A method for determining related substances in methotrexate

Abstract

The application discloses a method for determining related substances in methotrexate, which comprises the following steps: preparing an impurity C positioning solution, a system suitability solution and a test sample solution, and then respectively detecting the impurity C positioning solution, the system suitability solution, a reference substance solution and the test sample solution by using liquid chromatography, recording chromatograms, and quantitatively analyzing impurity C and other impurities in the test sample solution by comparing the chromatograms. The system suitability solution is prepared by destroying the main component reference substance solution, and the solution is a mixed solution containing impurity B, impurity C and methotrexate, so that impurity C can be positioned, and the separation degree between impurity B and impurity C should be not less than 1.5.

Description

Technical Field

[0001] This invention belongs to the field of drug quality control, and specifically relates to a method for determining related substances in methotrexate. Background Technology

[0002] Currently, the Chinese Pharmacopoeia (2025 Edition, Part II), the European Pharmacopoeia (Eur. Ph. 11.0), and the United States Pharmacopeia (USPMethotrexate Monographs, Official as of 01-Oct-2024) all include information on methotrexate-related substances, while the Japanese Pharmacopoeia (JP18) does not. A comparison of the impurity limits for methotrexate-related substances in the pharmacopoeias of various countries is shown in Table 1.

[0003] Table 1 Comparison of Impurity Limits for Relevant Substances in Various Quality Standards

[0004]

[0005] According to the quality standards of various national pharmacopoeias, a total of five known impurities are involved: impurity B, impurity C, impurity E, impurity I, and impurity H. European and American pharmacopoeias treat these five impurities as specific impurities, and each impurity is located using impurity reference standards. The Chinese pharmacopoeia standard does not specify specific impurities; instead, it controls the content of individual impurities and total impurities as unknown impurities.

[0006] As shown in Table 1, the limit for impurity C in European and American pharmacopoeias is 0.5%, significantly higher than that for other impurities. Impurity C is obtained by the hydrolysis of the 4-amino group of methotrexate, and it is easily generated during the synthesis of the active pharmaceutical ingredient and during the production, storage, and transportation of the finished product. The Chinese Pharmacopoeia does not classify impurity C as a specific impurity, but rather stipulates that a single impurity must not exceed 0.5%, thus making it impossible to accurately quantify impurity C and relaxing the limits for other individual impurities. Since impurity reference standards are expensive and take a long time to purchase, it is necessary to establish an efficient, rational, and accessible analytical method to accurately locate impurity C, thereby achieving effective control of related substances in methotrexate.

[0007] Table 2 shows a comparison of the methods for the related substances section of methotrexate in various quality standards:

[0008] Table 2 Comparison of Methods for Relevant Substances in Various Quality Standards

[0009]

[0010] Experiments have shown that the preparation methods for test solutions in the pharmacopoeia standards of various countries all have certain shortcomings: the Chinese Pharmacopoeia uses a mobile phase of pH 6.0 as the solvent, in which methotrexate is difficult to dissolve, and ultrasonic-assisted dissolution can easily lead to sample degradation; the European and American pharmacopoeias all use a pre-dissolution followed by dilution method, which is a rather cumbersome two-step operation, and the dimethyl sulfoxide used in the United States Pharmacopoeia has a certain degree of toxicity, while the European Pharmacopoeia uses a small amount of ammonia solution to dissolve the sample first, which can cause a local over-alkali effect that leads to the degradation of methotrexate. Summary of the Invention

[0011] This application provides a method for the determination of related substances in methotrexate.

[0012] The objective of this invention is achieved through the following technical solution:

[0013] The preparation method of the methotrexate impurity C localization solution includes the following steps:

[0014] Add an alkaline solution to the methotrexate reference solution until the pH of the reference solution is 12.5–13.2. After reacting for 20–30 minutes, add an acid solution to neutralize, and obtain a methotrexate impurity C localization solution.

[0015] The alkaline solution is at least one of sodium hydroxide solution and potassium hydroxide solution;

[0016] The acid solution is at least one of hydrochloric acid solution, sulfuric acid solution, and phosphoric acid solution.

[0017] The methotrexate impurity C localization solution was prepared by the above method.

[0018] A method for preparing a system suitability solution for the determination of related substances in methotrexate includes the following steps:

[0019] The above-mentioned impurity C positioning solution was irradiated with ultraviolet light for 20-30 minutes to obtain the solution.

[0020] The system suitability solution for the determination of related substances in methotrexate is prepared by the above method.

[0021] The method for determining related substances in methotrexate includes the following steps:

[0022] (1) Dissolve the methotrexate raw material or preparation in a solvent to prepare a test solution with a methotrexate concentration of 0.2~1 mg / mL;

[0023] The solvent is composed of phosphate buffer and acetonitrile in a volume ratio of 95:5, and the pH is adjusted to 7.5-8.0;

[0024] (2) Perform liquid chromatography detection on the impurity C positioning solution, system suitability solution, reference solution and test solution respectively, record the chromatograms, and perform quantitative analysis of impurity C and other impurities in the test solution by comparing the chromatograms;

[0025] The reference solution is a 0.2-1% methotrexate solution;

[0026] The other impurities mentioned are one or more of impurity B, impurity D, impurity E, impurity I, or impurity H;

[0027] The liquid chromatography described herein uses mobile phase A as phosphate buffer at pH 5.6 and mobile phase B as acetonitrile, employing gradient elution. The elution program is as follows:

[0028]

[0029] The liquid chromatography described herein has a mobile phase elution flow rate of 0.9~1.1 mL / min, preferably 1.0 mL / min; and a column temperature of 25~35℃, preferably 30℃.

[0030] The liquid chromatography detection also includes a sensitivity solution;

[0031] The sensitivity solution is a 0.2 μg / mL methotrexate solution;

[0032] The quantitative analysis method described is the principal component self-comparison method without correction factors, and impurity C is a specific impurity.

[0033] The present invention has the following advantages and effects compared with the prior art:

[0034] 1. The present invention uses a method of destroying the main component reference solution to prepare a system suitability solution. The solution is a mixed solution containing impurity B, impurity C and methotrexate. Impurity C can be located and the separation degree between impurity B and impurity C should be not less than 1.5.

[0035] 2. This invention optimizes chromatographic conditions by adjusting mobile phase pH, mobile phase ratio, column temperature, and gradient elution program, thereby improving the resolution between various impurities and establishing a scientifically sound method for determining impurities in methotrexate. Validation has shown that the method can simultaneously determine six known impurities and can quantify each impurity using a principal component self-comparison method without correction factors.

[0036] 3. Compared with the methods in domestic and international pharmacopoeias, the method of this invention has the following significant advantages: ① It eliminates the need for impurity reference standards, improving the economy and versatility of the method; ② It rationally prepares system suitability solutions, providing support for the accurate quantification of specific impurities and improving the specificity and accuracy of the method; ③ It optimizes the solvent of the test solution; ④ Compared with the United States Pharmacopeia, it optimizes the mobile phase pH and gradient elution procedure, resulting in better peak separation. Attached Figure Description

[0037] Figure 1 This is a chromatogram of a mixed reference solution under the chromatographic conditions described in the Chinese Pharmacopoeia.

[0038] Figure 2 This is a chromatogram of a mixed reference solution under the chromatographic conditions of the European Pharmacopoeia.

[0039] Figure 3 This is a chromatogram of a mixed reference solution under the chromatographic conditions of the United States Pharmacopeia.

[0040] Figure 4 This is a chromatogram of the pH value test.

[0041] Figure 5 This is a chromatogram of the mobile phase ratio determination experiment.

[0042] Figure 6 This is the chromatogram from the column temperature test.

[0043] Figure 7 This is a chromatogram of the flow rate test.

[0044] Figure 8 This is a column durability test for chromatography. Figure 1 .

[0045] Figure 9 This is a column durability test for chromatography. Figure 2 .

[0046] Figure 10 This is a chromatogram of a solution suitable for the Chinese Pharmacopoeia system.

[0047] Figure 11 These are chromatograms of test solutions supplied by various methotrexate raw material manufacturers.

[0048] Figure 12 These are chromatograms of test solutions supplied by various manufacturers of injectable methotrexate.

[0049] Figure 13 This is a graph showing the results of a forced degradation test.

[0050] Figure 14 These are comparative chromatograms of the solutions.

[0051] Figure 15These are comparative chromatograms of the localization solution in different chromatographic columns.

[0052] Figure 16 These are comparative chromatograms of the system suitability solution on different chromatographic columns.

[0053] Figure 17 This is a comparison of the spectra of two degradation impurity peaks in the system suitability solution (Huapu C18 column).

[0054] Figure 18 This is a chromatogram of a mixed reference solution.

[0055] Figure 19 This is a blank solvent chromatogram.

[0056] Figure 20 This is a chromatogram of a blank excipient solution.

[0057] Figure 21 This is a comparison chart of the results of methotrexate raw material determination using the proposed method and the ChP method.

[0058] Figure 22 This is a comparison chart of the results of methotrexate for injection determined using the proposed method and the ChP method.

[0059] In the above chromatograms, the elution order of the integrated peaks is: impurity B, impurity C, methotrexate, impurity D, impurity E, impurity I, and impurity H. Detailed Implementation

[0060] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0061] Example 1

[0062] Methods for the determination of related substances in methotrexate

[0063] (1) Selection of chromatographic conditions:

[0064] Preparation of the mixed reference solution: A mixed reference solution was prepared by taking methotrexate, impurity B, impurity C, impurity D, impurity E, impurity I, and impurity H reference standards for system suitability studies. The source information and prepared concentrations of each impurity are shown in Table 3. The impurity concentrations were prepared with reference to the limits specified in European and American pharmacopoeias. This solution was used for injection in all chromatographic condition optimization experiments to investigate the method's separation ability and robustness against each impurity.

[0065] Table 3. Preparation concentrations of the mixed reference solution and information on the sources of each reference standard.

[0066]

[0067] Note: CATO is the abbreviation for Guangzhou Jiatu Technology Co., Ltd.

[0068] (2) Comparison of chromatographic conditions in pharmacopoeias of various countries:

[0069] The Chinese Pharmacopoeia uses a C18 column with isocratic elution using an acetonitrile-7.0% citric acid solution-2.0% anhydrous disodium hydrogen phosphate solution (8.5:10:80) as the mobile phase (adjusted to pH 6.0 with 7.0% citric acid solution or 2.0% anhydrous disodium hydrogen phosphate solution). The mixed reference solution was injected according to the chromatographic conditions of the Chinese Pharmacopoeia; the chromatogram is shown in [Figure number missing]. Figure 1 .

[0070] Depend on Figure 1 It can be seen that under these chromatographic conditions, the elution time of methotrexate is approximately 10 minutes, impurity D cannot be separated from methotrexate, impurities B and C elute early and are poorly separated, and the elution times of impurities I and H are approximately 61 minutes and 130 minutes, respectively. According to the Chinese Pharmacopoeia, "the chromatogram should be recorded up to four times the retention time of the main component peak," which is 40 minutes; therefore, the peaks of impurities I and H cannot be collected.

[0071] Therefore, a gradient elution method is considered to ensure that all known impurities can be eluted within a suitable collection time.

[0072] The European Pharmacopoeia specifies the use of a C18 column with dimensions of 4.0 mm × 250 mm and a 5 μm depth; the United States Pharmacopoeia (USP) uses a C18 column with dimensions of 4.6 mm × 250 mm and a 5 μm depth. The mobile phase compositions of the European and USPs are similar, both employing gradient elution, but the pH of the aqueous phase and the gradient elution program differ. A mixed reference solution was injected under the chromatographic conditions specified in both the European and USPs. The chromatograms are shown in the figure. Figure 2 and Figure 3 .

[0073] As shown in the figure, under the chromatographic conditions of the United States Pharmacopeia, the peak shape and elution time of each peak are relatively suitable, and the column specifications (4.6 mm × 250 mm, 5 μm) are commonly used with low column pressure.

[0074] Therefore, we considered optimizing the chromatographic conditions based on the United States Pharmacopeia and investigated the effects of different pH values, mobile phase ratios, flow rates, column temperatures, and different brands of chromatographic columns on chromatographic separation.

[0075] (3) Optimization of chromatographic conditions and study of method robustness

[0076] pH value assessment:

[0077] A Waters C18 column (4.6 mm × 250 mm, 5 μm) was used. Phase A consisted of 0.34% anhydrous sodium dihydrogen phosphate solution (adjusted to pH 5.2, 5.4, 5.6, 5.8, and 6.0 with sodium hydroxide solution), and phase B consisted of acetonitrile. Gradient elution was performed according to Table 4. The flow rate was 1.0 mL / min, and the column temperature was 30 °C. The chromatogram of the mixed reference solution is shown in [Figure 4]. Figure 4 .

[0078] When the pH value of phase A is 5.2, the peaks of impurity E and impurity I cannot be separated; when the pH value of phase A is 6.0, the peaks of impurity D and methotrexate cannot be separated.

[0079] Depend on Figure 4 It can be seen that the peaks are well separated at pH values ​​of 5.4, 5.6 and 5.8. Considering that the pH value of the aqueous phase in the mobile phase can be adjusted within ±0.2 pH range of the original specified value as stipulated in Chinese Pharmacopoeia General Chapter 0512, pH 5.6 was selected as the final pH value of the aqueous phase to ensure the robustness of the method.

[0080] Table 4. Gradient elution procedure for pH value determination

[0081]

[0082] Investigation of the proportion of the mobile phase:

[0083] A Waters C18 column (4.6 mm × 250 mm, 5 μm) was used. Phase A consisted of 0.34% anhydrous sodium dihydrogen phosphate solution (pH adjusted to 5.6 with sodium hydroxide solution), and phase B consisted of acetonitrile. Referring to the gradient elution program in Table 4, the proportion of phase B for 30–34 minutes was adjusted to 22%, 25%, and 28%. The flow rate was 1.0 mL / min, and the column temperature was 30 °C. The chromatogram of the mixed reference solution is shown in [Figure number missing]. Figure 5 .

[0084] Depend on Figure 5 It is evident that when phase B is 25%, the peaks exhibit good separation, the analysis time is appropriate, and the method demonstrates good robustness.

[0085] Investigation of flow rate and column temperature:

[0086] A GL Science C18 column (4.6 mm × 250 mm, 5 μm) was used with 0.34% anhydrous sodium dihydrogen phosphate solution (pH adjusted to 5.6 with sodium hydroxide solution) as phase A and acetonitrile as phase B, following a gradient elution according to Table 4. The peak values ​​of the mixed reference solution were investigated at column temperatures of 25, 30, and 35 °C and flow rates of 0.9, 1.0, and 1.1 mL / min. The chromatograms are shown in [Table 4]. Figure 6 and Figure 7As shown in the figure, when the flow rate is 1.0 mL / min and the column temperature is 30℃, all peaks can be effectively separated, indicating that the method has good robustness.

[0087] Column robustness study:

[0088] Based on the above investigation and experiments, the chromatographic conditions were determined as follows: octadecylsilane-bonded silica gel was used as the packing material (4.6 mm × 250 mm, 5 μm or equivalent column); 0.34% anhydrous sodium dihydrogen phosphate solution (pH adjusted to 5.6 with sodium hydroxide solution) was used as mobile phase A, acetonitrile as phase B, and linear gradient elution was performed according to Table 4; the flow rate was 1.0 mL per minute; the column temperature was 30℃; the detection wavelength was 280 nm; and the injection volume was 20 μL.

[0089] To examine the robustness of the method, six C18 columns of different brands, all with specifications of "4.6mm × 250mm, 5μm", were selected for testing. The chromatograms are shown below. Figure 8 and Figure 9 The relative retention times and resolutions of each impurity are shown in Table 5. The results show that the elution order of the mixed reference solution in each column is: impurity B, impurity C, methotrexate, impurity D, impurity E, impurity I, and impurity H. The resolution of each peak is greater than 1.5, indicating that the method has good robustness.

[0090] Table 5 Summary of column durability test results

[0091]

[0092] Example 2

[0093] Methods for the determination of related substances in methotrexate

[0094] Investigation of solution preparation methods

[0095] (1) Test solution and solvent

[0096] The concentration of the test solution for related substances in the Chinese Pharmacopoeia was 1 mg / mL. The mobile phase used was acetonitrile-7.0% citric acid solution-2.0% anhydrous disodium hydrogen phosphate solution (8.5:10:80) (the pH was adjusted to 6.0 with 7.0% citric acid solution or 2.0% anhydrous disodium hydrogen phosphate solution). Experiments showed that methotrexate was difficult to dissolve in this solvent, and the solution remained slightly turbid even after sonication for more than half an hour.

[0097] The United States Pharmacopeia (USP) method for preparing test solutions involves first dissolving the sample in a small amount of dimethyl sulfoxide (DMSO), then diluting it with the initial proportioned mobile phase solution. DMSO is somewhat toxic. The European Pharmacopeia (EU) method involves first dissolving the sample in a dilute ammonia solution, then diluting it with the initial proportioned mobile phase solution. However, excessive alkalinity in some areas can lead to methotrexate degradation. Both the USP and EU pharmacopoeias employ a two-step dilution method, which is relatively cumbersome.

[0098] In this application, the concentration of the test solution is 0.4 mg / mL, and the solvent is "0.34% anhydrous sodium dihydrogen phosphate solution-acetonitrile (95:5) (adjusted to pH 8.0 with 1 mol / L sodium hydroxide solution)". The sample has good solubility, the operation is simple, and the test solution is stable within 24 hours as verified by the test.

[0099] (2) System suitability solution

[0100] The system suitability solution for the Chinese Pharmacopoeia is a mixed solution containing 0.1 mg / mL each of methotrexate and folic acid (system suitability requires a resolution greater than 8.0 between the two). Inject according to the chromatographic conditions of the Chinese Pharmacopoeia. The chromatogram is shown below. Figure 10 .

[0101] Folic acid is a structural analogue of methotrexate (both are pteridine compounds), but their retention behaviors in liquid chromatography differ significantly. Figure 10 It is evident that folic acid has a peak elution time of approximately 3 minutes, while methotrexate has a peak elution time of approximately 10 minutes. Using the separation degree between these two as a system suitability requirement cannot effectively control the separation degree between impurities and between adjacent impurities and the main peak.

[0102] The system suitability solutions of the United States Pharmacopeia and the European Pharmacopeia are prepared using impurity reference standards. However, the National Institutes for Food and Drug Control (NIFDC) does not have relevant impurity reference standards. Therefore, this invention, considering the need to improve the accessibility of reference standards and the general applicability of the method, will formulate the preparation method and system suitability requirements of the system suitability solution based on the actual test results of the samples and the results of the forced destruction test.

[0103] This invention collected samples from 5 raw material manufacturers and 5 injectable methotrexate manufacturers. Chromatograms of a batch of test solutions with high impurity content from each manufacturer were compared. (See attached figures.) Figure 11 and Figure 12 .

[0104] As shown in the figure: ① No impurity D with the retention time closest to the main peak was detected in either the raw material or the finished product samples; ② No impurities E, I, or H were detected; ③ Impurity B was detected in some manufacturers; ④ Impurity C was detected in all raw materials except for company E and in finished products except for company e, and all of them were the largest single impurities. Impurity C is obtained by hydrolysis of the 4-amino group of methotrexate. Impurity C is easily generated during the synthesis of raw materials and during the production, storage, and transportation of finished products.

[0105] Therefore, based on the sample test results, the preparation of the system suitability solution should focus on the location and detection of impurities B and C, and should be based on the column robustness test results ( Figure 8 , Figure 9 As shown in Table 5, the resolution between impurity B and impurity C varies greatly in different chromatographic columns, and requirements should be set for the resolution between their chromatographic peaks.

[0106] This invention examines the degradation mechanism of each impurity through forced degradation tests, and achieves the purpose of system suitability control by destroying the generated impurities B and C.

[0107] The methotrexate reference solution was subjected to destruction by high temperature (80℃ for 4 hours), oxidation (30% hydrogen peroxide solution for 10 minutes), alkali (1 mol / L sodium hydroxide solution for 1 hour), acid (1 mol / L hydrochloric acid solution for 2 hours), light (4000 lx irradiation for 4 hours), and ultraviolet light (254 nm irradiation for 30 minutes). The undestroyed and destroyed solutions were then analyzed. The chromatograms are shown below. Figure 13 .

[0108] The results showed that: ① Acid, alkali and high temperature degradation all produced impurity C, among which methotrexate was degraded more rapidly in alkali; ② Oxidative degradation produced impurity C and many unknown impurities; ③ Light degradation and ultraviolet light degradation both produced impurity B and a small amount of unknown impurities.

[0109] Table 6 shows the analysis of the six related sources of methotrexate:

[0110] Table 6 Information and Source Analysis of Known Substances

[0111]

[0112] Based on the results of the forced degradation test, this invention first uses an alkaline degradation solution as a positioning solution to locate impurity C, and then uses the solution after ultraviolet light degradation (producing impurity B) as a system suitability solution, requiring that the separation degree between impurity B and impurity C should not be less than 1.5.

[0113] Comparative chromatograms of the undamaged solution, alkali-damaged solution, UV-damaged solution, alkali-damaged solution followed by UV-damaged solution, and mixed reference solution are shown below. Figure 14 .

[0114] In addition, to ensure that the sensitivity of the proposed method meets the requirements, a solution with a methotrexate concentration of 0.2 μg / mL (0.05%) was prepared as a sensitivity solution.

[0115] In summary, the preparation methods and system suitability requirements for the impurity C positioning solution, system suitability solution, and sensitivity solution in this invention are as follows:

[0116] Impurity C positioning solution: Take about 8 mg of methotrexate reference standard, place it in a 20 mL volumetric flask, add 5 mL of solvent and shake to dissolve, add 2 mL of 1 mol / L sodium hydroxide solution (the pH of the solution after mixing is about 13.2), let it stand at room temperature for 30 minutes, add 2 mL of 1 mol / L hydrochloric acid solution, dilute to the mark with solvent, shake well, and obtain the impurity C positioning solution.

[0117] System suitability solution: Take an appropriate amount of the positioning solution and irradiate it under a UV lamp (254nm) for 30 minutes.

[0118] Sensitivity solution: Accurately measure an appropriate amount of the control solution and quantitatively dilute it with a solvent to prepare a solution containing approximately 0.2 μg per 1 mL.

[0119] System suitability requirements: In the positioning solution chromatogram, the retention time of the methotrexate peak should be 10–15 minutes, and the relative retention time of impurity C peak should be 0.7–0.8. In the system suitability solution chromatogram, the resolution between impurity C peak and impurity B peak should be no less than 1.5. In the sensitivity solution chromatogram, the signal-to-noise ratio of the methotrexate peak should be greater than 10.

[0120] Three different brands of C18 columns (all 4.6 mm × 250 mm, 5 μm) were used to analyze the C-targeting solution and the system suitability solution. The chromatograms are shown below. Figure 15 and Figure 16 The statistical results are shown in Table 7.

[0121] Table 7 Summary of analytical results of impurity C localization solution and system suitability solution in different chromatographic columns

[0122]

[0123] Experimental pair Figure 16 The two degradation impurity peaks with retention times of 7.924 min (peak 1) and 9.097 min (peak 2) produced by degradation in the chromatogram of the "Huapu C18 column" were spectrally scanned. The spectral comparison is shown in the figure below. Figure 17 .

[0124] Depend on Figure 17 It can be seen that the spectra of the two chromatographic peaks mentioned above are basically consistent with the spectra of the main peaks in the reference solutions of impurity B and impurity C.

[0125] Example 3

[0126] Methods for the determination of related substances in methotrexate

[0127] Investigation of known impurity correction factors

[0128] Take appropriate amounts of methotrexate reference standard and reference standards of each known impurity, prepare solutions of different concentrations, accurately measure appropriate amounts of each, inject and record the chromatogram, plot the regression curve of concentration versus peak area, calculate the correction factor of each impurity by the ratio of the slope of the regression line of methotrexate to the slope of the regression line of impurities, and the results are shown in Table 8.

[0129] The 2025 edition of the Chinese Pharmacopoeia, Volume IV, 9101, Analytical Method Validation Guidelines stipulate that when the correction factor is approximately equal to 1 (the relative response factor between the analyte and the standard is 0.8 to 1.2) or the amount of related substances has been overestimated, the correction factor may not be used for calculation.

[0130] As shown in Table 8, the correction factors for impurities B, C, E, and H are all in the range of 0.8–1.2; the content of impurity D is overestimated when calculated with a correction factor of 1; the correction factor for impurity I is 1.3, which is slightly higher than the above range, but it was not detected in the collected raw material and formulation samples and is not a degradation impurity. Therefore, this invention uses the principal component self-comparison method without correction factors to quantify each known impurity.

[0131] Table 8 Results of the Investigation of Known Impurity Correction Factors

[0132]

[0133] Example 4

[0134] A method for determining related substances in methotrexate and methotrexate for injection includes the following steps:

[0135] Solvent: Mobile phase A-acetonitrile (95:5) (adjusted to pH 8.0 with 1 mol / L sodium hydroxide solution).

[0136] Methotrexate raw material test solution: Take an appropriate amount of this product, dissolve and dilute it with solvent to prepare a solution containing approximately 0.4 mg of methotrexate per 1 mL.

[0137] Methotrexate for Injection Test Solution: Take an appropriate amount of the contents of this product, dissolve and dilute it with solvent to prepare a solution containing approximately 0.4 mg of methotrexate per 1 mL.

[0138] Control solution: Accurately measure an appropriate amount of the test solution and dilute it quantitatively with a solvent to prepare a solution containing approximately 2 μg per 1 mL.

[0139] Impurity C positioning solution: Take about 8 mg of methotrexate reference standard, place it in a 20 mL volumetric flask, add 5 mL of solvent and shake to dissolve, add 2 mL of 1 mol / L sodium hydroxide solution, let stand at room temperature for 30 minutes, add 2 mL of 1 mol / L hydrochloric acid solution, dilute to the mark with solvent, and shake well.

[0140] System suitability solution: Take an appropriate amount of the positioning solution and irradiate it under a UV lamp (254nm) for 30 minutes.

[0141] Sensitivity solution: Accurately measure an appropriate amount of the control solution and quantitatively dilute it with a solvent to prepare a solution containing approximately 0.2 μg per 1 mL.

[0142] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the packing material (4.6 mm × 250 mm, 5 μm or equivalent column); 0.34% anhydrous sodium dihydrogen phosphate solution (adjusted to pH 5.6 with 1 mol / L sodium hydroxide solution) was used as mobile phase A, and acetonitrile was used as phase B. Linear gradient elution was performed according to Table 4; the flow rate was 1.0 mL per minute; the column temperature was 30 °C; the detection wavelength was 280 nm; and the injection volume was 20 μL.

[0143] Methodological validation:

[0144] (1) Exclusivity

[0145] According to the method of this embodiment, the following solution was injected: mixed reference solution (chromatogram shown in the figure). Figure 18 ), blank solvent (chromatogram see) Figure 19 ), blank excipient solution (chromatogram shown) Figure 20 ), methotrexate raw material test solution (chromatogram shown in...) Figure 11 Methotrexate for Injection Test Solution (chromatogram shown) Figure 12 ) and forced degradation test solution (chromatogram shown) Figure 13 ).

[0146] As shown in the figure, the blank solvent, blank excipient, and degradation impurities do not interfere with the determination. The resolution between the known impurity peaks and the main component peak in the chromatogram of the mixed reference solution meets the requirements, indicating that the method of the present invention has good specificity.

[0147] (2) Linearity and Range

[0148] The results of the linearity and range tests are shown in Table 9. The results show that methotrexate and each known impurity have a good linear relationship with the concentration and peak area.

[0149] (3) Repeatability

[0150] Six test solutions were prepared in parallel using the method of this invention, based on sample E (batch number: 20221105) from enterprise E. The content of impurity C was 0.02% in all samples, the maximum content of other single impurities was between 0.21% and 0.23%, and the total amount of impurities was between 0.32% and 0.34%, indicating that the method has good repeatability.

[0151] (4) Other items

[0152] The results of the verification of injection precision, spiking recovery rate, detection limit, quantitation limit and 24-hour stability (the mixed reference solution was placed at room temperature and injected at 0, 1, 4, 7, 14, 18 and 24 hours) are shown in Table 9. All of them meet the requirements of related substance inspection.

[0153] Table 9 Summary of Results of Some Methodological Validation Tests

[0154]

[0155] Example 5

[0156] The method for determining related substances in methotrexate and methotrexate for injection shall be performed according to the method in Example 4:

[0157] Sample measurement results:

[0158] (1) Results of methotrexate raw material assay

[0159] The method of this invention and the method of the 2025 edition of the Chinese Pharmacopoeia, Part II (hereinafter referred to as the ChP method) were used to conduct relevant substance tests on 16 batches of samples collected from 5 companies.

[0160] The measurement results of the method of the present invention are shown in Table 10, and the comparison of the measurement results of the two methods is shown in Table 11 and Table 2. Figure 21 The results show that the maximum single impurity content measured by the two methods is basically the same. The total impurity content measured by the method of this invention is slightly higher than that measured by the ChP method for some manufacturers. This is because the method of this invention uses a gradient elution method, which allows for more thorough elution of impurities with stronger retention, and the selection of the detection wavelength is also more conducive to the detection of impurities.

[0161] Table 10 Results of related substance tests for methotrexate according to the proposed method

[0162]

[0163] Table 11 Comparison of related substance test results for methotrexate using the proposed method and the ChP method

[0164]

[0165] (2) Results of methotrexate for injection

[0166] Related substances were tested on seven batches of samples from five companies using the method of this invention and the ChP method. The results of the determination using the method of this invention are shown in Table 12, and a comparison of the results of the two methods is shown in Table 13. Figure 22 The results show that the results obtained by the two methods are basically consistent. The impurity content of some manufacturers measured by the method of this invention is slightly higher than that measured by the ChP method. This is because the method of this invention uses a gradient elution method, which allows for more thorough elution of impurities with stronger retention, and the selection of the detection wavelength is also more conducive to the detection of impurities.

[0167] Table 12 Results of related substance tests for methotrexate for injection according to the proposed method

[0168]

[0169] Table 13 Comparison of related substance test results for methotrexate for injection according to the proposed method and the ChP method.

[0170]

[0171] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing a system suitability solution for the determination of related substances in methotrexate, characterized in that... Includes the following steps: The solution for the methotrexate impurity C is obtained by irradiating it with ultraviolet light for 20-30 minutes; the system suitability solution is a mixed solution containing impurity B, impurity C and methotrexate. The methotrexate impurity C localization solution is prepared by the following steps: Add an alkaline solution to the methotrexate reference solution until the pH of the solution is 12.5-13.

2. After reacting for 20-30 minutes, add an acid solution to neutralize, and obtain a methotrexate impurity C positioning solution. In the system suitability solution, the separation degree between impurity B and impurity C is not less than 1.

5.

2. The preparation method according to claim 1, characterized in that: The alkaline solution is at least one of sodium hydroxide solution and potassium hydroxide solution; The acid solution is at least one of hydrochloric acid solution, sulfuric acid solution, and phosphoric acid solution.

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