Method for detecting multiple related impurities of Iguratimod tablets by utilizing HPLC (High Performance Liquid Chromatography)

Abstract

The invention discloses a method for detecting a plurality of related substances of Iguratimod tablets by utilizing HPLC (High Performance Liquid Chromatography), which comprises the following steps: carrying out gradient elution by adopting a chromatographic column which takes 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 according to a peak area by adopting an external standard method. The method is simple to operate, good in specificity, high in sensitivity and accuracy and capable of rapidly and accurately detecting the four related substances in the Iguratimod tablet.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis, and more specifically, relates to a method for detecting multiple impurities in ellamod tablets using HPLC. Background Technology

[0002] Iguratimod is a novel disease-modifying drug (DMARD) for the treatment of rheumatoid arthritis, jointly developed by Toyama Pharmaceutical Co., Ltd. and Eisai Pharmaceutical Co., Ltd. of Japan. It is a new type of nonsteroidal anti-inflammatory drug that selectively inhibits cyclooxygenase-2 and has antipyretic, analgesic, anti-arthritis, and immunomodulatory effects. It has a high anti-inflammatory efficacy against rheumatoid arthritis.

[0003] Currently, there is limited research on related substances in ailamod tablets. The literature "Determination of Related Substances in Ailamod Tablets by HPLC" (Shi Yuhong et al., Journal of Shenyang Pharmaceutical University, 2014, 31(9)) discloses the determination of related substances in ailamod tablets by HPLC. The article studied two impurities in ailamod tablets, but no reports were found on other impurities (including oxidative degradation impurities). In order to ensure the quality control and safety of ailamod tablets, it is urgent to develop a detection method that can effectively separate and detect degradation impurities in ailamod tablets. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a method for detecting multiple impurities in elamod tablets using HPLC. Through extensive scientific experiments and optimization of testing conditions, this invention establishes a scientifically effective detection method capable of simultaneously determining these impurities, thereby improving product quality control and possessing significant application value.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A method for detecting multiple related substances in ellamod tablets using HPLC employs an octadecyl-bonded silica column, a phosphate buffer solution as mobile phase A, an organic phase as mobile phase B, and gradient elution for detection; the impurities are the compounds shown below.

[0007] Table 1 Impurity compounds and their sources

[0008]

[0009]

[0010] Preferably, in the method, the phosphate buffer solution is a 0.04 mol / L ammonium dihydrogen phosphate solution, and the organic phase is acetonitrile.

[0011] Preferably, the gradient elution conditions are as follows:

[0012] Table 2 Elution Procedure

[0013] Time (min) A(％) B(％) 0 70 30 20 70 30 30 55 45 45 55 45 58 30 70 60 70 30 65 70 30

[0014] Preferably, the chromatographic column is a Phenomenex Gemini C18 with a column length of 250 mm, an inner diameter of 4.6 mm, and a packing particle size of 5 μm.

[0015] Because A1 and A9 have similar polarities, their elution times are similar, making them difficult to separate. Researchers unexpectedly discovered that the chromatographic column with the above parameters is highly targeted to the impurities involved in this invention, has a stronger sample loading capacity and retention capacity, and can effectively solve the problem of impurities A1 and A9 being unable to be separated due to their similar polarities. At the same time, it can also meet the detection and separation requirements of impurities A6 and A7, with a resolution greater than 1.5, achieving baseline separation, good resolution, and good peak shape.

[0016] Preferably, the detection wavelength is 268–272 nm.

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

[0018] Preferably, the injection volume is 20 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 the impurity peaks and the main peak is further ensured. A wavelength of 270 nm accommodates the UV absorption of both the main component and various impurities, improving the method's sensitivity. Combining these optimized parameters, the method can accurately, stably, and effectively detect related substances in Airamod tablets.

[0021] When quantitatively determining the above-mentioned substances, the content of each impurity is calculated by peak area using the external standard method.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] This method provides a detection method for other related substances (including oxidative degradation impurities) in Ailamod tablets. The method is simple to operate, has good specificity, and the main peak and various impurity peaks in the solution do not interfere with each other. The resolution is greater than 1.5, and each impurity can be effectively separated. It has high sensitivity and accuracy, good repeatability, and can quickly and accurately detect related substances in Ailamod tablets. Attached Figure Description

[0024] Figure 1 The high-performance liquid chromatogram of the blank solution described in Example 1;

[0025] Figure 2 The high-performance liquid chromatogram of the spiked solution described in Example 1;

[0026] Figure 3 The high-performance liquid chromatogram of impurity A6 described in Example 1;

[0027] Figure 4 The high-performance liquid chromatogram of impurity A7 described in Example 1;

[0028] Figure 5 The high-performance liquid chromatogram of impurity A9 described in Example 1;

[0029] Figure 6 The high-performance liquid chromatogram of impurity A1 described in Example 1;

[0030] Figure 7 The linear analysis chromatogram of impurity A6 described in Example 2;

[0031] Figure 8 The linear analysis chromatogram of impurity A7 described in Example 2;

[0032] Figure 9 The linear analysis chromatogram of impurity A9 described in Example 2;

[0033] Figure 10 The graph is a linear analysis diagram of impurity A1 as described in Example 2. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0035] The impurity reference standards used in the examples were self-made, and the reagents, ellamod reference standards and instruments could all be purchased through conventional commercial channels.

[0036] The sources of the four impurities and the ellamod reference standard described in the examples are shown in Table 3:

[0037] Table 3 Sources of various drugs

[0038] name factory Impurity A6 Self-made, Jiangsu Zhengda Qingjiang Pharmaceutical Co., Ltd. Impurity A7 Self-made, Jiangsu Zhengda Qingjiang Pharmaceutical Co., Ltd. Impurity A9 Self-made, Jiangsu Zhengda Qingjiang Pharmaceutical Co., Ltd. Impurity A1 Self-made, Jiangsu Zhengda Qingjiang Pharmaceutical Co., Ltd. Ailamod reference National Institutes for Food and Drug Control (NIFDC)

[0039] The chromatographic conditions for the methods implemented in the following examples are as follows:

[0040] Column: Phenomenex Gemini C18 (4.6*250mm, 5μm)

[0041] Mobile phase A: 0.04M ammonium dihydrogen phosphate solution

[0042] Mobile phase B: Acetonitrile

[0043] Gradient elution:

[0044] Table 4 Gradient elution program

[0045] Time (min) A(％) B(％) 0 70 30 20 70 30 30 55 45 45 55 45 58 30 70 60 70 30 65 70 30

[0046] Flow rate: 1.0 ml / min

[0047] Column temperature: 30℃

[0048] Detection wavelength: 270nm

[0049] Injection volume: 20 μl

[0050] Example 1: System Applicability and Specificity

[0051] 1.1 System Applicability

[0052] Solution preparation:

[0053] Diluent (blank solution): Acetonitrile-methanol-water (5:3:2)

[0054] Impurity stock solution: Take appropriate amounts of each of the impurities A1, A6, A7 and A9 of Ailamod, accurately weigh them, dissolve them with diluent and dilute them quantitatively to prepare a solution containing about 0.2 mg per ml, which is used as the impurity stock solution.

[0055] Mixed solution: Weigh approximately 10 mg of ellamod reference standard accurately and place it in a 20 ml volumetric flask. Accurately measure 0.1 ml of impurity stock solution, dissolve and dilute to the mark with diluent, and shake well.

[0056] Sample pretreatment: Take 20 tablets of Ailamod, weigh them accurately, and grind them into a fine powder.

[0057] Test solution: Take an appropriate amount of sample powder (approximately equivalent to 10 mg of ellamod), accurately weigh it, place it in a 20 ml volumetric flask, add solvent to dissolve and dilute to the mark, shake well, filter, and take the filtrate as the test solution.

[0058] Self-control solution: Accurately measure 1.0 ml of the test solution, place it in a 100 ml volumetric flask, dilute to the mark with diluent, and shake well.

[0059] Six injections of the self-control solution were performed under the above conditions, and the RSD% of the peak area of ​​each impurity was calculated. The statistical results are shown in Table 5.

[0060] Table 5 System Applicability Results

[0061]

[0062] Results: As shown in Table 3, the maximum peak area RSD of the self-reference solution after 6 repeated injections was 0.2% (the standard is no more than 2%), which meets the requirements.

[0063] 1.2 Specificity

[0064] Blank solution (diluent), impurity stock solution, and mixed solution were introduced into the chromatographic system under the chromatographic conditions described above. Statistical results are shown in Table 6, and chromatograms are shown below. Figures 1-6 .

[0065] Table 6 Specificity-Separation Results

[0066]

[0067] Results: Table 4 shows that the blank solution had no interfering peaks in the retention time of the main peak and each impurity peak. In the mixed solution, the main peak and each impurity peak did not interfere with each other, and the resolution was greater than 1.5, indicating that each impurity could be effectively separated.

[0068] Example 2: Linearity and Range

[0069] The mixed stock solution of each impurity reference standard was diluted to prepare solutions with linear concentrations ranging from LOQ to 200% of the limit for each component. These solutions were then introduced into the chromatographic system under the conditions described above. The results are shown in Tables 7-10, and the corresponding linearity graphs are shown below. Figures 7-10 .

[0070] Table 7 Linearity Results for Impurity A6

[0071]

[0072] Table 8. Linearity Results for Impurity A7

[0073]

[0074] Table 9 Linearity Results for Impurity A9

[0075]

[0076] Table 10 Linearity Results for Impurity A1

[0077]

[0078] Results: From Tables 7-10 and Figures 7-10 It can be seen that, within the LOQ-limit of 200% concentration range, the linear correlation coefficient R... 2 The minimum value is 0.9983, which meets the linearity requirement.

[0079] Example 3 Limit of Quantification

[0080] Take the LOQ linear solution and introduce it into the chromatographic system under the chromatographic conditions described above. The results are shown in Table 11.

[0081] Table 11 Results of Limit of Quantification

[0082] name Signal-to-noise ratio (S / N) Concentration (μg / ml) Relative concentration of the test sample (%) Impurity A6 13.3 0.13 0.02 Impurity A7 23.8 0.13 0.02 Impurity A9 22.8 0.13 0.02 Impurity A1 11.3 0.13 0.02

[0083] Results: As can be seen from Table 11, the analytical method of this invention has high sensitivity under these chromatographic conditions.

[0084] Example 4 Accuracy

[0085] Nine samples of the starting material of ellamod (concentration 0.5 mg / ml) were taken and spiked at three concentration levels (80%, 100%, and 120% limit) for each impurity for recovery. The samples were then introduced into the chromatographic system under the chromatographic conditions described above. The recovery results are shown in Tables 12-15.

[0086] Table 12 Accuracy Results for Impurity A6

[0087]

[0088] Table 13 Accuracy Results for Impurity A7

[0089]

[0090]

[0091] Table 14 Accuracy Results for Impurity A9

[0092]

[0093] Table 15 Accuracy Results for Impurity A1

[0094]

[0095] Results: As shown in Tables 12-15, for the nine samples at the three limit levels of LOQ, 100%, and 150%, the recovery rates of each impurity were all between 90% and 110%; the RSDs between the recovery rates were all less than 5%, indicating good accuracy.

[0096] Example 5 Repeatability

[0097] Take six samples of Ailamod tablets (concentration 0.5 mg / ml) and test them. The repeatability of the method is investigated using the spiked method (0.2% of the sample is known to be added). Precision is investigated in six parallel samples. The samples are introduced into the chromatographic system under the conditions described above. The recovery results are shown in Table 16.

[0098] Table 16 Results of Repeatability Experiments

[0099] sample Impurity A6 (%) Impurity A7 (%) Impurity A9 (%) Impurity A1 (%) Repeatability -1 0.202％ 0.206％ 0.206％ 0.206％ Repeatability-2 0.202％ 0.207％ 0.206％ 0.206％ Repeatability -3 0.199％ 0.206％ 0.205％ 0.220％ Repeatability -4 0.202％ 0.208％ 0.210％ 0.203％ Repeatability -5 0.199％ 0.207％ 0.206％ 0.212％ Repeatability - 6 0.202％ 0.207％ 0.211％ 0.201％ average value(%) 0.201％ 0.207％ 0.207％ 0.206％ RSD% 0.8 0.4 1.1 3.0

[0100] Results: As shown in Table 16, the RSD of each impurity detected in the 6 test sample solutions was less than 6.0%, indicating good precision.

[0101] Example 6 Durability

[0102] Take one sample of Aramod tablets (concentration 0.5 mg / ml) and one portion of the test sample. Use the spiked method (adding a known amount of 0.2%) to investigate the robustness of the method. The main changes in chromatographic conditions are shown in Table 17. Enter the chromatographic system under the above chromatographic conditions; the robustness results are shown in Table 18.

[0103] Table 17 Durability Test Parameters

[0104] Chromatographic conditions Specified value Range of variation Column temperature (°C) 30 ±2℃ Flow rate (ml / min) 1.0 ±0.2 wavelength 270 ±2

[0105] Table 18 Durability Test Results

[0106]

[0107]

[0108] Results: As shown in Table 18, when the chromatographic conditions change slightly, such as column temperature change of ±2℃, flow rate change of ±0.2ml / min, and wavelength change of ±2nm, the RSD of the impurity determination results is 1.5, which is less than 6%, indicating that this method has good robustness.

[0109] Example 7: Column Screening

[0110] The effects of different types of chromatographic columns, including Kromasil C18 (4.6*250mm, 5μm), Phenomenex Gemini C18 (4.6*250mm, 5μm), and Shimadzu Inersustain C18 (4.6*250mm, 5μm), on impurity resolution were investigated. The results showed that using Kromasil C18 (4.6*250mm, 5μm) and Shimadzu Inersustain C18 (4.6*250mm, 5μm), the resolution of impurities A9 and A1 was less than 1.5; using Phenomenex Gemini C18 (4.6*250mm, 5μm), the resolution of impurities A1 and A9 was greater than 1.5, and the peak shapes of both the main component and the impurities were better. Therefore, the Phenomenex Gemini C18 (4.6*250mm, 5μm) column was preferred.

[0111] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting multiple related substances in ellamod tablets using HPLC, characterized in that: A chromatographic column packed with octadecyl-bonded silica gel was used, with phosphate buffer solution as mobile phase A and organic phase as mobile phase B, and gradient elution was employed for detection. The impurities are compounds represented by impurities A6, A7, A9, and A1.

2. The method according to claim 1, characterized in that, The mobile phase A is a phosphate buffer solution of 0.04 mol / L ammonium dihydrogen phosphate solution; the mobile phase B is an organic phase of acetonitrile.

3. The method according to claim 2, characterized in that, The gradient elution procedure is as follows:

4. The method according to claim 1, characterized in that, The chromatographic column is a Phenomenex Gemini C18 with a length of 250 mm, an inner diameter of 4.6 mm, and a packing particle size of 5 μm.

5. The method according to claim 1, characterized in that, The detection wavelength is 268–272 nm.

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 was 20 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 A6, A7, A9, and A1, the content of each impurity is calculated based on the peak area using the external standard method.

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