Method for detecting residual amount of glacial acetic acid in crisaborole raw material by high performance liquid chromatography
By employing high-performance liquid chromatography and gradient elution technology, the problem of detecting residual glacial acetic acid in criborone raw materials has been solved, achieving highly sensitive and specific detection results to ensure drug quality and safety.
Patent Information
- Application Number
- CN202511419844.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-30
- Publication Date
- 2026-01-20
- Estimated Expiration
- Not applicable · inactive patent
AI Technical Summary
There is a lack of effective methods in the current technology to detect the residual amount of glacial acetic acid in criborone raw materials, which affects the quality control and safety of the drug.
High-performance liquid chromatography (HPLC) was employed, using a column packed with high-purity silica gel particles chemically bonded to octadecylsilane, and a mobile phase of 0.1% phosphoric acid aqueous solution and acetonitrile. The sample was processed by gradient elution and extraction to ensure the effective retention and detection of glacial acetic acid.
It achieves highly sensitive and specific detection of residual glacial acetic acid in criborone raw materials, with simple sample processing, ensuring the accuracy and safety of drug quality control.
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Figure CN121364262A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for detecting residual acetic acid in Crisaborole raw materials by high performance liquid chromatography. BACKGROUND
[0002] Crisaborole is a phosphodiesterase 4 (PDE4) inhibitor, and its molecular formula is C 14 H 10 BNO3, and its molecular weight is only 251.05 g / mol, and its structural formula is
[0003]
[0004] The drug can relieve skin itching, redness and other symptoms caused by atopic dermatitis by inhibiting the activity of phosphodiesterase 4 (PDE4) and reducing the release of inflammatory mediators, and improve the skin condition of patients. It is suitable for local treatment of mild to moderate atopic dermatitis in children and adults over 3 months old.
[0005] In the process of synthesizing Crisaborole raw materials, ice acetic acid is used as a solvent for purification and refining in the last step process. Since it cannot be completely removed in the process, according to the requirements of the 2020 edition of the Chinese Pharmacopoeia four-part general rules "0861 residual solvent determination method", the residual amount of ice acetic acid needs to be quantitatively detected. Ice acetic acid is a third type of solvent, and the limit is 0.5%.
[0006] At present, there is no related detection method for the residual ice acetic acid solvent in Crisaborole raw materials reported, so it is urgent to develop a related detection method to realize the quality control of Crisaborole raw materials, so as to ensure the safety and effectiveness of the finished compound and its preparation. SUMMARY
[0007] The application provides a method for determining the residual amount of ice acetic acid by high performance liquid chromatography, which is not interfered by a blank solvent, has simple sample treatment, strong specificity, high sensitivity and high accuracy.
[0008] In order to achieve the above application purposes, the technical solutions adopted by the application are as follows:
[0009] A method for detecting the residual amount of ice acetic acid in Crisaborole raw materials by high performance liquid chromatography, which adopts a chromatographic column with high-purity silica gel particles chemically bonded with octadecylsilane as a filler, 0.1% phosphoric acid aqueous solution as a mobile phase A, and acetonitrile as a mobile phase B for gradient elution, the impurity is ice acetic acid, and the specific structural formula is as shown in formula I:
[0010]
[0011] The elution gradient program is as follows:
[0012] Table 1 elution gradient program table
[0013]
[0014] When the test solution is prepared, dichloromethane is used for dissolution, and water is added in an equal volume to the dichloromethane for extraction, and the upper liquid is taken as the test solution.
[0015] Glacial acetic acid is an organic acid, and its retention mainly depends on the pH value and ionization degree of the mobile phase. In a neutral or weakly acidic mobile phase, glacial acetic acid exists in the form of a neutral molecule, has strong hydrophobicity, and has a short retention time; if the pH of the mobile phase is lowered to be acidic, the glacial acetic acid is protonated, the polarity is increased, and the retention time is prolonged. Therefore, the acid mobile phase system is used in the present application, the retention time of the glacial acetic acid is enhanced, and the glacial acetic acid can be effectively detected.
[0016] In addition, the raw material of crizotinib is difficult to dissolve in water, and the glacial acetic acid is difficult to dissolve in common organic solvents, therefore, the conventional test solution preparation method cannot meet the requirements of dissolving the raw material of crizotinib and dissolving the glacial acetic acid. The researchers accidentally found that by using the extraction method, the raw material of crizotinib is first dissolved in an organic solvent dichloromethane, and an equal volume of water is added for extraction, and the glacial acetic acid in crizotinib can be extracted for detection, thereby solving the detection problem caused by the different dissolution solvents of the two.
[0017] Preferably, the chromatographic column is YMC-Pack ODS-AQ, 150*4.6mm, 3μm or a chromatographic column with equivalent performance.
[0018] Preferably, the column flow rate is 0.8ml / min-1.2ml / min.
[0019] Preferably, the detection wavelength is 200nm-220nm.
[0020] Preferably, the column temperature is 20℃-30℃.
[0021] Preferably, the concentration of glacial acetic acid in the control solution is in the range of 1.3122-104.9749μg / ml.
[0022] Preferably, when the impurity glacial acetic acid is quantitatively determined, the impurity external standard method is used for calculation.
[0023] Preferably, when the impurity glacial acetic acid is quantitatively determined, the control limit is that the signal-to-noise ratio of glacial acetic acid in the quantitative limit solution should not be less than 10, and the RSD of the peak area should not be more than 10.0%.
[0024] The peak area and the concentration of the control sample solution are determined by using a known concentration of glacial acetic acid control sample solution with standard limits, a correction factor between the peak area and the concentration of the control sample solution is determined, then the peak area of the test sample solution is determined under the same detection conditions, and the content of glacial acetic acid in the test sample is calculated according to the peak area by the external standard method,
[0025] The present application has the advantages that:
[0026] The analysis method provided by the present application has the advantages of simple sample treatment, overcomes the defect that the raw material drug is difficult to extract due to the insolubility of glacial acetic acid in water, has no interference with the determination of glacial acetic acid for both the blank solution and the test sample, has strong specificity of the detection method, has good durability and high detection sensitivity, effectively realizes the quality control of the raw material of crisaborole, and ensures the safety and effectiveness of the finished compound and its preparation. BRIEF DESCRIPTION OF DRAWINGS
[0027] Figure 1 The HPLC spectrum of the spiked test sample solution is detected;
[0028] Figure 2 The HPLC spectrum of the glacial acetic acid quantitative limit is detected;
[0029] Figure 3 The HPLC spectrum of the glacial acetic acid detection limit is detected;
[0030] Figure 4 The linear graph of glacial acetic acid.
[0031] DETAILED EMBODIMENT
[0032] The present application is further described below through various embodiments, but these embodiments do not limit the protection scope of the present application.
[0033] Example 1: System applicability and specificity
[0034] Chromatographic conditions: chromatographic column with high-purity silica gel particles chemically bonded with octadecylsilane as filler (recommended column: YMC-Pack ODS-AQ, 150x4.6mm, 3μm or chromatographic column with equivalent performance)
[0035] Mobile phase A: 0.1% phosphoric acid aqueous solution; mobile phase B: acetonitrile;
[0036] Flow rate: 1.0ml / min;
[0037] Detection wavelength: 208nm;
[0038] Column temperature: 25℃;
[0039] Injection volume: 40μl;
[0040] Diluent: water
[0041] The gradient elution was carried out according to Table 2:
[0042] Table 2 Gradient elution procedure table
[0043] Time (min) Mobile phase A (%) Mobile phase B (%) 0 100 0 5 100 0 10 0 100 12 0 100 12.1 100 0 20 100 0
[0044] 1.1 System suitability test and specificity test
[0045] (1) Preparation of solutions
[0046] Blank solution: water
[0047] Reference solution: accurately weigh chloroborole reference substance, dissolve and dilute with water to prepare a solution containing about 0.25 mg per 1 ml, as a reference stock solution; accurately take 2 ml of the reference stock solution into a 10 ml flask, dilute to the mark with diluent and shake well, as the reference solution.
[0048] Chloroborole positioning solution: accurately weigh about 250 mg of chloroborole working reference substance into a 125 ml separatory funnel, dissolve with 25 ml of dichloromethane, then extract with 25 ml of water, and take the upper layer as the chloroborole positioning solution.
[0049] Test solution: accurately weigh about 250 mg of chloroborole into a 125 ml separatory funnel, dissolve with 25 ml of dichloromethane, then extract with 25 ml of water, and take the upper layer as the test solution.
[0050] Separation degree solution: accurately weigh about 250 mg of chloroborole into a 125 ml separatory funnel, dissolve with 25 ml of dichloromethane, accurately take 5 ml of the reference stock solution, then extract with 20 ml of water, and take the upper layer as the separation degree solution.
[0051] (2) Injection: analyze the blank solution, test solution, chloroborole positioning solution, and separation degree solution each time, and analyze the reference solution 5 times, and record the chromatogram; the specific test results are shown in Table 3 and Figure 1 .
[0052] Table 3 System suitability test results
[0053]
[0054] (3) Test results: The blank solution had no interference. The control solution was continuously injected for 5 times. The RSD of the peak area of the main component was 0.47%, which was less than 2.0%. The RSD of the retention time of the main peak was 0.17%, which was less than 1.0%. The theoretical plate number was 18889 according to the peak of ice acetic acid, which was greater than 5000. The separation degree between the peak of ice acetic acid and other peaks was greater than 1.5. The blank and clilorol peaks did not interfere with the detection of ice acetic acid. The system suitability of the method was good.
[0055] 1.2. Sensitivity of ice acetic acid (detection limit, quantification limit)
[0056] Detection limit solution: accurately take 5 ml of the quantification limit solution into a 10 ml volumetric flask, dilute to the mark with the diluent, shake well, and obtain the detection limit solution.
[0057] Quantification limit solution: accurately take the control solution, dilute to obtain a solution of about 1.25 ug / ml as the quantification limit solution.
[0058] The test results are shown in Tables 4, 5 and Figure 2 .
[0059] Table 4 Test results of the quantification limit 1
[0060]
[0061] Table 5 Test results of the quantification limit 2
[0062]
[0063] The test results showed that the detection limit of ice acetic acid was 0.6561 ug / ml, which was equivalent to 0.007% of the concentration of the test sample. The quantification limit was 1.3122 ug / ml, which was equivalent to 0.013% of the concentration of the test sample. The RSD of the peak area of the quantification limit solution of ice acetic acid was 1.8%, which was less than 10%.
[0064] Example 2-8
[0065] The chromatographic condition parameters were unchanged, and the following parameters were adjusted:
[0066] (1) The flow rate was adjusted to 0.9 ml / min and 1.1 ml / min based on Example 1.
[0067] (2) The column temperature was adjusted to 20°C and 30°C based on Example 1.
[0068] (3) The detection wavelength was adjusted to 206 nm and 210 nm based on Example 1.
[0069] (4) The initial proportion of the mobile phase was adjusted to A:B = 95:5 based on Example 1.
[0070] (5) The chromatographic parameters are based on those in Example 1, but the chromatographic column will be replaced.
[0071] High-performance liquid chromatography (HPLC) was performed under each of the aforementioned durability conditions. The blank solution was analyzed once, reference solution 1 was analyzed five times, reference solution 2 was analyzed twice, the test solution was analyzed once, and the spiked test solution was analyzed once. Specific test results are shown in Table 6.
[0072] Table 6. Durability Test Results
[0073]
[0074] The experimental results show that the RSD of the content data under various chromatographic conditions, including slight changes in wavelength, flow rate, column temperature, and mobile phase ratio, as well as different column batches, is 0.84%, which is less than 2.0%. This indicates that the method has good robustness under slight changes in chromatographic conditions such as wavelength, flow rate, column temperature, mobile phase ratio, and column.
[0075] Example 10: Quantitative determination of glacial acetic acid content
[0076] The chromatographic conditions remained unchanged, the same as in Example 1.
[0077] (1) Linearity and range of glacial acetic acid
[0078] In the analytical method for the content of glacial acetic acid in criborone, the concentration of the test solution is 10 mg / ml, the limit of glacial acetic acid is 0.5%, and the linearity validation range is 50% to 200% of the limit concentration. Five solutions with different concentrations are prepared within this concentration range for validation.
[0079] Solution preparation:
[0080] Blank solution: water.
[0081] Reference solution: Weigh an appropriate amount of glacial acetic acid reference standard accurately, dissolve and dilute it with water to prepare a solution containing approximately 0.25 mg per ml, which is used as the reference standard stock solution; accurately measure 2 ml of the above reference standard stock solution, place it in a volumetric flask, dilute it to the mark with diluent and shake well, which is used as the reference solution.
[0082] Linear stock solution: Accurately weigh an appropriate amount of glacial acetic acid reference standard, dissolve it in diluent, dilute to the mark, and shake well. Prepare according to the method in Table 7.
[0083] Table 7 Preparation of Linear Stock Solution
[0084] Control name Weighed (mg) Vial (ml) Linear stock concentration (mg / ml) Glacial acetic acid 25 100 0.25
[0085] Linear solution: Take the linear stock solution of glacial acetic acid and prepare a linear solution according to Table 8.
[0086] Table 8 Preparation of Linear Solutions
[0087]
[0088] The test results are shown in Table 9. Figure 4 .
[0089] Table 9 Linearity / Range Results for Glacial Acetic Acid
[0090] Percentage concentration 2.5％ 50％ 80％ 100％ 150％ 200％ Concentration (pg / ml) 1.3122 26.2437 41.9900 52.4875 78.7312 104.9749 Peak area 1 0.031 0.616 0.942 1.175 1.764 2.364 Peak area 2 0.030 0.606 0.946 1.175 1.764 2.373 Average peak area 0.0305 0.6110 0.9440 1.1750 1.7640 2.3685
[0091] Experimental results show that, according to Table 7 and Figure 4 The test results show that glacial acetic acid exhibits a good linear relationship between peak area and concentration in the concentration range of 1.3122–104.9749 μg / ml, with the linear equation being y = 0.0224x + 0.0054, R0. 2 The value is 0.9999, and the intercept deviation is 0.005.
[0092] (2) Calculation of glacial acetic acid content
[0093] Solution preparation
[0094] Blank solution: water
[0095] Reference stock solution: Take an appropriate amount of glacial acetic acid reference standard, accurately weigh it, dissolve and dilute it with water to prepare a solution containing about 0.25 mg per 1 ml, which is used as the reference stock solution.
[0096] Reference solution: Accurately measure 2 ml of the reference stock solution, place it in a volumetric flask, dilute to the mark with diluent and shake well to obtain the reference solution.
[0097] Test solution: Accurately weigh about 250 mg of criborone and place it in a 125 ml separatory funnel. Accurately add 25 ml of dichloromethane to dissolve it, then accurately add 25 ml of water to extract it. Take the supernatant as the test solution.
[0098] Calculation formula
[0099] The content of glacial acetic acid in the test sample was calculated based on peak area using the external standard method.
[0100]
[0101] In the formula:
[0102] m1—The sample weight of glacial acetic acid reference standard, in mg;
[0103] ρ—Content of glacial acetic acid reference standard, %;
[0104] m2—The weight of the test sample, in mg;
[0105] V1—dilution volume of control solution, ml;
[0106] V2—dilution volume of test solution, ml;
[0107] A1—peak area of glacial acetic acid in control solution;
[0108] A2—peak area of glacial acetic acid in test solution.
[0109] The content of glacial acetic acid in Crofelemer should be not more than 0.5%. The test results are shown in Table 10.
[0110] Table 10 Test results of glacial acetic acid content
[0111]
[0112] The experimental results show that the detected amount of glacial acetic acid in the sample meets the standard requirements.
[0113] (3) Recovery rate of glacial acetic acid
[0114] An appropriate amount of impurity glacial acetic acid reference substance was added to the Crofelemer raw material according to 2.5% to 120% of the limit, and the recovery rate of the impurity was calculated by the ratio of the measured amount minus the amount of impurity in the sample to the added amount. The test results are shown in Table 11.
[0115] Table 11 Test results of recovery rate of impurity glacial acetic acid
[0116]
[0117]
[0118] The test results show that according to the external standard method, the system applicability requirements are met; the recovery rate of glacial acetic acid with 80% concentration of the added sample is between 94.46% and 98.44%, the recovery rate of glacial acetic acid with 100% concentration of the added sample is between 93.85% and 97.83%, the recovery rate of glacial acetic acid with 120% concentration of the added sample is between 94.31% and 99.45%, the recovery rate of the limit of quantification is between 80.78% and 93.13%, the recovery rate of the limit of quantification concentration level is between 70% and 130%, and the recovery rate of each concentration level is between 80% and 120%; the RSD of the recovery rate of each concentration level is less than 10%, and the accuracy of the method is good.
Claims
1. A method for determining the residual amount of glacial acetic acid in criborone raw material by high performance liquid chromatography, characterized in that: A chromatographic column packed with high-purity silica gel particles chemically bonded to octadecylsilane was used. Gradient elution was performed with 0.1% phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B. The impurity was glacial acetic acid, and its specific structural formula is shown in Formula I. The elution gradient program is as follows: ； When preparing the test solution, dichloromethane is used for dissolution, followed by extraction with an equal volume of water to dichloromethane, and the supernatant is taken as the test solution.
2. The method according to claim 1, characterized in that, The chromatographic column is a YMC-Pack ODS-AQ column, 150×4.6mm, 3μm or equivalent in performance.
3. The method according to claim 1, characterized in that, The column flow rate was 0.8 ml / min to 1.2 ml / min.
4. The method according to claim 1, characterized in that, The detection wavelength is 200nm~220nm.
5. The method according to claim 1, characterized in that, The column temperature is 20℃~30℃.
6. The method according to claim 1, characterized in that, The concentration of glacial acetic acid in the reference solution was in the range of 1.3122–104.9749 μg / ml.
7. The method according to claim 1, characterized in that, When quantitatively determining the impurity glacial acetic acid, the external standard method for impurities shall be used.
8. The method according to claim 8, characterized in that, when quantitatively determining the impurity glacial acetic acid, the control limit is that, in the limit of quantitation solution, the signal-to-noise ratio of glacial acetic acid shall not be less than 10; and the RSD of the peak area shall not exceed 10.0%.