Inspection method for related substances of danazol hydroxylate
The separation and detection of related substance impurities of danazol hydroxylide by high performance liquid chromatography solves the problem that the existing technology cannot effectively separate them, achieves a highly sensitive and accurate detection effect, and is suitable for industrial quality monitoring.
Patent Information
- Application Number
- CN202511038906.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-28
- Publication Date
- 2025-10-17
AI Technical Summary
Existing technologies are unable to effectively separate and detect related substance impurities in danazol hydroxylide, and the existing liquid phase method has insufficient detection capabilities and cannot meet the requirements of the pharmacopoeia.
High performance liquid chromatography was used with a C18 column packed with octadecylsilane bonded silica gel, the mobile phase was 5-20 mmol/L ammonium acetate-water solution (pH 3.0-4.0) and acetonitrile, gradient elution, detection wavelength of 250-260 nm, column temperature of 25-35°C, flow rate of 0.8-1.2 ml/min, injection volume of 10-50 μL, and the diluent was acetonitrile-water or methanol-water mixed solution to achieve separation and quantification of hydroxyl compounds and their impurities.
The method achieves good peak separation of hydroxyl compounds and their main impurities, high-sensitivity detection, accurate and reliable results, strong applicability, cost-effectiveness, and is suitable for industrial quality monitoring.
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Figure CN120801557A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of medical analysis, and specifically provides a method for checking related substances of danazol hydroxyimine. BACKGROUND
[0002] Danazol hydroxyimine (hereinafter referred to as hydroxyimine) is a key intermediate for synthesizing danazol, and its chemical name is (17A)-17-hydroxy-2-(hydroxyimino)-pregn-4-ene-20-yne-3-ketone, the CAS number is 2787-02-2, and the structural formula is as follows:
[0003] Danazol is a kind of steroid drug, which is often used for treating endometriosis, fibrocystic breast disease, idiopathic thrombocytopenic purpura, hereditary angioedema, systemic lupus erythematosus and other diseases which are ineffective to other drugs. As a key intermediate of danazol, the limit level of the related substance impurities of hydroxyimine directly affects the quality of the raw drug. Danazol standards are collected in various pharmacopoeias, and the liquid phase method under the inspection item of danazol related substances cannot completely separate and control the key impurities, and cannot be used to control the related substance impurity level of hydroxyimine, and the impurity detection capacity of the method is insufficient.
[0004] Therefore, it is necessary to provide a method for detecting related substance impurities of hydroxyimine by high performance liquid chromatography, so as to solve the above technical problems. SUMMARY
[0005] In view of this, the present application provides a method for detecting related substance impurities of hydroxyimine by liquid chromatography, and the technical scheme adopted by the present application is as follows.
[0006] The technical scheme of the present application is realized as follows: the present application provides a method for detecting related substances of (17A)-17-hydroxy-2-(hydroxyimino)-pregn-4-ene-20-yne-3-ketone (hereinafter referred to as "hydroxyimine") by high performance liquid chromatography. The method can separate and quantitatively analyze hydroxyimine, process impurities and degradation products at one time, and the methodological verification result meets the requirements of ICH Q2(R1), and the method comprises the following steps: After the test sample is dissolved, the liquid chromatography is used for detection, and the content of the related substances is calculated by the peak area normalization method, and the liquid chromatography comprises the following steps: A chromatographic column filled with C18 chromatographic column with octadecylsilane bonded silica gel, with a specification of 4.6*250mm*5um; A mobile phase: the mobile phase A is 5-20mmol / L ammonium acetate-water solution or sodium acetate-water solution, and the pH is adjusted to 3.0-4.0 by phosphoric acid; the mobile phase B is acetonitrile; Detector: UV detector, detection wavelength is 250nm-260nm. Column temperature: 25-35℃. Flow rate: 0.8-1.2ml / min. Injection volume: 10-50μL. Gradient elution. Test solution: take (17A)-17-hydroxy-2-(hydroxymethylidene)-pregn-4-ene-20-yn-3-ketone test sample, dissolve and dilute to a concentration of 1.0-3.0mg / ml with diluent.
[0007] Chromatographic column: recommended Agilent ZORBAX SB-C18.
[0008] Mobile phase: A phase is 10mmol / L ammonium acetate aqueous solution (pH4.0 is adjusted by phosphoric acid); B phase is acetonitrile.
[0009] Gradient program: 0-20min, A linearly decreases from 65% to 50%; 20-50min, A linearly decreases from 50% to 20%; 50-53min, A maintains 20%; 53-53.01min, A linearly increases from 20% to 65%; 53.01-63min, A maintains 65%.
[0010] Flow rate: 1.0mL / min; column temperature: 30℃; detection wavelength: 254nm; injection volume: 20μL.
[0011] The diluent is a mixed solution of acetonitrile-water with a volume ratio of 7:3 or a mixed solution of methanol-water with a volume ratio of 7:3.
[0012] System suitability The blank solution has no interference at the retention time of the main peak; the main peak of the hydroxy radical has a theoretical plate number ≥8000, a tailing factor ≤1.2, and a separation degree of the main peak and adjacent impurity peak ≥2.0.
[0013] Determination steps The chromatograph is recorded by injecting the blank solution and the test solution in turn after the chromatograph is balanced to the baseline stability according to the above conditions. The content of each known impurity and total impurity is calculated by the peak area normalization method.
[0014] The present application has the following beneficial effects relative to the prior art: Strong specificity The hydroxylamine and its main impurities have good peak shapes, the blank has no interference, and the separation degree of the main peak and the adjacent impurities is ≥2.0, which meets the specificity requirement.
[0015] High sensitivity and accurate result The response value of the hydroxylamine under the detection wavelength of 254 nm is high, and the impurity at the level of 0.05% can be detected when the concentration of the test sample is 2 mg / mL; the purity RSD of 6 repeated tests is 0.12%, and the maximum single impurity RSD is 0.89%; the RSD of the precision of 6 consecutive sample injections is 0.04%.
[0016] Good durability When the buffer salt concentration is 5-20 mmol / L, the pH is 3.5-4.5, the column temperature is 25-35℃, the flow rate is 0.8-1.2 mL / min, and the detection wavelength fluctuates in the range of 250-260 nm, the system suitability indicators still meet the requirements, and the method is robust.
[0017] Economical and efficient The conventional C18 column and the ammonium acetate-acetonitrile system can realize rapid analysis, the total operation time is 63 min, the equilibration time is short, the testing cost is reduced, and the method is suitable for industrial quality monitoring. BRIEF DESCRIPTION OF DRAWINGS
[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.
[0019] Figure 1 The blank solvent chromatogram in Example 1 of the present application; Figure 2 The test sample solution chromatogram of the hydroxylamine in Example 1 of the present application; Figure 3 The chromatogram of the test sample of the hydroxylamine in Example 2 of the present application by selecting sodium acetate buffer salt system; Figure 4 The chromatogram of the test sample of the hydroxylamine in Example 2 of the present application by selecting buffer salt solution pH 3.0; Figure 5 The chromatogram of the test sample of the hydroxylamine in Example 2 of the present application by selecting methanol as the organic phase; Figure 6 The chromatogram of the test sample of the hydroxylamine in Example 2 of the present application by selecting methanol-water (7:3) as the diluent. DETAILED DESCRIPTION
[0020] The technical solutions in the embodiments of the present application will be clearly and completely described in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.
[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the application belong. If the definitions set forth in this section are contrary to or otherwise inconsistent with the definitions set forth in the patents, patent applications, published patent applications, and other publications that are herein incorporated by reference, the definitions set forth in this section control over the definitions that are incorporated by reference.
[0022] The methods used in the following examples are conventional unless otherwise stated. The materials, reagents and instruments used are conventional unless otherwise stated, and are available to those skilled in the art through commercial channels.
[0023] When a range, preferably a range or a series of upper preferred values and lower preferred values is used to express an equivalent, concentration or other value or parameter, it should be understood that all ranges formed by any pairing of an upper range limit or preferred value with a lower range limit or preferred value, whether or not the range is expressly disclosed, are specifically disclosed. For example, when the range "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its end values and all integers and fractions within the range. In the specification and claims of this application, range limitations can be combined and / or interchanged, unless otherwise stated, and these ranges include all sub-ranges contained therein.
[0024] A high performance liquid chromatography detection method of danazol hydroxy-substance: The instruments and reagents used in the examples are as follows: liquid chromatograph (Agilent 1260, Agilent), ultraviolet detector (Agilent), electronic balance (BS124S, Sartorius), Agilent ZORBAX SB-C18 chromatographic column (4.6 x 250 mm x 5 μm, Agilent), and ammonium acetate for chromatographic purity.
[0025] Example 1 1. High performance liquid chromatography analysis conditions: Chromatographic column: Agilent ZORBAX SB-C18 chromatographic column (4.6 x 250 mm x 5 μm, Agilent) The mobile phase A is a water solution containing ammonium acetate 10 mmol / L, and the pH is adjusted to 4.0 by phosphoric acid. The mobile phase B is acetonitrile, The column temperature of liquid chromatography is 30°C, and the detection wavelength is 254 nm. The flow rate of the mobile phase is 1.0 ml / min, and the gradient elution is performed
[0026] An ultraviolet detector is used, and the injection volume is 20 μL.
[0027] 2. Solution preparation: Preparation of mobile phase: Mobile phase A: Weigh ammonium acetate 0.77 g, dissolve and dilute to the mark with 1 L of water, and adjust the pH to 4.0 with phosphoric acid. Filter to obtain.
[0028] Mobile phase B: acetonitrile.
[0029] Preparation of test solution: Weigh 20 mg of hydroxy test sample, accurately weigh, into a 10 ml volumetric flask, dissolve with diluent, dilute to the mark, and shake well to obtain a test solution with a concentration of 2 mg / ml.
[0030] Blank solution: diluent 3. Determination The detection is performed by liquid chromatography, and the detection conditions are as described above.
[0031] Inject the blank and test solution into the liquid chromatograph, and record the chromatogram. The obtained chromatogram is shown in Figure 1 and Figure 2 The injection sequence is as follows:
[0032] Example 2 1. Method optimization 1.1 Selection of buffer salt: Take an appropriate amount of sodium acetate, respectively, and prepare 10 mmol / L buffer salt solution (adjust the pH to 4.0) with water as mobile phase A, and acetonitrile as mobile phase B; take the test solution of the product, and respectively inject to investigate the detection conditions of the two buffer salt systems.
[0033] Results: In the sodium acetate buffer salt system, the main component peak shows a forward extension, and in the ammonium acetate buffer salt system, the main component peak is slightly forward extended (see attached figure Figure 2 ), which has obvious advantages over the sodium acetate system; the sodium acetate system is not suitable for this method (see attached figure Figure 3 ).
[0034] 1.2 Selection of buffer salt pH: Take 10 mmol / L ammonium acetate buffer salt solution, adjust the pH to 3.0 and 4.0 with phosphoric acid respectively as mobile phase A, and acetonitrile as mobile phase B; take the test solution of the product, and respectively sample to investigate the detection conditions of the two pH buffer salt systems.
[0035] Results: In the pH 3.0 buffer salt system, the main component peak is broadened and significantly delayed and tailed, and the impurities of the product have a tendency to increase in the pH 3.0 elution environment, indicating that the product has poor stability in this system environment, and the pH 3.0 buffer salt system is not suitable for the detection of the product (see Figure Figure 4 ).
[0036] 1.3 Selection of organic phase: Take 10 mmol / L ammonium acetate buffer salt solution, adjust the pH to 4.0 with phosphoric acid as mobile phase A, and use methanol and acetonitrile as mobile phase B respectively; take the test solution of the product, and respectively sample to investigate the detection conditions of the two organic phase systems.
[0037] Results: In the methanol system, the overall peaks of each component are delayed, and the detection time is extended to 95 min, showing poor timeliness of the detection results, indicating that the methanol system is not suitable for the detection of the product (see Figure 5 ).
[0038] 1.4 Selection of diluent: Take 10 mmol / L ammonium acetate buffer salt solution, adjust the pH to 4.0 with phosphoric acid as mobile phase A, and acetonitrile as mobile phase B; take the test solution of the product, and respectively sample to investigate the detection conditions of the two diluent prepared test solution.
[0039] Results: The test solution prepared by using methanol-water (7:3) as diluent shows a new impurity after the main component peak (see Figure 6 ), indicating that the product has poor stability in the solution environment containing methanol, and the detection results may misjudge the quality of the product, therefore, the diluent acetonitrile-water (7:3) is selected for the related substance method of the product.
[0040] 2. Specificity (same as Example 1) 2.1 Solution preparation: Blank: water Test solution: Take about 20 mg of hydroxyamino, accurately weigh into a 10 ml volumetric flask, dissolve with water and dilute to the mark, shake well to obtain a hydroxyamino solution with a concentration of 2 mg / ml.
[0041] 2.2 Determination method Take the blank and test solution according to the following order, other conditions are the same as Example 1, record the chromatogram (see Figure Figures 1-2).
[0042] Injection sequence:
[0043] 2.3 Results: No interference of blank; main component peak type is good in test solution.
[0044] 3. Repeatability 3.1 Preparation of solution: Test solution: Take about 20 mg of hydroxyanisole test sample, accurately weigh, into a 10 ml volumetric flask, dissolve with water and dilute to the mark, shake well, to obtain a hydroxyanisole solution with a concentration of 2 mg / ml. Prepare 6 samples in the same way.
[0045] 3.2 Determination method Take the test solution, according to the injection detection method of Example 1, inject 2 needles each time, and record the chromatogram.
[0046] 3.3 Results Table II: Repeatability data table of hydroxyanisole detection method
[0047] Take the test sample, prepare 6 samples in the same way, the related substance purity of the 6 test solution is between 90.59%-90.88%, the RSD value of the related substance purity of the 6 solutions is 0.12%; the maximum single impurity content of the 6 solutions is 4.01%-4.11%, and the RSD value of the impurity content is 0.89%; it shows that the repeatability of the related substance detection method is good.
[0048] 4. Injection precision 4.1 Preparation of solution: Test solution: Take about 20 mg of hydroxyanisole test sample, accurately weigh, into a 10 ml volumetric flask, dissolve with water and dilute to the mark, shake well, to obtain a hydroxyanisole solution with a concentration of 2 mg / ml.
[0049] 4.2 Determination method Take the test solution, according to the injection detection method of Example 1, inject 6 needles continuously, and record the chromatogram.
[0050] 4.3 Results Table III: Injection precision data table of hydroxyanisole related substance method
[0051] Take one test solution of the product, inject 6 needles continuously, the RSD value of the test sample purity is 0.04%, which shows that the injection precision of the method is good.
[0052] The above merely provides the preferred embodiment of the present application, and is not used to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.
Claims
1. A method for detecting related substances of danazol-hydroxy compounds, characterized in that: After the test sample is dissolved, it is detected by liquid chromatography, and the content of related substances is calculated by peak area normalization method. The liquid chromatography method includes: Chromatographic column: C18 column filled with octadecylsilane bonded silica gel, size 4.6×250mm×5μm; Mobile phase: Mobile phase A is 5-20 mmol / L ammonium acetate-water or sodium acetate-water, pH adjusted to 3.0-4.0 with phosphoric acid; mobile phase B is methanol or acetonitrile; Detector: UV detector, detection wavelength is 250nm-260nm; Column temperature: 25-35°C; Flow rate: 0.8-1.2 ml / min; Injection volume: 10-50 μL; Gradient elution; Test solution: Take (17A)-17-hydroxy-2-(hydroxymethylene)-pregn-4-ene-20-yn-3-one test sample, dissolve it in diluent and dilute it to a concentration of 1.0-3.0 mg / ml.
2. The detection method according to claim 1, wherein The gradient elution procedure includes: 0–20 min, the volume percentage of mobile phase A decreased linearly from 65% to 50%; From 20 to 50 min, the volume percentage of mobile phase A was linearly reduced from 50% to 20%; 50–53 min, the volume percentage of mobile phase A was maintained at 20%; 53–53.01 min, the volume percentage of mobile phase A increased linearly from 20% to 65%; From 53.01 to 63 min, the volume percentage of mobile phase A was maintained at 65%.
3. The detection method according to claim 1, wherein The chromatographic column was Agilent ZORBAX SB-C18 4.6×250 mm, 5 μm.
4. The detection method according to claim 1, wherein The column temperature was 30°C.
5. The detection method according to claim 1, wherein The detection wavelength is 254 nm.
6. The detection method according to claim 1, wherein The flow rate was 1.0 mL / min.
7. The detection method according to claim 1, wherein The injection volume was 20 μL.
8. The detection method according to claim 1, wherein The mobile phase A is a 10 mmol / L ammonium acetate aqueous solution, pH 4.
0.
9. The detection method according to claim 1, wherein The concentration of the test solution is 2.0 mg / mL.
10. The detection method according to claim 1, wherein The diluent is a mixed solution of acetonitrile and water in a volume ratio of 7:3 or a mixed solution of methanol and water in a volume ratio of 7:3.