Detection method of folic acid related substances

Abstract

The invention provides a detection method of folic acid related substances, and belongs to the technical field of drug analysis and detection. The detection method comprises the following steps: S1, preparing a test solution, a contrast solution and a system applicability solution; s2, chromatographic conditions of a high performance liquid chromatograph are set, and a mobile phase A is composed of a mixed solution of monopotassium phosphate and dipotassium phosphate and methanol; the mobile phase B consists of a mixed solution of monopotassium phosphate and dipotassium phosphate and methanol; s3, a system applicability solution is injected, and whether the system applicability requirement is met or not is verified; s4, if the system applicability requirement is met, the test solution and the contrast solution are sequentially injected, and a chromatogram is recorded; and S5, processing the chromatogram, and calculating to obtain the content of the related substance impurities of the folic acid. According to the method, related substance impurities of folic acid can be presented in a primary chromatographic behavior, quality control of products in the production process is facilitated, and the method is simple, easy to implement, high in accuracy and precision and good in stability and reproducibility.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis and detection technology, and in particular relates to a method for detecting folic acid-related substances. Background Technology

[0002] Folic acid, chemically named N-[4-[(2-amino-4-oxo-1,4-dihydro-6-terpinen)methylamino]benzoyl]-L-glutamic acid, has the molecular formula C0. 19 H 19 N7O6, molecular weight 441.4. Its chemical structural formula is shown in Formula I: Formula I; Folic acid is a water-soluble vitamin essential for cell growth and reproduction. It functions in the body as tetrahydrofolate, participating in the synthesis and conversion of purine and pyrimidine nucleotides. Commercially available folic acid tablets are mainly available in 0.4 mg and 5 mg strengths. The 0.4 mg strength is primarily used for preventative administration during pregnancy and lactation to prevent neural tube defects in the fetus. The 5 mg strength is mainly used for megaloblastic anemia caused by folic acid deficiency. Due to its important clinical role, folic acid is widely used in daily life.

[0003] Due to the influence of the folic acid processing route, the resulting folic acid-containing products may contain a wide variety of process impurities and degradation products. For example, impurity A [N-(4-aminobenzoyl)-L-glutamic acid] increases significantly under alkaline, high-temperature, and light-degrading conditions; impurity M [2-amino-4-oxo-3,4-dihydropteridine-6-carboxylic acid] increases significantly under alkaline, oxidative, and light-degrading conditions; under alkaline degrading conditions, it degrades to produce impurity L [2-amino-6-methylpteridine-4(3H)-one]; under alkaline and light-degrading conditions, it degrades to produce impurity K [2-amino-6-(hydroxymethyl)pteridine-4(3H)-one]; impurity C [isoflavonic acid], impurity D [pteroic acid], impurity E [6-pterinic folic acid], and impurity F [2- The impurities G, H, and 4-aminobenzoic acid are all process impurities or degradation impurities that may be generated during the production of folic acid raw materials. However, due to different process conditions, the types of impurities in the samples obtained under different process conditions may be different.

[0004] Existing studies typically employ the HPLC method published in the Chinese Pharmacopoeia for related substance analysis. However, this method has a low organic phase ratio, making it ineffective in eluting and detecting various lipid-soluble impurities in the product. Furthermore, the separation between folic acid and impurity peaks, as well as between impurity peaks themselves, is poor. Summary of the Invention

[0005] This invention provides a method for detecting folic acid-related substances, which can simultaneously detect all folic acid-related substances that may be present in a product, and can effectively separate the main peak from adjacent impurity peaks and among each impurity peak. The method is simple to operate, has high precision, good stability, and good reproducibility.

[0006] This invention proposes a method for detecting folic acid-related substances, which uses reversed-phase high-performance liquid chromatography (RP-HPLC) and includes the following steps: S1. Prepare the test solution, control solution, and system suitability solution; S2. Set the chromatographic conditions for the high-performance liquid chromatograph, whereby... Mobile phase A consists of a mixed solution of potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and methanol; Mobile phase B consists of a mixed solution of potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and methanol; Perform gradient elution according to the table below:

[0007] S3. Sample system suitability solution, to verify whether it meets the system suitability requirements; S4. If the system suitability requirements are met, inject the test solution and the control solution sequentially, and record the chromatograms. S5. The above chromatograms were processed using a self-comparison method to calculate the content of folic acid-related impurities.

[0008] Further, in S2, the mobile phase A: the concentration of potassium dihydrogen phosphate is 11.16 g / L, the concentration of dipotassium hydrogen phosphate is 5.50 g / L, and the pH is adjusted to 5.7~6.0 with phosphoric acid; the volume ratio of the mixed solution to methanol is 90:10. Mobile phase B: In the mixed solution, the concentration of potassium dihydrogen phosphate is 11.16 g / L, the concentration of dipotassium hydrogen phosphate is 5.50 g / L, and the pH value is adjusted to 5.7~6.0 with phosphoric acid; the volume ratio of the mixed solution to methanol is 80:20.

[0009] Furthermore, in S2, the chromatographic conditions are as follows: Column temperature: 25℃~35℃; Flow rate: 0.9 ml / min~1.1 ml / min.

[0010] Furthermore, in S2, the chromatographic conditions are as follows: Column temperature: 30℃; Flow rate: 1.0 ml / min.

[0011] Furthermore, in S2, the chromatographic conditions are as follows: Column: The column is packed with octadecylsilane-bonded silica gel. Column dimensions: 250 × 4.6 mm 5 μm; Detection wavelength: 280nm; Injection volume: 20 μl; Running time: 45 minutes.

[0012] Furthermore, S2 also includes, after setting the chromatographic conditions, using a blank solution to perform high-performance liquid chromatography equilibration until two consecutive baselines coincide; The method for preparing the blank solution includes: diluting a sodium carbonate solution with water to obtain a blank solution.

[0013] Furthermore, in S3, the system suitability requirements include: in the chromatogram obtained from the injection system suitability solution, the theoretical plate number of each impurity shall not be less than 5000, the tailing factor shall not be greater than 2.0, and the resolution between each impurity peak and the folic acid main peak and other impurity peaks shall be greater than 1.0.

[0014] Furthermore, In S5, the folic acid-related impurities include, Impurity A: N-(4-aminobenzoyl)-L-glutamic acid Impurity C: Isoflavone acid, Impurity D: Pterygium acid, Impurity E: 6-Pterinol Folic Acid, Impurity F: 2-amino-7-(chloromethyl)pterin-4(1H)-one Impurity G: (2S)-[4-[(2-amino-7-methyl-4-oxo-1,4-dihydropteridin-6-yl)amino]benzamido]glutaric acid, Impurity H: (2S)-2-[4-[(4S)-4-[4-[(2-amino-4-oxy-1,4-dihydroxyanthocyanin-6-yl)methyl]amino]benzamide]-4-carboxybutylamine]benzamide]glutaraldehyde), Impurity K: 2-amino-6-(hydroxymethyl)pterin-4(3H)-one Impurity L: 2-Amino-6-methylpterin-4(3H)-one, Impurity M: 2-Amino-4-oxo-3,4-dihydropteridine-6-carboxylic acid Impurity: 4-Aminobenzoic acid.

[0015] Further, in S1, the method for preparing the test solution includes: dissolving the sample to be tested in sodium carbonate solution and then diluting it with water to obtain the test solution; The method for preparing the control solution includes: diluting the test solution with water to obtain the control solution; The method for preparing the system suitability solution includes: dissolving impurities A, C, D, E, F, G, H, K, 4-aminobenzoic acid, L, and M in sodium carbonate solution and then diluting with water to obtain stock solutions for each impurity; dissolving folic acid reference standard in sodium carbonate solution, adding the stock solutions for each impurity, and then diluting with water to obtain the system suitability solution.

[0016] Furthermore, the mass-to-volume ratio of the sodium carbonate solution is 2.86 g / 100 mL.

[0017] This invention has the following advantages: The folic acid-related substances detection method proposed in this invention, by adjusting the mobile phase system, elution gradient ratio, and related chromatographic conditions, allows impurities such as A, C, D, E, F, G, H, K, 4-aminobenzoic acid, L, and M to be detected in a single chromatographic operation. The specific gradient elution mode increases the elution capacity of the method for lipid-soluble impurities, facilitating product quality control during the production process. This method is simple to implement, has high accuracy and precision, and good stability and reproducibility. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a chromatogram of the system suitability solution obtained in Example 1 of the present invention; Figure 2 This is a chromatogram of the test sample and its self-control in Example 1 of the present invention; Figure 3 This is the chromatogram obtained under the chromatographic conditions of Comparative Example 1 of this invention; Figure 4 This is the chromatogram obtained under the chromatographic conditions of Comparative Example 2 of this invention; Figure 5 This is the chromatogram obtained under the chromatographic conditions of Comparative Example 3 of the present invention; Figure 6 This is the chromatogram obtained under the chromatographic conditions of Comparative Example 4 of this invention; Figure 7 This is the chromatogram of the spiked test solution for impurities in Comparative Example 5 of this invention. Detailed Implementation

[0020] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0021] This invention provides a method for detecting folic acid-related substances using reversed-phase high-performance liquid chromatography (RP-HPLC), comprising the following steps: S1. Prepare the test solution, control solution, and system suitability solution; S2. Set the chromatographic conditions for the high-performance liquid chromatograph, whereby... Mobile phase A consists of a mixed solution of potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and methanol; Mobile phase B consists of a mixed solution of potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and methanol; Perform gradient elution according to the table below:

[0022] S3. Sample system suitability solution, to verify whether it meets the system suitability requirements; S4. If the system suitability requirements are met, inject the test solution and the control solution sequentially, and record the chromatograms. S5. The above chromatograms were processed using a self-comparison method to calculate the content of folic acid-related impurities.

[0023] The folic acid-related substance detection method proposed in this invention analyzes the structural characteristics (such as pKa, solubility, and functional group polarity) of each impurity compound during the screening process. Based on these characteristics, suitable buffer salt types and concentrations, the organic phase ratio of mobile phase A and mobile phase B, and the pH value of the mobile phase are selected. By setting a reasonable gradient program, including the time nodes and the rate of ratio change of the gradient program, specific impurities are separated. At the same time, other chromatographic parameters such as column temperature, flow rate, and detection wavelength are used to assist in ensuring that all impurity compounds that need to be controlled can be effectively retained and separated under the same chromatographic conditions. This method is simple and easy to implement, with high accuracy and precision, and good stability and reproducibility.

[0024] In one embodiment of the present invention, in S2, Mobile phase A: In the mixed solution, the concentration of potassium dihydrogen phosphate is 11.16 g / L, the concentration of dipotassium hydrogen phosphate is 5.50 g / L, and the pH is adjusted to 5.7~6.0 with phosphoric acid; the volume ratio of the mixed solution to methanol is 90:10. Mobile phase B: In the mixed solution, the concentration of potassium dihydrogen phosphate is 11.16 g / L, the concentration of dipotassium hydrogen phosphate is 5.50 g / L, and the pH value is adjusted to 5.7~6.0 with phosphoric acid; the volume ratio of the mixed solution to methanol is 80:20.

[0025] In a preferred embodiment of the present invention, the pH value of both mobile phase A and mobile phase B is adjusted to 5.8 using phosphoric acid.

[0026] In one embodiment of the present invention, the chromatographic conditions in S2 are as follows: The specific model of the high performance liquid chromatograph is: welch Ultimate XB-C18, which is a conventional reversed-phase C18 analytical column manufactured by Yuexu Technology; Column: The column is packed with octadecylsilane-bonded silica gel. Column dimensions: 250 × 4.6 mm 5 μm; Detection wavelength: 280nm; Injection volume: 20 μl; Running time: 45 minutes.

[0027] In one embodiment of the present invention, the chromatographic conditions in S2 are as follows: Column temperature: 25℃~35℃; preferably, column temperature: 30℃; Flow rate: 0.9 ml / min to 1.1 ml / min; preferably, flow rate: 1.0 ml / min.

[0028] In one embodiment of the present invention, S2 further includes setting the chromatographic conditions of the high performance liquid chromatograph after completing the connection and initialization of the high performance liquid chromatograph.

[0029] In one embodiment of the present invention, S2 further includes, after setting the chromatographic conditions, using a blank solution to perform high performance liquid chromatography equilibration until two consecutive baselines coincide.

[0030] In one embodiment of the present invention, in S3, the system suitability requirements include: in the system suitability solution chromatogram, the theoretical plate number of each impurity shall not be less than 5000, the tailing factor shall not be greater than 2.0, and the resolution between each impurity peak and the folic acid main peak and other impurity peaks shall be greater than 1.0.

[0031] In one embodiment of the present invention, in step S5, the folic acid-related impurities include, Impurity A: N-(4-aminobenzoyl)-L-glutamic acid Impurity C: Isoflavone acid, Impurity D: Pterygium acid, Impurity E: 6-Pterinol Folic Acid, Impurity F: 2-amino-7-(chloromethyl)pterin-4(1H)-one Impurity G: (2S)-[4-[(2-amino-7-methyl-4-oxo-1,4-dihydropteridin-6-yl)amino]benzamido]glutaric acid, Impurity H: (2S)-2-[4-[(4S)-4-[4-[(2-amino-4-oxy-1,4-dihydroxyanthocyanin-6-yl)methyl]amino]benzamide]-4-carboxybutylamine]benzamide]glutaraldehyde), Impurity K: 2-amino-6-(hydroxymethyl)pterin-4(3H)-one Impurity L: 2-Amino-6-methylpterin-4(3H)-one, Impurity M: 2-Amino-4-oxo-3,4-dihydropteridine-6-carboxylic acid Impurity: 4-Aminobenzoic acid.

[0032] In this embodiment of the invention, the test solution, control solution, system suitability solution, and blank solution are all test solutions.

[0033] The method for preparing the blank solution includes: diluting the sodium carbonate solution with water to obtain the blank solution. Specifically, the method for preparing the blank solution includes: taking 1 ml of sodium carbonate solution, placing it in a 100 ml volumetric flask, diluting it with water to the mark, and shaking well.

[0034] The preparation method of the test solution includes: dissolving the sample to be tested in sodium carbonate solution and then diluting it with water to obtain the test solution. Specifically, the preparation of the test solution is as follows: take 20 mg of the sample to be tested, place it in a 100 ml volumetric flask, add 1 ml of sodium carbonate solution to dissolve it, dilute with water to the mark, and shake well. It should be prepared immediately before use.

[0035] The preparation method of the control solution includes: diluting the test solution with water to obtain the control solution. Specifically, the preparation of the control solution is as follows: take 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute with water to the mark, and shake well. The method for preparing the system suitability solution includes: dissolving impurities A, C, D, E, F, G, H, K, 4-aminobenzoic acid, L, and M in sodium carbonate solution and then diluting with water to obtain stock solutions for each impurity; dissolving pure folic acid in sodium carbonate solution, adding the stock solutions for each impurity, and then diluting with water to obtain the system suitability solution. Specifically, the preparation method of the system suitability solution includes: weighing 10 mg each of impurity A, impurity C, impurity D, impurity E, impurity F, impurity G, impurity H, impurity K, impurity 4-aminobenzoic acid, impurity L, and impurity M, placing them in separate 100 ml volumetric flasks, adding an appropriate amount of sodium carbonate solution to dissolve them, diluting with water to the mark, and shaking well to prepare the stock solutions for each impurity; weighing 20 mg of folic acid reference standard (folic acid pure product that meets the legal standard and whose purity has been standardized), placing it in a 100 ml volumetric flask, adding 1 ml of sodium carbonate solution to dissolve it, then adding the stock solutions for each impurity, diluting with water to the mark, and shaking well.

[0036] Preferably, the mass-to-volume ratio of the sodium carbonate solution is 2.86 g / 100 mL. In this embodiment of the invention, the addition of sodium carbonate solution is mainly to provide an alkaline environment, dissolve folic acid and impurities, and stabilize the solution system. It is a key reagent to ensure complete dissolution of the sample and accurate test results.

[0037] In one embodiment of the present invention, the test solutions, including blank solution, test sample solution, control solution, and system suitability solution, all require operation in the dark.

[0038] In one embodiment of the present invention, in S1, the self-comparison method is a conventional quantitative method. The content of folic acid-related impurities is calculated by processing the above chromatogram using the following formula:

[0039] In the formula, A 杂 Peak area of ​​impurities in the test solution; A 对 Peak area of ​​folic acid in the control solution.

[0040] The present invention will now be described in detail with reference to the embodiments.

[0041] Example 1 The detection method for folic acid-related substances employs reversed-phase high-performance liquid chromatography (RP-HPLC), and includes the following steps: S1. Prepare the test solution, control solution, system suitability solution, and blank solution; S2. After setting the chromatographic conditions of the high performance liquid chromatograph, use a blank solution to balance the high performance liquid chromatograph until two consecutive baselines coincide. S3. Sample system suitability solution, to verify whether it meets the system suitability requirements; S4. After meeting the system suitability requirements, inject the test solution and the control solution in sequence and record the chromatograms. S5. The above chromatograms were processed using a self-comparison method to calculate the content of folic acid-related impurities; among which, In S1, the test solution is prepared as follows: Blank solution: Measure 1.0 ml of 2.86 g / 100 mL sodium carbonate solution into a 100 ml volumetric flask, dilute with water to the mark, and shake well to obtain the blank solution.

[0042] Preparation of control solution: Take 1 ml of the test solution, place it in a 100 ml volumetric flask, dilute with water to the mark, and shake well.

[0043] Test solution: Take 20 mg of the sample to be tested, place it in a 100 ml volumetric flask, add 1 ml of sodium carbonate solution to dissolve it, dilute with water to the mark, shake well and it is ready to use.

[0044] Preparation of system suitability solutions: First, prepare stock solutions for each impurity: Impurity A stock solution: Weigh 20 mg of impurity A, place it in a 50 ml volumetric flask, add 5 ml of 2.86 g / 100 mL sodium carbonate solution to dissolve it, dilute with water to the mark, and shake well to obtain the solution.

[0045] Impurity 4-aminobenzoic acid stock solution: Weigh 10 mg of impurity 4-aminobenzoic acid, place it in a 100 ml volumetric flask, add 5 ml of 2.86 g / 100 mL sodium carbonate solution to dissolve it, dilute with water to the mark, and shake well to obtain the solution.

[0046] Impurity C stock solution: Weigh 12 mg of impurity C, place it in a 200 ml volumetric flask, add 5 ml of 2.86 g / 100 mL sodium carbonate solution to dissolve it, dilute with water to the mark, and shake well to obtain the solution.

[0047] Impurity D stock solution: Weigh 12 mg of impurity D, place it in a 100 ml volumetric flask, add 5 ml of 2.86 g / 100 mL sodium carbonate solution to dissolve it, dilute with water to the mark, and shake well to obtain the solution.

[0048] Stock solutions for impurities E, G, and H: The preparation method is the same as that for "Stock solution for impurity C" above.

[0049] Impurity F stock solution: Weigh 6 mg of impurity F and place it in a 100 ml volumetric flask. Add 5 ml of 2.86 g / 100 mL sodium carbonate solution to dissolve it. Dilute with water to the mark and shake well. Measure 10 ml of the solution and place it in a 100 ml volumetric flask. Dilute with water to the mark and shake well. Impurity K stock solution: Weigh 10 mg of impurity K and place it in a 50 ml volumetric flask. Add 5 ml of 2.86 g / 100 mL sodium carbonate solution to dissolve it. Dilute with water to the mark and shake well. Take 5 ml of the solution and place it in a 50 ml volumetric flask. Dilute with water to the mark and shake well. Impurity L stock solution: Weigh 10 mg of impurity L and place it in a 50 ml volumetric flask. Add 5 ml of 2.86 g / 100 mL sodium carbonate solution to dissolve it. Dilute with water to the mark and shake well. Measure 5 ml of the solution and place it in a 50 ml volumetric flask. Dilute with water to the mark and shake well.

[0050] Impurity M stock solution: Weigh 10 mg of impurity M, place it in a 100 ml volumetric flask, add 5 ml of 2.86 g / 100 mL sodium carbonate solution to dissolve it, dilute with water to the mark, and shake well to obtain the solution.

[0051] Then, weigh 20 mg of folic acid reference standard and place it in a 100 ml volumetric flask. Add 1 ml of 2.86 g / 100 mL sodium carbonate solution to dissolve it. Then add 1.0 ml each of the above impurity A, impurity C, impurity D, impurity E, impurity G, impurity K, impurity L, and impurity M stock solutions, 0.5 ml of impurity H stock solution, 3.0 ml of impurity F stock solution, and 0.2 ml of impurity 4-aminobenzoic acid stock solution. Dilute with water to the mark and shake well to obtain the final product.

[0052] In S2, the chromatographic conditions are as follows: Column: Octadecylsilane-bonded silica gel as packing material (Welch Ultimate XB-C18 250×4.6mm 5μm); Detection wavelength: 280 nm; Column temperature: 30 ℃; Flow rate: 1.0 ml / min; Injection volume: 20 μl; Run time: 45 min; Mobile phase A: a mixed solution of 11.16 g / L potassium dihydrogen phosphate and 5.50 g / L dipotassium hydrogen phosphate (adjusted to pH 5.8 with phosphoric acid) - methanol (90:10); Mobile phase B: a mixed solution of 11.16 g / L potassium dihydrogen phosphate and 5.50 g / L dipotassium hydrogen phosphate (adjusted to pH 5.8 with phosphoric acid) - methanol (80:20); Perform gradient elution according to Table 1: Table 1

[0053] The ratio of mobile phases A and B ("%" in the table) is a volume ratio (v / v) by default.

[0054] In S3, system suitability requirements include: in the system suitability solution chromatogram, the theoretical plate number of each impurity must not be less than 5000, the tailing factor must not be greater than 2.0, and the resolution between each impurity peak and the folic acid main peak and other impurity peaks should be greater than 1.0. The resulting chromatogram is shown below. Figure 1 The detailed analysis results are shown in Table 2.

[0055] Table 2. Results of system suitability test in Example 1

[0056] In S4, the chromatograms obtained from the injected test solution and its own control solution are shown in [the provided text]. Figure 2 The chromatograms were processed using a self-comparison method, and the contents of folic acid-related impurities were calculated, as shown in Table 3.

[0057] As shown in Table 3, due to the optimization of process conditions, the sample to be tested does not necessarily contain all 11 impurities. However, as long as the sample to be tested contains any one of the 11 impurities, this method can still detect them.

[0058] Table 3. Results of related substance detection for each batch of samples.

[0059] Example 2 Durability testing Under different durability test conditions (see Table 4), system suitability solutions were injected into the liquid chromatograph. The results are shown in Table 5.

[0060] Initial conditions: Column: Welch Ultimate XB-C18 250×4.6mm 5μm Mobile phase A: a mixed solution of 11.16 g / L potassium dihydrogen phosphate and 5.50 g / L dipotassium hydrogen phosphate (adjusted to pH 5.8 with phosphoric acid) - methanol (90:10) Mobile phase B: a mixed solution of 11.16 g / L potassium dihydrogen phosphate and 5.50 g / L dipotassium hydrogen phosphate (adjusted to pH 5.8 with phosphoric acid) - methanol (80:20) Perform gradient elution according to Table 1 above; Detection wavelength: 280nm; injection volume: 20μl; Running time: 45 minutes; The remaining conditions are shown in Table 4.

[0061] Table 4

[0062] Table 5 Durability test results

[0063] Comparative Example 1 Same as Example 1, except that the column temperature was 40°C. Results are shown below. Figure 3 Impurity F and impurity D cannot be separated, thus failing to meet the objective of detecting all impurities together.

[0064] Comparative Example 2 Same as Example 1, except that the flow rate was 1.3 ml / min. Results are shown below. Figure 4 The separation degree between impurity A and impurity M is 0.64, which is poor and cannot meet the purpose of detecting all impurities together.

[0065] Comparative Example 3 Same as Example 1, except that the pH value was 5.3. Results are shown below. Figure 5 Impurity E and impurity D cannot be separated, thus failing to meet the objective of detecting all impurities together.

[0066] Comparative Example 4 Same as Example 1, except that the pH value was 6.3. Results are shown below. Figure 6 Impurity M cannot be separated from impurity 4-aminobenzoic acid, and impurity E cannot be separated from impurity D, thus failing to meet the objective of detecting all impurities together.

[0067] Comparative Example 5 Currently, the publicly available methods for determining folic acid-related substances are... HPLC method (see Chinese Pharmacopoeia 2025 Edition, Part II), details are as follows: The relevant substances were determined by high performance liquid chromatography (General Rule 0512), and the operation was carried out in the dark.

[0068] For the test solution, take about 100 mg of this product, place it in a 100 ml volumetric flask, add about 1 ml of ammonia test solution to dissolve it, dilute to the mark with the mobile phase, and shake well.

[0069] Accurately measure 1 ml of the test solution into a 100 ml volumetric flask, dilute to the mark with the mobile phase, and shake well.

[0070] Impurity stock solution: Weigh approximately 1 mg of each of the following impurities: A, B, C, D, E, F, G, H, J, K, L, M, N, p-aminobenzoic acid, and folic acid isomer. Place each in a 25 ml volumetric flask, add 2 ml of 0.1 mol / L sodium carbonate to dissolve, dilute to the mark with the mobile phase, and shake well.

[0071] Impurity spiked test solution: Weigh about 10 ml of folic acid raw material, place it in a 100 ml volumetric flask, add 1 ml of ammonia test solution to dissolve it, add 1 ml of each impurity stock solution, dilute to the mark with the mobile phase, and shake well.

[0072] For system suitability solution, take 10 mg of dicofol and place it in a 100 ml volumetric flask. Add 5 ml of 0.1 mol / L sodium carbonate solution and 10 ml of the test solution. Dissolve and dilute to the mark with the mobile phase and shake well.

[0073] Chromatographic conditions: Octadecylsilane-bonded silica gel was used as the stationary phase; phosphate buffer (pH 5.0) was used as the mobile phase (2.0 g of potassium dihydrogen phosphate was dissolved in about 650 ml of water, 15 ml of 0.5 mol / L tetrabutylammonium hydroxide methanol solution, 7 ml of 1 mol / L phosphoric acid solution and 270 ml of methanol were added, cooled, and the pH was adjusted to 5.0 with 1 mol / L phosphoric acid solution or ammonia solution, and diluted with water to 1000 ml); the detection wavelength was 280 nm; the flow rate was 1.2 ml per minute; and the injection volume was 10 μl.

[0074] System suitability requirements: In the system suitability solution chromatogram, the resolution between the pteroic acid peak and the folic acid peak should be greater than 4.0.

[0075] For the assay, accurately measure the test solution and the reference solution, inject them separately into the liquid chromatograph, and record the chromatograms up to three times the retention time of the main peak.

[0076] In the chromatogram of the limit test solution, the peak areas of teicocyanic acid and any other individual impurity must not exceed 0.6 times (0.6%) the area of ​​the main peak in the control solution. The sum of the peak areas of all impurities except teicocyanic acid must not exceed twice (2.0%) the area of ​​the main peak in the control solution; peaks less than 0.05 times the area of ​​the main peak in the control solution are negligible. Results are shown below. Figure 7 .

[0077] Experimental results show that folic acid and some impurities have poor peak shapes, and impurity J and impurity K, impurity N and impurity L, impurity M and 4-aminobenzoic acid, and impurity A and impurity D cannot be effectively separated.

[0078] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting folic acid-related substances, characterized in that, The detection was performed using reversed-phase high-performance liquid chromatography, including the following steps: S1. Prepare the test solution, control solution, and system suitability solution; S2. Set the chromatographic conditions for the high-performance liquid chromatograph, whereby... Mobile phase A consists of a mixed solution of potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and methanol; Mobile phase B consists of a mixed solution of potassium dihydrogen phosphate and dipotassium hydrogen phosphate, and methanol; Perform gradient elution according to the table below: S3. Sample system suitability solution, to verify whether it meets the system suitability requirements; S4. If the system suitability requirements are met, inject the test solution and the control solution sequentially, and record the chromatograms. S5. The above chromatogram was processed using a self-comparison method to calculate the content of folic acid-related impurities.

2. The detection method according to claim 1, characterized in that, In S2, mobile phase A: the concentration of potassium dihydrogen phosphate is 11.16 g / L, the concentration of dipotassium hydrogen phosphate is 5.50 g / L, and the pH is adjusted to 5.7~6.0 with phosphoric acid; the volume ratio of the mixed solution to methanol is 90:

10. Mobile phase B: In the mixed solution, the concentration of potassium dihydrogen phosphate is 11.16 g / L, the concentration of dipotassium hydrogen phosphate is 5.50 g / L, and the pH value is adjusted to 5.7~6.0 with phosphoric acid; the volume ratio of the mixed solution to methanol is 80:

20.

3. The detection method according to claim 1, characterized in that, In S2, the chromatographic conditions are as follows: Column temperature: 25℃~35℃; Flow rate: 0.9 ml / min~1.1 ml / min.

4. The detection method according to claim 3, characterized in that, In S2, the chromatographic conditions are as follows: Column temperature: 30℃; Flow rate: 1.0 ml / min.

5. The detection method according to claim 1, characterized in that, In S2, the chromatographic conditions are as follows: Column: The column is packed with octadecylsilane-bonded silica gel. Column dimensions: 250 × 4.6 mm 5 μm; Detection wavelength: 280nm; Injection volume: 20 μl; Running time: 45 minutes.

6. The detection method according to claim 1, characterized in that, S2 also includes, after setting the chromatographic conditions, using a blank solution to perform high performance liquid chromatography equilibration until two consecutive baselines coincide; The method for preparing the blank solution includes: diluting a sodium carbonate solution with water to obtain a blank solution.

7. The detection method according to claim 1, characterized in that, In S3, the system suitability requirements include: in the chromatogram obtained from the injection system suitability solution, the theoretical plate number of each impurity shall not be less than 5000, the tailing factor shall not be greater than 2.0, and the resolution between each impurity peak and the folic acid main peak and other impurity peaks shall be greater than 1.

0.

8. The detection method according to claim 1, characterized in that, In S5, the folic acid-related impurities include, Impurity A: N-(4-aminobenzoyl)-L-glutamic acid Impurity C: Isoflavone acid, Impurity D: Pterygium acid, Impurity E: 6-Pterinol Folic Acid, Impurity F: 2-amino-7-(chloromethyl)pterin-4(1H)-one Impurity G: (2S)-[4-[(2-amino-7-methyl-4-oxo-1,4-dihydropteridin-6-yl)amino]benzamido]glutaric acid, Impurity H: (2S)-2-[4-[(4S)-4-[4-[(2-amino-4-oxy-1,4-dihydroxyanthocyanin-6-yl)methyl]amino]benzamide]-4-carboxybutylamine]benzamide]glutaraldehyde), Impurity K: 2-amino-6-(hydroxymethyl)pterin-4(3H)-one Impurity L: 2-Amino-6-methylpterin-4(3H)-one, Impurity M: 2-Amino-4-oxo-3,4-dihydropteridine-6-carboxylic acid Impurity: 4-Aminobenzoic acid.

9. The detection method according to claim 8, characterized in that, In S1, the preparation method of the test solution includes: dissolving the sample to be tested in sodium carbonate solution and then diluting it with water to obtain the test solution; The method for preparing the control solution includes: diluting the test solution with water to obtain the control solution; The method for preparing the system suitability solution includes: dissolving impurities A, C, D, E, F, G, H, K, 4-aminobenzoic acid, L, and M in sodium carbonate solution and then diluting with water to obtain stock solutions for each impurity; dissolving folic acid reference standard in sodium carbonate solution, adding the stock solutions for each impurity, and then diluting with water to obtain the system suitability solution.

10. The detection method according to claim 6 or 9, characterized in that, The mass-to-volume ratio of the sodium carbonate solution is 2.86 g / 100 mL.

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