Method for detecting impurities in adapalene starting material by HPLC (High Performance Liquid Chromatography) method

Abstract

The invention belongs to the technical field of chemical analysis, and particularly relates to a method for detecting adapalene starting material impurities by utilizing an HPLC (High Performance Liquid Chromatography) method, which adopts a conventional C18 chromatographic column, takes a 0.05-0.2% formic acid or acetic acid solution as a mobile phase A, takes a mixed solution of methanol and tetrahydrofuran or a mixed solution of acetonitrile and tetrahydrofuran as a mobile phase B, and adopts an HPLC (High Performance Liquid Chromatography) method to detect impurities in an adapalene starting material. The initial volume fraction of the mobile phase A is 10-40%, the initial volume fraction of the mobile phase B is 10-40%, carrying out gradient elution, and carrying out high performance liquid chromatography analysis on the adapalene initial material and impurities. By using the analysis method, effective detection and analysis of the adapalene starting material and various impurities can be realized, the analysis method is efficient, accurate, high in specificity, high in detection sensitivity, good in separation degree and low in detection limit and quantification limit, a method is provided for research of the adapalene starting material, and the analysis method is suitable for control of related substances and research of impurities.

Description

Technical Field

[0001] The invention belongs to the technical field of chemical analysis, and in particular relates to a method for detecting impurities of adapalene starting materials by utilizing an HPLC method. Background Art

[0002] Adapalene is mainly used to treat skin diseases of acne vulgaris, which is mainly manifested by acne, papules and pustules. It can also be used to treat acne on the face, chest and back. 6-Bromo-2-naphthoic acid is the starting material for the first step of the synthesis of adapalene, and its molecular formula is: C 11 H 7 B O 2 , molecular weight is 251.0761, structural formula is

[0003] The following three impurities are easily present in the starting materials of adapalene: AD-IMP-01: 2-bromonaphthalene, molecular formula C 10 H 7 Br, molecular weight is 207.067, structural formula is AD-IMP-03: 2-acetyl-6-bromonaphthalene, molecular formula C 12 H 9 BrO, molecular weight is 249.10300, structural formula is AD-IMP-04: 7-bromo-1-naphthoic acid, molecular formula C 11 H 7 B O 2 , molecular formula is 251.0761, structural formula is

[0004] Impurities A starting material for the synthesis of adapalene; impurity It is the previous intermediate of the starting material for the synthesis of adapalene. These three impurities are byproducts of the adapalene starting material. These three impurities have similar polarity to the adapalene starting material and are difficult to separate. There is no report on the detection and analysis of the above starting materials and impurities in the prior art. Therefore, it is particularly important to develop an analytical method with high detection sensitivity, strong specificity, and the ability to effectively separate the adapalene starting material and impurities. Summary of the invention

[0005] To solve the above problems, the present invention provides a method for effectively detecting and analyzing adapalene starting materials and various impurities. The analytical method is efficient and accurate, has strong specificity, high detection sensitivity, good separation, and low detection limit and quantitative limit. It provides a method for the study of adapalene starting materials and is suitable for the control of related substances and impurity research.

[0006] The specific technical solution adopted by the present invention is a method for detecting impurities in adapalene starting materials using an HPLC method, comprising the following steps:

[0007] Step 1. Prepare a blank solution consisting of water, tetrahydrofuran, methanol or acetonitrile, with a volume ratio of water:tetrahydrofuran:methanol or acetonitrile of 43:20:37;

[0008] Step 2. Chromatographic conditions: the chromatographic column is an octadecylsilane bonded silica gel column;

[0009] The column temperature is 30℃~40℃;

[0010] Mobile phase A is 0.05% to 0.2% formic acid solution or acetic acid solution;

[0011] Mobile phase B is a mixed solution of methanol and tetrahydrofuran or a mixed solution of acetonitrile and tetrahydrofuran;

[0012] The gradient elution program was:

[0013] 0-5min: the proportion of mobile phase B changes from 10% to 40% to 40%;

[0014] 15-12min: the proportion of mobile phase B changes from 40% to 58%;

[0015] 12-15min: the proportion of mobile phase B changes from 58% to 80%;

[0016] 15-18min: the proportion of mobile phase B changes from 80% to 90%;

[0017] 20-23min: the proportion of mobile phase B is a value between 10% and 40%, and is the same as the proportion of mobile phase B at 0min;

[0018] The flow rate of the mobile phase is 0.8-1.2 mL / min;

[0019] The injection volume is 5 to 20 μL;

[0020] Step 3. Sample testing: Dissolve the sample to be tested in a blank solution to prepare a test solution, inject it into a high performance liquid chromatography system, and analyze the type and content of impurities contained in the sample to be tested according to the chromatogram.

[0021] Moreover, the adapalene starting material impurity is the synthetic adapalene starting material C 11 H 7 B O 2 Process impurities and degradation impurities generated during the process.

[0022] Furthermore, the types of impurities contained in step 3 include

[0023] Moreover, in step 2, the length of the chromatographic column is 250 mm, and the particle size of the filler in the chromatographic column is 3.0 to 5.0 μm.

[0024] Moreover, in step 2, the column temperature of the chromatographic column is 35° C.; the flow rate of the mobile phase is 1.2 mL / min; and the injection volume is 20 μL.

[0025] Furthermore, in step 2, mobile phase A was 0.1% acetic acid aqueous solution, and mobile phase B was acetonitrile-tetrahydrofuran (65:35).

[0026] Compared with the prior art, the beneficial effects of this technical solution are:

[0027] 1. Good elution and separation effect, high detection sensitivity, strong specificity, and can separate all related substances in the sample under the same chromatographic conditions with high sensitivity. It can quickly, accurately and reliably detect relevant impurities in the sample By detecting the relevant impurities in the starting materials of adapalene, data assistance and adjustment direction can be provided for the quality control of the synthesis and preparation process, thereby effectively controlling the quality of adapalene.

[0028] 2. In order to achieve better determination results, the most suitable determination condition parameter range is provided, specifically: (1) Mobile phase A is formic acid solution or acetic acid solution, the baseline obtained is better, and it can ensure that the blank solvent has no interference with the detection of impurities; the concentration of acetic acid (v / v) is 0.05% to 0.2%, which can obtain better peak shape and separation effect; (2) The solvent of mobile phase B is selected from methanol-tetrahydrofuran or acetonitrile-tetrahydrofuran, so that the peak shape and separation effect are the best and the retention time is moderate; (3) The organic solvent is a mixture of tetrahydrofuran, methanol or acetonitrile and water to ensure that the sample is fully dissolved; and the ratio of the mobile phase to the initial elution mobile phase is similar to that of the mobile phase to prevent solvent effect; (4) The flow rate of the mobile phase is 0.8 to 1.2 mL / min, which can further improve the separation degree and improve the peak shape; (5) The column temperature of the chromatographic column is 30°C to 40°C, which can further improve the separation degree of each component. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : The test chromatogram of the blank solution in Example 1;

[0030] Figure 2 : The test chromatogram of the separation solution in Example 1;

[0031] Figure 3 : The chromatogram of the adapalene starting material and the impurity detection limit solution test in Example 2;

[0032] Figure 4: The chromatogram of the adapalene starting material and the impurity quantitative limit solution test in Example 2;

[0033] Figure 5 : The separation solution test chromatogram in Example 3;

[0034] Figure 6 : The separation solution test chromatogram in Example 4;

[0035] Figure 7 : Separation solution test chromatogram in Example 5. DETAILED DESCRIPTION

[0036] The present invention is described in detail below in conjunction with the accompanying drawings and examples, and the content of the present invention is not limited to the following examples. The adapalene starting materials and impurity reference substances used in the examples of the present invention are all purchased from outside. The reagents and materials, if not otherwise specified, can be obtained from commercial sources.

[0037] Adapalene starting material (purchased from outside, batch number: 2023021)

[0038] AD-IMP-01 reference substance (purchased from outside, batch number: AK2024-193-846-3)

[0039] AD-IMP-03 reference substance (purchased from outside, batch number: HZ2024-1246-06-1-1)

[0040] AD-IMP-05 reference substance (purchased from outside, batch number: DH23-13154-25-0)

[0041] The high performance liquid chromatograph used in the following examples is Agilent 1260 high performance liquid chromatograph.

[0042] Example 1: Method specificity test

[0043] Chromatographic column: Aglient Zorbax SB-C18 (250*4.6mm 5μm)

[0044] Mobile phase A: 0.1% acetic acid;

[0045] Mobile phase B: acetonitrile-tetrahydrofuran (65:35);

[0046] Gradient elution conditions:

[0047] Time / minute Mobile phase A / V% Mobile phase B / V% 0 90 10 5 60 40 12 42 58 15 20 80 18 10 90 20 90 10 23 90 10

[0048] Column temperature: 30°C;

[0049] Flow rate: 1.2 mL / min;

[0050] Injection volume: 20 μL;

[0051] Detection wavelength: 270nm;

[0052] The test steps are as follows:

[0053] Diluent: acetonitrile-tetrahydrofuran-water (37:20:43);

[0054] Blank solution: diluent;

[0055] Location solution: Weigh approximately 2.5 mg of adapalene starting material, AD-IMP-01, AD-IMP-03, and AD-IMP-05 respectively, accurately weigh, place in 100 ml volumetric flasks, add diluent to dissolve and dilute to scale, shake well, and obtain each impurity location solution;

[0056] Separation solution: accurately weigh 25 mg of adapalene starting material and place it in a 10 ml volumetric flask, add 0.1 ml of each impurity stock solution in turn, add diluent to dissolve and dilute to the scale, shake well, and the separation solution is obtained;

[0057] Take the blank solution, positioning solution and separation solution respectively, analyze them under the above HPLC conditions, and record the chromatogram. The blank solution results are shown in the attached Figure 1 , the separation solution chromatogram is attached Figure 2 The retention time and separation degree of each compound are shown in Table 1.

[0058] Table 4 Adapalene starting materials and impurity specificity test results

[0059]

[0060] in conclusion: Figure 1 This indicates that the blank solution has no interference with the detection of each impurity; Figure 2 This indicates that the separation between each impurity and the main component is good, so the method has strong specificity.

[0061] Example 2 Detection limit and quantification limit test

[0062] Take an appropriate amount of each component reference solution, accurately weigh it, dissolve it with diluent acetonitrile-tetrahydrofuran-water (37:20:43) and dilute it step by step. According to the chromatographic conditions of Example 1, the sample concentration when the signal-to-noise ratio is 3:1 is the detection limit concentration. The results are as follows: the detection limit concentration of AD-IMP-01 is 0.08μg / ml, the detection limit concentration of AD-IMP-03 is 0.08μg / ml, and the detection limit concentration of AD-IMP-05 is 0.08μg / ml. See the detection limit test spectrum Figure 3 .

[0063] Take an appropriate amount of each component reference solution, accurately weigh it, dissolve it with a diluent of acetonitrile-tetrahydrofuran-water (37:20:43) and dilute it step by step. According to the chromatographic conditions of Example 1, the sample concentration when the signal-to-noise ratio is 10:1 is the detection limit concentration. The results are as follows: the quantitative limit concentration of AD-IMP-01 is 0.25μg / ml, the quantitative limit concentration of AD-IMP-03 is 0.23μg / ml, and the quantitative limit concentration of AD-IMP-05 is 0.25μg / ml. The quantitative limit test spectrum is shown in Figure 4 .

[0064] Conclusion: At a detection limit of less than 0.08 μg / ml and a quantification limit of less than 0.25 μg / ml, the impurities related to each adapalene starting material can be well detected, which is helpful for quality control and yield improvement of subsequent products.

[0065] Example 3 Durability Test

[0066] Chromatographic column: Aglient Zorbax SB-C18 (250*4.6mm 5μm)

[0067] Mobile phase A: 0.1% acetic acid;

[0068] Mobile phase B: acetonitrile-tetrahydrofuran (65:35);

[0069] Gradient elution conditions:

[0070] Time / minute Mobile phase A / V% Mobile phase B / V% 0 80 20 5 60 40 12 42 58 15 20 80 18 10 90 20 80 20 23 80 20

[0071] Column temperature: 30°C;

[0072] Flow rate: 1.2 mL / min;

[0073] Injection volume: 20 μL;

[0074] Detection wavelength: 270nm;

[0075] The test steps are as follows:

[0076] Diluent: acetonitrile-tetrahydrofuran-water (37:20:43);

[0077] Location solution: Weigh approximately 2.5 mg of adapalene starting material, AD-IMP-01, AD-IMP-03, and AD-IMP-05 respectively, accurately weigh, place in 100 ml volumetric flasks, add diluent to dissolve and dilute to scale, shake well, and obtain each impurity location solution;

[0078] Separation solution: accurately weigh 25 mg of adapalene starting material and place it in a 10 ml volumetric flask, add 0.1 ml of each impurity stock solution in turn, add diluent to dissolve and dilute to the scale, shake well, and the separation solution is obtained;

[0079] Take the blank solution and separation solution respectively, analyze them under the above HPLC conditions, record the chromatogram, and the results are shown in the attached Figure 5 .

[0080] Conclusion: The blank solution has no interference with the detection of impurities and has good specificity; Figure 5 This indicates that the separation between each impurity and the main component is good;

[0081] Example 4 Durability Test

[0082] Chromatographic column: Aglient Zorbax SB-C18 (250*4.6mm 5μm)

[0083] Mobile phase A: 0.1% acetic acid;

[0084] Mobile phase B: acetonitrile-tetrahydrofuran (65:35);

[0085] Gradient elution conditions:

[0086]

[0087]

[0088] Column temperature: 30°C;

[0089] Flow rate: 1.2 mL / min;

[0090] Injection volume: 20 μL;

[0091] Detection wavelength: 270nm;

[0092] The test steps are as follows:

[0093] Diluent: acetonitrile-tetrahydrofuran-water (37:20:43);

[0094] Location solution: Weigh approximately 2.5 mg of adapalene starting material, AD-IMP-01, AD-IMP-03, and AD-IMP-05 respectively, accurately weigh, place in 100 ml volumetric flasks, add diluent to dissolve and dilute to scale, shake well, and obtain each impurity location solution;

[0095] Separation solution: accurately weigh 25 mg of adapalene starting material and place it in a 10 ml volumetric flask, add 0.1 ml of each impurity stock solution in turn, add diluent to dissolve and dilute to the scale, shake well, and the separation solution is obtained;

[0096] Take the blank solution and separation solution respectively, analyze them under the above HPLC conditions, record the chromatogram, and the results are shown in the attached Figure 6 .

[0097] Conclusion: The blank solution has no interference with the detection of impurities and has good specificity; Figure 6 This indicates that the separation between each impurity and the main component is good;

[0098] Example 5 Durability Test

[0099] Chromatographic column: Aglient Zorbax SB-C18 (250*4.6mm 5μm)

[0100] Mobile phase A: 0.1% formic acid;

[0101] Mobile phase B: acetonitrile-tetrahydrofuran (65:35);

[0102] Gradient elution conditions:

[0103] Time / minute Mobile phase A / V% Mobile phase B / V% 0 60 40 5 60 40 12 42 58 15 20 80 18 10 90 20 60 40 23 60 40

[0104] Column temperature: 30°C;

[0105] Flow rate: 1.2 mL / min;

[0106] Injection volume: 20 μL;

[0107] Detection wavelength: 270nm;

[0108] The test steps are as follows:

[0109] Diluent: acetonitrile-tetrahydrofuran-water (37:20:43);

[0110] Location solution: Weigh approximately 2.5 mg of adapalene starting material, AD-IMP-01, AD-IMP-03, and AD-IMP-05 respectively, accurately weigh, place in 100 ml volumetric flasks, add diluent to dissolve and dilute to scale, shake well, and obtain each impurity location solution;

[0111] Separation solution: accurately weigh 25 mg of adapalene starting material and place it in a 10 ml volumetric flask, add 0.1 ml of each impurity stock solution in turn, add diluent to dissolve and dilute to the scale, shake well, and the separation solution is obtained;

[0112] Take the blank solution and separation solution respectively, analyze them under the above HPLC conditions, record the chromatogram, and the results are shown in the attached Figure 7 .

[0113] Conclusion: The blank solution has no interference with the detection of impurities and has good specificity; Figure 7 This indicates that the separation between the impurities and the main component is good.

Claims

1. A method for detecting impurities in adapalene starting materials using HPLC method, characterized in that The following steps are involved: Step 1. Prepare a blank solution consisting of water, tetrahydrofuran, methanol or acetonitrile, with a volume ratio of water:tetrahydrofuran:methanol or acetonitrile of 43:20:37; Step 2. Chromatographic conditions: the chromatographic column is an octadecylsilane bonded silica gel column; The column temperature is 30℃~40℃; Mobile phase A is 0.05% to 0.2% formic acid solution or acetic acid solution; Mobile phase B is a mixed solution of methanol and tetrahydrofuran or a mixed solution of acetonitrile and tetrahydrofuran; The gradient elution program was: 0-5min: the proportion of mobile phase B changes from 10% to 40% to 40%; 15-12min: the proportion of mobile phase B changes from 40% to 58%; 12-15min: the proportion of mobile phase B changes from 58% to 80%; 15-18min: the proportion of mobile phase B changes from 80% to 90%; 20-23min: the proportion of mobile phase B is a value between 10% and 40%, and is the same as the proportion of mobile phase B at 0min; The flow rate of the mobile phase is 0.8-1.2 mL / min; The injection volume is 5 to 20 μL; Step 3. Sample testing: Dissolve the sample to be tested in a blank solution to prepare a test solution, inject it into a high performance liquid chromatography system, and analyze the type and content of impurities contained in the sample to be tested according to the chromatogram.

2. A method for detecting impurities in adapalene starting materials using HPLC method according to claim 1, characterized in that: The adapalene starting material impurity is the starting material C for synthesizing adapalene 11 Process impurities and degradation impurities generated in the H7BrO2 process.

3. A method for detecting impurities in adapalene starting materials using HPLC method according to claim 1, characterized in that: The types of impurities contained in step 3 include 4. A method for detecting impurities in adapalene starting materials using HPLC method according to claim 1, characterized in that: In step 2, the length of the chromatographic column is 250 mm, and the particle size of the filler in the chromatographic column is 3.0 to 5.0 μm.

5. A method for detecting impurities in adapalene starting materials using HPLC method according to claim 1, characterized in that: In step 2, the column temperature of the chromatographic column is 35° C.; the flow rate of the mobile phase is 1.2 mL / min; and the injection volume is 20 μL.

6. A method for detecting impurities in adapalene starting materials using HPLC method according to claim 1, characterized in that: In step 2, mobile phase A is 0.1% acetic acid aqueous solution, and mobile phase B is acetonitrile-tetrahydrofuran (65:35).

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