroepiandrosterone acetate and/or related substances by taking the concentration of the standard working solutions as abscissa and the peak areas as ordinate; substituting the chromatographic peak area of dehydroepiandrosterone acetate and/or related substances in the chromatographic detection result of the test solution into the standard curve of the dehydroepiandrosterone acetate and/or related substances, and calculating to obtain the content of the dehydroepiandrosterone acetate and/or related substances in the test solution.
8. The assay of claim 1 or 2, wherein the test sample comprises a material or a preparation comprising dehydroepiandrosterone acetate;
preferably, the method for preparing the test solution comprises: dissolving the sample by using the mobile phase B, and diluting by using a diluent; the diluent is a mobile phase A and/or a mobile phase B;
preferably, the volume ratio of the mobile phase a to the mobile phase B in the dilution is 100: 0 to 0: 100.
9. The detection method according to claim 1 or 2, wherein the detection conditions of the high performance liquid chromatography include:
the detection wavelength is 210 nm;
mobile phase A: a mixture of acetonitrile, water and triethylamine, the pH of the mixture being adjusted to 6.5; mobile phase B: acetonitrile;
in the mobile phase A, before the pH of the mixture is adjusted, the volume ratio of acetonitrile to water to triethylamine is 155: 844: 1; performing gradient elution by using the mobile phase A and the mobile phase B;
the flow rate of the gradient elution is 1.0 mL/min;
the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column, and the column temperature of the chromatographic column is 30 ℃.
10. Use of the method for detecting dehydroepiandrosterone acetate and related substances according to any one of claims 1-9 for quality control of dehydroepiandrosterone acetate starting materials or preparations.
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| CN115480015A (en) * | 2022-10-11 | 2022-12-16 | 山西振东制药股份有限公司 | Detection method and application of epiandrosterone-17-hydrazone related substance |
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