Method for determining related substances of folic acid tablets by high performance liquid chromatography

Abstract

The invention discloses a method for determining related substances of folic acid tablets by high performance liquid chromatography, and belongs to the technical field of medical analysis. The method comprises the following steps: 1) preparing a system applicability solution, a test solution and a self-control solution; and 2) setting high performance liquid detection conditions: adopting a C18 chromatographic column, taking a monopotassium phosphate and dipotassium phosphate solution as a mobile phase A, taking methanol as a mobile phase B, and carrying out gradient elution. And (3) respectively sucking the system applicability solution, the test solution and the contrast solution, injecting into a high performance liquid chromatograph, recording and analyzing a chromatogram, drawing a standard curve, and calculating the content of the related substances in the folic acid tablets. The content of known impurities and unknown impurities in the folic acid tablets can be rapidly, effectively, accurately and reliably separated and detected, the product quality of the folic acid tablets is improved, and the medication safety of patients is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical analysis, and particularly relates to a method for determining related substances in folic acid tablets using a high performance liquid chromatography method. Background Art

[0002] Folic acid is a vitamin-based nutritional medicine that has the functions of promoting cell growth, preventing fetal malformations and helping hematopoiesis. In terms of treatment, folic acid is mainly used to improve folic acid deficiency caused by long-term picky eating and poor digestion and absorption. Such conditions may cause anemia symptoms such as fatigue and dizziness.

[0003] The method under the related substances of folic acid tablets in the 2020 edition of the Chinese Pharmacopoeia is as follows: the chromatographic column uses octadecylsilane bonded silica gel as the filler; phosphate buffer (pH 5.0) (take 2.0g of potassium dihydrogen phosphate, add about 650ml of water to dissolve, add 15ml of 0.5mol / L tetrabutylammonium hydroxide methanol solution, 7ml of 1mol / L phosphoric acid solution and 270ml of methanol, let cool, adjust the pH value to 5.0 with 1mol / L phosphoric acid solution or ammonia test solution, and dilute to 1000ml with water) as the mobile phase; the detection wavelength is 280nm; the flow rate is 1.2ml per minute; the injection volume is 10μL, and the separation between each known impurity peak and the main component peak is low, and the detection accuracy is low. Therefore, it is necessary to improve the test method for related substances in folic acid tablets and establish a rapid and effective detection method, which will help improve the product quality of folic acid tablets and improve patient medication safety. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for determining related substances in folic acid tablets by high performance liquid chromatography, which can quickly, effectively, accurately and reliably detect related substances in folic acid tablets.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0006] A method for determining related substances in folic acid tablets by high performance liquid chromatography comprises the following steps:

[0007] 1) Prepare system suitability solution, test solution and self-control solution;

[0008] 2) Setting HPLC detection conditions: using a C18 column, potassium dihydrogen phosphate and dipotassium hydrogen phosphate solution as mobile phase A, methanol as mobile phase B, and performing gradient elution;

[0009] 3) Aspirate the system suitability solution, test solution and self-control solution separately, inject them into the high performance liquid chromatograph, record and analyze the chromatogram, draw the standard curve, and calculate the content of related substances in the folic acid tablets.

[0010] Furthermore, in step 1), the system suitability solution is prepared by accurately weighing an appropriate amount of folic acid test sample, adding an appropriate amount of impurity C, impurity D, impurity E, impurity G, impurity H, YS-J, YS-K, and YS-L stock solution, and dissolving and diluting with a solvent to make a solution containing about 1 mg of folic acid, about 5 μg of impurity C, about 4 μg of impurity D, about 3 μg of impurity E, about 3 μg of impurity G, about 2 μg of impurity H, and about 10 μg of YS-K per 1 ml. , YS-J is about 5μg, and YS-L is about 10μg; Preparation method of test solution: grind the test sample into fine powder, accurately weigh about 200mg of fine powder into a 10ml volumetric flask, dilute with solvent to the scale, shake for 30min, centrifuge at 5000rpm for 5min, filter the supernatant, and take the filtrate; Preparation method of self-control solution: transfer 1ml of test solution to a 100ml volumetric flask, dilute with solvent to the scale.

[0011] Furthermore, in step 2), the preparation method of mobile phase A is as follows: 11.16 g of potassium dihydrogen phosphate and 5.5 g of dipotassium hydrogen phosphate are weighed and dissolved in 1000 ml of water.

[0012] Furthermore, in step 2), the volume ratio of mobile phase A to mobile phase B is 90-93:10-7.

[0013] Furthermore, in step 2), the chromatographic column is Welch Ultimate Alk C18, with a specification of 4.6×250 mm and a filler particle size of 5 μm.

[0014] Furthermore, in step 2), the flow rate is 0.58-0.65 ml / min.

[0015] Furthermore, in step 2), the column temperature is 33-37°C.

[0016] Furthermore, in step 2), the injection volume is 5 μL.

[0017] Furthermore, in step 2), the detection wavelength is 280 nm.

[0018] Furthermore, in step 2), the gradient elution program is: 0-7 min, the volume content of mobile phase A is 92%, and the volume content of mobile phase B is 8%; 7-32 min, the volume content of mobile phase A is 92-80%, and the volume content of mobile phase B is 8-20%; 32-58 min, the volume content of mobile phase A is 80-74%, and the volume content of mobile phase B is 20-26%; 59-70 min, the volume content of mobile phase A is 74%, and the volume content of mobile phase B is 26%.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] (1) The present invention adopts a method for determining related substances in folic acid tablets by high performance liquid chromatography, which can effectively separate the main component from the impurities, and other impurities, blank solvents and blank excipients do not interfere with the determination of the impurities and the main component.

[0021] (2) The present invention has good durability and has no significant change in the amount of impurities detected when slight changes occur to liquid chromatography parameters such as column temperature, flow rate, and initial ratio of mobile phase and when the same type of chromatographic column is used.

[0022] (3) The present invention can rapidly, effectively, accurately and reliably detect folic acid tablet-related substances to meet the needs of drug quality control. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 A test chromatogram of the system suitability under the chromatographic conditions of this application;

[0024] Figure 2 This is the linearity and range diagram of impurity C tested under the liquid chromatography conditions of this application;

[0025] Figure 3 This is the linearity and range diagram of impurity D tested under the liquid chromatography conditions of this application;

[0026] Figure 4 The linearity and range diagram of impurity E tested under the liquid chromatography conditions of this application;

[0027] Figure 5 This is the linearity and range diagram of impurity G tested under the liquid chromatography conditions of this application;

[0028] Figure 6 This is the linearity and range diagram of impurity H tested under the liquid chromatography conditions of this application;

[0029] Figure 7 The linearity and range diagram of YS-J tested under the liquid chromatography conditions of this application;

[0030] Figure 8 The linearity and range diagram of YS-K tested under the liquid chromatography conditions of this application;

[0031] Figure 9 The linearity and range diagram of YS-L tested under the liquid chromatography conditions of this application;

[0032] Figure 10 The figure shows the linearity and range of folic acid tested under the liquid chromatography conditions of the present invention. DETAILED DESCRIPTION

[0033] The present invention will be further illustrated below with reference to specific examples. The examples are implemented based on the technical solutions of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0034] Example 1

[0035] A method for determining related substances in folic acid tablets by high performance liquid chromatography comprises the following steps:

[0036] (1) Instrument selection: HPLC: Agilent 1260

[0037] (2) Setting liquid chromatography detection conditions

[0038] Chromatographic column: Welch Ultimate Alk C18, size 4.6 × 250 mm, particle size 5 μm;

[0039] Flow rate: 0.6 ml / min;

[0040] Column temperature: 35°C;

[0041] Injection volume: 5 μL;

[0042] Sample plate temperature: 4°C;

[0043] Detection wavelength: 280nm;

[0044] Mobile phase: Prepare mobile phase A by weighing 11.16 g of potassium dihydrogen phosphate and 5.5 g of dipotassium hydrogen phosphate and dissolving them in 1000 ml of water. Mobile phase B is methanol. Prepare the solvent by diluting 20 ml of ammonia water to 1 L with purified water.

[0045] The gradient elution program is shown in Table 1:

[0046] Table 1 Gradient elution program

[0047] Time (min) Mobile phase A (%) Mobile phase B (%) 0 92 8 7 92 8 32 80 20 58 74 26 70 74 26

[0048] (3) Preparation of sample solution

[0049] System suitability solution: Accurately weigh an appropriate amount of folic acid test sample, add an appropriate amount of impurity C, impurity D, impurity E, impurity G, impurity H, YS-J, YS-K, and YS-L stock solutions, and dissolve and dilute with solvent to make a mixed solution containing approximately 1 mg of folic acid, approximately 5 μg of impurity C, approximately 4 μg of impurity D, approximately 3 μg of impurity E, approximately 3 μg of impurity G, approximately 2 μg of impurity H, approximately 10 μg of YS-K, approximately 5 μg of YS-J, and approximately 10 μg of YS-L per 1 ml;

[0050] Test solution: Grind the test sample into fine powder, accurately weigh about 200 mg of the fine powder into a 10 ml volumetric flask, dilute to the mark with solvent, shake for 30 minutes, centrifuge at 5000 rpm for 5 minutes, filter the supernatant, and take the filtrate;

[0051] Self-control solution: Pipette 1 ml of the test solution into a 100 ml volumetric flask and dilute to the mark with solvent.

[0052] (4) Sample testing method

[0053] Accurately measure 5 μL of the system suitability solution, test solution, and self-control solution, inject them into the liquid chromatograph, and record the chromatogram.

[0054] Depend on Figure 1 As can be seen from the system suitability chromatogram, the main component and impurities can be effectively separated, and other impurities, blank solvents and blank excipients do not interfere with the determination of impurities and the main component.

[0055] Example 2

[0056] The detection method of Example 1 was validated from several aspects, including system applicability, limit of quantification, limit of detection, solution stability, filter membrane adsorption, linear relationship, precision, repeatability, accuracy, and durability.

[0057] 1. System applicability

[0058] Blank solution: dilute 20 ml of ammonia water to 1 L with purified water.

[0059] Blank excipient: weigh about 190 mg of blank excipient into a 10 ml volumetric flask, add solvent to the mark, shake well, shake for 30 min, centrifuge at 5000 rpm for 5 min, filter the supernatant, and take the filtrate.

[0060] Impurity C localization solution: Take an appropriate amount of Impurity C reference substance, dissolve it in solvent and dilute it to prepare a stock solution containing approximately 0.2 mg of Impurity C per 1 ml. Take 0.5 ml of Impurity C stock solution and place it in a 20 ml volumetric flask, dissolve it in solvent and dilute it to prepare a stock solution containing approximately 5 μg of Impurity C per 1 ml.

[0061] Impurity D localization solution: Take an appropriate amount of Impurity D reference substance, dissolve it in solvent and dilute it to prepare a stock solution containing approximately 0.16 mg of Impurity D per 1 ml. Take 0.5 ml of Impurity D stock solution and place it in a 20 ml volumetric flask, dissolve it in solvent and dilute it to prepare a 1 ml stock solution containing approximately 4 μg of Impurity D.

[0062] Impurity E localization solution: Take an appropriate amount of Impurity E reference substance, dissolve it in solvent and dilute it to prepare a stock solution containing approximately 0.3 mg of Impurity E per 1 ml. Take 0.5 ml of Impurity E stock solution and place it in a 50 ml volumetric flask, dissolve it in solvent and dilute it to prepare a stock solution containing approximately 3 μg of Impurity E per 1 ml.

[0063] Impurity H localization solution: Take an appropriate amount of impurity H reference substance, dissolve it in solvent and dilute it to prepare a stock solution containing approximately 0.15 mg of impurity H per 1 ml. Take 0.5 ml of impurity H stock solution and place it in a 25 ml volumetric flask, dissolve it in solvent and dilute it to prepare a 1 ml impurity H localization solution containing approximately 3 μg.

[0064] Impurity G localization solution: Take an appropriate amount of Impurity G reference substance, dissolve it with solvent and dilute it to prepare a stock solution containing approximately 0.3 mg of Impurity G per 1 ml. Take 0.5 ml of Impurity G stock solution and place it in a 50 ml volumetric flask, dissolve it with solvent and dilute it to prepare a stock solution containing approximately 3 μg of Impurity G per 1 ml.

[0065] YS-J localization solution: Take an appropriate amount of YS-J reference substance, dissolve it in solvent and dilute it to make a YS-J stock solution containing approximately 0.2 mg per 1 ml. Take 0.5 ml of YS-J stock solution and place it in a 20 ml volumetric flask, dissolve it in solvent and dilute it to make a YS-J localization solution containing approximately 5 μg per 1 ml.

[0066] YS-K localization solution: Take an appropriate amount of YS-K reference substance, dissolve it in solvent and dilute it to make a YS-K stock solution containing approximately 0.2 mg per 1 ml. Take 0.5 ml of YS-K stock solution and place it in a 10 ml volumetric flask, dissolve it in solvent and dilute it to make a YS-K localization solution containing approximately 10 μg per 1 ml.

[0067] YS-L positioning solution: Take an appropriate amount of YS-L reference substance, dissolve it in solvent and dilute it to make a YS-L stock solution containing approximately 0.2 mg per 1 ml. Take 0.5 ml of YS-L stock solution and place it in a 10 ml volumetric flask, dissolve it in solvent and dilute it to make a YS-L positioning solution containing approximately 10 μg per 1 ml.

[0068] System practical solution: Weigh an appropriate amount of folic acid, add appropriate amount of impurity C, impurity D, impurity E, impurity G, impurity H, YS-J, YS-K, and YS-L stock solutions, and dissolve and dilute with solvent to make a mixed solution containing approximately 1 mg of folic acid, approximately 5 μg of impurity C, approximately 4 μg of impurity D, approximately 3 μg of impurity E, approximately 3 μg of impurity G, approximately 2 μg of impurity H, approximately 10 μg of YS-K, approximately 5 μg of YS-J, and approximately 10 μg of YS-L per 1 ml.

[0069] 5 μL of blank solution, system suitability solution and impurity location solution were injected into the high performance liquid chromatograph at a detection wavelength of 280 nm. The chromatograms were recorded to examine the separation of the components. The results are shown in Table 2.

[0070] Table 2 System suitability results

[0071]

[0072] From the results in Table 2, it can be seen that the main component and the impurities can be effectively separated under the chromatographic conditions, and other impurities, blank solvents and blank excipients do not interfere with the determination of the impurities and the main component.

[0073] 2. Limit of quantification and limit of detection

[0074] Appropriate amounts of folic acid reference substance and impurity C, impurity D, impurity E, impurity G, impurity H, YS-J, YS-K, and YS-L reference substances were prepared into a mixed solution containing approximately 10 μg of folic acid, approximately 3 μg of impurity C, 4 μg of impurity D, approximately 3 μg of impurity E, approximately 3 μg of impurity G, approximately 1.5 μg of impurity H, approximately 5 μg of YS-J, approximately 2 μg of YS-K, and approximately 4 μg of YS-L per 1 ml. The limit of quantification (S / N ≥ 10) and the limit of detection (S / N ≥ 3) were determined by the serial dilution method. The results are shown in Table 3.

[0075] Table 3 Results of quantification limit and detection limit tests

[0076]

[0077] From the results in Table 3, it can be seen that low concentrations of various known impurities can be effectively detected under these chromatographic conditions.

[0078] 3. Injection precision

[0079] Take appropriate amounts of folic acid reference substance and impurity C, impurity D, impurity E, impurity G, impurity H, YS-J, YS-K, and YS-L reference substances to prepare a mixed solution containing approximately 10 μg of folic acid, approximately 3 μg of impurity C, 4 μg of impurity D, approximately 3 μg of impurity E, approximately 3 μg of impurity G, approximately 1.5 μg of impurity H, approximately 5 μg of YS-J, approximately 2 μg of YS-K, and approximately 5 μg of YS-L per 1 ml. This solution serves as the injection precision solution. A 5 μL volume was injected into the liquid chromatograph and injected six times continuously. The peak areas were recorded. The results are shown in Table 4.

[0080] Table 4 Injection precision results

[0081]

[0082] From the results in Table 4, it can be seen that the chromatographic conditions have good injection precision.

[0083] 4. Solution stability

[0084] Take appropriate amounts of folic acid reference substance and impurity C, impurity D, impurity E, impurity G, impurity H, YS-J, YS-K, and YS-L reference substances to prepare a mixed solution containing approximately 10 μg of folic acid, approximately 3 μg of impurity C, 4 μg of impurity D, approximately 3 μg of impurity E, approximately 3 μg of impurity G, approximately 1.5 μg of impurity H, approximately 5 μg of YS-J, approximately 2 μg of YS-K, and approximately 5 μg of YS-L per 1 ml. As the reference solution, the samples were injected at different times and the peak areas were recorded. The results are shown in Table 5.

[0085] Table 5 Stability results of reference solution

[0086]

[0087]

[0088] From the results in Table 5, it can be seen that the reference solution is stable within 52 hours (RSD≤2%).

[0089] Grind the test sample to a fine powder. Place approximately 200 mg of the powder in a 10 mL volumetric flask. Dissolve and dilute to volume with solvent. Shake for 30 minutes and centrifuge at 5000 rpm for 5 minutes. Filter the supernatant and prepare a solution containing approximately 1 mg of folic acid per mL of the filtrate. This solution is used as the test solution and injected at different times. Calculate the impurity content using the normalization method. The results are shown in Table 6.

[0090] Table 6 Stability results of test solution

[0091]

[0092] From the results in Table 6, it can be seen that the test solution is stable within 52 hours (the maximum deviation from 0H is less than 0.050%).

[0093] 5. Filter membrane adsorption

[0094] According to the proposed method for the determination of related substances, the product powder was weighed, dissolved in solvent, and diluted to a constant volume to produce a solution containing approximately 1 mg of folic acid per 1 ml. The solution was shaken for 30 minutes and centrifuged at 5000 rpm for 5 minutes. The supernatant was collected and centrifuged again at 5000 rpm for 5 minutes. The supernatant was used as the centrifuged sample. The supernatant from this centrifugation was filtered, and different volumes of the filtrate were discarded. The filtrate was then collected as a filtration sample and compared with the centrifuged sample. The membrane adsorption of the homemade sample was investigated, and the results are shown in Table 7.

[0095] Table 7 Filter membrane adsorption test results

[0096]

[0097] From the results in Table 7, it can be seen that the membrane adsorption meets the requirements (adsorption rate ≤ 2.0%).

[0098] 6. Linearity and Range

[0099] Take appropriate amounts of folic acid reference substance and impurity C, impurity D, impurity E, impurity G, impurity H, YS-J, YS-K, and YS-L reference substances to prepare a mixed solution containing approximately 12 μg of impurity C, approximately 16 μg of impurity D, approximately 12 μg of impurity E, approximately 12 μg of impurity G, approximately 6 μg of impurity H, approximately 20 μg of YS-J, approximately 20 μg of YS-K, approximately 20 μg of YS-L, and approximately 40 μg of folic acid per 1 ml. This solution serves as the linearization stock solution. Dilute these solutions with solvents as shown in the table below to prepare linearization solutions of various concentrations. Prepare the linearization solutions according to Table 8.

[0100] Table 8 Linear solution preparation

[0101]

[0102] Accurately measure 5 μL of each of the above solutions and inject them into the high performance liquid chromatograph, record the chromatogram, determine the peak area, and perform linear regression with the peak area A as the ordinate and the concentration C as the abscissa. The results are shown in Table 9-17. Figure 2-10 .

[0103] Table 9 Impurity C linear determination results

[0104]

[0105] Table 10 Impurity D linearity determination results

[0106]

[0107] Table 11 Impurity E linearity determination results

[0108]

[0109] Table 12 Impurity G linearity determination results

[0110]

[0111]

[0112] Table 13 Impurity H linear determination results

[0113]

[0114] Table 14YS-J linear determination results

[0115]

[0116] Table 15YS-K linear determination results

[0117]

[0118] Table 16YS-L linear determination results

[0119]

[0120]

[0121] Table 17 Folic acid linear determination results

[0122]

[0123] From Table 9-17, Figure 2-10 The results show that under the present chromatographic conditions, the linear results of each known impurity and folic acid are good.

[0124] 6. Repeatability

[0125] Repeatability stock solution: Take appropriate amounts of impurity C, impurity D, impurity E, impurity G, impurity H, YS-J, YS-K, and YS-L reference substances to prepare a mixed solution containing approximately 25 μg of impurity C, 20 μg of impurity D, approximately 15 μg of impurity E, approximately 15 μg of impurity G, approximately 10 μg of impurity H, approximately 25 μg of YS-J, approximately 40 μg of YS-K, and approximately 50 μg of YS-L per 1 ml as the repeatability stock solution.

[0126] Test solution: Grind the test sample to fine powder, take about 200 mg of the fine powder and place it in a 10 ml volumetric flask, transfer 2 ml of the reproducible stock solution, add solvent to dissolve and dilute to volume, shake well, shake for 30 min, centrifuge at 5000 rpm for 5 min, filter the supernatant, and take the filtrate to prepare a mixed solution containing about 1 mg of folic acid, about 5 μg of impurity C, about 4 μg of impurity D, about 3 μg of impurity E, about 3 μg of impurity G, about 2 μg of impurity H, about 8 μg of YS-K, about 10 μg of YS-L, and about 5 μg of YS-J per 1 ml. This solution is used as the test solution and prepared in 6 parallel portions.

[0127] Self-control solution: Take 1 ml of the test solution and place it in a 100 ml volumetric flask, then add solvent to dilute to the mark.

[0128] According to the proposed method for the determination of related substances, 5 μL of each of the above-mentioned repeatability stock solution, test solution, and self-control solution was injected into the high performance liquid chromatograph, the chromatogram was recorded, and the determination was repeated 6 times according to the relevant substance inspection method. The results are shown in Table 18.

[0129] Table 18 Repeatability test results

[0130]

[0131] As shown in Table 18, there was no significant change in the impurities in the test results of the six repeatable samples, indicating good repeatability.

[0132] 7. Accuracy

[0133] Recovery rate stock solution: Take appropriate amounts of impurity C, impurity D, impurity E, impurity G, impurity H, YS-J, YS-K, and YS-L reference substances to prepare a mixed solution containing approximately 25 μg of impurity C, 20 μg of impurity D, approximately 15 μg of impurity E, approximately 15 μg of impurity G, approximately 10 μg of impurity H, approximately 25 μg of YS-J, approximately 40 μg of YS-K, and approximately 50 μg of YS-L per 1 ml as the recovery rate stock solution.

[0134] Accurately pipette the above stock solution and dilute it to 0%, 25%, 50%, 100%, and 200% stock solutions according to Table 19 below. Prepare three replicates for each concentration. Prepare one replicate of the 0% solution at the limiting concentration.

[0135] Self-control solution: Take 1 ml of the test solution and place it in a 100 ml volumetric flask, then add solvent to dilute to the mark.

[0136] Table 19 Recovery rate stock solution preparation

[0137]

[0138]

[0139] 5 μL of each of the above samples was injected, the peak area of ​​each known impurity was recorded, and the recovery rate and RSD of each known impurity were calculated. The results are shown in Tables 20-27.

[0140] Table 20 Impurity C recovery test results

[0141]

[0142] Table 21 Impurity D recovery test results

[0143]

[0144]

[0145] Table 22 Impurity E recovery test results

[0146]

[0147] Table 23 Impurity G recovery test results

[0148]

[0149] Table 24 Impurity H recovery test results

[0150]

[0151] Table 25YS-J recovery test results

[0152]

[0153] Table 26YS-K recovery test results

[0154]

[0155] Table 27YS-L recovery test results

[0156]

[0157] From the results in Tables 20-27, it can be seen that the recovery rate of each impurity and the average recovery rate are all in the range of 92% to 105%, with RSD ≤ 10%, and the recovery rate is good.

[0158] 8. Intermediate precision

[0159] Different operators, at different times and using different instruments, conducted inspections of relevant substances on the same batch of samples according to the repeatability test method. The results are shown in Table 28.

[0160] Table 28 Intermediate precision determination results

[0161]

[0162] As shown in Table 28, different operators tested the samples at different times and with different instruments. There was no significant change in the impurity content of the 12 samples (RSD≤5.0%), and the intermediate precision was good.

[0163] 9. Durability

[0164] Following the reproducible sample preparation method, validation was performed using different flow rates, column temperatures, ratios, and columns. The chromatographic conditions are shown in Table 29, and the results are shown in Table 30.

[0165] Table 29 Chromatographic conditions

[0166]

[0167] Table 30 Durability test results

[0168]

[0169]

[0170] The results in Table 30 show that the main component and impurities can be effectively separated even with slight changes in column temperature, flow rate, and initial mobile phase ratio, and when using the same model but different batches of chromatographic columns. Furthermore, other impurities, blank solvents, and blank excipients do not interfere with the determination of the impurities and main component, and the test results do not change significantly. This method has good robustness.

[0171] Compared to the methods for folic acid tablets listed in the Chinese Pharmacopoeia, the United States Pharmacopoeia, and the Japanese Pharmacopoeia, this method differs in its control of related substances. The USP and the Japanese Pharmacopoeia do not control related substance inspection items, while the Chinese Pharmacopoeia does, but there are certain differences in the types and limits of controlled impurities. The Chinese Pharmacopoeia controls one known impurity, D, while this method simultaneously separates and detects eight known impurities: impurity C, impurity D, impurity E, impurity G, impurity H, YS-J, YS-K, and YS-L. This method controls a large number of impurities, is comprehensively researched, and has sufficient methodology validation, making it a reliable inspection method for controlling related substances in folic acid tablets.

[0172] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for determining related substances in folic acid tablets by high performance liquid chromatography, characterized in that: The following steps are involved: 1) Prepare system suitability solution, test solution and self-control solution; 2) Setting HPLC detection conditions: Using a C18 column, potassium dihydrogen phosphate and dipotassium hydrogen phosphate solutions as mobile phase A, methanol as mobile phase B, and gradient elution; 3) Aspirate the system suitability solution, test solution and self-control solution separately, inject them into the high performance liquid chromatograph, record and analyze the chromatogram, draw the standard curve, and calculate the content of related substances in the folic acid tablets.

2. The method for determining related substances in folic acid tablets by high performance liquid chromatography according to claim 1, characterized in that: In step 1), the system suitability solution is prepared by accurately weighing an appropriate amount of folic acid test sample, adding an appropriate amount of impurity C, impurity D, impurity E, impurity G, impurity H, YS-J, YS-K, and YS-L stock solution, and dissolving and diluting with a solvent to prepare a mixed solution containing approximately 1 mg of folic acid, approximately 5 μg of impurity C, approximately 4 μg of impurity D, approximately 3 μg of impurity E, approximately 3 μg of impurity G, approximately 2 μg of impurity H, approximately 10 μg of YS-K, approximately 5 μg of YS-J, and approximately 10 μg of YS-L per 1 ml; the test solution is prepared by grinding the test sample to a fine powder, accurately weighing approximately 200 mg of the fine powder into a 10 ml volumetric flask, diluting the volume with solvent to the mark, shaking for 30 min, centrifuging at 5000 rpm for 5 min, filtering the supernatant, and taking the filtrate; the self-control solution is prepared by transferring 1 ml of the test solution to a 100 ml volumetric flask and diluting the volume with solvent to the mark.

3. The method for determining related substances in folic acid tablets by high performance liquid chromatography according to claim 1, characterized in that: In step 2), the preparation method of mobile phase A is as follows: 11.16 g of potassium dihydrogen phosphate and 5.5 g of dipotassium hydrogen phosphate are weighed and dissolved in 1000 ml of water.

4. The method for determining related substances in folic acid tablets by high performance liquid chromatography according to claim 1, characterized in that: In step 2), the volume ratio of mobile phase A to mobile phase B is 90-93:10-7.

5. The method for determining related substances in folic acid tablets by high performance liquid chromatography according to claim 1, characterized in that: In step 2), the chromatographic column is Welch Ultimate Alk C18, with a size of 4.6×250 mm and a filler particle size of 5 μm.

6. The method for determining related substances in folic acid tablets by high performance liquid chromatography according to claim 1, characterized in that: In step 2), the flow rate is 0.58-0.65 ml / min.

7. The method for determining related substances in folic acid tablets by high performance liquid chromatography according to claim 1, characterized in that: In step 2), the column temperature is 33-37°C.

8. The method for determining related substances in folic acid tablets by high performance liquid chromatography according to claim 1, characterized in that: In step 2), the injection volume was 5 μL.

9. The method for determining related substances in folic acid tablets by high performance liquid chromatography according to claim 1, characterized in that: In step 2), the detection wavelength is 280 nm.

10. The method for determining related substances in folic acid tablets by high performance liquid chromatography according to claim 1, characterized in that: In step 2), the gradient elution program is as follows: from 0 to 7 min, the volume content of mobile phase A is 92%, and the volume content of mobile phase B is 8%; from 7 to 32 min, the volume content of mobile phase A is 92 to 80%, and the volume content of mobile phase B is 8 to 20%; from 32 to 58 min, the volume content of mobile phase A is 80 to 74%, and the volume content of mobile phase B is 20 to 26%; from 59 to 70 min, the volume content of mobile phase A is 74%, and the volume content of mobile phase B is 26%.

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