Method for detecting related substances in folic acid tablets

Abstract

The invention relates to the technical field of pharmaceutical analysis, in particular to a method for detecting related substances in folic acid tablets. In the long-term stability lofting process of the folic acid tablets, unknown impurities are generated, the content of the unknown impurities is in a rising trend and exceeds the identification limit of 0.2% of a preparation, the invention discloses a detection method of related substances in the folic acid tablets, the related substances in the folic acid tablets are detected by adopting a high performance liquid chromatography, and the related substances are impurities shown in the formula 1; the detection method for the related substances in the folic acid tablets is used for detecting the content of the impurities shown in the formula 1 in the folic acid tablets, the method is high in specificity, accurate, precise and sensitive, and the content of the impurities shown in the formula 1 in the folic acid tablets can be effectively controlled.

Description

Technical Field

[0001] The present application relates to the technical field of drug analysis, and in particular to a method for detecting related substances in folic acid tablets. Background Art

[0002] Folic acid tablets (0.4mg) are a drug with folic acid as the main ingredient. They can be used to prevent congenital neural tube defects in the fetus. They are preventive drugs for pregnant and lactating women. Studies have shown that pregnant women taking folic acid tablets during pregnancy can reduce the rate of neural tube defects in the fetus by 85%. This result has been widely used in more than 50 countries around the world. Impurity research is a key pharmaceutical research content throughout the life cycle of drugs. As a drug taken by pregnant women, the study of its impurity spectrum is particularly critical.

[0003] In order to ensure the safety and effectiveness of the drug, a drug stability test was carried out. During the long-term stability sampling of folic acid tablets, an impurity with a relative retention time of about 4.5 times that of folic acid was detected. The content showed an increasing trend and exceeded the identification threshold of 0.2% for the preparation. The ICH guidelines indicate that the amount of impurities exceeding the 0.2% limit has reached the limit that may cause harm to the human body, and the impurity should be structurally identified and its content controlled.

[0004] At present, this impurity is an unknown impurity, and there is no analytical method to control this unknown impurity. Therefore, how to effectively control the analytical method of this impurity has become a problem that needs to be solved urgently. Summary of the invention

[0005] As described in the background technology, folic acid tablets produce unknown impurities in the stability test, and the content shows an upward trend and exceeds the identification threshold of 0.2% of the preparation. Currently, there is no detection method for the content of this unknown impurity in folic acid tablets. The method of this application is used to separate and purify folic acid tablets to obtain the target impurity, named impurity 3, and then the infrared spectrum, mass spectrum and nuclear magnetic resonance spectrum of the impurity are comprehensively analyzed (see the mass spectrum for details). Figure 1 ), confirming that the impurity structure is as follows: In view of the deficiencies in the prior art, the present application provides a HPLC detection method for detecting the content of related substances (impurity 3) in folic acid tablets. The HPLC detection method is highly specific, accurate, precise and sensitive, and can be used for quality control in the research and development and production of folic acid tablets, to improve the quality and safety of drugs and ensure the safety of clinical drug use.

[0006] The present application provides a method for detecting related substances in folic acid tablets, which adopts the following technical scheme: A method for detecting related substances in folic acid tablets, using high performance liquid chromatography to detect related substances in folic acid tablets, wherein the related substances are impurities shown in formula 1 (i.e., impurity 3);

[0007] Preferably, the chromatographic conditions of the high performance liquid chromatography are as follows: Preferably, the chromatographic column: a chromatographic column using octylsilane bonded silica gel as a filler; The mobile phase is a mixture of component A and component B; Wherein, component A is a mixed solution of 1% (g / mL)-1.2% (g / mL) potassium dihydrogen phosphate and 0.4% (g / mL)-0.7% (g / mL) dipotassium hydrogen phosphate; the pH value is adjusted to 6.4±0.2 with phosphoric acid or 5 mol / L potassium hydroxide; Component B is methanol; Elution mode: isocratic elution.

[0008] Preferably, in the mobile phase, the volume ratio of component A to component B is (85-90):(15-10).

[0009] Further preferably, in the mobile phase, the volume ratio of component A to component B is selected from 85:15, 86:14, 87:13, 88:12, 89:11 or 90:10; more preferably, in the mobile phase, the volume ratio of component A to component B is 90:10.

[0010] Preferably, in the mobile phase, component A is a mixed solution of 1.116% (g / mL) potassium dihydrogen phosphate and 0.55% (g / mL) dipotassium hydrogen phosphate.

[0011] Preferably, when high performance liquid chromatography is used for detection, the detection wavelength is 275-285 nm.

[0012] Further preferably, the detection wavelength is selected from 275nm, 276nm, 277nm, 278nm, 279nm, 280nm, 281nm, 282nm, 283nm, 284nm, and 285nm; more preferably, the detection wavelength is 280nm.

[0013] Preferably, when high performance liquid chromatography is used for detection, the injection volume is 10-20 μl.

[0014] Further preferably, the injection volume is selected from 10 μl, 11 μl, 12 μl, 13 μl, 14 μl, 15 μl, 16 μl, 17 μl, 18 μl, 19 μl or 20 μl; more preferably, the injection volume is 10 μl.

[0015] Preferably, when high performance liquid chromatography is used for detection, the column temperature of the chromatographic column is 25-30°C.

[0016] Further preferably, the column temperature of the chromatographic column is selected from 25°C, 26°C, 27°C, 28°C, 29°C or 30°C; more preferably, the column temperature of the chromatographic column is 25°C.

[0017] Preferably, the flow rate of the mobile phase is 0.5-1 mL / min.

[0018] Further preferably, the mobile phase flow rate is selected from 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min or 1 mL / min; more preferably, the mobile phase flow rate is 0.6 mL / min.

[0019] Preferably, the chromatographic column is Merck 100RP-8; column length 250 mm, diameter 4.0 mm, filler particle size 5 μm; or equivalent column.

[0020] Preferably, a method for detecting related substances in folic acid tablets comprises the following steps: (1) Preparation of system suitability solution: Take appropriate amounts of the impurity shown in Formula 1 and folic acid reference substance, add 2.86% (g / mL) sodium carbonate solution into a brown volumetric flask, sonicate to dissolve, then add mobile phase to dilute to a solution containing approximately 0.5 mg of folic acid and 2.5 μg of the impurity shown in Formula 1 per 1 mL, which is used as the system suitability solution; (2) Preparation of test solution: Take an appropriate amount of fine powder of this product (equivalent to about 10 mg of folic acid) and place it in a 20 mL brown volumetric flask. Add 5 mL of 2.86% (g / mL) sodium carbonate solution, dissolve it by ultrasonication, dilute it to the mark with mobile phase, shake well, centrifuge (4000 rpm) for 10 minutes, filter the supernatant, and take the filtrate as the test solution; (3) Prepare the control solution: accurately measure 1 mL of the test solution, place it in a 100 mL brown bottle, and dilute to the mark with the mobile phase as the control solution; (4) The system suitability solution, the test solution and the control solution are injected into a high performance liquid chromatograph to detect the content of the impurity shown in Formula 1.

[0021] In a specific embodiment, a method for detecting related substances in folic acid tablets comprises the following steps: (1) Solution preparation: (operation should be carried out in a dark place) System suitability solution: Take appropriate amount of impurity 3 (the impurity shown in Formula 1) and folic acid reference substance, add 2.86% (g / mL) sodium carbonate solution into a brown volumetric bottle, sonicate to dissolve, then add mobile phase to dilute into a solution containing about 0.5 mg of folic acid and 2.5 μg of impurity 3 per 1 mL, as the system suitability solution; Test solution: Take an appropriate amount of sample powder (equivalent to about 10 mg of folic acid) and place it in a 20 mL brown volumetric flask. Add 5 mL of 2.86% (g / mL) sodium carbonate solution and sonicate for 10 min to dissolve it. Dilute to the scale with mobile phase, shake well, centrifuge (4000 rpm) for 10 minutes, filter the supernatant, and take the filtrate as the test solution.

[0022] Control solution: Accurately measure 1 mL of the test solution and place it in a 100 mL brown volumetric flask. Dilute to the mark with the mobile phase and shake well as the control solution.

[0023] (2) Chromatographic conditions: Chromatographic column: Octylsilane bonded silica gel as filler, Merck 100RP-8 (250mm×4.0mm, 5μm) or equivalent column; Mobile phase: a mixed solution of 1.116% (g / mL) potassium dihydrogen phosphate and 0.55% (g / mL) potassium dihydrogen phosphate-methanol, wherein the volume ratio of the mixed solution to methanol is 90:10; the pH value of the mixed solution is adjusted to 6.4±0.2 with phosphoric acid or 5 mol / L potassium hydroxide; Detection wavelength: 280nm; injection volume: 10μl; column temperature: 25℃; flow rate: 0.6mL / min; acquisition time: 7 times the retention time of the main component peak; Sample dissolving solvent: 2.86% (g / mL) sodium carbonate solution (take about 2.86 g of anhydrous sodium carbonate, dissolve it in water and dilute it to 100 mL); elution method: isocratic elution.

[0024] (3) The calculation formula is as follows: Impurity 3 content % = (A×f) / A s ×1%; Where: A is the peak area of ​​impurity 3 in the test solution; A s is the main peak area in the control solution; f is the correction factor of impurity 3; the correction factor is shown in the method linearity verification results in the embodiment; (4) Limit In the chromatogram of the test sample, the content of impurity 3 does not exceed 0.5%.

[0025] In summary, the present application includes at least one of the following beneficial technical effects: The present application provides a method for detecting related substances in folic acid tablets, which is used to detect the content of impurity 3 in folic acid tablets. The method is highly specific, accurate, precise and sensitive, and can effectively control the content of impurity 3 in folic acid tablets. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1is the mass spectrum of impurity 3; Figure 2 is the chromatogram of the specific system suitability solution 1; Figure 3 is the chromatogram of the specific system suitability solution 2; Figure 4 This is the chromatogram of the precision of the analytical method for related substance impurity 3 in folic acid tablets; Figure 5 This is the chromatogram of the accuracy-reference solution in the accuracy verification experiment of the analytical method for related substance impurity 3 in folic acid tablets; Figure 6 This is the chromatogram of the accuracy-background solution in the accuracy verification experiment of the analytical method for related substance impurity 3 in folic acid tablets; Figure 7 This is the chromatogram of the accuracy of the 20% accuracy solution in the accuracy verification experiment of the analytical method for related substance impurity 3 in folic acid tablets; Figure 8 This is the chromatogram of the accuracy of the 100% accuracy solution in the accuracy verification experiment of the analytical method for related substance impurity 3 in folic acid tablets; Fig. 9 This is the chromatogram of the accuracy of the 150% accuracy solution in the accuracy verification experiment of the analytical method for related substance impurities 3 in folic acid tablets. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further illustrated by specific embodiments below. The specific embodiments do not limit the protection scope of the present invention; some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0028] The raw materials involved in this application are all commercially available products, and the application is further described in detail below in conjunction with the embodiments and comparative examples.

[0029] Example 1: Folic acid tablet sample detection This method was used to test three batches of homemade folic acid tablets with stability up to 18 months under 0-day and long-term test conditions (temperature 25℃±2℃, relative humidity 60%±5%), and two batches of folic acid tablets of reference preparations with near-expiry date (stored at 25℃ in dark place).

[0030] 1. Main instruments and reagents: Instruments: High performance liquid chromatograph, model: Agilent 1260; electronic analytical balance; acidity meter; high-speed desktop centrifuge.

[0031] Reagents: potassium dihydrogen phosphate, anhydrous dipotassium hydrogen phosphate, anhydrous sodium carbonate, phosphoric acid, methanol (chromatographic grade), purified water.

[0032] Preparation of 2.86% (g / mL) sodium carbonate solution: Take about 2.86 g of anhydrous sodium carbonate, dissolve it in water and dilute it to 100 mL.

[0033] Preparation of 1.116% (g / mL) potassium dihydrogen phosphate solution: Take about 1.116 g of potassium dihydrogen phosphate, dissolve it in water and dilute it to 100 mL.

[0034] Preparation of 0.55% (g / mL) dipotassium hydrogen phosphate solution: Take about 0.55 g of dipotassium hydrogen phosphate, dissolve it in water and dilute it to 100 mL.

[0035] 2. Reference substances and test substances: Homemade folic acid tablets for testing, batch numbers: Y200601, Y200602, Y200603, source: Jiangsu Yabang Epson Pharmaceutical Co., Ltd.; Reference preparation test product (folic acid tablets), batch number: 19FA255, 19FA406, purchased from Merus Labs Luxco II Sàrl; Folic acid reference substance, batch number: DY23210101, purity: 92.7%, was purchased from Hebei Jiheng (Group) Pharmaceutical Co., Ltd.; impurity 3 reference substance, batch number: 20210315, purity: 96.55%, was purchased from Nanjing Mingjie Biomedical Testing Co., Ltd.

[0036] 3. The detection method is as follows: Solution preparation: (1) System suitability solution: Take an appropriate amount of impurity 3 (the impurity shown in Formula 1) and folic acid reference substance, dissolve them in 2.86% (g / mL) sodium carbonate solution, and then add mobile phase to dilute to a solution containing 0.5 mg of folic acid and 2.5 μg of the impurity shown in Formula 1 per 1 mL, which is used as the system suitability solution; (2) Test solution: Take an appropriate amount of folic acid tablet powder (equivalent to about 10 mg of folic acid) and place it in a 20-mL brown volumetric flask. Add 5 mL of 2.86% (g / mL) sodium carbonate solution and sonicate for 10 min to dissolve it. Dilute to the scale with mobile phase, shake well, and centrifuge (4000 rpm) for 10 min. Filter the supernatant and use the filtrate as the test solution.

[0037] (3) Control solution: Accurately measure 1 mL of the test solution and place it in a 100 mL brown volumetric flask. Dilute to the mark with the mobile phase and shake well. This is used as the control solution.

[0038] (4) Chromatographic conditions: Chromatographic column: Merck 100RP-8 (250mm×4.0mm, 5μm); Mobile phase: a mixture of component A and component B; the volume ratio of component A to component B is 90:10; Component A is a mixed solution of 1.116% (g / mL) potassium dihydrogen phosphate and 0.55% (g / mL) dipotassium hydrogen phosphate; the pH value is adjusted to 6.4±0.2 with phosphoric acid or 5 mol / L potassium hydroxide; Component B: methanol; Detection wavelength: 280nm; injection volume: 10μl; column temperature: 25℃; flow rate: 0.6mL / min; acquisition time: 7 times the retention time of the main component peak; sample solvent: 2.86% sodium carbonate solution; Elution mode: isocratic elution.

[0039] (5) Inject the system suitability solution, test solution, and control solution into the liquid chromatograph and record the chromatogram. Figure 1 The results of impurity 3 content are shown in Table 1.

[0040] The calculation formula is as follows: Impurity 3 content % = (A×f) / A s ×1%; Where: A is the peak area of ​​impurity 3 in the test solution; A s is the main peak area in the control solution; f is the correction factor of impurity 3 (obtained from Example 4, the correction factor is 1.94); Table 1 Content of impurity 3 in three batches of homemade folic acid tablets and two batches of reference preparations Experimental results: The method of the present invention can effectively detect the content of impurity 3. The content of impurity 3 shows an increasing trend in the stability test, and the impurity content exceeds the identification threshold of 0.2% of the preparation.

[0041] Example 2: Specificity of the method 1. The main instruments and reagents are the same as those in Example 1; 2. Reference substances: Folic acid reference substance, batch number: DY23210101, purity: 92.7%, source: Hebei Jiheng (Group) Pharmaceutical Co., Ltd.; impurity reference substance: Impurity A ((2S)-2-(4-aminobenzoyl)glutaric acid), batch number: Batch2.0, source: EP; Impurity D (pteroic acid), batch number: Batch4.0, source: EP; 6-Aldopterin, batch number: FA176621701, purity: 97.3%, source: Carbosynth; Impurity 3, batch number: 20210315, purity: 96.55%, source: Nanjing Mingjie Biomedical Testing Co., Ltd.

[0042] 3. Detection method: (1) Chromatographic conditions: same as in Example 1; (2) Preparation of specific solution: Impurity 3 localization solution: Weigh an appropriate amount of impurity 3 reference substance, add an appropriate amount of 2.86% (g / mL) sodium carbonate solution to dissolve it, and add the mobile phase to prepare an impurity 3 localization solution (the concentration of impurity 3 in the solution is about 25 μg / mL); System suitability solution 1: Take appropriate amount of folic acid reference substance and impurity 3 reference substance, add appropriate amount of 2.86% (g / mL) sodium carbonate solution to dissolve, and add mobile phase to prepare system suitability solution 1 (the folic acid concentration in system suitability solution 1 is about 0.5 mg / mL, and the impurity 3 concentration is about 2.5 μg / mL); System suitability solution 2: Take appropriate amounts of impurity A, pteroic acid, 6-formopterin, and folic acid reference substance, add appropriate amount of 2.86% (g / mL) sodium carbonate solution to dissolve, and add mobile phase to prepare system suitability solution 2 (the folic acid concentration in system suitability solution 2 is about 0.5 mg / mL, the impurity A concentration is about 10 μg / mL, the pteroic acid concentration is about 3 μg / mL, and the 6-formopterin concentration is about 5 μg / mL).

[0043] Take the prepared solution and inject it into the liquid chromatograph and record the chromatogram Figure 2 and Figure 3 , the specific results are shown in the table below.

[0044] Table 2 Specificity of the method 3. Result analysis: According to Table 2, it can be seen that in system suitability solution 1, the separation degree of folic acid and impurity 3 meets the requirements, and in system suitability solution 2, each impurity has no interference with impurity 3, indicating that the detection method of the present application has strong specificity.

[0045] Example 3. Sensitivity of the method 1. Chromatographic conditions are the same as in Example 2; 2. Sensitivity detection: Using the signal-to-noise ratio method, take folic acid reference substance and impurity 3 reference substance respectively, add appropriate amount of 2.86% (g / mL) sodium carbonate solution to dissolve, dilute to a certain concentration with mobile phase, inject the sample, and observe the signal-to-noise ratio S / N.

[0046] The specific results are shown in Table 3 below: Table 3 Sensitivity test results of detection limit solution As shown in Table 3, the analytical method of the present invention has high sensitivity, with the detection limit concentration equivalent to 0.001% of the test sample concentration and the quantitative limit concentration equivalent to 0.005% of the test sample concentration. This method can detect impurities 3 at relatively low concentrations.

[0047] Example 4: Linearity of the method 1. Chromatographic conditions are the same as in Example 2; 2. Preparation of linear solution: Linear stock solution: Weigh 10.03 mg of impurity 3 reference substance, place in a 20 mL brown volumetric flask, add 5 mL of 2.86% (g / mL) sodium carbonate solution, dissolve by ultrasound, dilute to the scale with mobile phase, shake well, and use it as impurity 3 stock solution (the concentration of impurity 3 in the impurity 3 stock solution is 484.198 μg / mL); Take 10.09 mg of folic acid reference substance, accurately weigh, place in a 20 mL brown volumetric flask, add 5 mL of 2.86% sodium carbonate solution to dissolve, dilute to the scale with mobile phase, shake well, and use it as folic acid reference substance stock solution (the folic acid concentration in the folic acid reference substance stock solution is 467.671 μg / mL); Take 1 mL each of impurity 3 stock solution and folic acid reference stock solution, place in the same 20 mL brown volumetric flask, add mobile phase to dilute to the scale, shake well, and use it as linear stock solution (the folic acid concentration in the linear stock solution is 23.384 μg / mL, and the impurity 3 concentration is 24.210 μg / mL).

[0048] 200% linear solution: accurately measure 2 mL of linear stock solution, place in a 10 mL brown volumetric flask, add mobile phase to make up to volume, and shake well; 150% linear solution: accurately measure 3 mL of linear stock solution, place in a 20 mL brown volumetric flask, add mobile phase to make up to volume, and shake well; 100% linear solution: accurately measure 2 mL of linear stock solution, place in a 20 mL brown volumetric flask, add mobile phase to make up to volume, and shake well; 50% linear solution: accurately measure 1 mL of linear stock solution, place in a 20 mL brown volumetric flask, add mobile phase to make up to volume, and shake well; 10% linear solution: Accurately measure 1 mL of 100% linear solution, place it in a 10 mL brown volumetric flask, add mobile phase to make up the volume, and shake well; inject the linear solution into the liquid chromatograph, record the chromatogram, use the sample mass concentration as the abscissa and the peak area as the ordinate, perform linear regression calculation, and obtain the linear equation and correlation coefficient. The specific results are shown in Tables 4 and 5 below.

[0049] Table 4 Linear equation and correlation coefficient of folic acid Table 5 Impurity 3 linear equation and correlation coefficient 3. Experimental results: The folic acid concentration is in the range of 0.023-4.677 μg / mL, R=0.9996, the impurity 3 is in the range of 0.242-4.842 μg / mL, R=0.9996, the absolute value of the intercept in the linear equation of folic acid and impurity 3 is less than 10% of the 100% concentration peak area, the correlation coefficient R is greater than 0.990, the linear relationship is good, and the linear range meets the daily detection requirements.

[0050] The correction factor of the linear equation slope of folic acid / the linear equation slope of impurity 3 is 1.94.

[0051] Example 5. Precision of the method 1. Chromatographic conditions are the same as in Example 2; 2. Precision testing Preparation of injection precision solution: Weigh an appropriate amount of impurity 3 reference substance, add an appropriate amount of 2.86% (g / mL) sodium carbonate solution to dissolve it, and add the mobile phase to obtain the injection precision solution (the concentration of impurity 3 in the injection precision solution is about 2.5 μg / mL).

[0052] The precision solution was measured 6 times continuously and the chromatogram was recorded. Figure 4 The peak area and retention time results of 6 measurements are shown in the following table: Table 6 Injection precision 3. Experimental results: The RSD of the peak area of ​​impurity 3 was 0.24%, and the RSD of the retention time was 0.02%, both of which were less than 2.0%, indicating that the determination method of the present invention has precision.

[0053] Example 6. Accuracy of the method 1. The detection conditions are the same as those in Example 2; 2. Preparation of accuracy solution: Accuracy stock solution: Accurately measure 5 mL of the impurity 3 stock solution of Example 4, place it in a 100 mL brown volumetric flask, add mobile phase to dilute to the scale, shake well, and use it as the accuracy stock solution (the concentration of impurity 3 in the accuracy stock solution is 24.210 μg / mL).

[0054] Reference solution: Accurately measure an appropriate amount of accuracy stock solution, add mobile phase to dilute 10 times, and use it as the reference solution (the concentration of impurity 3 in the reference solution is 2.421 μg / mL).

[0055] Background solution: Take 202.87 mg (equivalent to 10 mg folic acid) of the test sample fine powder (folic acid tablets batch number: Y200603), accurately weigh, place in a 20 mL brown volumetric flask, add 5 mL of 2.86% (g / mL) sodium carbonate solution, dissolve by ultrasonication, dilute to the scale with mobile phase, shake well, centrifuge, filter the supernatant, and take the filtrate as the background solution.

[0056] Accuracy solution: Take about 200 mg (equivalent to 10 mg folic acid) of the test sample fine powder (folic acid tablet batch number: Y200603), accurately weigh, place in three 20 mL brown volumetric bottles, add appropriate amount of 2.86% (g / mL) sodium carbonate solution, ultrasonically dissolve, accurately add 3 mL, 2 mL, and 0.4 mL of accuracy stock solution, dilute to scale with mobile phase, shake well, centrifuge for 10 minutes, filter the supernatant, take the filtrate as the test sample solution, and obtain 150% accuracy solution, 100% accuracy solution, and 20% accuracy solution respectively. Repeat the same operation twice, that is, prepare 3 copies of each concentration level in the same way.

[0057] Take the reference solution and inject it 5 times, inject the other solutions once, and record the chromatogram. Figure 5-Figure 9 The accuracy test results are shown in Table 7.

[0058] Table 7 Accuracy test results 3. Experimental conclusion: It can be seen from Table 7 that the recovery rates of the solutions at each level of accuracy are between 90% and 110%, and the RSD of the recovery results is less than 5.0%. This method has high accuracy, and the measured values ​​are close to the theoretical values.

[0059] The present application provides a method for detecting related substances in folic acid tablets, which is used to detect the content of impurity 3 in folic acid tablets. The method is highly specific, accurate, precise and sensitive, and can effectively control the content of impurity 3 in folic acid tablets.

Claims

1. A method for detecting related substances in folic acid tablets, characterized in that: The related substances in the folic acid tablets were detected by high performance liquid chromatography, wherein the related substances were impurities shown in Formula 1; 2. The method for detecting related substances in folic acid tablets according to claim 1, characterized in that: The chromatographic conditions of HPLC are as follows: Chromatographic column: a column filled with octylsilane bonded silica gel; The mobile phase is a mixture of component A and component B; Wherein, component A is a mixed solution of 1% (g / mL)-1.2% (g / mL) potassium dihydrogen phosphate and 0.4% (g / mL)-0.7% (g / mL) dipotassium hydrogen phosphate, with a pH value of 6.4±0.2; Component B is methanol; Elution mode: isocratic elution.

3. The method for detecting related substances in folic acid tablets according to claim 2, characterized in that: In the mobile phase, the volume ratio of component A to component B is (85-90):(15-10).

4. The method for detecting related substances in folic acid tablets according to claim 3, characterized in that: In the mobile phase, the volume ratio of component A to component B is 90:

10.

5. The method for detecting related substances in folic acid tablets according to claim 2, characterized in that: In the mobile phase, component A is a mixed solution of 1.116% (g / mL) potassium dihydrogen phosphate and 0.55% (g / mL) dipotassium hydrogen phosphate.

6. The method for detecting related substances in folic acid tablets according to claim 2, characterized in that: In the mobile phase, phosphoric acid or 5 mol / L potassium hydroxide is used to adjust the pH value of component A.

7. The method for detecting related substances in folic acid tablets according to claim 2, characterized in that: When high performance liquid chromatography is used for detection, the detection wavelength is 275-285 nm; the injection volume is 10-20 μl; the column temperature of the chromatographic column is 25-30° C.; and the flow rate of the mobile phase is 0.5-1 mL / min.

8. The method for detecting related substances in folic acid tablets according to claim 7, characterized in that: When high performance liquid chromatography was used for detection, the detection wavelength was 280 nm; the injection volume was 10 μl; the column temperature was 25° C.; and the mobile phase flow rate was 0.6 mL / min.

9. The method for detecting related substances in folic acid tablets according to claim 2, characterized in that: The chromatographic column is Merck 100RP-8; the length of the chromatographic column is 250 mm, the diameter is 4.0 mm, and the filler particle size is 5 μm.

10. A method for detecting related substances in folic acid tablets according to any one of claims 1 to 9, characterized in that: The detection method steps are as follows: (1) Preparation of system suitability solution: Take the impurity shown in Formula 1 and the folic acid reference substance, dissolve them in 2.86% (g / mL) sodium carbonate solution, and then add the mobile phase to dilute to a solution containing 0.5 mg of folic acid and 2.5 μg of the impurity shown in Formula 1 per 1 mL, which is used as the system suitability solution; (2) Preparation of the test solution: Take a sample powder containing 10 mg of folic acid, place it in a 20 mL volumetric flask, add 5 mL of 2.86% (g / mL) sodium carbonate solution to dissolve it, dilute it to the mark with the mobile phase, shake well, centrifuge, filter the supernatant, and take the filtrate as the test solution; (3) Prepare the control solution: accurately measure 1 mL of the test solution, place it in a 100 mL volumetric flask, and dilute to the mark with the mobile phase as the control solution; (4) The system suitability solution, the test solution and the control solution are injected into a high performance liquid chromatograph to detect the content of the impurity shown in Formula 1.

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