Method for detecting content of photodegradable impurities in bifonazole solution

Abstract

The invention discloses a method for detecting the content of photodegradation impurities in a bifonazole solution. The photodegradation impurities in the bifonazole solution comprise N-[(4-biphenyl)-phenyl methyl]-formamide (impurity I), an impurity II ((4-biphenyl)-phenyl methylamine) and an impurity III (4-biphenyl)-phenyl ketone). According to the method, a high performance liquid chromatography method is adopted, amide group embedded octadecylsilane chemically bonded silica is used as a filler, acetonitrile-phosphoric acid solutions with different volume ratios are respectively used as a mobile phase A and a mobile phase B, and a gradient elution mode is adopted; wherein the pH value range of the phosphoric acid solution is 3.0-3.4; in the range, good separation of the bifonazole photodegradation impurity I from an adjacent peak can be ensured, and effective and accurate content detection of the photodegradation impurity is facilitated; and meanwhile, the quantitative detection of the impurity II and the impurity III can be realized, and a comprehensive quality control method is provided for the bifonazole photodegradation impurity.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical impurity analysis technology, specifically relating to a method for detecting the content of photodegradation impurities in bifonazole solution. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Bifonazole is a broad-spectrum antifungal active ingredient developed by Bayer AG, Germany, and is effective in treating various skin infections. According to the Japanese IF document (Pharmaceutical Product Information Sheet, based on the Japan Hospital Pharmacists Association's "Information Sheet Writing Guidelines 2018" (2019 Update), this product will produce impurities I, II, and III under light exposure, whose names and structures are as follows:

[0004]

[0005] The commonly used synthetic routes for bifonazole, as disclosed in "Research on the Two-Step Synthesis of Bifonazole" and other literature, are as follows:

[0006] In this synthetic route, the final intermediate (4-phenyldiphenylchloromethane, referred to as impurity IV in this patent) is mixed with acetone and imidazole, heated under reflux, decolorized, filtered and crystallized to obtain the crude product, which is then purified by post-treatment to obtain bifonazole.

[0007] The related substances method for bifonazole is included in the pharmacopoeia. The pharmacopoeia method was used to control impurities in bifonazole solution. However, when using the pharmacopoeia method to detect photodegradation impurities, it was found that the intermediate (impurity IV) and photodegradation impurity I in the bifonazole synthesis process could not be separated, which affected the accurate detection of photodegradation impurities.

[0008] To improve the intermediate process control of pharmaceuticals, effectively analyze drug quality, and ensure medication safety, it is necessary to develop a convenient and effective analytical method for detecting the content of photodegradation impurities in bifonazole. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for detecting the content of photodegradation impurities in bifonazole solution.

[0010] The present invention achieves the above objectives through the following technical solution: In a first aspect, the present invention provides a method for detecting the content of photodegradation impurities in bifonazole solution: The photodegradation impurities in bifonazole solution include: N-[(4-biphenyl)-phenylmethyl]-formamide (impurity I); High-performance liquid chromatography (HPLC) was used with octadecylsilane-bonded silica gel containing amide groups as the packing material. Acetonitrile-phosphoric acid solutions with different volume ratios were used as mobile phase A and mobile phase B, respectively, and a gradient elution method was employed. The pH range of the phosphoric acid solution was 3.0–3.4. Within this range, the resolution between bifonazole photodegradation impurity I and adjacent peaks, especially impurity IV, was greater than 1.5.

[0011] Preferably, the chromatographic column used is an Agilent ZORBAX Bonus-RP, 4.6 mm × 150 mm, 3.5 μm. Chromatographic chromatograms obtained using this column show high peak resolution and good peak shape.

[0012] Preferably, mobile phase A is an acetonitrile-phosphoric acid solution (20:80), and mobile phase B is an acetonitrile-phosphoric acid solution (80:20).

[0013] The gradient program was as follows: from 0 to 25 min, the volume percentage of mobile phase A was maintained at 75%; from 25 to 30 min, the volume percentage of mobile phase A was linearly and gradually changed from 75% to 15%; from 30 to 40 min, the volume percentage of mobile phase A was maintained at 15%; from 40 to 40.1 min, the volume percentage of mobile phase A was linearly and gradually changed from 15% to 75%; and from 40.1 to 45 min, the volume percentage of mobile phase A was maintained at 75%.

[0014] Optionally, the phosphoric acid solution is an aqueous solution of phosphoric acid with a volume concentration of 0.15% to 0.25%, and the pH value is adjusted to 3.0 to 3.4 with triethylamine.

[0015] In some embodiments of the present invention, the pH value of the phosphoric acid solution in the mobile phase is 3.2. The preparation method is as follows: prepare an aqueous solution of phosphoric acid with a volume concentration of 0.2%, and adjust the pH value to 3.2 using triethylamine.

[0016] Optionally, an ultraviolet detector is used with a detection wavelength of 254±2nm.

[0017] Optionally, the column temperature is 40±5℃. Comparative analysis shows that a column temperature within the range of 35~45℃ ensures a resolution greater than 1.5 between impurity I and adjacent peaks. Furthermore, at a column temperature of 40℃, the resolution between impurity I and adjacent peaks is greater than 2.0. To extend the column's lifespan, a column temperature of 40℃ is preferred.

[0018] Optionally, the mobile phase flow rate is 1.0~1.3 ml / min. Within this range, the separation degree between impurity I and the adjacent peak can be guaranteed to be greater than 1.5. More preferably, the flow rate is 1.2 ml / min, at which point the separation degree between impurity I and the adjacent peak is the highest.

[0019] Comparative analysis revealed that the separation effect was optimal at a flow rate of 1.2 ml / min. Decreasing or increasing column temperature, or decreasing or increasing the pH of the phosphoric acid solution, reduced the separation. Based on the robustness test results of this method, the proposed chromatographic column for this method is an Agilent ZORBAX Bonus-RP, 4.6 mm × 150 mm, 3.5 μm, with a flow rate of 1.2 ml / min, a column temperature of 40 °C, and a phosphoric acid solution pH of 3.2 in the mobile phase.

[0020] Furthermore, through tests on linearity and range, limit of quantitation, limit of detection, and accuracy, this method can also accurately detect two other photodegradation impurities: II ((4-biphenyl)-phenylmethylamine) and III (4-biphenyl)-phenylmethyl ketone.

[0021] As can be seen, the method provided by this invention can detect the content of the three previously disclosed photodegradable impurities.

[0022] Furthermore, the detection method also includes sample preparation, the method being: Solvent: Anhydrous ethanol; Test solution: A solution of bifonazole with a concentration of 0.5 ± 0.05 mg / ml; Control solution: A solution with a bifonazole concentration of 1 ± 0.1 μg / ml; System suitability solution: Prepare a solution containing impurities I, II, and III at a concentration of 1 ± 0.1 μg / ml and bifonazole at a concentration of 0.5 ± 0.05 mg / ml; Assay: Accurately measure the test solution, control solution and system suitability solution, inject them into the liquid chromatograph, and record the chromatograms.

[0023] Furthermore, in the detection method, the system suitability meets the following requirements: in the system suitability solution chromatogram, impurity II, bifonazole, impurity I, and impurity III elute in sequence, the resolution between impurity I and adjacent peaks should not be less than 1.5, and the theoretical plate number, calculated based on bifonazole, should not be less than 2000.

[0024] Furthermore, in the detection method, the impurity content is calculated as follows: the contents of impurities I, II, and III are calculated using a self-comparison method with a correction factor, and the calculation formula is: ; In the formula: A 杂 Peak areas of impurities I, II, and III in the test solution; A 对 : Area of ​​the main peak in the control solution; f: Correction factor for impurities I, II, and III.

[0025] Secondly, the present invention provides the application of the method for detecting the content of photodegradable impurities in bifonazole solution described in the first aspect in the detection of photodegradable impurities in bifonazole preparations.

[0026] In some embodiments of the present invention, it was found that the content of photodegradation impurities in bifonazole solutions using different packaging materials varied. Based on this, the detection method of the present invention can be used to detect photodegradation impurities in bifonazole solutions using different packaging materials, and to monitor the stability of bifonazole formulations.

[0027] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: 1. This invention provides a method for detecting the content of photodegradation impurities in bifonazole, which can achieve good separation of impurity I from bifonazole intermediate impurities, and helps to effectively and accurately detect the content of photodegradation impurities; at the same time, it can also achieve quantitative detection of impurities II and III, providing a comprehensive quality control method for photodegradation impurities in bifonazole raw materials and preparations. 2. Methodological validation experiments have demonstrated that the method provided by this invention has good specificity, high sensitivity, high repeatability and accuracy, and strong robustness; it can achieve efficient detection of photodegradation impurities in bifonazole, providing a powerful means for drug stability monitoring. Attached Figure Description

[0028] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0029] Figure 1 This is the chromatogram of the test solution of batch YF24122001 sample in Example 1.

[0030] Figure 2 The chromatograms are from the specificity test in Example 3; from bottom to top, they are the chromatograms of diluent, blank solution, impurity II positioning solution, impurity I positioning solution, impurity III positioning solution, test solution, and mixed solution.

[0031] Figure 3 The chromatograms are for the durability test under the condition of adjusted flow rate (0.8 ml / min to 1.3 ml / min); where a is the flow rate of 0.8 ml / min, b is 1.0 ml / min, c is 1.2 ml / min, and d is 1.3 ml / min.

[0032] Figure 4 The chromatograms are obtained under the adjusted (35℃~45℃) conditions during the durability test; where a represents the column temperature of 35℃ and b represents the column temperature of 45℃.

[0033] Figure 5The chromatograms are for the mobile phase pH values ​​adjusted (pH 2.8–3.4) during the durability test; where a represents mobile phase pH 2.8, b represents mobile phase pH 3.0, and c represents mobile phase pH 3.4.

[0034] Figure 6 Chromatograms under different column conditions for different batches; batch number B22488.

[0035] Figure 7 The following are chromatograms under different column conditions in Comparative Method 1; where a is Waters XBridge C18, and from bottom to top are impurity I localization solution and test solution; b is Agilent Eclipse plus C18; and c is Agilent Poroshell 120 EC-C18.

[0036] Figure 8 The chromatogram is shown under the conditions of comparative method 2, where, from bottom to top, it represents the impurity I localization solution and the test solution.

[0037] Figure 9 This is the chromatogram under the conditions of comparative method 3.

[0038] Figure 10 This is an LC-MS chromatogram of impurity IV in bifonazole solution. Detailed Implementation

[0039] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0040] The present invention will be further described below with reference to the embodiments.

[0041] Bifonazole solution sample information: Specification 1%, sample batch numbers: YF24122001, YF24122002, YF24122003, packaged in transparent glass bottles; YF25051301, packaged in brown glass bottles.

[0042] The prescription for bifonazole solution is as follows: Component Amount (g) Percentage (% w / v) Action Bifonazole 0.1 1 Active ingredient Isopropyl myristate 5.06 50.6 Solvent, emollient Ethanol 3.216 32.20 Solvent, bacteriostatic agent Example 1 1. Chromatographic conditions Test method: ChP2025 General Rules 0512 High Performance Liquid Chromatography.

[0043] Column: Agilent ZORBAX Bonus-RP, 4.6 mm × 150 mm, 3.5 μm; Mobile phase A: Acetonitrile-pH 3.2 phosphoric acid solution (20:80); Mobile phase B: Acetonitrile-pH 3.2 phosphoric acid solution (80:20); pH 3.2 phosphoric acid solution: Take 2.0 ml of phosphoric acid, add 1000 ml of water, shake well, and adjust the pH value to 3.2 with triethylamine.

[0044] Detection wavelength: 254nm; Column temperature: 40℃; Flow rate: 1.2 ml / min; Injection volume: 10 μl; Gradient procedure: Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 25 75 25 30 15 85 40 15 85 40.1 75 25 45 75 25 2. Solution preparation Diluent: Anhydrous ethanol.

[0045] Stock solution of impurities I, II, and III: Weigh approximately 2 mg each of impurities I, II, and III, place them in the same 100 ml volumetric flask, add diluent, sonicate to dissolve and dilute to the mark, shake well, and the solution is ready.

[0046] System suitability solution: Accurately measure 1 ml of impurity I, II, and III stock solution and place it in a 20 ml volumetric flask containing 1 ml of this product solution. Dilute to the mark with diluent and shake well to obtain the solution.

[0047] Test solution: Accurately measure 1 ml of the drug solution and place it in a 20 ml volumetric flask. Dilute to the mark with diluent and shake well to obtain the test solution.

[0048] Control solution: Accurately measure 1 ml of the test solution and place it in a 50 ml volumetric flask. Dilute to the mark with diluent and shake well. Then accurately measure 2 ml of the solution and place it in a 20 ml volumetric flask. Dilute to the mark with diluent and shake well.

[0049] Sensitivity solution: Accurately measure 5 ml of the control solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0050] 3. Determination method After the system is balanced, inject the diluent 2-3 times and each of the other solutions 1 time according to the chromatographic conditions, record the chromatogram, and calculate impurities I, II and III according to the self-comparison method with correction factors.

[0051] 4. System applicability requirements In the sensitivity solution chromatogram, the signal-to-noise ratio of the main peak should be greater than 10; in the system suitability solution chromatogram, impurity II, bifonazole, impurity I, and impurity III should elute in sequence, the resolution between impurity I and the adjacent peak should be not less than 1.5, and the theoretical plate number based on bifonazole should not be less than 2000.

[0052] 5. Calculate the relevant substances using the self-comparison method with correction factors. The calculation formula is as follows: ; In the formula: A 杂 Peak areas of impurities I, II, and III in the test solution; A 对 : Area of ​​the main peak in the control solution; f: Correction factor for impurities I, II, and III.

[0053] 6. Acceptable Standards If the chromatogram of the test solution contains chromatographic peaks with retention times consistent with those of impurities I, II, and III in the system suitability chromatogram, the correction factors and limits for each impurity peak are calculated using the self-comparison method with correction factors, as shown in Table 1.

[0054] Table 1 Correction factors and limits for impurities Impurity name Correction factor Limit (%) Impurity I 1.0 ≤0.2 Impurity II 1.4 ≤0.2 Impurity III 1.5 ≤0.2 7. The results of the sample impurity content test are shown in Table 2 and... Figure 1 As shown.

[0055] Table 2 Detection results of photodegradation impurities in samples

[0056] The results show that packaging the samples in brown bottles helps reduce the generation of photodegradation impurities.

[0057] Example 2 Light damage test Accurately measure 1 ml of this product (batch YF25051301) solution, place it in a quartz petri dish, irradiate under a xenon lamp for 30 hours, then transfer the sample completely to a 20 ml volumetric flask, dilute to the mark with diluent, and shake well. The results are shown in Table 3 using the chromatographic conditions of Example 1. Table 3 Results of photodegradation impurity content in light damage test

[0058] Under other acid, alkali, oxidative, and high-temperature destructive conditions, none of the above-mentioned photodegradable impurities were degraded or increased.

[0059] Example 3 Method validation was performed using YF25051301 batch of bifonazole solution with low levels of impurities I, II, and III. Specificity tests were conducted to locate other potential impurities in the bifonazole solution (impurities A, B, C, and D, which were investigated in other methods) to verify that their presence does not affect the detection of photodegradation impurities. 1. Exclusivity Diluent: Anhydrous ethanol.

[0060] Blank solution: Accurately measure 1 ml of blank excipient solution (a blank excipient without bifonazole prepared according to the prescription ratio), place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the blank solution.

[0061] Stock solutions of impurities I, II, and III: Weigh approximately 5 mg each of I, II, and III accurately, place them in separate 50 ml volumetric flasks, add diluent, sonicate to dissolve and dilute to the mark, shake well, and the solution is ready.

[0062] Impurity mixed stock solution: Accurately measure 5 ml of each of the impurity I, II and III stock solutions, place them in the same 50 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0063] Stock solutions of impurities A and C: Weigh approximately 5 mg each of impurities A and C, place them in separate 50 ml volumetric flasks, add diluent, sonicate to dissolve and dilute to the mark, shake well, and the solution is ready.

[0064] Stock solutions for impurities B and D: Weigh approximately 5 mg each of impurities B and D, place them in separate 20 ml volumetric flasks, add diluent, sonicate to dissolve and dilute to the mark, shake well, and the solution is ready.

[0065] Mixed solution: Accurately measure 0.2 ml of each of the stock solutions of impurities A and C, and stock solutions of impurities B and D, and 2 ml of the mixed stock solution of impurities, and place them in a 20 ml volumetric flask containing 1 ml of the drug solution of this product. Dilute to the mark with diluent and shake well to obtain the solution.

[0066] Positioning solutions for impurities I, II, and III: Accurately measure 1 ml of each of the impurity I, II, and III stock solutions and place them in separate 100 ml volumetric flasks. Dilute to the mark with diluent and shake well to obtain the solution.

[0067] For the positioning solutions of other impurities: accurately measure 0.2 ml each of the stock solutions of impurities A and C, and stock solutions of impurities B and D, place them in different 20 ml volumetric flasks, dilute to the mark with diluent, and shake well to obtain the solution.

[0068] Test solution: Accurately measure 1 ml of the drug solution and place it in a 20 ml volumetric flask. Dilute to the mark with diluent and shake well to obtain the test solution.

[0069] Take 10 μl of each of the above solutions, inject it into the liquid chromatograph, and record the chromatogram.

[0070] The results of the specificity test are shown in Table 4 and Figure 2 As shown.

[0071] Table 4. Results of Specificity Tests for Method Validation of Impurities I, II, and III

[0072] The structure of impurity IV was confirmed, such as Figure 10 As shown, the peak with a mass-to-charge ratio of 243.11830, as measured by LC-MS, represents the molecular ion [M-Cl] of this impurity. + Peak, and its molecular weight (C 19 H 15 (Cl, Exact Mass: 278.0862) is consistent.

[0073] Note: This method examines three photodegradation impurities in bifonazole solution. Therefore, other impurities in the bifonazole solution (impurity A with a retention time of 30.269 min, impurity B with a retention time of 3.133 min, impurity C with a retention time of 37.448 min, and impurity D with a retention time of 31.148 min; located using their respective impurity localization solutions) are only localized in specificity studies to demonstrate that they do not interfere with the examination of the three photodegradation impurities.

[0074] It is evident that the diluent, blank excipient, and other impurity solutions do not interfere with the testing of impurities I, II, and III in this product; the purity angles of impurity I, II, and III peaks in the mixed solution chromatogram are all less than the threshold, and the purity of each impurity peak meets the requirements; the minimum resolution between impurity I, II, and III peaks and adjacent impurity peaks in the mixed solution chromatogram appears between impurity I and impurity IV, which is 1.97, greater than 1.5, meeting the acceptable standard, indicating that this method has good specificity.

[0075] 2. Limit of detection and limit of quantitation Diluent: Anhydrous ethanol.

[0076] Impurity mixed stock solution: Same as 1, under the specificity item.

[0077] Impurity I, II, III limit of quantitation solution: Accurately measure 1 ml of the impurity mixed stock solution, place it in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0078] Detection limit solution: Accurately measure 3 ml of the quantitation limit solution for impurities I, II, and III, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0079] Inject 2-3 doses of the diluent, 1 dose of the limit of detection solution, and 6 consecutive doses of the limit of quantitation solution, and record the chromatograms. The results are shown in Tables 5 and 6. Table 5. Results of Detection Limit Tests for Methodology Validation of Impurities I, II, and III Name Signal-to-noise ratio Concentration (pg / ml) Equivalent to percentage of test sample concentration (%) Impurity I 19.2 0.0291 0.006 Impurity II 54.6 0.0287 0.006 Impurity III 44.8 0.0294 0.006 Conclusion: The S / N ratio of impurity peak I was 19.2, and the detection limit was 0.0291 μg / ml, equivalent to 0.006% of the sample concentration. The S / N ratio of impurity II peak was 54.6, and the detection limit was 0.0287 μg / ml, equivalent to 0.006% of the sample concentration. The S / N ratio of impurity III peak was 44.8, and the detection limit was 0.0294 μg / ml, equivalent to 0.006% of the sample concentration. The results meet the acceptable standard, indicating that impurities I, II, and III with a content ≥0.006% can be detected, demonstrating the good sensitivity of this method.

[0080] Table 6 Results of Limit of Quantitation Tests for Method Validation of Impurities I, II, and III

[0081] Conclusion: The S / N ratio of impurity I peak was 72.1–87.2, the peak area RSD was 0.5%, and the limit of quantitation concentration was 0.0969 μg / ml, equivalent to 0.02% of the test sample concentration. Impurity II peak S / N ranged from 178.1 to 211.0, peak area RSD was 1.1%, and limit of quantitation was 0.0957 μg / ml, equivalent to 0.02% of the test sample concentration. Impurity III peak S / N was 145.6–172.5, peak area RSD was 0.8%, and limit of quantitation (LOQ) was 0.0979 μg / ml, equivalent to 0.02% of the test sample concentration. The results meet the acceptable criteria, indicating that impurities I, II, and III with a content ≥0.02% can be accurately quantified, demonstrating good method sensitivity.

[0082] 3. Linearity and Range Diluent: Anhydrous ethanol.

[0083] Bifonazole stock solution: Weigh approximately 5 mg of bifonazole reference standard accurately, place it in a 50 ml volumetric flask, add diluent, sonicate to dissolve and dilute to the mark, shake well, and the solution is ready.

[0084] Impurity I, II, III stock solutions: Same as 1, under the specificity item.

[0085] Linear stock solution: Accurately measure 5 ml each of bifonazole stock solution and impurity I, II and III stock solutions, place them in the same 50 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0086] Linear solution ① (equivalent to 10% of the limit concentration): Accurately measure 1 ml of the linear stock solution, place it in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0087] Linear solution ② (equivalent to 50% of the limit concentration): Accurately measure 1 ml of the linear stock solution, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0088] Linear solution ③ (equivalent to 100% of the limit concentration): Accurately measure 2 ml of the linear stock solution, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0089] Linear solution ④ (equivalent to 150% of the limit concentration): Accurately measure 3 ml of the linear stock solution, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0090] Linear solution ⑤ (equivalent to 200% of the limit concentration): Accurately measure 2 ml of the linear stock solution, place it in a 10 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0091] Take 10 μl of each of the above linear solutions, inject it into the liquid chromatograph, and record the chromatogram. Plot the concentration C (µg / ml) on the x-axis and the peak area A on the y-axis, perform linear regression using the least squares method, and calculate the correction factor for each impurity.

[0092] As a result, the correlation coefficient r of bifonazole was 0.9999 in the concentration range of 0.1035 μg / ml to 2.0706 μg / ml. Impurity I, within the concentration range of 0.0968 μg / ml to 1.9357 μg / ml (equivalent to 200% of the LOQ), has a correlation coefficient r = 0.9999 and a correction factor of 0.89. Impurity II, within the concentration range of 0.0957 μg / ml to 1.9141 μg / ml (equivalent to 200% of the LOQ), had a correlation coefficient r = 0.9998 and a correction factor of 1.4. Impurity III, within the concentration range of 0.0977 μg / ml to 1.9948 μg / ml (equivalent to 200% of the LOQ), had a correlation coefficient r = 0.9999 and a correction factor of 1.5. All met the acceptable standards, indicating that bifonazole and impurities I, II, and III all showed good linear relationships between concentration and peak area within a certain concentration range.

[0093] 4. Accuracy (1) Recovery rate Diluent: Anhydrous ethanol.

[0094] Impurity mixed stock solution: Same as 1, under the specificity item.

[0095] Test solution: Accurately measure 1 ml of the drug solution and place it in a 20 ml volumetric flask. Dilute to the mark with diluent and shake well. Prepare 3 portions using the same method.

[0096] Impurity reference solution: Accurately measure 2 ml of the impurity mixed stock solution, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0097] 50% recovery solution: Accurately measure 1 ml of the impurity mixture stock solution and place it in a 20 ml volumetric flask containing 1 ml of the drug solution already accurately added. Dilute to the mark with diluent and shake well. Prepare 3 portions using the same method.

[0098] 100% recovery solution: Accurately measure 2 ml of the impurity mixture stock solution and place it in a 20 ml volumetric flask containing 1 ml of the drug solution already accurately added. Dilute to the mark with diluent and shake well. Prepare 3 portions using the same method.

[0099] 150% recovery solution: Accurately measure 3 ml of the impurity mixture stock solution and place it in a 20 ml volumetric flask containing 1 ml of the drug solution already accurately added. Dilute to the mark with diluent and shake well. Prepare 3 portions using the same method.

[0100] Control solution: Accurately measure 1 ml of each of the above 100% recovery solutions, place them in a 50 ml volumetric flask, dilute to the mark with diluent, shake well, then accurately measure 2 ml of each solution, place them in a 20 ml volumetric flask, dilute to the mark with diluent, shake well, and the solution is ready.

[0101] Take 10 μl of each of the above solutions, inject it into the liquid chromatograph, and record the chromatogram.

[0102] Injection requirements: inject the diluent 2-3 times, the impurity reference solution 2 times, and each sample solution 1 time. Calculate the recovery rate using the following formula; the results are shown in Table 7.

[0103] Calculation formula: Amount added:

[0104]

[0105]

[0106] Measured quantity:

[0107] Recovery rate:

[0108] In the formula: A 对 The average peak areas of impurities I, II, and III in the impurity reference solution (two consecutive injections); W 对 Weigh (mg) the samples of impurities I, II, and III in the impurity reference solution. A 杂 Peak areas of impurities I, II, and III in sample solutions with different recovery rates; A 供 The average peak area of ​​impurities I, II, and III in the three test sample solutions.

[0109] Table 7. Results of Accuracy Tests for Methodology Validation of Impurities I, II, and III

[0110] Conclusion: Within the range of 50% to 150% of the limit concentration, The individual recoveries of impurity I ranged from 99.0% to 101.8%, with an average of 99.6% and an RSD of 0.9%. The individual recoveries of impurity II ranged from 98.4% to 101.2%, with a mean of 100.0% and an RSD of 1.1%. The individual recoveries of impurity III ranged from 99.0% to 100.8%, with a mean of 100.1% and an RSD of 0.7%. All met the acceptable standards, indicating that this method is accurate for testing impurities I, II, and III in this product.

[0111] (2) Comparison of different calculation methods The contents of each known impurity in three 100% recovery solutions were calculated using both the external standard method and the self-comparison method with correction factors. The absolute values ​​of the differences in impurity contents obtained by the two methods were also calculated to confirm the reasonable calculation method. The results are shown in Table 8.

[0112] Table 8 Comparison of different calculation methods for the validation of impurity I, II, and III inspection methodologies

[0113] As can be seen, the contents of impurities I, II, and III in three 100% recovery solutions were calculated using both the external standard method and the self-comparison method with correction factors. The absolute values ​​of the differences between the contents obtained by the two calculation methods were 0.001% to 0.006%, which meets the acceptable standard, indicating that the correction factors for each impurity are accurate under this method.

[0114] The linearity and range results show that the correction factors for impurities I, II, and III are 0.89, 1.4, and 1.5, respectively. The correction factor for impurity I is between 0.8 and 1.2, so no correction is needed and the correction factor is set at 1.0. The correction factors for impurities II and III are both greater than 1.2 and are calculated according to the self-comparison method with correction factors.

[0115] 5. Precision (1) Repeatability Diluents, system suitability solutions, and sensitivity solutions: Same as 1, under the specificity category.

[0116] Repeatability solution: Same as 4, 100% recovery solution under accuracy. Prepare 6 portions using the same method.

[0117] Control solution: Accurately measure 1 ml of the repeatability solution and place it in a 50 ml volumetric flask. Dilute to the mark with diluent and shake well. Then accurately measure 2 ml of the solution and place it in a 20 ml volumetric flask. Dilute to the mark with diluent and shake well.

[0118] Take 10 μl of each of the above solutions, inject it into the liquid chromatograph, and record the chromatogram.

[0119] (2) Intermediate precision The operation under item (1) was repeated by different personnel using the same batch of samples at different times and with different instruments, and the RSD of the impurity I, II and III contents of the two individuals was calculated (n=12).

[0120] The results are shown in Table 9.

[0121] Table 9. Results of Precision Tests for Methodology Validation of Impurities I, II, and III

[0122] As can be seen, the RSDs of impurity I, II, and III contents in the 6 repeatable solutions were 0.8%, 1.0%, and 0.8%, respectively. These meet the acceptable standard, indicating that this method has good repeatability for testing the contents of impurities I, II, and III in this product. The RSDs of impurity I, II, and III contents in the 12 repeatable solutions were 1.6%, 6.4%, and 2.8%, respectively. These also meet the acceptable standard, indicating that this method has good intermediate precision for testing the contents of impurities I, II, and III in this product.

[0123] 7. Solution stability Diluent: Anhydrous ethanol.

[0124] Impurity mixed stock solution: Same as 1, under the specificity item.

[0125] Impurity reference solution: Accurately measure 2 ml of the impurity mixed stock solution, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0126] Test solution: Accurately measure 1 ml of this product (batch YF24122001) solution, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the test solution.

[0127] Note: The test solution was prepared using a bifonazole solution with high levels of impurities I, II, and III for stability testing.

[0128] The above-mentioned test solution and impurity reference solution were placed at room temperature, and each was injected once at an appropriate time point to examine the stability of the solution within 48 hours and record the chromatogram.

[0129] The results of the stability tests on the impurity reference solution and the test solution are shown in Table 10.

[0130] Table 10. Results of Stability Tests on Impurity Reference Solutions and Test Solutions for Method Validation of Impurity I, II, and III Inspection Methods

[0131] Conclusion: The results show that after 48 hours at room temperature, the ratio of the peak area of ​​each impurity to the peak area at 0 h for both the impurity reference solution and the test solution is between 0.99 and 1.01, which meets the acceptable standard. This indicates that the impurity I, II, and III reference solutions and the test solution have good stability after 48 hours at room temperature.

[0132] 8. Durability Diluent: Anhydrous ethanol.

[0133] Mixed solutions: Same as 1, mixed solutions under the specificity category.

[0134] Control solution: Accurately measure 1 ml of the mixed solution and place it in a 50 ml volumetric flask. Dilute to the mark with diluent and shake well. Then accurately measure 2 ml of the mixed solution and place it in a 20 ml volumetric flask. Dilute to the mark with diluent and shake well.

[0135] Based on the chromatographic conditions in Example 1, robustness tests were conducted by changing one chromatographic condition at a time. Under normal conditions (column batch number B24372), column temperatures (35℃, 45℃), flow rates (0.8 ml / min, 1.0 ml / min, 1.3 ml / min), different mobile phase pH values ​​(pH 2.8, pH 3.0, pH 3.4), and with a different batch of the same brand column (column batch number B22488), two injections of diluent and one injection of each of the other sample solutions were performed, and the chromatograms were recorded. The resolution results of impurity I and impurity IV in the mixed solution are shown in Table 11 and...Figure 3~6 As shown.

[0136] Note: The flow rate (0.8 ml / min, 1.0 ml / min, 1.3 ml / min) gradient program is as follows: Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 25 75 25 30 15 85 40 15 85 40.1 75 25 50 75 25 Other chromatographic condition gradient programs are as follows: Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 25 75 25 30 15 85 40 15 85 40.1 75 25 45 75 25 The two gradient programs are basically the same, with the same gradient for the first 40 minutes to ensure that all impurity peaks emerge. The only difference is the time it takes to return to the initial gradient equilibrium after 40 minutes.

[0137] Table 11 Results of Chromatographic Condition Robustness Tests for Method Validation of Impurity I, II, and III Detection Methods Different flow rates Separation results 0.8 ml / min Impurity I and Impurity IV cannot be completely separated, no separation degree is shown 1.0 ml / min Impurity I and Impurity IV separation degree 1.84, greater than 1.5, meet the requirements 1.2 ml / min Impurity I and Impurity IV separation degree 2.07, greater than 1.5, meet the requirements 1.3 ml / min Impurity I and Impurity IV separation degree 1.73, greater than 1.5, meet the requirements Different column temperatures Results Column temperature 35℃ Impurity I and Impurity IV separation degree 1.66, greater than 1.5, meet the requirements Column temperature 40℃ Impurity I and Impurity IV separation degree 2.07, greater than 1.5, meet the requirements Column temperature 45℃ Impurity I and Impurity IV separation degree 1.87, greater than 1.5, meet the requirements Different phosphoric acid solutions pH Results pH 2.8 Impurity I and Impurity IV separation degree 1.40, less than 1.5, do not meet the requirements pH 3.0 Impurity I and Impurity IV separation degree 2.13, greater than 1.5, meet the requirements pH 3.2 Impurity I and Impurity IV separation degree 2.07, greater than 1.5, meet the requirements pH 3.4 Impurity I and Impurity IV separation degree 1.82, greater than 1.5, meet the requirements Different batch numbers of chromatographic columns Results B22488 Impurity I and Impurity IV separation degree 1.97, greater than 1.5, meet the requirements Conclusion: The method exhibits good robustness when adjusting flow rate (1.0 ml / min~1.3 ml / min), column temperature (35℃~45℃), different mobile phase pH values ​​(pH 3.0~3.4), and different batches of the same brand, model, and specification of chromatographic columns. Due to the significant variation in resolution between pH 3.0 and pH 2.8, the pH range of 3.0~3.4, which provides more stable resolution, was selected. Furthermore, the optimal separation effect was observed at a flow rate of 1.2 ml / min. Resolution decreased with both decreasing and increasing column temperature and increasing pH of the phosphoric acid solution. The proposed chromatographic column for this method is an Agilent ZORBAX Bonus-RP, 4.6 mm × 150 mm, 3.5 μm, with a flow rate of 1.2 ml / min, a column temperature of 40℃, and a phosphoric acid solution pH of 3.2 in the mobile phase.

[0138] Comparative Example During the method exploration phase, the separation degree between impurity I and impurity IV under different chromatographic conditions was investigated. Since impurity IV is a bifonazole intermediate, a small amount of residue was detected. Through comparison, it was found that when other chromatographic columns were used or the mobile phase ratio and gradient were adjusted, complete separation of impurity IV and impurity I could not be achieved, which would affect the detection and accurate quantification of impurities.

[0139] Sample solution preparation: Diluent: Anhydrous ethanol.

[0140] Stock solutions of impurities I, II, and III: Weigh approximately 5 mg each of impurities I, II, and III accurately, place them in separate 500 ml volumetric flasks, add diluent, sonicate to dissolve and dilute to the mark, and shake well to obtain the stock solution.

[0141] For each impurity positioning solution: accurately measure 2 ml of each impurity stock solution, place it in a 20 ml volumetric flask, dilute to the mark with diluent, and shake well to obtain the solution.

[0142] Test solution: Accurately measure 1 ml of the drug solution and place it in a 20 ml volumetric flask. Dilute to the mark with diluent and shake well to obtain the test solution.

[0143] Mixed solution: Accurately measure 2 ml of each impurity stock solution and place it in a 20 ml volumetric flask containing 1 ml of the drug solution already accurately added. Dilute to the mark with diluent and shake well to obtain the solution.

[0144] Method 1, different chromatographic columns Using the chromatographic conditions provided in Example 1, to shorten the test run time, the gradient program was as follows (the latest elution times of impurity I and impurity IV are both before 35 min; before 35 min, this gradient program is consistent with the gradient program in Example 1). Different packed columns were used, and the above solutions were injected. The results are shown in Table 12 and... Figure 7 As shown: Gradient procedure: Time (min) Mobile phase A (%) Mobile phase B (%) 0 75 25 25 75 25 30 15 85 35 15 85 35.1 75 25 40 75 25 Table 12 Separation effect of different chromatographic columns on impurity I and impurity IV Different chromatographic columns Results Waters XBridge C18, 4.6 mm x 150 mm, 3.5 pm Impurity I and Impurity IV cannot be completely separated Agilent Eclipse plus C18 4.6 mm x 150 mm, 3.5 pm Impurity I and Impurity IV separation degree 1.25, less than 1.5, do not meet the requirements Agilent Poroshell 120 EC-C18 3.0 x 150 mm, 2.7 pm Impurity I and Impurity IV are coincident, and Impurity I is out of shoulder peak, cannot be completely separated It is evident that neither conventional C18 columns nor core-shell columns could achieve the separation of impurity I from impurity IV in the test solution.

[0145] Method 2, Bifonazole Raw Material ChP2025, Part II, Related Substances Method for Bifonazole Raw Material The chromatographic conditions are as follows: Chromatographic column: Waters XBridge C18, 4.6 mm × 250 mm, 5 μm; Mobile phase A: pH 3.2 phosphoric acid solution; Mobile phase B: Acetonitrile; Mobile phase C: Methanol; Detection wavelength: 254nm; Column temperature: 30℃; Flow rate: 1.0 ml / min; Injection volume: 10 μl; Gradient procedure: Time (min) Mobile phase A (%) Mobile phase B (%) Mobile phase C (%) 0 55 35 10 12 55 35 10 20 25 65 10 29 25 65 10 30 10 80 10 404152 105555 803535 101010 The above-mentioned impurity I positioning solution and test sample solution were injected, and the results are as follows: Figure 8 As shown: Conclusion: Using the ChP2025 method for related substances of bifonazole feedstock, impurity I and impurity IV in the test solution are essentially the same and cannot be separated.

[0146] Method 3, patent CN 114252536 B, provides "A high-performance liquid chromatography method for determining related substances in bifonazole raw material". High-performance liquid chromatography (HPLC) was used, and the detection conditions were as follows: Column: Waters XBridge C18, 4.6 mm × 250 mm, 5 μm Mobile phase: Methanol-tetrahydrofuran-10mM ammonium dihydrogen phosphate (pH adjusted to 3.0 with phosphoric acid) (84:1:15); Detection wavelength: 254nm; Column temperature: 25℃; Flow rate: 1.0 ml / min; Injection volume: 10 μl; The above mixed solution was injected, and the results are as follows: Figure 9 As shown: Using the patent CN 114252536 B "A high performance liquid chromatography method for determining related substances in bifonazole raw material", impurity I and impurity IV in the mixed solution completely overlap and cannot be separated.

[0147] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting the content of photodegradable impurities in a bifonazole solution, wherein the photodegradable impurities in the bifonazole solution include N-[(4-biphenyl)-phenylmethyl]-formamide; characterized in that, High-performance liquid chromatography (HPLC) was used with octadecylsilane-bonded silica gel containing amide groups as the packing material. Acetonitrile-phosphoric acid solutions with different volume ratios were used as mobile phase A and mobile phase B, respectively, and gradient elution was employed. The pH range of the phosphoric acid solution was 3.0–3.

4.

2. The method for detecting the content of photodegradation impurities in bifonazole solution according to claim 1, characterized in that, The chromatographic column used was an Agilent ZORBAX Bonus-RP, 4.6 mm × 150 mm, 3.5 μm.

3. The method for detecting the content of photodegradation impurities in bifonazole solution according to claim 1, characterized in that, Mobile phase A is an acetonitrile-phosphoric acid solution of 20:80, and mobile phase B is an acetonitrile-phosphoric acid solution of 80:

20.

4. The method for detecting the content of photodegradation impurities in bifonazole solution according to claim 1, characterized in that, The gradient elution program is as follows: During the 0-25 min period, the volume percentage of mobile phase A is maintained at 75%; during the 25-30 min period, the volume percentage of mobile phase A is linearly and gradually changed from 75% to 15%; during the 30-40 min period, the volume percentage of mobile phase A is maintained at 15%. Within 40 to 40.1 min, the volume percentage of mobile phase A gradually changed linearly from 15% to 75%; within 40.1 to 45 min, the volume percentage of mobile phase A remained constant at 75%.

5. The method for detecting the content of photodegradation impurities in bifonazole solution according to claim 1, characterized in that, The phosphoric acid solution is an aqueous solution of phosphoric acid with a volume concentration of 0.15% to 0.25%, and the pH value is adjusted to 3.0 to 3.4 with triethylamine.

6. The method for detecting the content of photodegradation impurities in bifonazole solution according to claim 1, characterized in that, An ultraviolet detector was used, with a detection wavelength of 254±2nm.

7. The method for detecting the content of photodegradation impurities in bifonazole solution according to claim 1, characterized in that, The column temperature was 40±5℃.

8. The method for detecting the content of photodegradation impurities in bifonazole solution according to claim 1, characterized in that, The mobile phase flow rate is 1.0~1.3 ml / min.

9. The method for detecting the content of photodegradation impurities in bifonazole solution according to claim 1, characterized in that, The detection method also includes sample preparation, the method being: Solvent: Anhydrous ethanol; Test solution: A solution of bifonazole with a concentration of 0.5 ± 0.05 mg / ml; Control solution: A solution with a bifonazole concentration of 1 ± 0.1 μg / ml; System suitability solution: Prepare a solution containing impurities I, II, and III at a concentration of 1 ± 0.1 μg / ml and bifonazole at a concentration of 0.5 ± 0.05 mg / ml; Assay: Accurately measure the test solution, control solution and system suitability solution, inject them into the liquid chromatograph, and record the chromatograms; The method for calculating impurity content is as follows: The contents of impurities N-[(4-biphenyl)-phenylmethyl]-formamide, (4-biphenyl)-phenylmethylamine, and (4-biphenyl)-phenylmethyl ketone are calculated using a self-comparison method with a correction factor. The calculation formula is as follows: In the formula: A 杂 Peak areas of impurities I, II, and III in the test solution; A 对 : Area of ​​the main peak in the control solution; f: Correction factor for impurities I, II, and III.

10. The method for detecting photodegradation impurities in bifonazole solution according to any one of claims 1 to 9 is used in the detection of photodegradation impurities in bifonazole preparations.

Images