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

Abstract

The invention discloses a method for detecting a plurality of related impurities of Iguratimod by utilizing HPLC (High Performance Liquid Chromatography), which comprises the following steps: weighing impurity reference substances with known concentrations, respectively preparing a plurality of mixed standard solutions, carrying out high performance liquid chromatography analysis on the mixed standard solutions at each concentration level, and recording the retention time and peak area of each impurity; for each impurity, drawing a relational graph between the peak area and the known concentration; performing high performance liquid chromatography analysis on the to-be-detected sample according to the same condition, and recording the retention time and peak area of all peaks; comparing the retention time of each peak in the to-be-detected sample with the retention time of the known impurities in the standard solution, and determining the types of the impurities in the to-be-detected sample; and calculating the corresponding concentration by using the drawn relational graph according to the peak area of each impurity peak in the sample to be detected. The method is simple to operate, good in specificity, high in sensitivity and accuracy, and capable of rapidly and accurately detecting 16 related substances in Iguratimod.

Description

Technical Field

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

[0002] Aridocarpine is an organic compound with a molecular formula of C17H14N2O6S and a molecular weight of 374.37. It is a white crystalline powder. It is a drug used to treat rheumatic diseases and has multiple biological activities such as anti-inflammatory, inhibition of immunoglobulin and cytokine production, anti-bone resorption and promotion of bone formation.

[0003] The chemical structure of AMD is shown below: .

[0004] Its chemical name is: N-[3-(formylamino)-4-oxo-6-phenoxy-4H-1-benzopyran-7-yl]methanesulfonamide.

[0005] According to the synthetic process of Aguratimod, there are 16 known impurities that may exist (see Table 1 of the instructions for specific impurity information). At present, the Chinese Pharmacopoeia (current edition), the United States Pharmacopoeia (current edition), the Japanese Pharmacopoeia (JP17) and the European Pharmacopoeia (EP10.5) have not included the relevant standards for Aguratimod APIs and their preparations. Literature search found that there are few research reports on the detection methods of Aguratimod-related substances. In order to ensure the quality control of Aguratimod products and to ensure their effectiveness and safety, it is urgent to develop a detection method that can effectively separate and detect these 16 known impurities. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a method for detecting multiple impurities related to iguratimod using HPLC. The present invention establishes a set of scientific and effective detection methods through a large number of scientific experiments and optimization of test conditions, which can simultaneously determine these impurities, thereby improving the level of product quality control and having important application value.

[0007] The purpose of the present invention is achieved through the following technical solutions: A method for detecting multiple impurities of iguratimod using HPLC, comprising the following steps: (1) Weigh each impurity reference substance of known concentration and prepare multiple mixed standard solutions. Perform HPLC analysis on the mixed standard solutions of each concentration level and record the retention time and peak area of ​​each impurity. For each impurity, draw a graph of the relationship between the peak area and the known concentration; (2) Perform HPLC analysis on the samples under the same conditions and record the retention time and peak area of ​​all peaks.

[0008] (3) Compare the retention time of each peak in the sample to be tested with the retention time of the known impurities in the standard solution in step (1) to determine the type of impurities in the sample to be tested; use the relationship diagram drawn in step (1) to calculate the corresponding concentration based on the peak area of ​​each impurity peak in the sample to be tested.

[0009] Preferably, in step (1), the structure of each impurity reference substance is as follows: .

[0011] Preferably, in step (1), the conditions for high performance liquid chromatography analysis are: using a chromatographic column with octadecyl bonded silica as a filler, using phosphate buffer solution and organic phase as mobile phases, performing gradient elution, a detection wavelength of 200-250 nm, a column temperature of 25-35° C., and an injection volume of 10-25 μL.

[0012] Preferably, the chromatographic column is Agilent Zorbax SB-C18. The specifications of the chromatographic column are: column length 250 mm, inner diameter: 4.6 mm, filler particle size: 5 μm.

[0013] The chromatographic column has strong selectivity and can effectively separate and detect these impurities in Iguratimod. Generally speaking, the longer the column, the higher the resolution, but the longer the separation time; the smaller the particle size, the faster the separation speed and the higher the column efficiency, but the column pressure will also increase, the column is easy to be contaminated, resulting in a decrease in column life. For this product, a chromatographic column with a length of 250mm and a particle size of 5μm has better analytical capabilities.

[0014] Preferably, the phosphate buffer solution is 0.01 mol / L potassium dihydrogen phosphate buffer, and the pH value is adjusted to 4.5-6.0 with sodium hydroxide solution. More preferably, the pH value is adjusted to 5.5 with sodium hydroxide solution.

[0015] Low concentration of buffer salt can not only reduce the pressure of the chromatographic column, but also improve the peak shape. At pH 5.5, each impurity peak has good peak shape and retention on the chromatographic column.

[0016] Preferably, the organic phase is acetonitrile, because acetonitrile has a stronger elution ability than methanol and acetonitrile can reduce the pressure of the chromatographic column.

[0017] Preferably, the flow rate of the mobile phase is 0.8-1.2 mL / min.

[0018] Preferably, the detection wavelength is 220 nm and the flow rate of the mobile phase is 1.0 mL / min.

[0019] Preferably, the column temperature is 30°C.

[0020] Preferably, the injection volume is 15 μL.

[0021] Under the flow rate and column temperature conditions, the best separation between each impurity peak and the main peak can be ensured. The wavelength of 220 nm takes into account the ultraviolet absorption of the main component and each impurity, improving the sensitivity of the method. Combining the above-mentioned preferred parameters, impurities in the iguratimod sample can be accurately, stably and effectively detected.

[0022] Preferably, the procedure of gradient elution is: .

[0024] Compared with the prior art, the beneficial effects of the present invention include: (1) This method is simple to operate, low in cost, has good specificity, high sensitivity and accuracy, and can quickly and accurately detect related substances in elamod.

[0025] (2) This method can detect 16 possible impurities, with a wide impurity spectrum and strong applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the linear analysis diagram of impurity AMD-IMP-1.

[0027] Figure 2 This is the linear analysis diagram of impurity AMD-IMP-2.

[0028] Figure 3 This is the linear analysis diagram of impurity AMD-IMP-4.

[0029] Figure 4 This is the linear analysis diagram of impurity AMD-IMP-5.

[0030] Figure 5 This is the linear analysis diagram of impurity AMD-IMP-6.

[0031] Figure 6 This is the linear analysis diagram of impurity AMD-IMP-7.

[0032] Figure 7 This is the linear analysis diagram of impurity AMD-IMP-8.

[0033] Figure 8 This is the linear analysis diagram of impurity AMD-IMP-9.

[0034] Fig. 9 This is the linear analysis diagram of impurity AMD-IMP-10.

[0035] Fig.10 This is the linear analysis diagram of impurity AMD-IMP-11.

[0036] Fig.11 This is the linear analysis diagram of impurity AMD-IMP-12.

[0037] Fig.12 This is the linear analysis diagram of impurity AMD-IMP-13.

[0038] Fig.13 This is the linear analysis diagram of impurity AMD-IMP-14.

[0039] Fig.14 This is the linear analysis diagram of impurity AMD-IMP-15.

[0040] Fig.15 This is the linear analysis diagram of impurity AMD-IMP-16.

[0041] Fig.16 This is the linear analysis diagram of the main peak AMD.

[0042] Fig.17 The HPLC chromatogram of the blank solution described in Example 2 is shown in FIG.

[0043] Fig.18 The HPLC chromatogram of the spiked solution described in Example 2 is shown in FIG.

[0044] Fig.19 This is the high performance liquid chromatogram of the sample to be tested described in Example 2. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution 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 used to limit the present invention.

[0046] The iguratimod reference substance and test substance used in the examples were homemade, and other reagents, impurity reference substances and instruments can be purchased through conventional channels in the market.

[0047] The sources of the 14 impurities, the iguratimod reference substance and the test substance described in the examples are shown in Table 1: Table 1 Sources of various drugs

[0048] Among them, the iguratimod reference substance and the test substance were prepared according to the method disclosed in Chinese patent publication number CN116891450A.

[0049] Example 1 1. Chromatographic system: Chromatographic column: Agilent Zorbax SB-C18, 250*4.5mm, 5μm Mobile phase A: 0.01M potassium dihydrogen phosphate solution (sodium hydroxide solution adjusted to pH 5.5) Mobile phase B: acetonitrile Gradient elution conditions:

[0050] Flow rate: 1.0ml / min Column temperature: 30°C Detection wavelength: 220nm Injection volume: 15 μL 2. Experimental steps: 2.1 Dilution: 70% acetonitrile solution 2.2 Mixed stock solution of impurity reference substances: Weigh 6 mg of each impurity reference substance into a 10 ml volumetric flask, add appropriate amount of acetonitrile to dissolve, add diluent to dilute to the scale, and shake well; then transfer 1 ml to a 100 ml volumetric flask, add diluent to dilute to the scale, and shake well.

[0051] 2.3 Impurity reference solution: Accurately pipette 1.0 ml of the impurity reference mixed stock solution into a 10 ml volumetric flask, add diluent to dilute to the mark, and shake well.

[0052] 2.4 Separation solution: Weigh 12 mg of ilamod reference substance and place it in a 20 ml volumetric flask. Add appropriate amount of acetonitrile to dissolve it. Then accurately add 2.0 ml of impurity reference substance mixed stock solution to the volumetric flask, dilute to the scale with diluent, and shake well.

[0053] 2.5 Test sample solution: Weigh 12 mg of the test sample accurately, place it in a 20 ml volumetric flask, dissolve it with diluent and dilute it to the scale, and shake well.

[0054] 3. Test method verification results: 3.1 System Applicability Take the impurity reference solution and inject it three times continuously under the above conditions, calculate the RSD% of each impurity peak area, and the statistical results are shown in Table 2.

[0055] Table 2 System suitability results

[0056] It can be seen from Table 2 that the impurity reference solution was injected three times repeatedly, and the maximum RSD of each impurity peak area was 1.67% (the standard is not more than 5%), which meets the requirements.

[0057] 3.2 Exclusivity The blank solution (i.e. 70% acetonitrile solution) and the separation solution were introduced into the chromatographic system according to the above chromatographic conditions. The statistical results are shown in Table 3.

[0058] Table 3 Specificity-resolution results

[0059] It can be seen from Table 3 that the blank solution has no interfering peaks at the retention times of the main peak and each impurity peak, the main peak and each impurity peak in the separation solution do not interfere with each other, and each impurity can be effectively separated.

[0060] 3.3 Linearity and range The mixed stock solutions of each impurity reference substance were diluted to prepare solutions with a linear concentration range of 0.1~1.2μg / mL for each component, and then introduced into the chromatographic system under the above chromatographic conditions. The results are shown in Tables 4~19, and the corresponding linear relationship diagrams are shown in Figures 1 to 16 .

[0061] Table 4 Impurity AMD-IMP-1 linear results

[0062] Table 5 Impurity AMD-IMP-2 linear results

[0063] Table 6 Impurity AMD-IMP-4 linear results

[0064] Table 7 Impurity AMD-IMP-5 linear results

[0065] Table 8 Impurity AMD-IMP-6 linear results

[0066] Table 9 Impurity AMD-IMP-7 linear results

[0067] Table 10 Impurity AMD-IMP-8 linear results

[0068] Table 11 Impurity AMD-IMP-9 linear results

[0069] Table 12 Impurity AMD-IMP-10 linearity results

[0070] Table 13 Impurity AMD-IMP-11 linear results

[0071] Table 14 Impurity AMD-IMP-12 linearity results

[0072] Table 15 Impurity AMD-IMP-13 linear results

[0073] Table 16 Impurity AMD-IMP-14 Linearity Results

[0074] Table 17 Impurity AMD-IMP-15 Linearity Results

[0075] Table 18 Impurity AMD-IMP-16 linear results

[0076] Table 19 Main Peak AMD Linearity Results

[0077] Depend on Figures 1 to 16 It can be seen that the linear correlation coefficient R 2 The minimum value is 0.9819, and the linearity meets the requirements.

[0078] 3.4 Limit of quantitation Take 0.02% linear solution and enter the chromatographic system according to the above chromatographic conditions. The results are shown in Table 20: Table 20 Quantitation limit results

[0079] It can be seen from Table 20 that under this chromatographic condition, the quantitative limit of each impurity and main component is 0.1µg / mL (0.02% level), and the analytical method of the invention has high sensitivity.

[0080] 3.5 Accuracy Take 9 samples of iguratimod (concentration 0.6 mg / mL) and add the impurities at three concentration levels of 0.05% limit (0.3µg / mL), 0.1% limit (0.6µg / mL) and 0.2% limit (1.2µg / mL) respectively, and enter the chromatographic system according to the above chromatographic conditions. The recovery results are shown in Tables 21~34: Table 21 AMD-IMP-1 accuracy results

[0081] Table 22AMD-IMP-2 accuracy results

[0082] Table 23 AMD-IMP-4 accuracy results

[0083] Table 24 AMD-IMP-5 accuracy results

[0084] Table 25 AMD-IMP-6 accuracy results

[0085] Table 26 AMD-IMP-7 accuracy results

[0086] Table 27 AMD-IMP-9 accuracy results

[0087] Table 28 AMD-IMP-10 accuracy results

[0088] Table 29 AMD-IMP-11 accuracy results

[0089] Table 30 AMD-IMP-12 accuracy results

[0090] Table 31 AMD-IMP-13 accuracy results

[0091] Table 32 AMD-IMP-14 accuracy results

[0092] Table 33 AMD-IMP-15 accuracy results

[0093] Table 34 AMD-IMP-16 accuracy results

[0094] It can be seen from Tables 21 to 34 that the recovery rates of the 9 samples with three limit levels of 0.05%, 0.1% and 0.2% are all between 80% and 120%, and the total RSD between the recovery rates is no more than 15%. The recovery rate meets the requirements and the accuracy is good.

[0095] Example 2 The blank solution (i.e. 70% acetonitrile solution), spiked solution (i.e. resolution solution) and the sample to be tested were subjected to HPLC analysis according to the above chromatographic conditions. The corresponding chromatograms are shown in Figures 17 to 19 .

[0096] contrast Fig.17 and 18 It can be seen that the blank solution has no interfering peaks at the retention time of the main peak and each impurity peak, indicating that this method has good specificity.

[0097] contrast Fig.19 and Fig.18 , it can be seen that the sample to be tested does not contain any impurities and has a high purity.

[0098] The specific implementation of the present invention described above does not constitute a limitation on the protection scope 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 protection scope of the claims of the present invention.

Claims

1. A method for detecting multiple impurities of iguratimod using HPLC, characterized in that: The steps include: (1) Weigh each impurity reference substance of known concentration and prepare multiple mixed standard solutions. Perform HPLC analysis on the mixed standard solutions of each concentration level and record the retention time and peak area of ​​each impurity. For each impurity, plot the peak area versus known concentration; (2) Perform HPLC analysis on the samples under the same conditions and record the retention time and peak area of ​​all peaks; (3) Compare the retention time of each peak in the sample to be tested with the retention time of the known impurities in the standard solution described in step (1) to determine the type of impurities in the sample to be tested; use the relationship diagram drawn in step (1) to calculate the corresponding concentration based on the peak area of ​​each impurity peak in the sample to be tested.

2. The method for detecting multiple impurities of iguratimod using HPLC according to claim 1, characterized in that: The structures of the impurity reference substances in step (1) are as follows: 。 3. The method for detecting multiple impurities of iguratimod by HPLC according to claim 1, characterized in that: The conditions for the high performance liquid chromatography analysis in step (1) are: using a chromatographic column with octadecyl bonded silica as a filler, using phosphate buffer solution and organic phase as mobile phases, performing gradient elution, a detection wavelength of 200-250 nm, a column temperature of 25-35° C., and an injection volume of 10-25 μL.

4. The method for detecting multiple impurities of iguratimod by HPLC according to claim 3, characterized in that: The chromatographic column is Agilent Zorbax SB-C18.

5. The method for detecting multiple impurities of iguratimod by HPLC according to claim 3, characterized in that: The phosphate buffer solution is 0.01 mol / L potassium dihydrogen phosphate buffer salt, and the pH value is adjusted to 4.5-6.0 with sodium hydroxide solution.

6. The method for detecting multiple impurities of iguratimod by HPLC according to claim 5, characterized in that: The organic phase is acetonitrile.

7. The method for detecting multiple impurities of iguratimod by HPLC according to claim 3, characterized in that: The flow rate of the mobile phase is 0.8-1.2 mL / min.

8. The method for detecting multiple impurities of iguratimod by HPLC according to claim 7, characterized in that: The detection wavelength was 220 nm, and the flow rate of the mobile phase was 1.0 mL / min.

9. The method for detecting multiple impurities of iguratimod by HPLC according to claim 3, characterized in that: The column temperature was 30° C., and the injection volume was 15 μL.

10. The method for detecting multiple impurities of iguratimod by HPLC according to claim 6, characterized in that: The procedure of the gradient elution is: 。

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