Method for detecting residual acetic acid in crocetinate raw material by high performance liquid chromatography

Abstract

The application belongs to the technical field of pharmaceutical analysis, and relates to a method for detecting residual acetic acid in a clilborol raw material by high performance liquid chromatography. An octadecylsilane bonded silica gel is used as a filling agent of a chromatographic column, 0.1% phosphoric acid aqueous solution is used as a mobile phase A, and acetonitrile is used as a mobile phase B to perform gradient elution. The detection method has the advantages of simple sample processing, strong specificity, good durability, and high detection sensitivity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a method for detecting the residual amount of glacial acetic acid in criborone raw materials using high performance liquid chromatography. Background Technology

[0002] Crisaborole, a phosphodiesterase 4 (PDE4) inhibitor, has the molecular formula C2. 14 H 10 BNO3 has a molecular weight of only 251.05 g / mol and its structural formula is as follows.

[0003] .

[0004] This medication reduces the release of inflammatory mediators by inhibiting the activity of phosphodiesterase 4 (PDE4), thereby alleviating symptoms such as itching and redness caused by atopic dermatitis and improving the patient's skin condition. It is suitable for the topical treatment of mild to moderate atopic dermatitis in children aged 3 months and older and adults.

[0005] The currently disclosed synthetic routes for criborone, such as those in literature CN108366958A, require the use of glacial acetic acid as a solvent for purification. Since complete removal is not achieved in the process, according to the "0861 Determination of Residual Solvents" in General Chapter IV of the 2020 edition of the Chinese Pharmacopoeia, glacial acetic acid, as a Class III solvent, must be subject to quality control of its residual amount, with a control limit of 0.5%.

[0006] Currently, there are no reported detection methods for residual glacial acetic acid solvent in criborone raw materials. Therefore, it is urgent to develop relevant detection methods to achieve quality control of criborone raw materials, thereby ensuring the safety and efficacy of the finished compound and its formulations. Summary of the Invention

[0007] This invention provides a high-performance liquid chromatography method for determining the residual amount of glacial acetic acid, which is not affected by blank solvents, and has simple sample preparation, strong specificity, and high sensitivity and accuracy.

[0008] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0009] A method for detecting residual glacial acetic acid in criborone raw material by high performance liquid chromatography involves dissolving the sample in dichloromethane, then extracting it with an equal volume of water, and using the supernatant as the test solution.

[0010] A chromatographic column packed with octadecylsilane-bonded silica gel was used, with 0.1% phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B, for gradient elution. The impurity was glacial acetic acid, and its specific structural formula is shown in Formula I.

[0011] ;

[0012] The elution gradient program is as follows:

[0013] Table 1 Elution gradient program

[0014] .

[0015] As an organic acid, the retention of glacial acetic acid depends primarily on the pH of the mobile phase and the degree of ionization. In neutral or weakly acidic mobile phases, glacial acetic acid exists as a neutral molecule, exhibiting strong hydrophobicity and a short retention time. If the mobile phase pH decreases to acidic levels, glacial acetic acid protonates, increasing its polarity and prolonging its retention time. Therefore, this invention utilizes an acidic mobile phase system to enhance the retention time of glacial acetic acid, enabling its effective detection.

[0016] Furthermore, criborone is poorly soluble in water, and glacial acetic acid is poorly soluble in common organic solvents. Therefore, conventional sample preparation methods are insufficient to dissolve both criborone and glacial acetic acid. Researchers unexpectedly discovered that by using an extraction method—first dissolving the criborone in the organic solvent dichloromethane, then adding an equal volume of water for extraction—the glacial acetic acid in criborone can be extracted for detection, thus resolving the detection problem caused by the different dissolving solvents.

[0017] Preferably, the chromatographic column is a YMC-Pack ODS-AQ, 150×4.6mm, 3μm.

[0018] Preferably, the column flow rate is 0.8 ml / min to 1.2 ml / min.

[0019] Preferably, the detection wavelength is 200nm to 220nm.

[0020] Preferably, the column temperature is 20℃~30℃.

[0021] Preferably, the concentration of glacial acetic acid in the reference solution is in the range of 1.3122 to 104.9749 μg / ml.

[0022] Preferably, when quantitatively determining the impurity glacial acetic acid, the external standard method for impurities is used.

[0023] Preferably, when quantitatively determining the impurity glacial acetic acid, the control limits are as follows: in the quantitation limit 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%.

[0024] A high-performance liquid chromatography (HPLC) method for determining the residual amount of glacial acetic acid in criborone raw materials involves measuring the peak area of ​​a glacial acetic acid reference solution of known concentration at the standard limit, determining the correction factor between the peak area and the reference concentration, and then measuring the peak area of ​​the test sample solution under the same detection conditions. The content of glacial acetic acid in the test sample is then calculated based on the peak area using the external standard method.

[0025] The advantages of this invention are:

[0026] The analytical method provided by this invention is simple to process. By using extraction pretreatment, it overcomes the defect that the active pharmaceutical ingredient is difficult to extract due to its insolubility in water and the difficulty in extracting glacial acetic acid. The blank solution, criborone, and other components do not interfere with the determination of glacial acetic acid, and the detection method has strong specificity. At the same time, it also has the characteristics of good durability and high detection sensitivity, which effectively realizes the quality control of criborone raw material, thereby ensuring the safety and efficacy of criborone raw material and its preparations. Attached Figure Description

[0027] Figure 1 The HPLC chromatogram of the spiked test solution in Example 1 is shown below.

[0028] Figure 2 The linear graph of glacial acetic acid in Example 7;

[0029] Figure 3 The HPLC chromatogram for the detection limit of glacial acetic acid in Example 9 is shown.

[0030] Figure 4 The HPLC chromatogram for the limit of quantitation detection of glacial acetic acid in Example 9 is shown below.

[0031] Figure 5 The chromatogram for the detection of glacial acetic acid content in KLP-2023033101 in Example 10 is shown.

[0032] Figure 6 The chromatogram for the detection of glacial acetic acid content in KLP-2023061101 in Example 10 is shown.

[0033] Figure 7 The image shows the chromatogram for detecting the content of glacial acetic acid in KLP-2023061301 in Example 10. Detailed Implementation

[0034] The present invention will be further described through the following embodiments, but these embodiments do not limit the scope of protection of the present invention.

[0035] In the following examples, the relevant information and chromatographic conditions are as follows:

[0036] The instruments used were Thermo Fisher Ultimate 3000, Thermo Fisher Vanquish, and Agilent 1260.

[0037] The reagents used, including phosphoric acid, acetonitrile, and dichloromethane, were all commercially available chromatographic grade reagents.

[0038] Information on the reference standards and samples used:

[0039] Table 2 Information on Reference Standards and Samples

[0040]

[0041] Chromatographic conditions: YMC-Pack ODS-AQ, 150×4.6mm, 3μm, SN: 120XB20029;

[0042] Mobile phase A: 0.1% aqueous phosphoric acid solution (V / V); Mobile phase B: Acetonitrile;

[0043] Flow rate: 1.0 ml / min;

[0044] Detection wavelength: 208nm;

[0045] Column temperature: 25℃;

[0046] Injection volume: 40 μl;

[0047] Diluent: Water;

[0048] Perform gradient elution as shown in Table 3:

[0049] Table 3 Gradient elution program table

[0050] .

[0051] Example 1: System Applicability and Specificity

[0052] (1) Solution preparation:

[0053] Blank solution: water.

[0054] Reference solution: Weigh an appropriate amount of glacial acetic acid reference standard accurately, dissolve and dilute it with water to prepare a solution containing about 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 10 ml volumetric flask, dilute it to the mark with diluent and shake well, which is used as the reference solution.

[0055] Crizoborone localization solution: Accurately weigh approximately 250 mg of crizoborone working reference standard, place it in a 125 ml separatory funnel, accurately add 25 ml of dichloromethane to dissolve it, then accurately add 25 ml of water for extraction, and take the supernatant as the crizoborone localization solution.

[0056] Test solution: Accurately weigh approximately 250 mg of criborone raw material (batch number KLP-2023033101), 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, and take the supernatant as the test solution.

[0057] Spiked solution for test sample: Accurately weigh approximately 250 mg of criborone raw material (batch number KLP-2023033101), place it in a 125 ml separatory funnel, accurately add 25 ml of dichloromethane to dissolve it, accurately measure 5 ml of the reference stock solution, then accurately add 20 ml of water for extraction, and take the supernatant as the spiked solution for test sample.

[0058] (2) Injection: The blank solution, test solution, criborone positioning solution, and spiked test solution were each analyzed once, and the reference solution was analyzed five times. Chromatograms were recorded. Specific experimental results are shown in Table 4 and... Figure 1 .

[0059] Table 4 System Suitability Test Results

[0060]

[0061] (3) Experimental results: The blank solution showed no interference. With five consecutive injections of the reference solution, the RSD of the main component peak area was 0.47%, less than 2.0%, and the RSD of the main peak retention time was 0.17%, less than 1.0%. The theoretical plate number, calculated based on the glacial acetic acid peak, was 18889, greater than 5000. The resolution between the glacial acetic acid peak and other peaks was greater than 1.5. Crizotinol and other components in the blank solution and the test sample did not interfere with the detection of glacial acetic acid. This method has good system applicability.

[0062] Examples 2-6 Durability

[0063] With the chromatographic conditions unchanged, the following parameters are adjusted:

[0064] (1) Based on Example 1, the flow rates were adjusted to 0.9 ml / min and 1.1 ml / min, respectively;

[0065] (2) Based on Example 1, the column temperature was adjusted to 20℃ and 30℃ respectively;

[0066] (3) Based on Example 1, the detection wavelengths were adjusted to 206 nm and 210 nm respectively;

[0067] (4) Based on Example 1, the initial ratio of the mobile phase was adjusted to A:B = 95:5;

[0068] (5) The chromatographic parameters will be based on those in Example 1, but the chromatographic column will be changed.

[0069] 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 5.

[0070] Table 5. Durability test results.

[0071]

[0072] 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 batches of chromatographic columns, 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 chromatographic column.

[0073] Example 7: Linearity and Range

[0074] 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.

[0075] Solution preparation:

[0076] Blank solution: water.

[0077] Reference solution: Weigh an appropriate amount of glacial acetic acid reference standard accurately, dissolve and dilute it with water to prepare a solution containing about 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 10 ml volumetric flask, dilute it to the mark with diluent and shake well, which is used as the reference solution.

[0078] 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 6.

[0079] Table 6. Preparation of linear stock solutions.

[0080]

[0081] Linear solution: Take the linear stock solution of glacial acetic acid and prepare a linear solution according to Table 7.

[0082] Table 7. Preparation of linear solutions.

[0083]

[0084] The test results are shown in Table 8. Figure 4 .

[0085] Table 8 Linearity / Range Results for Glacial Acetic Acid

[0086]

[0087] Experimental results show that, according to Table 8 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.

[0088] Example 8 Recovery rate

[0089] Take an appropriate amount of glacial acetic acid reference standard (2.5%–120% of the limit) and add it to the criborone raw material. Calculate the recovery rate of the impurity by subtracting the amount of impurity in the sample from the measured amount and then adding the added amount. The test results are shown in Table 9.

[0090] Table 9 Results of the test on the recovery rate of glacial acetic acid as an impurity

[0091]

[0092] The experimental results show that, calculated using the external standard method, the system meets the suitability requirements. The recoveries of glacial acetic acid at 80% concentration ranged from 94.46% to 98.44%, at 100% concentration from 93.85% to 97.83%, at 120% concentration from 94.31% to 99.45%, at the limit of quantitation (LOQ) from 80.78% to 93.13%, and at the LQ concentration level from 70% to 130%. The recoveries at other concentration levels ranged from 80% to 120%. The RSD of the recoveries at each concentration level was less than 10%, indicating good accuracy of the method.

[0093] Example 9 Sensitivity (Limit of Detection, Limit of Quantification)

[0094] Detection limit solution: Accurately measure 5 ml of the quantitation limit solution into a 10 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0095] Limit of Quantitation Solution: Accurately measure an appropriate amount of the reference solution and dilute it to prepare a solution of approximately 1.25 ug / ml as the limit of quantitation solution.

[0096] The test results are shown in Tables 10 and 11. Figure 2 and Figure 3 .

[0097] Table 10 Results of Limit of Quantitation Test 1

[0098]

[0099] Table 11 Results of Limit of Quantitation Test 2

[0100]

[0101] The experimental results showed that the detection limit of glacial acetic acid was 0.6561 μg / ml, equivalent to 0.007% of the sample concentration, and the quantitation limit was 1.3122 μg / ml, equivalent to 0.013% of the sample concentration. The RSD of the peak area of ​​the 5 needles for the quantitation limit of glacial acetic acid was 1.8%, which is less than 10%.

[0102] Example 10: Determination of glacial acetic acid content in keriborone raw material

[0103] Solution preparation:

[0104] Blank solution: water.

[0105] 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.

[0106] Reference solution: Accurately measure 2 ml of the reference stock solution and place it in a 10 ml volumetric flask. Dilute to the mark with diluent and shake well to obtain the reference solution.

[0107] Test solution: Accurately weigh about 250 mg of criborone raw material, 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, and take the supernatant as the test solution.

[0108] Calculation formula: The content of glacial acetic acid in the test sample is calculated based on the peak area using the external standard method.

[0109]

[0110] In the formula:

[0111] m1—The sample weight of glacial acetic acid reference standard, in mg;

[0112] Content of ρ-glacial acetic acid reference standard, %

[0113] m2—The weight of the test sample, in mg;

[0114] V1—Dilution volume of the reference solution, ml;

[0115] V2—Dilution volume of the test solution, ml;

[0116] A1—Peak area of ​​glacial acetic acid in the reference solution;

[0117] A2—Peak area of ​​glacial acetic acid in the test solution.

[0118] According to the pharmacopoeia standard, the residual solvent content of glacial acetic acid in criborone raw material should not exceed 0.5%. Three batches of criborone raw material (KLP-2023033101, KLP-2023061101 and KLP-2023061301) were tested for glacial acetic acid content. The test results are detailed in Table 12.

[0119] Table 12 Results of glacial acetic acid content determination.

[0120]

[0121] The experimental results showed that the amount of glacial acetic acid detected in the samples all met the standard requirements.

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: 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 for determining the residual amount of glacial acetic acid in criborone raw material by high performance liquid chromatography according to claim 1, uses an octadecylsilane-bonded silica gel column as the packing material, and performs gradient elution with 0.1% phosphoric acid aqueous solution as mobile phase A and acetonitrile as mobile phase B. in, The elution gradient program is as follows: 。 3. The method according to claim 1, characterized in that, The chromatographic column was a YMC-Pack ODS-AQ, 150×4.6mm, 3μm.

4. The method according to claim 1, characterized in that, The column flow rate was 0.8 ml / min to 1.2 ml / min.

5. The method according to claim 1, characterized in that, The detection wavelength is 200nm~220nm.

6. The method according to claim 1, characterized in that, The column temperature is 20℃~30℃.

7. 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.

8. 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.

9. 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%.

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