Method for detecting related substances of iguratimod starting material by HPLC (High Performance Liquid Chromatography)

Abstract

The invention discloses a method for detecting related impurities of an iguratimod starting material (4-chloro-3-nitroanisole) by HPLC (High Performance Liquid Chromatography), which comprises the following steps: carrying out isocratic elution by adopting a chromatographic column taking octadecyl bonded silica gel as a filler and taking a phosphate buffer solution and an organic phase as mobile phases, and calculating the content of each impurity by peak area by adopting an external standard method. The method is easy to operate, good in specificity, high in sensitivity and accuracy, capable of rapidly and accurately detecting related substances in 4-chloro-3-nitroanisole and good in separation degree which is larger than 1.5.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical analysis, and in particular relates to a method for detecting impurities related to iguratimod starting materials by HPLC. Background Art

[0002] Iguratimod is a new type of disease-modifying drug (DMARDs) for the treatment of rheumatoid arthritis jointly developed by Toyama Pharmaceuticals and Eisai Pharmaceuticals of Japan. It is a new non-steroidal anti-inflammatory drug that selectively inhibits cyclooxygenase-2. It has antipyretic and analgesic, anti-arthritic and immunomodulatory effects, and has a high anti-inflammatory effect on rheumatism and rheumatoid arthritis.

[0003] 4-Chloro-3-nitroanisole is the starting material for the synthesis of iguratimod, with a molecular formula of C7H6ClNO3, a molecular weight of 187.58, and an appearance of a yellow to dark brown solid.

[0004] The chemical structure is shown below:

[0005]

[0006] At present, there are few studies on the starting materials of iguratimod. The document "Determination of genotoxic impurities in iguratimod starting materials by high performance liquid chromatography" (Zhu Xiaohua et al., Physical and Chemical Testing, 2021, 57(8)) discloses a high performance liquid chromatography method for determining genotoxic impurities in iguratimod starting materials, but no reports have been found on the determination of other related substances in iguratimod starting materials. In order to ensure the controllable quality of the starting materials of iguratimod and to ensure their safety, it is urgent to develop a detection method that can effectively separate and detect related substances in the starting materials. Summary of the Invention

[0007] In response to the shortcomings of the existing technology, the present invention aims to provide a method for detecting impurities in the starting material of elaboramic acid using HPLC. Through extensive scientific experiments and optimization of testing conditions, the present invention has established a scientific and effective detection method that can simultaneously determine these impurities, thereby improving the level of product quality control and having important application value.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A method for detecting related substances of iguratimod starting materials by high performance liquid chromatography, using a chromatographic column with octadecyl bonded silica as a filler, a phosphate buffer solution and an organic phase as mobile phases, and performing isocratic elution.

[0010] The impurities are compounds represented by impurity I and impurity II:

[0011]

[0012] In the method, preferably, the phosphate buffer solution is a 0.04 mol / L ammonium dihydrogen phosphate solution, and the pH value is adjusted to 2.0-3.0 with a phosphoric acid solution; further preferably, the pH value is adjusted to 2.5 with phosphoric acid, and the organic phase is acetonitrile.

[0013] Preferably, the volume ratio of phosphate buffer solution: organic phase = 55:45.

[0014] Preferably, the chromatographic column is of Thermo Hypersil Gold model, with specifications of column length 250 mm, inner diameter 4.6 mm, and filler particle size 5 μm.

[0015] The chromatographic conditions are highly selective for impurities I and II, and can effectively separate and detect impurities I and II in the iguratimod starting material. The separation between the starting material and the impurities, and between impurities I and II is greater than 1.5, achieving baseline separation, good separation, and good peak shape.

[0016] Preferably, the detection wavelength is 218-228 nm.

[0017] Preferably, the column temperature is 25-35°C.

[0018] Preferably, the injection volume is 10 to 25 μl.

[0019] Preferably, the flow rate of the mobile phase is 0.8-1.2 ml / min.

[0020] Under these flow rate and column temperature conditions, optimal separation between impurity peaks and the main peak was achieved. A wavelength of 220 nm balanced the UV absorption of both the main component and impurities, improving the sensitivity of the method. Combined with these optimized parameters, impurities in 4-chloro-3-nitroanisole, the starting material for iguratimod, can be accurately, stably, and effectively detected.

[0021] When quantitatively determining impurities I and II, the external standard method is used to calculate the content of each impurity based on the peak area.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The method provides a method for detecting related substances in 4-chloro-3-nitroanisole, a starting material of iguratimod. The method is simple to operate and has good specificity. The main peak and the impurity peaks in the solution do not interfere with each other. The separation degrees are greater than 1.5, and the impurities can be effectively separated. The method has high sensitivity and accuracy, good repeatability, and can quickly and accurately detect related substances in the starting material of iguratimod. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1is the HPLC chromatogram of the blank solution described in Example 1;

[0025] Figure 2 HPLC chromatogram of the spiked solution described in Example 1;

[0026] Figure 3 The HPLC chromatogram of the sample to be tested described in Example 1;

[0027] Figure 4 This is the linear analysis diagram of impurity Ⅰ;

[0028] Figure 5 This is the linear analysis diagram of impurity II;

[0029] Figure 6 This is the chromatogram when the pH value of mobile phase A is 2.5 in Example 7;

[0030] Figure 7 This is the chromatogram when the pH value of mobile phase A in Example 7 is 3.0. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0032] The starting material reference substances used in the examples were homemade, and the test substances, reagents, impurity reference substances and instruments were all purchased through conventional commercial channels.

[0033] The sources of the two impurities and starting material reference substances described in the examples are shown in Table 1:

[0034] Table 1 Sources of various drugs

[0035] name factory Impurity I Homemade, Jiangsu Zhengda Qingjiang Pharmaceutical Co., Ltd. Impurity II Tokyo Chemical Industry Co., Ltd. (TCI) Starting material reference Jiangsu Zhengda Qingjiang Pharmaceutical Co., Ltd.

[0036] The chromatographic conditions for the following embodiments are as follows:

[0037] Chromatographic column: Thermo Hypersil Gold (4.6*250mm, 5μm)

[0038] Mobile phase A: 0.04 M ammonium dihydrogen phosphate solution (adjust pH to 2.5 with phosphoric acid)

[0039] Mobile phase B: acetonitrile

[0040] Isocratic elution: mobile phase A: mobile phase B = 55:45

[0041] Flow rate: 1.0ml / min

[0042] Column temperature: 30°C

[0043] Detection wavelength: 223nm

[0044] Injection volume: 20 μl

[0045] Example 1 System Applicability and Specificity

[0046] 1.1 System Applicability

[0047] Solution configuration:

[0048] Diluent: Mobile phase A (0.04 mol / L ammonium dihydrogen phosphate solution, pH adjusted to 2.5 with phosphoric acid): acetonitrile (75:25)

[0049] Mixed stock solution of impurity reference substances: Weigh 10 mg of each impurity reference substance into a 100 ml volumetric flask, add diluent to dissolve and dilute to the scale, and shake well.

[0050] Impurity reference solution: Accurately pipette 1.0 ml of the impurity reference mixed stock solution into a 20 ml volumetric flask, add diluent to dilute to the scale, and shake well.

[0051] Separation solution: Weigh 10 mg of iguratimod starting material reference substance and place it in a 20 ml volumetric flask. Add appropriate amount of acetonitrile to dissolve it. Then accurately add 1.0 ml of impurity reference substance mixed stock solution to the volumetric flask, dilute to the scale with diluent, and shake well.

[0052] Test solution: Weigh 10 mg of the test sample accurately, place it in a 20 ml volumetric flask, dissolve it with diluent and dilute to the scale, and shake well.

[0053] The impurity reference solution was injected 6 times continuously under the above conditions, and the RSD% of the peak area of ​​each impurity was calculated. The statistical results are shown in Table 2.

[0054] Table 2 System suitability results

[0055]

[0056] Results: As can be seen from Table 2, the impurity reference solution was injected 6 times, and the maximum RSD of each impurity peak area was 0.4% (the standard is not more than 2%), which meets the requirements.

[0057] 1.2 Exclusivity

[0058] Take blank solution (mobile phase A: acetonitrile (75:25)) and separation solution into the chromatographic system according to the above chromatographic conditions. The statistical results are shown in Table 3 and the spectrum is shown in Figures 1 to 3 .

[0059] Table 3 Specificity-resolution results

[0060]

[0061] Results: It can be seen from Table 3 that the blank solution has no interfering peaks at the retention time of the main peak and each impurity peak, the main peak and each impurity peak in the solution do not interfere with each other, the separation degrees are all greater than 1.5, and each impurity can be effectively separated.

[0062] Example 2 Linearity and Range

[0063] The mixed stock solutions of each impurity reference substance were diluted to prepare solutions with linear concentration range of each component in the range of LOQ to 200% of the limit, and then introduced into the chromatographic system according to the above chromatographic conditions. The results are shown in Tables 4 and 5, and the corresponding linear relationship diagrams are shown in Figures 4-5 .

[0064] Table 4 Impurity I linearity results

[0065]

[0066] Table 5 Impurity II linearity results

[0067]

[0068] Results: From Tables 4 to 5 and Figures 1-2 It can be seen that when the concentration of each component is within the LOQ-limit 200%, the linear correlation coefficient R 2 The minimum is 0.9996, and the linearity meets the requirements.

[0069] Example 3 Limit of Quantitation

[0070] Take the LOQ linear solution and enter the chromatography system according to the above chromatographic conditions. The results are shown in Table 6:

[0071] Table 6 Quantitation limit results

[0072] name Signal-to-noise ratio (S / N) Concentration (μg / ml) Relative test sample concentration (%) Impurity I 13.3 0.102 0.02 Impurity II 23.8 0.106 0.02

[0073] Results: As can be seen from Table 6, under this chromatographic condition, the analytical method of the invention has high sensitivity.

[0074] Example 4 Accuracy

[0075] Nine samples of the iguratimod starting material (concentration 0.5 mg / ml) were taken and spiked with each impurity at three concentration levels of 50%, 100% limit, and 150% limit, and then injected into the chromatographic system under the above chromatographic conditions. The recovery results are shown in Tables 7 and 8:

[0076] Table 7 Impurity I accuracy results

[0077]

[0078] Table 8 Impurity II accuracy results

[0079]

[0080]

[0081] Results: As can be seen from Tables 7 and 8, the recovery rates of the 9 samples at the three limit levels of LOQ, 100% and 150% were all between 90% and 108%; the RSDs between the recoveries were all less than 5%, indicating good accuracy.

[0082] Example 5 Repeatability

[0083] The repeatability of the method was investigated by spiked samples (adding a known amount of impurity II 1.0% and impurity I 0.2%) of iguratimod starting material sample (concentration 0.3 mg / ml) and 6 samples of the test sample. The precision was investigated by 6 replicates and the samples were loaded into the chromatographic system according to the above chromatographic conditions. The recovery results are shown in Table 9:

[0084] Table 9 Repeatability test results

[0085] sample Impurity I (%) Impurity II (%) Single maximum (%) Repeatability -1 1.03 0.27 0.15 Repeatability -2 0.95 0.26 0.14 Repeatability -3 0.94 0.28 0.15 Repeatability -4 1.01 0.27 0.15 Repeatability -5 0.98 0.26 0.14 Repeatability -6 0.96 0.28 0.15 average value(%) 0.98 0.27 0.15 RSD% 3.5 3.3 2.8

[0086] Results: It can be seen from Table 9 that the RSDs of the detected amounts of each impurity in the 6 test solutions were all less than 6.0%, indicating good precision.

[0087] Example 6 Durability

[0088] The robustness of the method was investigated using a spiked method (adding known amounts of impurity II (1.0%) and impurity I (0.2%)) of iguratimod starting material (concentration 0.3 mg / ml) and a test sample. The main changes in chromatographic conditions are shown in Table 10. The chromatographic system was loaded according to the above chromatographic conditions. The robustness results are shown in Table 11:

[0089] Table 10 Durability test parameters

[0090]

[0091] Table 11 Durability test results

[0092]

[0093] Results: As can be seen from Table 11, when the chromatographic conditions change slightly, such as column temperature changes by ±2°C, flow rate changes by ±0.2ml / min, and wavelength changes by ±2nm, the absolute deviation of the impurity determination results is no more than 20% of the quality standard limit, and this method has good durability.

[0094] Example 7 pH screening

[0095] The effect of mobile phase A with different pH values ​​on separation and peak shape was investigated. Mobile phase A was prepared at pH 2.5, pH 3.0, and pH 4.0, while other chromatographic conditions remained unchanged. Separation and peak shape were then investigated.

[0096] The results showed that when the pH of mobile phase A was 4.0, the separation between the main component and impurities was less than 1.0, and the peak shapes of impurities and main peaks were poor; when the pH of mobile phase A was 2.5, the separation between the main component and impurities was 2.75, and the peak shape was better; when the pH was 3.0, the separation between the main component and impurities was 2.45, and the peak shape was better. The separation at pH 2.5 was better than that at pH 3.0, so pH 2.5 was preferred.

[0097] Example 8 Chromatographic column screening

[0098] The effects of different types of chromatographic columns, such as Shimadzu InertSustain C18 (4.6*250mm, 5μm) and ThermoHypersil Gold (4.6*250mm, 5μm), on the main peak shape and impurity peak shape were investigated. The results showed that the main peak and impurity peak shapes of the Shimadzu column were tailing, which affected the separation. The Thermo Hypersil Gold (4.6*250mm, 5μm) column had better main peak and impurity I peak shapes, and was therefore preferred.

[0099] The specific embodiments of the present invention described above do not limit the scope of protection of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A method for detecting related substances of iguratimod starting materials by high performance liquid chromatography, characterized in that: The detection was performed using a chromatographic column filled with octadecyl bonded silica gel and isocratic elution with mobile phase A, phosphate buffer solution and mobile phase B, organic phase, in proportion. The impurities are compounds represented by impurity I and impurity II:

2. The method according to claim 1, characterized in that The mobile phase A phosphate buffer solution is a 0.04 mol / L ammonium dihydrogen phosphate solution, and the pH value is adjusted to 2.0-3.0 with phosphoric acid; or preferably, the pH value is adjusted to 2.5 with phosphoric acid; the mobile phase B organic phase is acetonitrile.

3. The method according to claim 2, characterized in that The volume ratio of mobile phase A:mobile phase B is 55:

45.

4. The method according to claim 1, wherein The chromatographic column model is Thermo Hypersil Gold, and the chromatographic column specifications are: column length 250 mm, inner diameter 4.6 mm, and filler particle size 5 μm.

5. The method according to claim 1, characterized in that The detection wavelength is 218~228nm.

6. The method according to claim 1, characterized in that The column temperature is 25-35℃.

7. The method according to claim 1, characterized in that The injection volume is 10-25 μl.

8. The method according to claim 1, characterized in that The flow rate of the mobile phase is 0.8-1.2 ml / min.

9. The method according to claim 1, characterized in that When quantitatively determining impurities I and II, the external standard method is used to calculate the content of each impurity based on the peak area.

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