High performance liquid chromatography detection method of clofazimine intermediate

Abstract

The invention belongs to the technical field of medical intermediate detection, and particularly relates to a high performance liquid chromatography detection method of a clofazimine intermediate. The method comprises the following steps: detecting a test sample by adopting a high performance liquid chromatography to obtain a high performance liquid chromatography detection result of the test sample; the test sample is a clofazimine intermediate, and the clofazimine intermediate comprises an intermediate 1 or an intermediate 2; the detection conditions of the high performance liquid chromatography are as follows: the elution mode is isocratic elution; the mobile phase is methanol and water, and the volume ratio of methanol to water is (75-85): (15-25); and the detection wavelength is 215 to 225 nm. Compared with a titration method for measuring the content, the detection method provided by the invention has the advantages of more accurate and visual detection result, good repeatability and high stability.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical intermediate detection, and particularly relates to a high performance liquid chromatography detection method for a clofazimine intermediate. Background Art

[0002] Clofazimine (Cfz, chemical structure as follows), also known as chlorphenamine, chlorphenamine, etc., is a relatively effective anti-leprosy drug. It is used in combination with dapsone as the first choice for the treatment of lepromatous leprosy. It can also be used together with other drugs to treat drug-resistant tuberculosis.

[0003]

[0004] At present, the pharmaceutical intermediates for preparing clofazimine include intermediate 1 and intermediate 2, wherein the chemical name of intermediate 1 is 2-nitro-4'-chlorodiphenylamine, and the chemical name of intermediate 2 is 2-amino-4'-chlorodiphenylamine. The chemical structural formulas of the two intermediates are as follows:

[0005]

[0006] In order to prevent unknown impurities from remaining in the clofazimine finished product, causing the impurity content of the clofazimine finished product to exceed the standard, it is necessary to perform quality inspection and control on the clofazimine intermediates 1 and 2. At present, the titration method is usually used to determine the content of organic compounds. This method is relatively cumbersome and is greatly affected by temperature and the operator, while the high performance liquid chromatography method has the advantages of good repeatability and simple operation. However, due to the large difference in the ultraviolet absorption curves of intermediates 1 and 2, it is difficult to determine their contents using the same liquid phase conditions, resulting in the complicated operation of the liquid chromatography detection method of intermediates 1 and 2, and the multi-pump mobile phase is prone to uneven mixing during operation, poor sample baseline stability, and poor repeatability. Summary of the invention

[0007] The object of the present invention is to provide a high performance liquid chromatography detection method for a clofazimine intermediate. The high performance liquid chromatography detection method provided by the present invention can use the same liquid phase conditions to detect intermediate 1 and intermediate 2, thereby simplifying the liquid phase detection method. Compared with the titration method for content determination, the detection result is more accurate and intuitive, and has good repeatability and high stability.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a high performance liquid chromatography detection method for a clofazimine intermediate, comprising the following steps:

[0010] The test sample is tested by high performance liquid chromatography to obtain a high performance liquid chromatography test result of the test sample; the test sample is a clofazimine intermediate, and the clofazimine intermediate includes intermediate 1 or intermediate 2; the chemical structural formulas of intermediate 1 and intermediate 2 are respectively:

[0011]

[0012] The detection conditions of the high performance liquid chromatography method include: the elution mode is isocratic elution; the mobile phase is methanol and water, and the volume ratio of the methanol to water is (75-85): (15-25); the detection wavelength is 215-225nm.

[0013] Preferably, the flow rate of the mobile phase is 0.8 to 1.2 mL / min.

[0014] Preferably, the column temperature of the chromatographic column used in the high performance liquid chromatography is 25-35°C.

[0015] Preferably, the chromatographic column used in the high performance liquid chromatography method comprises a C8 liquid chromatography column or a C18 liquid chromatography column.

[0016] Preferably, the volume ratio of methanol to water is 80:20.

[0017] Preferably, the detection wavelength is 220 nm.

[0018] Preferably, the flow rate of the mobile phase is 1.0 mL / min.

[0019] Preferably, the column temperature of the chromatographic column used in the high performance liquid chromatography is 30°C.

[0020] Preferably, the specification of the C8 liquid chromatography column is 250 mm×4.6 mm, and the filler particle size is 5 μm.

[0021] Preferably, the C18 liquid chromatography column has a specification of 150 mm×4.6 mm and a filler particle size of 5 μm.

[0022] The present invention provides a high performance liquid chromatography detection method for clofazimine intermediates, comprising the following steps: using high performance liquid chromatography to detect a sample to obtain a high performance liquid chromatography detection result of the sample; the sample is a clofazimine intermediate, and the clofazimine intermediate includes intermediate 1 or intermediate 2; the detection conditions of the high performance liquid chromatography include: the elution mode is isocratic elution; the mobile phase is methanol and water, and the volume ratio of the methanol and water is (75-85): (15-25); the detection wavelength is 215-225nm. The high performance liquid chromatography detection method provided by the present invention can detect intermediate 1 and intermediate 2 using the same liquid phase condition by optimizing the detection conditions, simplifying the liquid phase detection conditions, without changing the mobile phase ratio during operation, configuring the mobile phase during the high performance liquid chromatography detection process, fixing the running pipeline and the unit pump to detect intermediate 1 and intermediate 2, avoiding the problem of uneven mixing of the mobile phase of the multi-pump, and ensuring the stability of the sample baseline. Therefore, compared with the titration method for content determination, the detection method provided by the present invention has more accurate and intuitive detection results, good repeatability and high stability.

[0023] The present invention uses a high performance liquid chromatography method to accurately detect the purity of intermediate 1 and intermediate 2, thereby ensuring that the content of intermediate 1 and intermediate 2 is greater than 98%, reducing unknown impurities introduced into the final clofazimine product and affecting the quality of the clofazimine product. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a liquid chromatogram of a blank solution (i.e., mobile phase) obtained in the specificity investigation experiment of the intermediate 1 obtained in Example 1;

[0025] Figure 2 This is a liquid chromatogram of the intermediate 1 obtained in the specificity investigation experiment of the intermediate 1 obtained in Example 1;

[0026] Figure 3 This is a liquid chromatogram of a blank solution (i.e., mobile phase) obtained in the specificity investigation experiment of the intermediate 2 obtained in Example 1;

[0027] Figure 4 This is the liquid chromatogram of the intermediate 1 obtained in the specificity investigation experiment of the intermediate 2 obtained in Example 1;

[0028] Figure 5 is the linear investigation curve of the intermediate 1 obtained in Example 1;

[0029] Figure 6 is the linear investigation curve of the intermediate 2 obtained in Example 1;

[0030] Figure 7 is the liquid chromatogram of the intermediate 1 obtained in Comparative Example 1;

[0031] Figure 8 It is the liquid chromatogram of intermediate 2 obtained in Comparative Example 1. DETAILED DESCRIPTION

[0032] The present invention provides a high performance liquid chromatography detection method for a clofazimine intermediate, comprising the following steps:

[0033] The test sample is tested by high performance liquid chromatography to obtain a high performance liquid chromatography test result of the test sample; the test sample is a clofazimine intermediate, and the clofazimine intermediate includes intermediate 1 or intermediate 2; the chemical structural formulas of intermediate 1 and intermediate 2 are respectively:

[0034]

[0035] The detection conditions of the high performance liquid chromatography method include: the elution mode is isocratic elution; the mobile phase is methanol and water, and the volume ratio of the methanol to water is (75-85): (15-25); the detection wavelength is 215-225nm.

[0036] In the present invention, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0037] In the present invention, the chemical name of intermediate 1 is 2-nitro-4'-chlorodiphenylamine, and the chemical name of intermediate 2 is 2-amino-4'-chlorodiphenylamine. The chemical structural formulas of the two intermediates are as follows:

[0038]

[0039] In the present invention, the water is preferably pure water. The volume ratio of the methanol to water is preferably 80:20. In the present invention, the volume content of methanol in the mobile phase is preferably 75-85%, and in the embodiment it can be 75%, 80% or 85%. The volume of the water is preferably 15-25%, and in the embodiment it can be 15%, 20% or 25%. The detection wavelength is preferably 220nm. The flow rate of the mobile phase is preferably 0.8-1.2mL / min, more preferably 1.0mL / min, and in the embodiment it can be 0.8mL / min, 0.9mL / min, 1.0mL / min, 1.1mL / min or 1.2mL / min. The column temperature of the chromatographic column used in the high performance liquid chromatography is preferably 25-35°C, more preferably 30°C, and in the embodiment it can be 25°C, 28°C, 30°C, 32°C, 34°C or 35°C.

[0040] In the present invention, the chromatographic column used in the high performance liquid chromatography method preferably includes a C8 liquid chromatography column or a C18 liquid chromatography column. The specification of the C8 liquid chromatography column is preferably 250 mm×4.6 mm, and the filler particle size is preferably 5 μm. The specification of the C18 liquid chromatography column is preferably 150 mm×4.6 mm, and the filler particle size is preferably 5 μm.

[0041] In the embodiment of the present invention, the instrument used in the high performance liquid chromatography method is a high performance liquid chromatograph Agilent 1260 Infinity or waters-e2695. The chromatographic column is Caprisil C18-P (250 mm×4.6 mm, 5 μm). The injection volume is preferably 20 μL.

[0042] The present invention preferably uses an external standard method to process the HPLC test results of the test sample. The external standard method includes a standard curve method and a direct comparison method. In an embodiment of the present invention, the reference substances used in the external standard method, including the reference substances of intermediate 1 and intermediate 2, are manufactured by Sinopharm Chemical Reagent Co., Ltd.

[0043] Compared with the titration method for content determination, the high performance liquid chromatography detection method provided by the present invention is more accurate and intuitive; at the same time, the present invention simplifies the liquid phase detection method, and the RSD value of each experiment is less than 2%, with good repeatability and high stability.

[0044] In order to further illustrate the present invention, the technical solutions provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] Example 1

[0046] (1) Experimental materials and instrument conditions

[0047] Experimental materials: methanol; manufacturer: Tianjin Kangkede Technology Co., Ltd.; purified water: homemade; intermediate 1 and intermediate 2 test samples, manufacturer: Shanxi Liye Pharmaceutical Co., Ltd.; intermediate 1 (batch number: 20230821, content: 97%), intermediate 2 (batch number: 20230821, content: 97%) reference substances, manufacturer: Sinopharm Chemical Reagent Co., Ltd.; microporous filter membrane (0.45 μm), manufacturer: Luer Syringe Filter.

[0048] Instruments: High performance liquid chromatograph: Agilent 1260 Infinity; electronic balance: Sartorius SQP; chromatographic column: Caprisil C18-P (250 mm×4.6 mm, 5 μm).

[0049] (2) Experimental procedures

[0050] ① Specificity investigation experiment: Blank solution (i.e. mobile phase): methanol: water (v:v) = 80:20; Test sample positioning solution: Take 10 mg of each intermediate 1 and intermediate 2 test sample, accurately weigh, place in a 50 mL volumetric flask, add mobile phase to make a concentration of 0.2 mg / mL solution, take an appropriate amount of each sample, filter through a 0.45 μm filter membrane, inject into a 1.5 mL Agilent liquid phase vial, and inject the sample with an injection volume of 20 μL. HPLC detection conditions: detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 mL / min, and the column temperature is 30 ° C. Record the liquid chromatogram of the blank solution when testing intermediate 1 ( Figure 1 ), liquid chromatogram of intermediate 1 ( Figure 2 ), liquid chromatogram of blank solution during the test of intermediate 2 ( Figure 3 ), liquid chromatogram of intermediate 2 ( Figure 4 ).Depend on Figure 2 It can be seen that the retention time of intermediate 1 is 12.688min. Figure 4 It can be seen that the retention time of intermediate 2 is 11.369 min. Figure 1 to Figure 4 It can be seen that the blank solution (i.e., mobile phase) has no interference.

[0051] ②Linearity investigation experiment: Mobile phase: methanol: water (v: v) = 80:20. Intermediate 1, intermediate 2 reference solution: Take 20 mg of each intermediate 1 and intermediate 2 reference, accurately weigh, place in a 50 mL volumetric flask, add mobile phase to make a concentration of 0.40 mg / mL solution, dilute with mobile phase to different concentrations, namely intermediate 1, intermediate 2 reference solution 2, 3, 4, 5, 6, 7, the concentrations are 0.40760, 0.32608, 0.24456, 0.20380, 0.16304, 0.08152, 0.04076 mg / mL, respectively, take appropriate amount of each reference solution, filter through 0.45 μm filter membrane, inject into 1.5 mL Agilent liquid phase vial, inject, the injection volume is 20 μL. HPLC detection conditions: detection wavelength 220nm, mobile phase flow rate 1.0mL / min, column temperature 30℃. Record the chromatogram, and the obtained linear curve is as follows: Figure 5 and Figure 6 As shown by Figure 5 and Figure 6 It can be seen that the correlation coefficient R 2 They are 0.9998 and 0.9997 respectively, with good linearity.

[0052] ③Instrument precision investigation experiment: Take the intermediate 1 and 2 test sample solution 4 in the ② linear investigation experiment, inject 6 times continuously, and the HPLC detection conditions are: the detection wavelength is 220nm, the flow rate of the mobile phase is 1.0mL / min, and the column temperature is 30℃. Record the retention time and peak area. The test results are shown in Tables 1 and 2. Table 1 is the instrument precision test results of intermediate 1, and Table 2 is the instrument precision test results of intermediate 2. It can be seen from Tables 1 and 2 that the RSD values ​​of the test results are less than 0.5%, and the instrument precision is good.

[0053] Table 1 Instrument precision results of intermediate 1

[0054] sample Retention time (min) Peak area 1 12.745 10163.6 2 12.718 10150.6 3 12.693 10152.9 4 12.688 10157.3 5 12.687 10161.4 6 12.704 10163.3 RSD(%) 0.18 0.05

[0055] Table 2 Instrument precision results of intermediate 2

[0056] sample Retention time (min) Peak area 1 11.331 13767.7 2 11.288 13721.8 3 11.267 13702.5 4 11.262 13667.6 5 11.309 13631.0 6 11.299 13609.9 RSD(%) 0.23 0.43

[0057] ④Stability investigation experiment: Mobile phase: methanol: water (v:v) = 80:20. Take 10 mg of each of the intermediate 1 test sample and reference sample, intermediate 2 test sample and reference sample, place them in a 50 mL volumetric flask, add mobile phase to dissolve and dilute to the scale, shake well, and inject. HPLC detection conditions: detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 mL / min, and the column temperature is 30 ° C. Table 3 is the test results of intermediate 1, and Table 4 is the test results of intermediate 2. It can be seen from Tables 3 and 4 that the peak area and retention time RSD values ​​within 24 hours at room temperature are less than 1%, indicating that the samples have good stability.

[0058] Table 3 Intermediate 1 stability test results

[0059]

[0060] Table 4 Intermediate 2 stability test results

[0061]

[0062]

[0063] ⑤ Repeatability experiment: Mobile phase: methanol: water (v:v) = 80:20. Take 10 mg of each intermediate 1 and 2 test sample, accurately weigh, place in a 50 mL volumetric flask, add mobile phase to dissolve and dilute to the scale, shake well, prepare 6 copies in parallel, and inject. HPLC detection conditions: detection wavelength is 220 nm, flow rate of mobile phase is 1.0 mL / min, column temperature is 30 ° C. Calculate the content by external standard method. Table 5 is the external standard method test results of intermediate 1, and Table 6 is the external standard method test results of intermediate 2. It can be seen from Tables 5 and 6 that the RSD value is less than 1%, and the repeatability is good.

[0064] Table 5 Intermediate 1 external standard test results

[0065] sample content(%) 1 98.88 2 98.82 3 98.65 4 98.51 5 99.01 6 99.15 Average content (%) 98.84 RSD(%) 0.24

[0066] Table 6 Intermediate 2 external standard test results

[0067] sample content(%) 1 98.87 2 98.71 3 99.12 4 98.8 5 98.6 6 98.95 Average content (%) 98.84 RSD(%) 0.19

[0068] ⑥Intermediate precision experiment: 2 experimenters measured the content according to the repeatability experimental scheme on different instruments at different times. The HPLC detection conditions were as follows: the detection wavelength was 220 nm, the flow rate of the mobile phase was 1.0 mL / min, and the column temperature was 30°C. Table 7 shows the intermediate precision experimental results of intermediate 1, and Table 8 shows the intermediate precision experimental results of intermediate 2. It can be seen from Tables 7 and 8 that the RSD value is less than 1%, and the intermediate precision is good.

[0069] Table 7 Intermediate precision test results of intermediate 1

[0070]

[0071] Table 8 Intermediate precision test results of intermediate 2

[0072]

[0073] ⑦ Accuracy experiment: Mobile phase: methanol: water (v:v) = 80:20. Reference solution: Take appropriate amount of intermediate 1 and intermediate 2 reference substances, add solvent (mobile phase) to dissolve and dilute to make a solution containing about 0.2 mg per 1 mL, as reference solution. Reference stock solution: Take appropriate amount of intermediate 1 and intermediate 2 reference substances, add solvent (mobile phase) to dissolve and dilute to make a solution containing about 1 mg per 1 mL, as reference stock solution. 80% solution: Accurately measure 1.6 mL of intermediate 1 and intermediate 2 reference stock solutions, place them in a 10 mL volumetric flask, dilute to the mark with solvent, shake well, and get it. Prepare 3 copies in parallel. 100% spiked solution: Accurately measure 2 mL of intermediate 1 and intermediate 2 reference stock solutions, place them in a 10 mL volumetric flask, dilute to the mark with solvent, shake well, and get it. Prepare 3 copies in parallel. 120% spiked solution: Accurately measure 2.4 mL of intermediate 1 and intermediate 2 reference stock solutions respectively, place in a 10 mL volumetric flask, add solvent to dilute to the scale, shake well, and obtain. Prepare 3 copies in parallel. HPLC detection conditions: detection wavelength is 220 nm, flow rate of mobile phase is 1.0 mL / min, column temperature is 30 ° C. The content is calculated by external standard method. Table 9 is the recovery rate of intermediate 1, and Table 10 is the recovery rate of intermediate 2. It can be seen from Tables 9 and 10 that the average recovery rate of intermediate 1 is 98.83%, and the average recovery rate of intermediate 2 is 100.1%, and the RSD values ​​are all less than 1%.

[0074] Table 9 Recovery of intermediate 1

[0075]

[0076] Table 10 Recovery rate of intermediate 2

[0077]

[0078]

[0079] ⑧Durability experiment: The durability was examined by adjusting the flow rate, mobile phase ratio, column temperature, column type, detection wavelength and other chromatographic conditions. The specific experimental conditions are shown in Table 11, and the test results are shown in Table 12 and Table 13. Table 12 shows the durability experiment test results of intermediate 1, and Table 13 shows the durability experiment test results of intermediate 2.

[0080] Table 11 Durability test conditions

[0081]

[0082] Table 12 Durability test results of intermediate 1

[0083]

[0084]

[0085] Table 13 Durability test results of intermediate 2

[0086]

[0087] Comparative Example 1

[0088] The experimental materials and instruments are the same as those in Example 1.

[0089] The mobile phase is methanol and water. Take 10 mg of each test sample of intermediate 1 and intermediate 2, weigh accurately, place in a 50 mL volumetric flask, add mobile phase (methanol: water (v:v) = 95:5) to make a concentration of 0.2 mg / mL solution, take an appropriate amount of sample, filter through a 0.45 μm filter membrane, inject into a 1.5 mL Agilent liquid phase vial, inject, and the injection volume is 20 μL. HPLC detection conditions: detection wavelength is 220 nm, the flow rate of the mobile phase is 1.0 mL / min, and the column temperature is 30 ° C. Use gradient elution, and the gradient elution program is shown in Table 14:

[0090] Table 14 Mobile phase gradient program

[0091] Time (minutes) Methanol(%) water(%) 0 5 95 5 95 5 19 95 5 35 5 95 40 5 95

[0092] The liquid chromatogram of the intermediate 1 obtained is as follows Figure 7 As shown, the liquid chromatogram of intermediate 2 is as follows Figure 8 As shown. Figure 7 and Figure 8 It can be seen that when the gradient elution program shown in Table 14 is used, the baseline of the obtained liquid chromatogram drifts (drifts upward), affecting the accuracy of the measurement results.

[0093] From the above embodiments, it can be seen that the high-performance liquid chromatography detection method provided by the present invention can use the same liquid phase condition to detect intermediate 1 and intermediate 2 by optimizing the detection conditions, simplifying the liquid phase detection conditions, and there is no need to change the mobile phase ratio during operation. During the high-performance liquid chromatography detection process, the mobile phase is configured, and the fixed operation pipeline and unit pump can be used to detect intermediate 1 and intermediate 2, avoiding the problem of uneven mixing of the mobile phase of the multi-element pump, and ensuring that the sample baseline is stable. Therefore, compared with the titration method for determining the content, the detection method provided by the present invention is more accurate and intuitive, and has good repeatability and high stability.

[0094] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A high performance liquid chromatography detection method for clofazimine intermediates, characterized in that: The following steps are involved: The test sample is tested by high performance liquid chromatography to obtain a high performance liquid chromatography test result of the test sample; The test sample is a clofazimine intermediate, and the clofazimine intermediate includes intermediate 1 or intermediate 2; the chemical structural formulas of intermediate 1 and intermediate 2 are respectively: The detection conditions of the high performance liquid chromatography method include: the elution mode is isocratic elution; the mobile phase is methanol and water, and the volume ratio of the methanol to water is (75-85): (15-25); the detection wavelength is 215-225nm.

2. The high performance liquid chromatography detection method according to claim 1, characterized in that: The flow rate of the mobile phase is 0.8-1.2 mL / min.

3. The high performance liquid chromatography detection method according to claim 1, characterized in that: The column temperature of the chromatographic column used in the high performance liquid chromatography is 25-35°C.

4. The high performance liquid chromatography detection method according to claim 1 or 3, characterized in that: The chromatographic column used in the high performance liquid chromatography method includes a C8 liquid chromatography column or a C18 liquid chromatography column.

5. The high performance liquid chromatography detection method according to claim 1, characterized in that: The volume ratio of methanol to water is 80:

20.

6. The high performance liquid chromatography detection method according to claim 1, characterized in that: The detection wavelength is 220 nm.

7. The high performance liquid chromatography detection method according to claim 2, characterized in that: The flow rate of the mobile phase was 1.0 mL / min.

8. The high performance liquid chromatography detection method according to claim 3, characterized in that: The column temperature of the chromatographic column used in the high performance liquid chromatography was 30°C.

9. The high performance liquid chromatography detection method according to claim 4, characterized in that: The specification of the C8 liquid chromatography column is 250 mm×4.6 mm, and the filler particle size is 5 μm.

10. The high performance liquid chromatography detection method according to claim 4, characterized in that: The specification of the C18 liquid chromatography column is 150 mm×4.6 mm, and the filler particle size is 5 μm.

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