CN111693628B - Method for detecting dehydroepiandrosterone-17-hydrazone and related substances thereof and application thereof - Google Patents
Method for detecting dehydroepiandrosterone-17-hydrazone and related substances thereof and application thereof 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-17-hydrazone and related substances thereof. The method for detecting dehydroepiandrosterone-17-hydrazone 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 202-220 nm; mobile phase A: a salt-containing aqueous solution having a pH of 6.3 to 6.7; the salt comprises any one or more of ammonium salt of phosphoric acid, potassium salt of phosphoric acid, sodium salt of phosphoric acid, ammonium salt of carbonic acid, potassium salt of carbonic acid, sodium salt of carbonic acid, ammonium salt of acetic acid, potassium salt of acetic acid and sodium salt of acetic acid; mobile phase B: acetonitrile; and carrying out gradient elution by using the mobile phase A and the mobile phase B. The method can efficiently separate the dehydroepiandrosterone-17-hydrazone and related substances thereof at one time, and has high sensitivity, specificity and durability.
Description
Technical Field
The invention relates to the technical field of chemical drug analysis methods, in particular to a method for detecting dehydroepiandrosterone-17-hydrazone and related substances thereof and application thereof.
Background
Dehydroepiandrosterone-17-hydrazone, the compound 5-androstene-3 β -ol-17-hydrazone, is a key intermediate for the synthesis of the drug abiraterone acetate, and its structure is shown below:
dehydroepiandrosterone-17-hydrazone is an important part for synthesizing abiraterone acetate, if the dehydroepiandrosterone-17-hydrazone contains impurities, the impurities or conversion products of the impurities can enter subsequent reactions when the dehydroepiandrosterone-17-hydrazone is used for producing a raw material drug, so that the production quality of the raw material drug is influenced, and the quality of the raw material drug can be influenced. Therefore, the impurities of the dehydroepiandrosterone-17-hydrazone are accurately known, and the impurities are controlled by adopting a proper analysis method on the basis, so that the method has important significance.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first objective of the present invention is to provide a method for detecting dehydroepiandrosterone-17-hydrazone and its related substances, which can separate 2 related substances of dehydroepiandrosterone-17-hydrazone at a time and with high efficiency.
The second purpose of the invention is to provide the application of the detection method of dehydroepiandrosterone-17-hydrazone and related substances thereof in the quality control of raw materials or preparations of the dehydroepiandrosterone-17-hydrazone.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
the method for detecting dehydroepiandrosterone-17-hydrazone 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 202-220 nm;
mobile phase A: a salt-containing aqueous solution having a pH of 6.3 to 6.7; the salt comprises any one or more of ammonium salt of phosphoric acid, potassium salt of phosphoric acid, sodium salt of phosphoric acid, ammonium salt of carbonic acid, potassium salt of carbonic acid, sodium salt of carbonic acid, ammonium salt of acetic acid, potassium salt of acetic acid and sodium salt of acetic acid;
mobile phase B: acetonitrile;
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 70: 30 to 10: 90 within 0-35 min; and the volume ratio of the mobile phase A to the mobile phase B is 10: 90 within 35-40 min.
Further, the volume ratio of mobile phase a to mobile phase B was changed from 70: 30 to 10: 90 at 0-35 min.
The dehydroepiandrosterone-17-hydrazone can generate some degradation impurities and the like after being placed, and the detection method provided by the invention has good specificity, repeatability and accuracy on the basis of ensuring the efficient separation of various related substances and the dehydroepiandrosterone-17-hydrazone.
In a specific embodiment of the present invention, the related substances include I and II, which have the following structural formulas:
related substance I of dehydroepiandrosterone-17-hydrazone has a molecular formula of C19H28O2Molecular weight is 288; the related substance II has a molecular formula of C21H30O3And the molecular weight is 330. The polarity difference of related substances I and II is very small, and a detection method for dehydroepiandrosterone-17-hydrazone and related substances thereof does not exist at present, and a detection method for the two related substances with the very small polarity difference, which can be used for separating and detecting the dehydroepiandrosterone-17-hydrazone at one time, also does not exist.
By adopting the detection conditions, the invention can realize one-time high-efficiency separation of the 2 related substances of dehydroepiandrosterone-17-hydrazone and can effectively control the quality of the dehydroepiandrosterone-17-hydrazone.
In a specific embodiment of the present invention, the preparation method of the mobile phase a comprises: and adjusting the pH value of the saline water solution to 6.3-6.7 by adopting phosphoric acid, acetic acid or ammonia water to obtain the mobile phase A. Further, in the salt-containing aqueous solution, the concentration of salt is 0.0005-0.01 mol/L. Further, the pH was adjusted to 6.5. In practice, a salt-containing aqueous solution with a concentration of 0.0005 to 0.01mol/L is prepared in advance, and the pH is adjusted to 6.3 to 6.7, for example, 6.5, by phosphoric acid, acetic acid or ammonia water.
As in the various embodiments, the concentration of salt in the aqueous salt solution may be 0.0005, 0.001, 0.0015, 0.002, 0.0025, 0.003, 0.0035, 0.004, 0.0045, 0.005, 0.0055, 0.006, 0.0065, 0.007, 0.0075, 0.008, 0.0085, 0.009, 0.0095, 0.01, etc.
In a particular embodiment of the invention, the ammonium salt of phosphoric acid comprises any one or more of monoammonium phosphate, diammonium phosphate and ammonium phosphate; the potassium salt of phosphoric acid comprises any one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate and potassium phosphate; the sodium salt of phosphoric acid comprises any one or more of sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium phosphate; the ammonium salt of carbonic acid comprises ammonium bicarbonate and/or ammonium carbonate; the potassium salt of carbonic acid comprises potassium bicarbonate and/or potassium carbonate; the sodium salt of carbonic acid comprises sodium bicarbonate and/or sodium carbonate; the ammonium salt of acetic acid comprises ammonium acetate; the potassium salt of acetic acid comprises potassium acetate; the sodium salt of acetic acid comprises sodium acetate.
In a preferred embodiment of the invention, the salt comprises any one or more of monoammonium phosphate, diammonium phosphate and ammonium bicarbonate, preferably monoammonium phosphate.
By adopting the mobile phase A and matching with the mobile phase B, the dehydroepiandrosterone-17-hydrazone and related substances I and II thereof can be efficiently separated under the condition of gradient elution.
In various embodiments of the present invention, the detection wavelength may be 202nm, 203nm, 204nm, 205nm, 206nm, 207nm, 208nm, 209nm, 210nm, 211nm, 212nm, 213nm, 214nm, 215nm, 216nm, 217nm, 218nm, 219nm, 220nm, etc., preferably 205 to 215nm, and more preferably 210 nm.
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, the column may be a Phenomenex SB-C18 (250X 4.6mm, 5 μm) column, or a column with comparable performance.
In a specific embodiment of the invention, the detected column temperature is 25-40 ℃, preferably 25-30 ℃, and more preferably 30 ℃.
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 sample solution may be 10 to 20 μ L, such as 10 μ L, 15 μ L or 20 μ L.
In a preferred embodiment of the present invention, the detection conditions include:
the detection wavelength is 210 nm;
mobile phase A: 0.01mol/L ammonium dihydrogen phosphate aqueous solution with pH value adjusted to 6.5; mobile phase B: acetonitrile;
the flow rate of the gradient elution is 1.0 mol/L;
the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column, and the column temperature is 30 ℃.
In a specific embodiment of the present invention, the method for preparing the test solution comprises: and dissolving the test sample by adopting a diluent. The diluent is a mixed liquid of the mobile phase A and the mobile phase B, and preferably the mixed liquid of the mobile phase A and the mobile phase B is a mixed liquid of the mobile phase A and the mobile phase B in a volume ratio of 1: 1. In various embodiments, the concentration of dehydroepiandrosterone-17-hydrazone in the test solution is 0.1-5 mg/L, such as 2 mg/mL. In practical operation, the concentration of dehydroepiandrosterone-17-hydrazone in the sample solution is not limited to this, and the concentration x sample injection volume is more than the quantitative limit of each impurity.
In a specific embodiment of the present invention, the test article comprises a material or formulation comprising dehydroepiandrosterone-17-hydrazone.
In a preferred embodiment of the present invention, the content of dehydroepiandrosterone-17-hydrazone and/or related substances in the test solution is calculated by an external standard method.
The invention can effectively separate the effective components and the related substances by the detection method, thereby further carrying out quality control.
The invention also provides the application of the detection method of any dehydroepiandrosterone-17-hydrazone and related substances thereof in the quality control of raw materials or preparations of the dehydroepiandrosterone-17-hydrazone.
Compared with the prior art, the invention has the beneficial effects that:
(1) the detection method can efficiently separate the dehydroepiandrosterone-17-hydrazone and related substances thereof at one time, and has high sensitivity, good specificity and durability;
(2) the detection method can be used for the quality control of the raw material or preparation of dehydroepiandrosterone-17-hydrazone.
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 chromatogram corresponding to mixed sample injection in the specificity test of Experimental example 1 of the present invention;
FIG. 2 is a chromatogram corresponding to the separate injection of dehydroepiandrosterone-17-hydrazone in the specificity test of Experimental example 1 of the present invention;
FIG. 3 is a chromatogram corresponding to a related substance I injected separately in a specificity test in Experimental example 1 of the present invention;
FIG. 4 is a chromatogram corresponding to a related substance II injected separately in a specificity test in Experimental example 1 of the present invention. The retention time of dehydroepiandrosterone-17-hydrazone and its related substances corresponding to the chromatogram is described in the detailed description.
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, precision, 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 version of the pharmacopoeia of the people's republic of China.
The test solution in the following examples is mainly prepared from dehydroepiandrosterone-17-hydrazone and related substances I and II, and in actual detection, the raw material or preparation to be detected containing the dehydroepiandrosterone-17-hydrazone is dissolved and diluted to prepare the test solution.
Example 1
The embodiment provides a method for detecting dehydroepiandrosterone-17-hydrazone and related substances thereof, which comprises the following steps:
(1) test solution (containing substances I and II)
Preparation of test solution
Precisely measuring 0.5mL of solution of a related substance I and 0.5mL of solution of a related substance II, placing the solutions in a 50mL volumetric flask, precisely weighing about 25mg of a dehydroepiandrosterone-17-hydrazone sample, placing the sample in the volumetric flask, adding 10mL of acetonitrile for dissolution, then fixing the volume to a scale by using a diluent, and preparing to obtain 0.5mg of solution containing 10 mu g of each of the related substances I and II and 0.5mg of dehydroepiandrosterone-17-hydrazone in each 1mL of the solution;
the preparation method of the solution of the related substance I comprises the following steps: weighing about 10mg 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 1mL of a related substance I solution containing 1.0mg of the related substance I in the solution; the preparation method of the solution of the related substance II comprises the following steps: weighing about 10mg of a related substance II sample, placing the sample in a 10mL volumetric flask, adding 5mL of acetonitrile for dissolution, then using diluent for constant volume to scale, shaking up, and preparing 1mL of a related substance II solution containing 1.0mg in the solution;
the diluent is a mixed solvent of mobile phase A and mobile phase B at a volume ratio of 1: 1.
(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: phenomenex SB-C18(250 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: preparing 0.01mol/L ammonium dihydrogen phosphate aqueous solution, and adding ammonia water to adjust the pH value of the aqueous solution to be 6.5 to obtain mobile phase A; 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 | 70 | 30 |
| 35 | 10 | 90 |
| 40 | 10 | 90 |
(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, the peaks are dehydroepiandrosterone-17-hydrazone, related substance I and related substance II in sequence, the retention time is 10.39min, 18.07min and 31.18min respectively, and the separation degree among the related substances, the separation degree between the related substances and the dehydroepiandrosterone-17-hydrazone 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 dehydroepiandrosterone-17-hydrazone and related substances I and II of examples 1-3 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: a 0.0005mol/L aqueous solution of potassium hydrogencarbonate was prepared, and the pH of the aqueous solution was adjusted to 6.5 with addition of phosphoric acid to obtain mobile phase a.
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: preparing 0.0015mol/L ammonium acetate aqueous solution, and adding acetic acid to adjust the pH of the aqueous solution to 6.5 to obtain a mobile phase A.
The degrees of separation of dehydroepiandrosterone-17-hydrazone and related substances, and the degrees of separation of related substances, 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 the dehydroepiandrosterone-17-hydrazone in a diluent, and performing constant volume to obtain a sample solution to be detected containing the dehydroepiandrosterone-17-hydrazone, wherein the sample solution is about 0.5mg/mL, and performing detection.
Experimental example 1
Specificity
Resolution analysis process
(1) Solution preparation
Dehydroepiandrosterone-17-hydrazone test solution: precisely weighing 25.2mg of dehydroepiandrosterone-17-hydrazone sample, placing the sample in a 50mL volumetric flask, adding the diluent in the same example 1, dissolving, fixing the volume to scale, uniformly mixing, and preparing a test solution containing about 0.5mg of dehydroepiandrosterone-17-hydrazone in 1mL of solution.
Localization solution of related substance I: precisely weighing 25mg of a related substance I sample, placing the sample in a 50mL volumetric flask, adding the diluent in the same example 1, dissolving, fixing the volume to scale, uniformly mixing, and preparing a test solution containing 0.5mg of the related substance I in 1mL of solution.
Substance II localization solution: precisely weighing 25mg of a related substance II sample, placing the sample in a 50mL volumetric flask, adding the diluent in the same example 1, dissolving, fixing the volume to scale, uniformly mixing, and preparing a test solution containing 0.5mg of the related substance II in 1mL of solution.
Mixing the solution: test solution containing related substance
Accurately taking 0.5mL of dehydroepiandrosterone-17-hydrazone test solution, related substance I positioning solution and related substance II positioning solution respectively, and mixing to obtain a mixed solution.
(2) Detection of
Samples were injected in the order of Table 5 below, and detection was carried out under the detection conditions in example 1, and the chromatogram was recorded. Wherein, the blank solution is: the diluent in example 1.
TABLE 5 sample introduction sequence
| Sample name | Number of samples introduced |
| Blank solution | 1 |
| Dehydroepiandrosterone-17-hydrazone test sample solution | 1 |
| Related substance I | 1 |
| Related substance II | 1 |
| Mixed solution | 1 |
(3) Results
The specificity results are shown in table 6.
TABLE 6 Special Attribution data
The related chromatogram is shown in FIG. 1-FIG. 4, which are chromatogram of mixed sample solution, chromatogram of dehydroepiandrosterone-17-hydrazone, chromatogram of related substance I and chromatogram of related substance II. As can be seen from the specificity data of Table 6, the separation degree between related substance I, related substance II and dehydroepiandrosterone-17-hydrazone according to the detection method of the present invention meets the requirements, and the impurities of dehydroepiandrosterone-17-hydrazone can be stably and effectively detected.
Experimental example 2
Stability of solution
(1) Method of producing a composite material
Accurately taking the dehydroepiandrosterone-17-hydrazone stock solution, the relevant substance I stock solution and the relevant substance II stock solution, mixing in equal volume, and shaking uniformly to obtain a test solution;
wherein, the preparation of the dehydroepiandrosterone-17-hydrazone stock solution refers to a preparation method of a test solution of the dehydroepiandrosterone-17-hydrazone in a specificity test; preparing a related substance I stock solution by a preparation method of a related substance I positioning solution in a reference specificity test; the preparation of the related substance II stock solution refers to the preparation method of the related substance II positioning solution in the specificity test.
After leaving at room temperature, 20. mu.L of the test solution was precisely measured at 0, 2, 4, 6, 8, 10, and 1 hour, and the resulting solution was injected into a liquid chromatograph under the detection conditions of example 1 for analysis, and then a chromatogram was recorded.
(2) Results
TABLE 7 solution stability data (peak area)
| Stability of the solution (h) | Dehydroepiandrosterone-17-hydrazone | Related substance I | Related substance II |
| 0 | 2657.9 | 1271.3 | 1427.2 |
| 2 | 2703.9 | 1311.3 | 1401.9 |
| 4 | 2704 | 1240 | 1443.7 |
| 6 | 2801 | 1289.3 | 1441 |
| 8 | 2724.1 | 1303.1 | 1454.6 |
| 10 | 2740.3 | 1294.6 | 1432.9 |
| 12 | 2805 | 1286.3 | 1442.7 |
| Mean value of | 2733.74 | 1285.13 | 1434.86 |
| RSD(％) | 1.96 | 1.84 | 1.18 |
The stability of the solution was examined by applying the test solution over a period of 12 hours. From the above results, it was found that RSD (%) of the peak area change of each substance within 12 hours was within 2.0%, and therefore, it was confirmed that the test solution was satisfactory for detection within 12 hours.
Experimental example 3
Repeatability of
(1) Precisely weighing about 25mg of the same batch of dehydroepiandrosterone-17-hydrazone, placing the same batch of dehydroepiandrosterone-17-hydrazone into a 50mL volumetric flask, adding an appropriate amount of diluent to perform ultrasonic dissolution, fixing the volume of the diluent (the diluent is the same as the diluent in example 1), shaking up, and preparing 6 parts in parallel to serve as a repeated test solution.
Precisely measuring 6 parts of the test solution by 10. mu.L each, injecting into a liquid chromatograph according to the chromatographic detection conditions in example 1, recording the chromatogram, and calculating the RSD (%) of the main peak content in the test solution, the results are shown in Table 8.
Table 8 repeatability data
| Name (R) | Main peak area (%) |
| Repetitive test solution 1 | 98.11 |
| Repetitive test solution 2 | 97.53 |
| Repetitive test solution 3 | 98.21 |
| Repetitive test solution 4 | 97.05 |
| |
98.03 |
| Repetitive test solution 6 | 98.02 |
| Mean value of | 97.83 |
| RSD(％) | 0.46 |
From the repeatability data, the content RSD (%) of the dehydroepiandrosterone-17-hydrazone is less than or equal to 1%, the repeatability is good, and the standard is met.
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 (20)
1. The method for detecting dehydroepiandrosterone-17-hydrazone 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 202-220 nm;
the chromatographic column of the high performance liquid chromatography is an octadecylsilane chemically bonded silica chromatographic column;
mobile phase A: a salt-containing aqueous solution having a pH of 6.3 to 6.7; the salt comprises any one or more of ammonium salt of phosphoric acid, potassium salt of phosphoric acid, sodium salt of phosphoric acid, ammonium salt of carbonic acid, potassium salt of carbonic acid, sodium salt of carbonic acid, ammonium salt of acetic acid, potassium salt of acetic acid and sodium salt of acetic acid;
mobile phase B: acetonitrile;
performing gradient elution by using the mobile phase A and the mobile phase B;
the process of gradient elution includes: the volume ratio of the mobile phase A to the mobile phase B is changed from 70: 30 to 10: 90 within 0-35 min; the volume ratio of the mobile phase A to the mobile phase B is 10: 90 within 35-40 min;
the related substances comprise I and II, and the structural formulas are respectively as follows:
2. the detection method according to claim 1, wherein the ammonium salt of phosphoric acid comprises any one or more of ammonium dihydrogen phosphate, ammonium hydrogen phosphate, and ammonium phosphate; the potassium salt of phosphoric acid comprises any one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate and potassium phosphate; the sodium salt of phosphoric acid comprises any one or more of sodium dihydrogen phosphate, disodium hydrogen phosphate and sodium phosphate; the ammonium salt of carbonic acid comprises ammonium bicarbonate and/or ammonium carbonate; the potassium salt of carbonic acid comprises potassium bicarbonate and/or potassium carbonate; the sodium salt of carbonic acid comprises sodium bicarbonate and/or sodium carbonate; the ammonium salt of acetic acid comprises ammonium acetate; the potassium salt of acetic acid comprises potassium acetate; the sodium salt of acetic acid comprises sodium acetate.
3. The detection method according to claim 2, wherein the salt comprises any one or more of ammonium dihydrogen phosphate, diammonium hydrogen phosphate, and ammonium hydrogen carbonate.
4. The detection method according to claim 3, wherein the salt is ammonium dihydrogen phosphate.
5. The detection method according to any one of claims 1 to 4, wherein the preparation method of the mobile phase A comprises the following steps: and adjusting the pH value of the saline water solution to 6.3-6.7 by adopting phosphoric acid, acetic acid or ammonia water.
6. The detection method according to claim 5, wherein the pH of the salt-containing aqueous solution is adjusted to 6.5.
7. The detection method according to claim 5, wherein the concentration of the salt in the salt-containing aqueous solution is 0.0005 to 0.01 mol/L.
8. The detection method according to any one of claims 1 to 4, wherein the detection wavelength is 205 to 215 nm.
9. The detection method according to claim 8, wherein the detection wavelength is 210 nm.
10. The detection method according to any one of claims 1 to 4, wherein the column temperature of the high performance liquid chromatography is 25 to 40 ℃.
11. The detection method according to claim 10, wherein the column temperature is 30 ℃.
12. The detection method according to any one of claims 1 to 4, wherein the flow rate of the gradient elution is 0.8 to 1.2 mL/min.
13. The detection method according to claim 12, wherein the flow rate of the gradient elution is 0.9 to 1.1 mL/min.
14. The detection method according to claim 12, wherein the flow rate of the gradient elution is 1.0 mL/min.
15. The detection method according to any one of claims 1 to 4, wherein the preparation method of the test solution comprises: dissolving a test sample by adopting a diluent; the diluent is a mixed solution of the mobile phase A and the mobile phase B.
16. The method of claim 15, wherein the test sample comprises a material or formulation comprising dehydroepiandrosterone-17-hydrazone.
17. The detection method according to claim 15, wherein the volume ratio of the mobile phase a to the mobile phase B in the diluted solution is 1: 1.
18. The detection method according to any one of claims 1 to 4, wherein the detection condition includes:
the detection wavelength is 210 nm;
mobile phase A: 0.01mol/L ammonium dihydrogen phosphate aqueous solution with pH value adjusted to 6.5; mobile phase B: acetonitrile;
the flow rate of the gradient elution is 1.0 mol/L;
the chromatographic column is an octadecylsilane chemically bonded silica chromatographic column, and the column temperature is 30 ℃.
19. The method as set forth in claim 1, wherein the content of dehydroepiandrosterone-17-hydrazone and/or a related substance in the test solution is calculated by an external standard method.
20. Use of the method of detecting dehydroepiandrosterone-17-hydrazone and its related substances according to any one of claims 1 to 19 for the quality control of a raw material or a preparation of dehydroepiandrosterone-17-hydrazone.
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| Synthesis, characterization and antitumor activities of some steroidal derivatives with side chain of 17-hydrazone aromatic heterocycle;Jianguo Cui et al.;《Steroids》;20150109;第32-38页 * |
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