Efficient liquid chromatography method for analysis of isomers of aliflamol and its application

Abstract

This invention provides a high-performance liquid chromatography (HPLC) method for the analysis of isomers of iefluconazole and its application. The method enables effective separation and quantitative determination of isomers in iefluconazole raw materials and formulations. It uses the external standard method to calculate the content of each impurity in the test solution, providing a rapid, accurate, and convenient analytical method for the detection of iefluconazole isomers. This method is highly specific, accurate, and sensitive, and reproducible, effectively quantifying the content of isomers in the drug, which is of great significance for the quality control of iefluconazole formulations.

Description

Technical Field

[0001] This invention discloses an efficient high-performance liquid chromatography method for the separation of isomers of icoconazole and its application, belonging to the field of normal phase chromatography separation technology. Background Technology

[0002] Efinaconazole (trade name Jublia) is the first topical triazole antifungal drug developed by Dow Pharmaceuticals of Canada. It was approved by the US FDA on June 6, 2014. It is a topical medication containing 10% (w / w) finaconazole. Its formulation consists of finaconazole, disodium EDTA, butylated hydroxytoluene (BHT) / butylated hydroxyanisole (BHA), anhydrous citric acid, purified water, and C. 12-15 Composed of alkyl lactate, diisopropyl adipate, cyclomethyl silicone, and ethanol, it is mainly used to treat tinea manuum and tinea pedis (i.e., onychomycosis) caused by Trichophyton rubrum and Trichophyton mentagrophytes. Its chemical name is (2R,3R)-2-(2,4-difluorophenyl)-3-(4-methylene-1-piperidinyl)-1-(1H-1,2,4-triazol-1-yl)-2-butanol (Formula 1), which has two chiral centers and three isomers (Formulas 2, 3, and 4). Ifluconazole's mechanism of action is to inhibit fungal 14α-demethylase, thereby interfering with ergosterol synthesis. This enzyme converts lanosterol to ergosterol, an important component of fungal cell membranes, thus affecting cell membrane function and integrity.

[0003]

[0004] Li Peijie et al. developed an HPLC method for the determination of four optical isomers in the active pharmaceutical ingredient iefluconazole (China Pharmaceutical Industry Impurities, 2016, 47(11):1442-1444). Although this method can separate iefluconazole from its three optical isomers, this separation is based on the premise that the concentration of iefluconazole and its three isomers is 25 μg / mL. For iefluconazole topical preparations, in order to meet the requirements for the detection and quantification of iefluconazole isomers, the concentration of iefluconazole in the test sample needs to be set to 2 mg / mL, while the corresponding concentration of the isomers is only about 0.02 mg / mL or lower. The method described by Li Peijie et al. was applied to the analysis of iefluconazole liniment (the test solution contained approximately 2 mg of iefluconazole per 1 mL). The results showed that the main component iefluconazole was not effectively separated from iefluconazole impurity 37 and iefluconazole enantiomer impurities, with a resolution of less than 1.5, which did not meet the requirements. Therefore, quantitative detection of isomer impurities in iefluconazole liniment was not possible. Patent CN107167546A describes an analytical method for the reverse detection of iefluconazole isomers using a C18 column, which involves L-threonine and D-threonine, but the operation is relatively cumbersome. Currently, iefluconazole raw material and its preparations are not included in the current editions of pharmacopoeias of various countries.

[0005] Existing separation methods are either ineffective at separating the aforementioned isomers in iefluconazole formulations or are cumbersome to operate. Therefore, developing a separation and analysis method for isomers in iefluconazole raw materials and formulations is of positive significance for the quality control of iefluconazole formulations. Summary of the Invention

[0006] The present invention aims to provide an effective method for the separation and quantitative determination of three optical isomers in iefluconazole raw materials and preparations, thereby ensuring the quality control of iefluconazole.

[0007] To achieve the above objectives, the present invention provides a high-performance liquid chromatography (HPLC) method for the analysis of isomers of icoconazole, the method comprising the following chromatographic parameters:

[0008] The stationary phase of the chromatographic column is amylose-tris[(S)-α-tolylcarbamate]-bonded silica gel;

[0009] Mobile phase A is n-hexane, and mobile phase B is anhydrous ethanol;

[0010] The volume ratio of anhydrous ethanol to n-hexane is 2–5:95–98;

[0011] Isocratic elution;

[0012] The flow rate is 0.4 mL / min to 0.6 mL / min.

[0013] According to an embodiment of the present invention, the test sample of the method includes: eloxonazole raw material and eloxonazole preparation. According to an embodiment of the present invention, the eloxonazole preparation includes: eloxonazole liniment. According to an embodiment of the present invention, the eloxonazole liniment includes the following components: eloxonazole and butylated hydroxyanisole (BHA). According to an embodiment of the present invention, the eloxonazole liniment includes the following components: eloxonazole, disodium ethylenediaminetetraacetate (EDTA), butylated hydroxytoluene (BHT) / butylated hydroxyanisole (BHA), citric acid, C 12-15 Alkyl lactate, diisopropyl adipate, cyclomethyl silicone, etc.; preferably, the iefluconazole liniment consists of iefluconazole, disodium ethylenediaminetetraacetate (EDTA), butylated hydroxytoluene (BHT) / butylated hydroxyanisole (BHA), anhydrous citric acid, C 12-15 The product comprises alkyl lactate, diisopropyl adipate, cyclomethyl silicone, purified water, and ethanol; preferably, the proportion of aniconazole in the aniconazole liniment is 10% (w / w).

[0014] According to an embodiment of the present invention, the chromatographic column is of the Chiralpak AS-H type. According to an embodiment of the present invention, the column dimensions are 4.6 mm × 250 mm, 5 μm.

[0015] According to an embodiment of the present invention, the isomers of icoconazole include: the enantiomer of icoconazole (2S,3S-configuration) shown in formula (2), the icoconazole impurity 3 (2S,3R-configuration) shown in formula (3), and the icoconazole impurity 37 (2R,3S-configuration) shown in formula (4).

[0016]

[0017] According to a preferred embodiment of the present invention, the volume ratio of anhydrous ethanol to n-hexane is 2:98, 3:97, 4:96 or 5:95.

[0018] According to an embodiment of the present invention, the column temperature is 25℃~35℃; preferably, the column temperature is 28℃~35℃; for example, the column temperature is 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, or 35℃.

[0019] According to an embodiment of the present invention, the flow rate is 0.4 mL / min, 0.45 mL / min, 0.5 mL / min, 0.55 mL / min, or 0.6 mL / min.

[0020] According to an embodiment of the present invention, the injection volume is 5 μL-10 μL; for example, 5 μL, 6 μL, 8 μL, 10 μL.

[0021] According to an embodiment of the present invention, the running time is 30-50 minutes, for example, 30 minutes, 35 minutes, 40 minutes, 45 minutes, or 50 minutes.

[0022] According to an embodiment of the present invention, the detection wavelength is 205nm-215nm; for example, 205nm, 208nm, 210nm, 213nm, 215nm.

[0023] According to an embodiment of the present invention, the method further includes a process for preparing a mixed reference solution of isomers. According to an embodiment of the present invention, the process for preparing the mixed reference solution of isomers is as follows: the enantiomer of iefluconazole shown in formula (2), iefluconazole impurity 37 shown in formula (4), and iefluconazole impurity 3 shown in formula (3) are mixed with a diluent to obtain the mixed reference solution of isomers. According to an embodiment of the present invention, the concentrations of the enantiomer of iefluconazole shown in formula (2), iefluconazole impurity 37 shown in formula (4), and iefluconazole impurity 3 shown in formula (3) are 0.001 mg / mL to 0.04 mg / mL, preferably 0.01 mg / mL to 0.04 mg / mL, for example 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, and 0.04 mg / mL.

[0024] According to an embodiment of the present invention, the method further includes a process for preparing an iefluconazole test solution. According to an embodiment of the present invention, the preparation process of the iefluconazole test solution is as follows: an iefluconazole sample is taken and mixed with a diluent to obtain the test solution.

[0025] According to an embodiment of the present invention, the concentration of iefluconazole in the test sample is 1.5 mg / mL to 3 mg / mL; preferably, the concentration of iefluconazole in the test sample is 2 mg / mL. The theoretical maximum daily total permeation of iefluconazole liniment is 102.58 μg. Referring to ICH Q3B(R2) (Guideline for Quality Control and Quality Assurance of Pharmaceuticals for Human Use) established by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use), the reporting limit for isomers in iefluconazole liniment can be preliminarily determined to be 0.1%, and the limit is 1.0%. Based on the reporting limit of 0.1%, the minimum required concentration of the test sample is 1 mg / mL. To meet the requirements for the detection and quantification of iefluconazole isomers, the concentration of iefluconazole in the test sample in the embodiment of the present invention is set to 1.5 mg / mL to 3 mg / mL. Preferably, the concentration of iefluconazole in the test sample is 2 mg / mL.

[0026] According to an embodiment of the present invention, the method further includes a process for preparing a system suitability solution. According to an embodiment of the present invention, the process for preparing the system suitability solution is as follows: Efluconazole reference standard, the enantiomer of efluconazole shown in formula (2), efluconazole impurity 37 shown in formula (4), and efluconazole impurity 3 shown in formula (3) are mixed with a diluent to obtain the system suitability solution. According to an embodiment of the present invention, the concentrations of the enantiomer of efluconazole shown in formula (2), efluconazole impurity 37 shown in formula (4), and efluconazole impurity 3 shown in formula (3) are 0.001 mg / mL to 0.04 mg / mL, preferably 0.01 mg / mL to 0.04 mg / mL, for example, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, and 0.04 mg / mL. According to an embodiment of the present invention, the concentration of the efluconazole reference standard is 1.5 mg / mL to 3 mg / mL. According to an embodiment of the present invention, the concentration ratio of the effluconazole reference standard to the effluconazole enantiomer shown in formula (2), the effluconazole impurity 37 shown in formula (4), and the effluconazole impurity 3 shown in formula (3) is 150-300:0.1-4:0.1-4:0.1-4, preferably 80-150:0.5-3:0.5-3:0.5-3, for example 100:1:1:1. According to an embodiment of the present invention, the diluent is selected from ethanol, methanol, etc.

[0027] This invention also provides the application of the above-mentioned high-performance liquid chromatography analysis method in the effective separation and quantitative determination of icoconazole isomers.

[0028] Preferably, the application includes the effective separation and quantitative determination of iefluconazole isomers in iefluconazole raw materials and iefluconazole preparations. Preferably, the iefluconazole preparation is an iefluconazole liniment.

[0029] According to an embodiment of the present invention, the isomer of ivermectin has the foregoing definition.

[0030] Beneficial effects

[0031] This invention provides a high-performance liquid chromatography (HPLC) method for the analysis of isomers of iefluconazole. The method enables the effective separation and quantitative determination of isomers in iefluconazole raw materials and formulations (e.g., liniments). It uses an external standard method to calculate the content of each impurity in the test solution, providing a rapid, accurate, and simple analytical approach for the detection of iefluconazole isomers. This method is highly specific, accurate, and sensitive, and reproducible, effectively quantifying the content of isomers in the drug, which is of great significance for the quality control of iefluconazole formulations. Attached Figure Description

[0032] Figure 1: High-performance liquid chromatogram obtained according to the specificity test in Example 1, wherein,Figure 1A This is the chromatogram of the blank solution. Figure 1B This is the chromatogram of the blank formulation solution. Figure 1C This is a chromatogram of the system suitability solution. Figure 1D It is a chromatogram of the mixed reference solution. Figure 1E It is the chromatogram of the test solution;

[0033] Figure 2: High-performance liquid chromatogram obtained according to Example 2, wherein, Figure 2A The chromatogram is shown at a flow rate of 0.4 mL / min. Figure 2B The chromatogram is shown at a flow rate of 0.6 mL / min.

[0034] Figure 3: High-performance liquid chromatogram obtained according to Example 3, wherein, Figure 3A This is the chromatogram at 28℃. Figure 3B The chromatogram is at 32℃;

[0035] Figure 4: High-performance liquid chromatogram obtained according to Example 4, wherein, Figure 4A The chromatogram is for the mobile phase n-hexane-anhydrous ethanol (96:4). Figure 4B The chromatogram is for the mobile phase n-hexane-anhydrous ethanol (98:2);

[0036] Figure 5 : The high performance liquid chromatogram obtained according to Example 5;

[0037] Figure 6 : The high performance liquid chromatogram obtained according to Example 6;

[0038] Figure 7: High-performance liquid chromatogram obtained according to the specificity test in Example 7, wherein, Figure 7A This is the chromatogram of the blank solution. Figure 7B This is a chromatogram of the system suitability solution. Figure 7C It is a chromatogram of the mixed reference solution. Figure 7D It is the chromatogram of the test solution. Figure 7E It is a chromatogram of the spiked test solution;

[0039] Figure 8 : The high performance liquid chromatogram obtained according to Comparative Example 1;

[0040] Figure 9 : The high performance liquid chromatogram obtained according to Comparative Example 2;

[0041] Figure 10 : The high performance liquid chromatogram obtained according to Comparative Example 3;

[0042] Figure 11 : The high performance liquid chromatogram obtained according to Comparative Example 4;

[0043] Figure 12 The high performance liquid chromatogram obtained according to Comparative Example 5;

[0044] Figure 13 : The high performance liquid chromatogram obtained according to Comparative Example 6;

[0045] Figure 14 : The high performance liquid chromatogram obtained according to Comparative Example 7;

[0046] Figure 15 : The high performance liquid chromatogram obtained according to Comparative Example 8;

[0047] Figure 16 The high-performance liquid chromatogram obtained according to Comparative Example 9;

[0048] Figure 17 : The high performance liquid chromatogram obtained according to Comparative Example 10;

[0049] Figure 18 : The high performance liquid chromatogram obtained according to Comparative Example 11. Detailed Implementation

[0050] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0051] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0052] Example 1

[0053] I. Implementation Materials

[0054] Shimadzu LC-20AD high-performance liquid chromatography system and workstation; Waters e2695 high-performance liquid chromatography system and workstation;

[0055] XPE56 electronic balance, XSE205DU electronic balance (Mettler);

[0056] Chromatographic column: Chiralpak AS-H, 4.6mm×250mm, 5μm (Daicel Pharmaceutical Chiral Technology (Shanghai) Co., Ltd.);

[0057] Efluconazole reference standard (batch number E21091500, content 96.56%, Shenzhen Dinglicheng Biotechnology);

[0058] Ifluconazole enantiomer (batch number E20114001, content 96.15%, Shenzhen Dinglicheng Biotechnology);

[0059] Ifluconazole impurity 3 (batch number E20110301, content 95.51%, Shenzhen Dinglicheng Biotechnology);

[0060] Ifluconazole impurity 37 (batch number E20113801, content 90.83%, Shenzhen Dinglicheng Biotechnology);

[0061] Efluconazole Liniment (batch number 230002, specification 10%, the formula consists of evaconazole, disodium EDTA, BHA, anhydrous citric acid, C12-15 alkanol lactate, diisopropyl adipate, cyclomethyl silicone, ethanol and purified water, Taitai Pharmaceutical Co., Ltd.);

[0062] Blank preparation (batch number 230001, Taitai Pharmaceutical Co., Ltd.).

[0063] II. Experimental Methods and Results

[0064] 1. Solution preparation

[0065] Blank solution / diluent: anhydrous ethanol.

[0066] Preparation of elofconazole isomer stock solutions: Accurately weigh approximately 5 mg each of elofconazole enantiomer reference standard, elofconazole impurity 37 reference standard, and elofconazole impurity 3 reference standard, and place them in 25 mL volumetric flasks. Dissolve and dilute to the mark with diluent, and shake well to prepare solutions containing approximately 0.2 mg each of elofconazole impurity 3, elofconazole impurity 37, and elofconazole enantiomer per mL.

[0067] Preparation of system suitability solution: Accurately weigh approximately 20 mg of icoconazole reference standard and place it in a 10 mL volumetric flask. Accurately transfer 1 mL each of the icoconazole enantiomer stock solution, icoconazole impurity 37 stock solution, and icoconazole impurity 3 stock solution into the volumetric flask. Dilute to the mark with diluent to prepare a solution containing approximately 2 mg of icoconazole and approximately 0.02 mg of each of icoconazole impurity 3, icoconazole impurity 37, and icoconazole enantiomer per mL.

[0068] Preparation of blank preparation solution: Weigh 450 mg of blank preparation (without ciproconazole), place it in a 25 mL volumetric flask, add diluent to dissolve and dilute to the mark, and shake well.

[0069] Preparation of mixed reference solution: Accurately transfer 2 mL each of the stock solution of icoconazole enantiomer, icoconazole impurity 37, and icoconazole impurity 3 into the same 20 mL volumetric flask, and dilute to the mark with diluent to prepare a solution containing approximately 0.02 mg of each of icoconazole impurity 3, icoconazole impurity 37, and icoconazole enantiomer per mL.

[0070] Preparation of test solution: Weigh approximately 500 mg of ciprofloxacin lotion accurately, place it in a 25 mL volumetric flask, add diluent to dissolve and dilute to the mark, shake well to prepare a solution containing approximately 2 mg of ciprofloxacin per mL.

[0071] 2. Chromatographic conditions

[0072] Chromatographic column: Chiralpak AS-H, 4.6 mm × 250 mm, 5 μm;

[0073] Mobile phase: anhydrous ethanol-n-hexane, ratio 3:97, isocratic elution;

[0074] Mobile phase flow rate: 0.5 mL / min;

[0075] Column temperature: 30℃;

[0076] Detection wavelength: 210nm;

[0077] Injection volume: 10 μL.

[0078] 3. Specificity test

[0079] Blank solution (anhydrous ethanol), blank formulation solution, system suitability solution, mixed reference solution, and test solution were eluted and detected under the above conditions. The detection results are shown in Figure 1. Among them, Figure 1A This is the chromatogram of the blank solution. Figure 1B This is the chromatogram of the blank formulation solution. Figure 1C This is a chromatogram of the system suitability solution. Figure 1D It is a chromatogram of the mixed reference solution. Figure 1E This is the chromatogram of the test solution.

[0080] The experimental results showed that the blank solution did not interfere with the sample detection. In the system suitability solution, icofol, icofol impurity 37, icofol enantiomer, and icofol impurity 3 eluted sequentially. The chromatograms obtained under this elution program had stable baselines, good peak shapes, and resolutions greater than 1.5.

[0081] 4. Linearity and Range Test

[0082] Solutions of iefluconazole impurity 37, iefluconazole enantiomer, and iefluconazole impurity 3 at various concentrations were prepared as shown in Table 1, and their linearity was tested. The experimental results are shown in Table 2. The linearity of each impurity at each concentration was good, and the correlation coefficient was 1.000 for all impurities.

[0083] Table 1. Linear concentration gradient dilution steps

[0084]

[0085]

[0086] Table 2. Linearity of each isomer

[0087]

[0088] 5. Accuracy Test

[0089] Accurately weigh approximately 400 mg of effluconazole liniment and place it in a 20 mL volumetric flask. Add stock solutions of each isomer to prepare spiked solutions of test samples with limit concentrations of 10%, 100%, and 150%. The test results are shown in Table 3. The recoveries were all between 82.36% and 97.18%, and the RSDs of the nine impurities were 7.8%, 5.1%, and 2.1%, respectively, which met the requirements.

[0090] Table 3. Results of Accuracy Recovery Rate Test

[0091]

[0092] 6. Repeatability test

[0093] Accurately weigh approximately 400 mg of ieconazole liniment and place it in a 20 mL volumetric flask. Accurately transfer 2 mL each of the ieconazole enantiomer stock solution, ieconazole impurity 37 stock solution, and ieconazole impurity 3 stock solution into the volumetric flask. Dissolve and dilute to the mark with diluent to prepare a solution containing approximately 2 mg of ieconazole and 0.02 mg each of ieconazole impurity 3, ieconazole impurity 37, and ieconazole enantiomer per mL. Prepare six parallel solutions. The results are shown in Table 4. The recovery rate of ieconazole isomers was 100.3%–101.5%, and the RSD was 0.3%–0.5%, which met the requirements.

[0094] Table 4 Results of Repeatability Tests

[0095]

[0096] 7. Solution stability

[0097] The mixed reference solution and the spiked test solution were repeatedly analyzed at room temperature at 0h, 3h, 6h, 14.5h, 22.5h, 36h, 57h, and 74h. The mixed reference solution and the spiked test solution were stored in a refrigerator at 2–8℃, sealed, and analyzed repeatedly at 3, 5, and 7 days. The results showed that the absolute values ​​of the change rates of isomer peak areas in both the mixed reference solution and the spiked test solution were no greater than 5.0%. Therefore, both the mixed reference solution and the spiked test solution showed good stability within 74h at room temperature and good stability within 7 days of sealed storage at 2–8℃.

[0098] Example 2

[0099] Instrument: Waters e2695 high performance liquid chromatography system and workstation.

[0100] The chromatographic conditions were as follows: mobile phase flow rate: 0.4 mL / min, 0.6 mL / min; other chromatographic conditions were the same as in Example 1.

[0101] The experimental results showed that at a flow rate between 0.4 mL / min and 0.6 mL / min, iefluconazole could be effectively separated from its isomers and excipients. The liquid chromatograms are shown below. Figure 2A (The retention times of iefluconazole, its enantiomer, impurity 37, and impurity 3 were 15.586 min, 22.212 min, 25.029 min, and 28.060 min, respectively.) Figure 2B (The retention times of icoconazole, its enantiomer, impurity 37, and impurity 3 were 10.441 min, 14.885 min, 16.770 min, and 18.973 min, respectively).

[0102] Example 3

[0103] Instrument: Waters e2695 high performance liquid chromatography system and workstation.

[0104] The chromatographic conditions were as follows: column temperature: 28℃, 32℃, and other chromatographic conditions were the same as in Example 1.

[0105] Experimental results show that when the column temperature is between 28℃ and 32℃, iefluconazole can be effectively separated from its isomers and excipients. (See liquid chromatogram for details.) Figure 3A (The retention times of iefluconazole, its enantiomer, impurity 37, and impurity 3 were 12.842 min, 18.382 min, 20.874 min, and 23.657 min, respectively.) Figure 3B (The retention times of icoconazole, the enantiomer, impurity 37, and impurity 3 were 12.446 min, 17.621 min, 19.694 min, and 22.323 min, respectively).

[0106] Example 4

[0107] Waters e2695 high performance liquid chromatography system and workstation.

[0108] Chromatographic conditions and mobile phase ratios: n-hexane-anhydrous ethanol (96:4); n-hexane-anhydrous ethanol (98:2), other chromatographic conditions are the same as in Example 1.

[0109] Experimental results show that when the mobile phase ratio is between hexane and anhydrous ethanol (96-98:2-4), iefluconazole can be effectively separated from its isomers and excipients. The liquid chromatogram is shown below. Figure 4A(The retention times of iefluconazole, its enantiomer, impurity 37, and impurity 3 were 11.341 min, 15.393 min, 17.134 min, and 18.906 min, respectively.) Figure 4B (The retention times of icoconazole, the enantiomer, impurity 37, and impurity 3 were 14.647 min, 22.243 min, 25.176 min, and 29.210 min, respectively).

[0110] Example 5

[0111] Instrument: Shimadzu LC-20AD high performance liquid chromatography system and workstation.

[0112] Chromatographic conditions: Mobile phase ratio: n-hexane-anhydrous ethanol (95:5), other chromatographic conditions are the same as in Example 1.

[0113] The experimental results showed that elucazole eluted at 10.934 min, elucazole impurity 37 at 13.559 min, the elucazole enantiomer at 15.049 min, and elucazole impurity 3 at 17.112 min. The resolution between the elucazole isomer and each of the isomer impurities was greater than 1.5, meeting the requirements. The liquid chromatogram results are shown below. Figure 5 .

[0114] Example 6

[0115] Instrument: Shimadzu LC-20AD high performance liquid chromatography system and workstation.

[0116] The chromatographic conditions were the same as in Example 1: column temperature 35°C.

[0117] The experimental results show that when the column temperature is 35℃, iefluconazole can be effectively separated from its isomers and excipients, with a resolution of not less than 1.5, which meets the requirements. The liquid chromatogram is shown below. Figure 6 (The retention times of elconazole, the enantiomer, impurity 37, and impurity 3 were 12.291 min, 16.660 min, 18.363 min, and 21.283 min, respectively).

[0118] Example 7

[0119] I. Implementation Materials

[0120] Shimadzu LC-20AD High Performance Liquid Chromatography System and Workstation;

[0121] XPE56 electronic balance, XSE205DU electronic balance (Mettler);

[0122] Chromatographic column: Chiralpak AS-H, 4.6mm×250mm, 5μm (Daicel Pharmaceutical Chiral Technology (Shanghai) Co., Ltd.);

[0123] Efluconazole reference standard (batch number WS20240311, content 99.8%, Shenzhen Haibin Pharmaceutical Co., Ltd.);

[0124] Ifluconazole enantiomer (batch number 46886, purity 98.78%, QUALITY CONTROL SOLUTIONS LTD);

[0125] Ifluconazole impurity 3 (batch number 45223, content 92.73%, QUALITY CONTROL SOLUTIONS LTD);

[0126] Ifluconazole impurity 37 (batch number 46762, content 92.70%, QUALITY CONTROL SOLUTIONS LTD);

[0127] Efluconazole Liniment (batch number 230003, specification 10%, formula composed of evaconazole, disodium EDTA, BHT, anhydrous citric acid, C12-15 alkanol lactate, diisopropyl adipate, cyclomethyl silicone, ethanol and purified water, Taitai Pharmaceutical Co., Ltd.).

[0128] II. Experimental Methods and Results

[0129] 1. Solution preparation

[0130] Same as Example 1

[0131] 2. Chromatographic conditions

[0132] Same as Example 1

[0133] 3. Specificity test

[0134] Blank solution, system suitability solution, mixed reference solution, and test solution were eluted and detected under the chromatographic conditions described above. The detection results are shown in Figure 7. Among them, Figure 7A This is the chromatogram of the blank solution. Figure 7B This is a chromatogram of the system suitability solution. Figure 7C It is a chromatogram of the mixed reference solution. Figure 7D It is the chromatogram of the test solution. Figure 7E This is the chromatogram of the spiked test solution.

[0135] The experimental results showed that the blank solution did not interfere with the sample detection. In the system suitability solution, icofol, icofol impurity 37, icofol enantiomer, and icofol impurity 3 eluted sequentially. The chromatograms obtained under this elution program had stable baselines, good peak shapes, and resolutions greater than 1.5.

[0136] 4. Accuracy Test

[0137] The test method was the same as in Example 1. The test results are shown in Table 5. The recovery rates were all between 88.4% and 110.6%. The RSDs of the nine impurities were 5.0%, 7.0%, and 4.0%, respectively, which met the requirements.

[0138] Table 5. Results of Accuracy Recovery Rate Test

[0139]

[0140] 5. Repeatability test

[0141] The test method was the same as in Example 1. The test results are shown in Table 6. The recovery rate of icofol isomers in the six spiked test solutions was 95.2% to 103.2%, and the RSD of the recovery rate was 0.61% to 0.75%, which met the requirements.

[0142] Table 6 Results of Repeatability Tests

[0143]

[0144]

[0145] 7. Solution stability

[0146] Spiked test solutions were incubated at room temperature for 0 h, 11.5 h, 15 h, 18.5 h, 24 h, 36.5 h, 48 h, 60.5 h, and 67 h before being sampled and analyzed to examine the stability of each solution at room temperature. The results showed that after 67 h at room temperature, compared to the initial point, the absolute values ​​of the peak area changes for iefluconazole impurity 37, iefluconazole enantiomer, and iefluconazole impurity 3 ranged from 0.44 to 4.69, not exceeding 5.0%. Therefore, the mixed spiked test solutions exhibited good stability at room temperature for at least 67 h.

[0147] Comparative Example 1

[0148] Instrument: Waters e2695 high performance liquid chromatography system and workstation.

[0149] During the development of analytical methods for related substances of icoconazole, it was found that icoconazole and icoconazole impurity 3 could be baseline separated under the chromatographic conditions shown in the table below. Therefore, the chromatographic conditions were used to analyze a mixed reference solution of icoconazole, icoconazole enantiomers, icoconazole impurity 37, and icoconazole impurity 3 to investigate the separation of icoconazole from its three isomers.

[0150] Chromatographic column: Waters Symmetry-C18, 5μm, 4.6*250mm.

[0151] The mobile phase was acetonitrile and 0.01 mol / L sodium dihydrogen phosphate buffer solution (adjusted to pH 5.76 with triethylamine).

[0152] The elution method is gradient elution, and the elution procedure is as follows:

[0153]

[0154] The running time was 45 min and the flow rate was 1 mL / min.

[0155] The remaining chromatographic conditions were the same as in Example 1.

[0156] The experimental results showed that iefluconazole impurity 37 and iefluconazole impurity 3 (both with a retention time of 24.058 min), and iefluconazole and its enantiomer completely overlapped (both with a retention time of 25.182 min), and could not be effectively separated. The liquid chromatography chromatograms are shown below. Figure 8 .

[0157] Comparative Example 2

[0158] Instrument: Shimadzu LC-20AD high performance liquid chromatography system and workstation.

[0159] The mobile phase was anhydrous ethanol-n-hexane (1:99), with isocratic elution.

[0160] The running time is 40 minutes.

[0161] The remaining chromatographic conditions were the same as in Example 1.

[0162] The experimental results showed that only three chromatographic peaks could be eluted, namely ieconazole (retention time 7.157 min), ieconazole impurity 37 (retention time 20.533 min), and ieconazole enantiomer (retention time 35.146 min). Effective separation and analysis of ieconazole and its three structurally similar optical isomers could not be achieved. The liquid chromatography chromatograms are shown below. Figure 9 .

[0163] Comparative Example 3

[0164] Instrument: Shimadzu LC-20AD high performance liquid chromatography system and workstation.

[0165] The elution method is gradient elution, and the elution procedure is as follows:

[0166]

[0167] The running time is 50 minutes.

[0168] The remaining chromatographic conditions were the same as in Example 1.

[0169] The experimental results showed that the chromatographic peaks of the various isomers could be well separated. However, the peak width of the enantiomer of iefluconazole (Rt = 41.150 min) was relatively broad, and the peak of iefluconazole impurity 3 (Rt = 47.216 min) was located at a position of sharp baseline change, which was not conducive to the detection and quantification of the isomers. The liquid chromatography chromatogram is shown in [reference needed]. Figure 10 .

[0170] Comparative Example 4

[0171] Instrument: Shimadzu LC-20AD high performance liquid chromatography system and workstation.

[0172] The elution method is gradient elution, and the elution procedure is as follows:

[0173]

[0174] The running time is 34 minutes.

[0175] The remaining chromatographic conditions were the same as in Example 1.

[0176] The experimental results showed that the peak shapes of iefluconazole impurity 37 and its enantiomers were improved, but the peak of iefluconazole impurity 3 (Rt = 30.015 min) remained at the position of sharp baseline change. The liquid chromatography chromatogram is shown below. Figure 11 .

[0177] Comparative Example 5

[0178] Instrument: Shimadzu LC-20AD high performance liquid chromatography system and workstation.

[0179] Since the baseline change time point in Comparative Example 4 was 29 min, and the proportion of anhydrous ethanol in the mobile phase changed from 10% to 2% during the elution gradient period of 28 min to 29 min, while the retention time of the chromatographic peak of icorconazole impurity 3 was 30.015 min, considering that the elution time of impurity 3 falling within the time period of a fixed mobile phase proportion might solve the above problem, the time of the anhydrous ethanol-n-hexane (10:90) portion of the gradient was extended to 32 min. The elution gradient is shown in the table below:

[0180]

[0181] The running time is 38 minutes.

[0182] The remaining chromatographic conditions were the same as in Example 1.

[0183] The experimental results showed that both iefluconazole impurity 3 (Rt = 26.842 min) and the iefluconazole enantiomer (Rt = 25.783 min) were located at positions of sharp baseline change, and effective separation was not achieved between the two chromatographic peaks. (See the liquid chromatography chromatogram below.) Figure 12 .

[0184] Comparative Example 6

[0185] Instrument: Shimadzu LC-20AD high performance liquid chromatography system and workstation.

[0186] The mobile phase was n-hexane-anhydrous ethanol (85:15), and isocratic elution was performed.

[0187] The remaining chromatographic conditions were the same as in Example 1.

[0188] The experimental results showed that elucazole eluted at 13.176 min, and elucazole impurity 37 eluted at 14.511 min. However, the enantiomer of elucazole (Rt = 15.945 min) and elucazole impurity 3 (Rt = 16.354 min) could not be effectively separated. (See liquid chromatography results). Figure 13 .

[0189] Comparative Example 7

[0190] Instrument: Shimadzu LC-20AD high performance liquid chromatography system and workstation.

[0191] The mobile phase was n-hexane-anhydrous ethanol (90:10), with isocratic elution at a flow rate of 0.3 mL / min.

[0192] The remaining chromatographic conditions were the same as in Example 1.

[0193] The experimental results showed that elucazole eluted at 14.488 min, elucazole impurity 37 at 16.808 min, the elucazole enantiomer at 18.618 min, and elucazole impurity 3 at 19.579 min. The resolution between the elucazole isomer and elucazole impurity 3 was 1.138, less than 1.5, which does not meet the requirements. See the liquid chromatography results below. Figure 14 .

[0194] Comparative Example 8

[0195] Instrument: Shimadzu LC-20AD high performance liquid chromatography system and workstation.

[0196] The mobile phase was n-hexane-anhydrous ethanol (93:7), with isocratic elution at a flow rate of 0.4 mL / min.

[0197] The remaining chromatographic conditions were the same as in Example 1.

[0198] Experimental results showed that the resolution between iefluconazole and the adjacent excipient peak BHA was 0.519, which is less than 1.5 and does not meet the requirements. The liquid chromatogram is shown below. Figure 15 .

[0199] Comparative Example 9

[0200] The method disclosed in Reference 1 (Li Peijie et al., HPLC determination of four optical isomers in effluconazole. China Pharmaceutical Industry Impurities, 2016, 47(11):1442-1444) was used to detect effluconazole liniment. The chromatographic conditions are as follows:

[0201] Instruments: Shimadzu LC-20AD high performance liquid chromatography system and workstation;

[0202] Chromatographic column: Daicel Chiralpak IC, 4.6*250mm, 5μm;

[0203] Mobile phase: n-hexane-anhydrous ethanol (85:15), isocratic elution;

[0204] Flow rate: 1.0 mL / min;

[0205] Column temperature: 25℃;

[0206] Detection wavelength: 210nm;

[0207] Injection volume: 10 μL;

[0208] The concentration of the test solution is 2 mg / mL.

[0209] The experimental results showed that elofconazole eluted at 7.182 min, elofconazole impurity 37 at 6.921 min, the elofconazole enantiomer at 7.905 min, and elofconazole impurity 3 at 9.529 min. The resolution between elofconazole and elofconazole impurity 37, as well as the elofconazole enantiomer, was less than 1.5, which does not meet the requirements. Therefore, this method is not suitable for the quantitative detection of elofconazole and its isomers in elofconazole lotion. The liquid chromatography results are shown in the figure. Figure 16 .

[0210] Comparative Example 10

[0211] Based on Comparative Example 9, the mobile phase ratio was adjusted to n-hexane-anhydrous ethanol (90:10), column temperature: 30℃, and isocratic elution was performed.

[0212] The remaining chromatographic conditions were the same as those in Comparative Example 9.

[0213] The experimental results showed that efconazole and efconazole impurity 37 overlapped. The superimposed HPLC chromatograms of the mixed reference solution and the spiked test solution are shown in the figure. Figure 17 .

[0214] Comparative Example 11

[0215] Based on Comparative Example 10, the mobile phase ratio was adjusted to n-hexane-anhydrous ethanol (95:5), and isocratic elution was performed.

[0216] The remaining chromatographic conditions were the same as those in Comparative Example 10.

[0217] The experimental results showed that icoconazole and icoconazole impurity 37 could not be effectively separated. The superimposed HPLC chromatograms of icoconazole and the spiked test solution and the mixed sample solution are shown in the figure. Figure 18 .

[0218] In summary, iefluconazole possesses the active 2R,3R configuration, and its physicochemical properties are extremely similar to those of its three optical isomers. Through the embodiments and comparative examples of this invention, it was found that iefluconazole and its three isomers elute sequentially. Even minor changes in column temperature, flow rate, elution gradient, and mobile phase ratio significantly alter the peak positions and shapes, confirming the difficulty in separating iefluconazole and its isomers as impurities. This invention, through research experiments, screened a reasonable chromatographic condition range suitable for separating the optical isomers of iefluconazole, and through validation experiments, proved that the analytical method is accurate, reliable, and simple.

[0219] The above description is merely an embodiment of the present invention and is not intended to limit the invention. For those skilled in the art, the present invention can have various modifications and variations. 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. In summary, the detection method described in this invention can effectively separate iefluconazole from its isomers and excipients in formulations. It is suitable for the quantitative detection of isomers of iefluconazole raw materials and formulations, and has strong specificity, high sensitivity, simple operation, and is easy to promote.

Claims

1. A high-performance liquid chromatography (HPLC) method for the analysis of isomers of ivermectin, the method comprising the following chromatographic parameters: The stationary phase of the chromatographic column is amylose-tris[(S)-α-tolylcarbamate]-bonded silica gel; Mobile phase A is n-hexane, and mobile phase B is anhydrous ethanol; The volume ratio of anhydrous ethanol to n-hexane is 2–5:95–98; Isocratic elution; The flow rate is 0.4 mL / min to 0.6 mL / min.

2. The method according to claim 1, characterized in that, The test samples of the method include: icoconazole raw material and icoconazole preparation; preferably, the icoconazole preparation includes: icoconazole liniment.

3. The method according to claim 1 or 2, characterized in that, The chromatographic column is of the Chiralpak AS-H type; preferably, the column size is 4.6mm × 250mm, 5μm.

4. The method according to any one of claims 1-3, characterized in that, The isomers of icoconazole include: the enantiomer of icoconazole shown in formula (2), icoconazole impurity 3 shown in formula (3), and icoconazole impurity 37 shown in formula (4).

5. The method according to any one of claims 1-4, characterized in that, The volume ratio of anhydrous ethanol to n-hexane is 2:98, 3:97, 4:96, or 5:95; And / or, the column temperature is 25℃~35℃; preferably, the column temperature is 28℃~35℃.

6. The method according to any one of claims 1-5, characterized in that, The injection volume is 5 μL-10 μL; And / or, the running time is 30-50 minutes; And / or, the detection wavelength is 205nm-215nm.

7. The method according to any one of claims 1-6, characterized in that, The method also includes a process for preparing a mixed reference solution of isomers; Preferably, the preparation process of the mixed reference solution of isomers is as follows: the enantiomer of efconazole shown in formula (2), the efconazole impurity 37 shown in formula (4), and the efconazole impurity 3 shown in formula (3) are mixed with a diluent to obtain the mixed reference solution of isomers. Preferably, the concentrations of the enantiomer of efconazole shown in formula (2), the efconazole impurity 37 shown in formula (4), and the efconazole impurity 3 shown in formula (3) are 0.001 mg / mL to 0.04 mg / mL.

8. The method according to any one of claims 1-6, characterized in that, The method also includes the preparation process of the icoconazole test solution; Preferably, the preparation process of the effluconazole test solution is as follows: take an effluconazole sample, mix it with a diluent, and the test solution is obtained; Preferably, the concentration of the icoconazole test solution is 1.5 mg / mL to 3 mg / mL; more preferably, the concentration of the icoconazole test solution is 2 mg / mL.

9. The method according to any one of claims 1-6, characterized in that, The method also includes a process for preparing a system-suitable solution; Preferably, the preparation process of the system suitability solution is as follows: take erucic acid reference standard, erucic acid enantiomer shown in formula (2), erucic acid impurity 37 shown in formula (4), and erucic acid impurity 3 shown in formula (3), and mix them with diluent to obtain the system suitability solution; Preferably, the concentrations of the enantiomer of icoconazole shown in formula (2), the impurity 37 of icoconazole shown in formula (4), and the impurity 3 of icoconazole shown in formula (3) are 0.001 mg / mL to 0.04 mg / mL; Preferably, the concentration of the icoconazole reference standard is 1.5 mg / mL to 3 mg / mL; Preferably, the concentration ratio of the efconazole reference standard to the efconazole enantiomer shown in formula (2), the efconazole impurity 37 shown in formula (4), and the efconazole impurity 3 shown in formula (3) is 150-300:0.1-4:0.1-4:0.1-4.

10. The application of the method according to any one of claims 1-9 in the effective separation and quantitative determination of icoconazole isomers; Preferably, the application includes the effective separation and quantitative determination of icoconazole isomers in icoconazole raw materials and icoconazole preparations; Preferably, the erucic acid preparation is an erucic acid liniment.

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