Method for detecting related substances in pemebenzoic acid by HPLC (High Performance Liquid Chromatography) method

Abstract

The invention discloses a method for detecting related substances in pemebenzoic acid by HPLC (High Performance Liquid Chromatography), and belongs to the technical field of chemical engineering and pharmacy, the related substances in pemebenzoic acid are detected by high performance liquid chromatography, a phosphate buffer solution (a potassium dihydrogen phosphate solution with the concentration of 0.04 mol / L and the pH value of a sodium hydroxide solution with the concentration of 1 mol / L is regulated to 5.5) is used as a mobile phase A, acetonitrile is used as a mobile phase B, gradient elution is performed, the flow rate is 0.8 ml / min, the column temperature is 20 DEG C, and the detection wavelength is 250nm. According to the present invention, the column temperature is 30 DEG C, the detection wavelength is 250 nm, the contents of 2, 4, 6-trihydroxypyrimidine, N-methyl-2, 4-diamino-6-hydroxypyrimidine and 4-(4-butyric acid) methyl benzoate in pemebenzoic acid can be effectively separated and quantitatively detected, the method has characteristics of simple operation, strong specificity, high sensitivity, high accuracy and good linearity, and the method can be widely used in the field of detection of pemebenzoic acid. The quality of the pemebenzoic acid product can be effectively controlled, and the method is suitable for pemebenzoic acid quality control.

Description

Technical Field

[0001] This invention relates to the fields of chemical and pharmaceutical technology, specifically to an HPLC method for detecting related substances in pemetrexed benzoic acid. Background Technology

[0002] Pemetrexed benzoic acid is a key starting material for the preparation of pemetrexed disodium. Pemetrexed disodium, as a multi-target antifolate drug, inhibits tumor cell proliferation by blocking the normal folate-dependent metabolic mechanism within tumor cells. It can be used to treat non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM). Numerous clinical trials have shown that pemetrexed disodium can significantly prolong patient survival, reduce the risk of tumor progression, has relatively mild adverse reactions, is easy to use, and has significant clinical efficacy. Pemetrexed benzoic acid, as a key structural unit of pemetrexed disodium, is an organic synthesis intermediate and pharmaceutical intermediate, which can be used in laboratory research and development processes and chemical and pharmaceutical synthesis, showing broad application prospects in the fields of pharmaceutical therapy and chemical synthesis.

[0003] The impurity content of pemetrexed disodium is an important factor affecting the quality of pemetrexed disodium products. Currently, there is no publicly available method for detecting related substances in pemetrexed disodium. Therefore, it is essential to develop an HPLC method that can effectively detect related substances in pemetrexed disodium. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide an HPLC method for detecting related substances in benzoic acid, thereby solving the technical problems mentioned in the background section.

[0005] The above-mentioned technical objective of the present invention is achieved through the following technical solution: a method for detecting related substances in pemetrexed benzoic acid by HPLC, comprising the following steps:

[0006] 1) Instrument selection: High-performance liquid chromatograph; chromatographic column: Shim-Pack GIST C18-AQ, 4.6mm × 150mm, 5μm or equivalent column; mobile phase A: 0.04mol / L potassium dihydrogen phosphate solution, pH adjusted to 5.5 with 1mol / L sodium hydroxide solution; mobile phase B: acetonitrile; UV detector: detection wavelength 200~300nm; mobile phase flow rate: 0.5~1.5ml / min; column temperature: 25~45℃; column length: 100~250mm.

[0007] 2) Prepare solutions. Prepare the following solutions:

[0008] a. Diluent: 0.01 mol / L borax solution - acetonitrile (95:5);

[0009] b. Blank solution: Take an appropriate amount of diluent;

[0010] c. System suitability solution: Weigh appropriate amounts of impurities 2,4,6-trihydroxypyrimidine, N-methyl-2,4-diamino-6-hydroxypyrimidine, methyl 4-(4-butyric acid)benzoate reference standards and pemetrexed benzoate reference standards, accurately weigh them, dissolve them with diluent and quantitatively dilute to prepare a mixed solution containing 0.5 μg each of impurities 2,4,6-trihydroxypyrimidine, N-methyl-2,4-diamino-6-hydroxypyrimidine, and methyl 4-(4-butyric acid)benzoate and 0.25 mg of pemetrexed benzoate per 1 ml;

[0011] d. Test solution: Take an appropriate amount of pemetrexed benzoic acid test sample, accurately weigh it, add diluent to dissolve it and quantitatively dilute it to prepare a solution containing about 0.25 mg per ml;

[0012] e. Control solution: Accurately measure an appropriate amount of the test solution and dilute it quantitatively with diluent to prepare a solution containing 0.8 μg per ml;

[0013] Inject samples according to the following sequence list:

[0014]

[0015] Related substances in the pemetrexed benzoic acid test solution were calculated using the self-comparison method.

[0016] Preferably, an ultraviolet detector is used, with a detection wavelength of 250 nm.

[0017] Preferably, the optimal flow rate of the mobile phase is 0.8 ml / min.

[0018] Preferably, the column temperature is 30℃.

[0019] Preferably, the chromatographic column is Shim-Pack GIST C18-AQ, 4.6mm×150mm, 5μm; the column length is 150mm.

[0020] The present invention has the following advantages: The present invention provides a method for simultaneously detecting the content of impurities such as 2,4,6-trihydroxypyrimidine, N-methyl-2,4-diamino-6-hydroxypyrimidine, and methyl 4-(4-butyric acid)benzoate in pemetrexed benzoic acid. The method is simple to operate, highly specific, and has high sensitivity and accuracy, thereby ensuring the quality control of pemetrexed benzoic acid. Attached Figure Description

[0021] Figure 1 This is the spectrum of the blank solution in Example 1 of the present invention;

[0022] Figure 2 This is a system suitability solution spectrum from Example 1 of the present invention;

[0023] Figure 3 This is the detection limit solution chromatogram in Example 1 of the present invention;

[0024] Figure 4 This is the spectrum of the low-concentration spiked solution in Example 1 of the present invention;

[0025] Figure 5 This is the spectrum of the medium-concentration spiked solution in Example 1 of the present invention;

[0026] Figure 6 This is the spectrum of the high-concentration spiked solution in Example 1 of the present invention;

[0027] Figure 7 This is the chromatogram of the test solution in Example 1 of the present invention;

[0028] Figure 8 This is the chromatogram of the control solution in Example 1 of the present invention;

[0029] Figure 9 This is a spectrum of the mixed solution in the comparative example of this invention. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] Example 1,

[0032] A method for detecting related substances in pemetrexed benzoic acid includes the following steps: Instruments and chromatographic conditions: Instrument: High performance liquid chromatograph; chromatographic column: Shim-Pack GIST C18-AQ, 4.6 mm × 150 mm, 5 μm; Mobile phase A: 0.04 mol / L potassium dihydrogen phosphate solution, pH adjusted to 5.5 with 1 mol / L sodium hydroxide solution; Mobile phase B: acetonitrile; Gradient elution: Gradient elution conditions are shown in Table 1, wavelength: 250 nm, column temperature: 30 ℃, flow rate: 0.8 ml / min, injection volume: 20 μL, run time: 40 min.

[0033] Gradient elution is shown in Table 1 below:

[0034]

[0035] Stock solutions for each impurity: Weigh 12.5 mg each of impurities 2,4,6-trihydroxypyrimidine, N-methyl-2,4-diamino-6-hydroxypyrimidine, and methyl 4-(4-butyric acid)benzoate, place them in separate 50 ml volumetric flasks, dissolve and dilute to the mark with diluent, and shake well.

[0036] For each impurity positioning solution: accurately measure 1 ml of each of the above impurity stock solutions and place them in different 50 ml volumetric flasks. Dilute to the mark with diluent and shake well. Accurately measure 1 ml of each of the above impurity stock solutions and place them in different 10 ml volumetric flasks. Dilute to the mark with diluent and shake well.

[0037] System suitability solution: Weigh appropriate amounts of impurities 2,4,6-trihydroxypyrimidine, N-methyl-2,4-diamino-6-hydroxypyrimidine, methyl 4-(4-butyric acid)benzoate reference standards and pemetrexed benzoate reference standards, accurately weigh them, dissolve them with diluent and quantitatively dilute to prepare a pemetrexed benzoate solution containing 0.5 μg each of impurities 2,4,6-trihydroxypyrimidine, N-methyl-2,4-diamino-6-hydroxypyrimidine, and methyl 4-(4-butyric acid)benzoate per 1 ml. The pemetrexed benzoate content in this solution is 0.25 mg.

[0038] Mixed solution: Take appropriate amounts of impurities 2,4,6-trihydroxypyrimidine, N-methyl-2,4-diamino-6-hydroxypyrimidine, methyl 4-(4-butyric acid)benzoate, 2,4-diamino-6-hydroxypyrimidine, pemetrexed oxide, N-methylpemetrexed benzoate, T4, methyl p-bromobenzoate, and pemetrexed benzoate reference standard, accurately weigh them, dissolve them with diluent and quantitatively dilute to prepare a solution containing 0.5 µg of each impurity and 0.25 mg of pemetrexed benzoate reference standard per 1 ml.

[0039] 1.1 Specificity Examination:

[0040] Accurately measure 20 μL each of the blank solution, the localization solution for each impurity, and the system suitability solution, and inject them into the liquid chromatograph. Record the liquid chromatogram, as follows: Figure 2 As shown in the table below, the specificity test results are shown in the table below. The minimum resolution between each impurity and each peak of pemetrexed benzoic acid in the table is 20.8, which is greater than 1.5, indicating that the method has good specificity.

[0041] The following table shows the results of the specificity test:

[0042]

[0043] 1.2 Detection limit assessment:

[0044] Accurately transfer appropriate amounts of each impurity stock solution, dilute with diluent to prepare a detection limit solution, inject 20 μL of the detection limit solution into the liquid chromatograph, and record the chromatogram. Figure 3As shown in the table below, the detection limits for impurities 2,4,6-trihydroxypyrimidine, N-methyl-2,4-diamino-6-hydroxypyrimidine, and methyl 4-(4-butyric acid)benzoate are all less than 0.01% of the concentration of the test sample, indicating that the method has good sensitivity.

[0045] The following table shows the test results for the detection limit:

[0046]

[0047] 1.3 Accuracy assessment:

[0048] Accurately transfer 10 ml each of the stock solutions of 2,4,6-trihydroxypyrimidine, N-methyl-2,4-diamino-6-hydroxypyrimidine, and methyl 4-(4-butyric acid)benzoate to 50 ml volumetric flasks, dilute to the mark with diluent, and mix well to prepare the impurity mixed stock solutions. Accurately weigh 25 mg of pemetrexed sample, dividing it into nine portions, and place each portion in a 100 ml volumetric flask. Dilute three portions to each flask, adding 0.5 ml, 1.0 ml, and 2.5 ml of the impurity mixed stock solution to each flask at low, medium, and high concentrations, respectively. Dissolve and dilute to the mark with diluent, mix well, and inject for analysis. Record the chromatograms and calculate the recoveries. The results showed that the recoveries of each impurity were within the range of 92%–108%, with RSDs of 0.3%, 1.4%, and 0.6%, respectively. All RSDs were less than 5%, indicating good accuracy of the method. The chromatograms are shown below. Figures 4-6 As shown, the results are presented in the table below:

[0049] The following table shows the accuracy test results:

[0050]

[0051] The results showed that the method had good specificity, sensitivity and accuracy, and was suitable for the detection of pemetrexed related substances.

[0052] Comparative Example 1:

[0053] The detection method is exactly the same as the liquid chromatography conditions in Example 1, except that the chromatographic column is an ACE C18 column (4.6 mm × 250 mm, 5 μm), and all other conditions remain unchanged; the mixed solution from Example 1 is injected for detection, and the chromatogram is recorded; as follows Figure 9 As shown.

[0054] The experimental results show that in the mixed solution, 2,4,6-trihydroxypyrimidine is interfered with by the blank solvent, affecting the detection of related substances in pemetrexed benzoic acid.

[0055] This invention is simple to operate, convenient to use, and suitable for widespread promotion and application. Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for detecting related substances in pemetrexed benzoic acid by HPLC, characterized in that, Includes the following steps: 1) Preparation of diluent: 0.01 mol / L borax solution - acetonitrile (95:5); 2) Preparation of blank solution: Take an appropriate amount of diluent; 3) Preparation of system suitability solution: Weigh appropriate amounts of impurity 2,4,6-trihydroxypyrimidine, N-methyl-2,4-diamino-6-hydroxypyrimidine, methyl 4-(4-butyric acid)benzoate reference standard and pemetrexed reference standard, accurately weigh them, dissolve them with diluent and quantitatively dilute to prepare a mixed solution containing 0.5 μg each of impurity 2,4,6-trihydroxypyrimidine, N-methyl-2,4-diamino-6-hydroxypyrimidine, and methyl 4-(4-butyric acid)benzoate and 0.25 mg of pemetrexed benzoate per 1 ml; 4) Preparation of test solution: Take an appropriate amount of pemetrexed benzoic acid test sample, accurately weigh it, add diluent to dissolve it and quantitatively dilute it to prepare a solution containing about 0.25 mg per ml; 5) Preparation of control solution: Accurately measure an appropriate amount of the test solution and dilute it quantitatively with diluent to prepare a solution containing 0.5 μg per ml; 6) Determination of substances: Accurately measure blank solution, system suitability solution, control solution and test solution and inject them into the liquid chromatograph. Record the liquid chromatogram and calculate the result by peak area according to the self-comparison method. The liquid chromatography conditions include: detector: ultraviolet detector; Chromatographic column: The column is packed with octadecylsilane-bonded silica gel.

2. The method for HPLC detection of related substances in pemetrexed benzoic acid according to claim 1, characterized in that, The chromatographic column selected in step 1) is Shim-Pack GIST C18-AQ or a column with equivalent performance, and the column length is 100~250mm.

3. The method for HPLC detection of related substances in pemetrexed benzoic acid according to claim 1, characterized in that, In high-performance liquid chromatography, an ultraviolet detector is used for detection at a wavelength of 200-300 nm.

4. The method for detecting related substances in pemetrexed benzoic acid by HPLC according to claim 1, characterized in that, Mobile phase A is a 0.04 mol / L potassium dihydrogen phosphate solution, with the pH adjusted to 5.5 using 1 mol / L sodium hydroxide; mobile phase B is acetonitrile.

5. The method for detecting related substances in pemetrexed benzoic acid by HPLC according to claim 1, characterized in that, The mobile phase flow rate is 0.5~1.5 ml / min.

6. The method for detecting related substances in pemetrexed benzoic acid by HPLC according to claim 1, characterized in that, Gradient elution was used, and the gradient elution table is as follows: 。 7. The method for detecting related substances in pemetrexed benzoic acid by HPLC according to claim 1, characterized in that, The column temperature is 25~45℃.

8. The method for detecting related substances in pemetrexed benzoic acid by HPLC according to claim 1, characterized in that, The injection volume was 20 μL.

9. The method for detecting related substances in pemetrexed benzoic acid by HPLC according to claim 1, characterized in that, The substances determined in the substance determination step include at least one or a combination of two or more of 2,4,6-trihydroxypyrimidine, N-methyl-2,4-diamino-6-hydroxypyrimidine, and methyl 4-(4-butyric acid)benzoate.

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