A method for detecting trace amounts of folic acid impurity a in multivitamin formulations

Abstract

The application discloses a method for detecting trace folic acid impurity A in various vitamin preparations and belongs to the field of pharmaceutical analysis chemistry. The method adopts high performance liquid chromatography, and a phosphate and tetrabutylammonium hydrogen sulfate solution, methanol and isopropyl alcohol are used to prepare mobile phase A and mobile phase B. Through gradient elution, the content of trace folic acid impurity A with potential genetic toxicity in various vitamin preparations can be effectively detected. The detection method provided by the application has high sensitivity, good separation degree and good repeatability, can realize accurate quantitative detection of trace folic acid impurity A in various vitamin preparations, and ensures the safety of various vitamin preparation products.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analytical chemistry, specifically relating to a detection method for determining trace folic acid impurity A [(2S)-2-[(4-aminobenzoyl)amino]glutaric acid] in various vitamin preparations. Background Technology

[0002] Vitamins are essential micronutrients for maintaining normal physiological functions in the human body, playing an irreplaceable role in key processes such as energy metabolism, immune function, and cell repair. Based on their solubility, they are divided into water-soluble vitamins and fat-soluble vitamins. Water-soluble vitamins are essential nutrients that the human body cannot synthesize on its own and must be obtained through food and supplements. Major water-soluble vitamins include: vitamin C, dextropanthenol, riboflavin sodium phosphate, vitamin B6, vitamin B1, coenzyme tetrahydrate, folic acid, and niacinamide. They mainly participate in energy metabolism, nervous system regulation, and immune enhancement. Because they are easily soluble in water, they cannot be stored for long periods and need to be replenished daily. Fat-soluble vitamins include: vitamin D3, vitamin A, and vitamin E. They are absorbed through fat dissolution and mainly participate in physiological processes such as bone health, antioxidation, and blood clotting. Like water-soluble vitamins, they must be obtained through diet or supplements; excessive intake may lead to accumulation and toxicity.

[0003] To ensure market nutritional needs, research on multivitamins is being conducted. Currently, multivitamin injections under production or research include multivitamins for injection (12), 12-compound vitamins for injection, water-soluble vitamins for injection, multivitamin injection solution (13), multivitamins for injection (13), pediatric multivitamin injection solution (13), pediatric multivitamins for injection (13), and water-soluble vitamins for injection. Taking multivitamins for injection (12) as an example, this product is the only lyophilized powder injection product in China and abroad that simultaneously contains water-soluble and fat-soluble multivitamins. Its biggest feature is that fat-soluble and water-soluble vitamins can be dissolved in the same container. The specification is 5mL / vial. The formula contains 12 main ingredients and 6-7 excipients. The content of each component is different and all are trace amounts. For example, the specification of folic acid is 414μg / 5mL, which is 82.8μg / mL.

[0004] Vitamins are unstable and easily degraded by light and heat, producing impurities that directly affect the safety of clinical use of drugs, especially when supplementing multivitamins through intravenous nutrition. Therefore, the control of impurities is a key aspect of product development and quality control.

[0005] Folic acid is extremely unstable and easily destroyed under acidic conditions. It is also easily oxidized by light and heat, and even at room temperature. Experiments have shown that folic acid degrades upon contact with metal ions, resulting in a variety of impurities that can pose certain health risks, especially for medications taken by pregnant women. Therefore, it is necessary to study the degradation impurities of each component and control their content within a safe and effective range. Folic acid and its preparations are included in the pharmacopoeias of many countries. The EP / BP pharmacopoeia sets the limit for impurity A in folic acid raw materials at 0.5%, while the BP pharmacopoeia sets the limit for impurity A in folic acid injection at 2.0%.

[0006] Folic acid impurity A is (2S)-2-[(4-aminobenzoyl)amino]glutaric acid; its molecular formula is C 12 H 14 N2O5, molecular weight 266.09; structural formula:

[0007]

[0008] The warning structures of genotoxic and carcinogenic impurities have been listed in the "Warning Structures of Genotoxic Impurities" released by the Center for Drug Evaluation of the State Food and Drug Administration at the New Drug R&D Forum ([1] Ma Lei, Ma Yunan, Chen Zhen, et al. Warning structures of genotoxic impurities [J]. Chinese Journal of New Drugs, 2014, 23(18):2106-2111.), among which, primary aromatic amines or groups that can generate aromatic amines; aryl-substituted hydroxylamines and their derivative esters are all warning structures. Folic acid impurity A contains an aromatic amine group and has a potential genotoxic warning structure. Therefore, a highly sensitive detection method is particularly important for the accuracy of controlling the content of folic acid impurity A. Most of the pharmacopoeias and literature at home and abroad are methods for controlling a single raw material drug, or methods for controlling individual impurities in three or four multivitamin preparations. There are few reports on effective methods for controlling impurities in 12 or more multivitamin preparations.

[0009] Injectable multivitamins have complex compositions, with low levels of each component and even lower levels of impurities, including lower limits for genotoxic impurities. Most detection methods still utilize LC-MS or GC-MS. However, due to the high cost of LC-MS or GC-MS instruments, most pharmaceutical companies' QC departments lack the necessary equipment. Therefore, developing conventional liquid chromatography methods for detecting genotoxic impurities is more practical. Existing conventional detection methods lack sufficient sensitivity, or, considering the polarity of folic acid impurity A, use buffer solutions that are nearly pure salts as the mobile phase, all of which face limitations in storage and use.

[0010] In the prior art, CN 115047083 A discloses a method for detecting folic acid and its eight impurities. This method uses octadecylsilane-bonded silica gel as the packing material, methanol-ammonium acetate solution (12:88) as the mobile phase, a column temperature of 25℃, and a detection wavelength of 280nm to analyze folic acid and its eight impurities. The results show that the peak elution time of folic acid impurity A is around 5.0 min, and the peak elution time of folic acid is around 10-15 min. However, the method does not disclose its limit of quantification and limit of detection. Therefore, this method is not suitable for the accurate quantitative detection of folic acid impurity A in multivitamin preparations.

[0011] CN11507852B discloses a method for separating and detecting 10 folic acid impurities using Waters ultra-high performance liquid chromatography (UHPLC). This method uses an octadecylsilane-bonded silica column as the packing material. The mobile phase consists of mobile phase A (ammonium acetate aqueous solution and methanol, volume ratio 90:10) and mobile phase B (ammonium acetate aqueous solution and methanol, volume ratio 70:30), performing gradient elution. The limit of detection (LOD) for folic acid impurity A is 0.09 μg / mL, and the limit of quantitation (LOQ) is 0.17 μg / mL. (See attached instruction manual for details.) Figure 1 It is known that the peak elution time of folic acid impurity A is 2~3 min, which is easily affected by other highly polar components and impurities in the formulation, resulting in poor method specificity.

[0012] Li Xuelian et al. disclosed a high-performance liquid chromatography (HPLC) method for determining the content of related substances in folic acid tablets. The method used a Besil C18-B column (4.6 mm × 250 mm, 3.5 μm) or a column with equivalent performance. The mobile phase A was phosphate buffer (10.0 g of potassium dihydrogen phosphate and 5.0 g of dipotassium hydrogen phosphate dissolved in water and diluted to 1000 mL, with the pH adjusted to 5.5 using phosphoric acid), and the mobile phase B was methanol, with gradient elution. The column temperature was 35℃; the flow rate was 0.8 mL / min; a UV detector (for all related substances except impurity K) was used, with a detection wavelength of 280 nm. Impurity K was determined using a fluorescence detector, with an excitation wavelength of 280 nm and an emission wavelength of 450 nm; the injection volume was 5 μL. As can be seen from the detection graph, the retention time of impurity A is 6.200 min, and the peak time has not been improved. Moreover, the mobile phase A is a high pH pure salt buffer solution, which is prone to bacterial growth, resulting in abnormal peaks and making it unsuitable for long-term storage and use (Li Xuelian, Xiao Xueju, Cui Delin, Han Yahui, Yi Bin. Determination of related substances in folic acid tablets by high performance liquid chromatography [J]. Central South Pharmacy, 2024, 22(9):2429-2432).

[0013] Pan Zhaohui et al. used the following chromatographic column: Kromasil C18 (250mm×4.6mm×5μm); mobile phase: 0.05M potassium dihydrogen phosphate solution (adjusted to pH 5.5±0.03 with 5M sodium hydroxide); flow rate: 1.5mL / min; column temperature: room temperature; detection wavelength: 269nm; injection volume: 20μL. The mobile phase was still a high pH pure salt buffer, which was not suitable for long-term storage and use. The peak of impurity A was not well retained (Pan Zhaohui, Zhang Lihong, Xiao Hongming. Determination of hydrolysis products of folic acid in ferrous folic acid tablets by high performance liquid chromatography [J]. Northern Pharmacy, 2014, 11(4):5-5).

[0014] CN 109239230 B discloses an analytical method for folic acid impurity A, folic acid impurity D, p-aminobenzoic acid, and 3-aminopropanol in a multivitamin preparation. The chromatographic conditions are as follows: a column switching method is used, with an octadecylsilane-bonded silica gel column (Merck, Lichrospher, 125 mm × 4 mm, 5 μm) as the pretreatment column and an octadecylsilane-bonded silica gel column (250 mm × 4 mm, 5 μm) as the analytical column; the mobile phase A is acetate solution (60 g of anhydrous sodium acetate is dissolved in water and diluted to 1000 mL, pH is adjusted to 6.0 with glacial acetic acid, 1.5 mL of tetrahydrofuran is added, mixed, and filtered through a 0.22 μm filter membrane), and the mobile phase B is methanol. Gradient elution is performed at a flow rate of 1.5 mL / min and a detection wavelength of 380 nm; the column temperature is 40 °C. This method employs column switching, requiring derivatization of the test and reference solutions, which is cumbersome. Furthermore, the concentration of sodium acetate buffer in the mobile phase is as high as 0.7 mL / L, combined with a high pH value, making it unsuitable for long-term storage and use. The retention time of folic acid impurity A in the chromatogram is only 13 min, which is close to adjacent peaks. Considering the impact on robustness, there is a risk of poor resolution. In addition, under these chromatographic conditions, the limit of quantification for folic acid impurity A is only 10 ng, i.e., the limit of quantification concentration is 1 ng / μL, resulting in low sensitivity.

[0015] CN113092618A discloses a detection method for the separation and control of six impurities in 13 compound vitamins for injection: vitamin A palmitate degradation impurity 1, vitamin A palmitate degradation impurity 2, vitamin A palmitate degradation impurity 3, vitamin E degradation impurity 4, vitamin E process impurity 5, and vitamin E process impurity 6. The chromatographic conditions are as follows: YMC Carotenoid C30 (150mm×4.6mm, 3μm) column; acetonitrile-ethanol-0.01% tetrahydrofuran water (60:10:30) as mobile phase A and methanol-acetonitrile-ethanol (6:3:1) as mobile phase B. Because the mobile phase contains a very high proportion of organic phase, it is suitable for the detection of fat-soluble components. However, folic acid impurity A is highly polar and cannot be retained on the chromatographic column, so it cannot be used for the quantitative detection of folic acid impurity A.

[0016] CN 118501298 B provides a method for detecting 2,3-diketo-L-gulonic acid in multivitamin preparations. This method employs high-performance liquid chromatography (HPLC) under the following chromatographic conditions: an octadecylsilane-bonded silica column; mobile phase A: phosphate buffer solution containing tetrabutylammonium bromide-acetonitrile; mobile phase B: acetonitrile-water; mobile phases A and B are run in a gradient program; column temperature: 40–50 °C; flow rate: 1.0 mL / min; detection wavelength: 200–250 nm; injection volume: 20 μL. This method is not suitable for detecting folic acid impurity A in multivitamin preparations, as it may exhibit peak broadening or tailing, leading to inaccurate results.

[0017] CN115561371A describes a method for detecting folic acid content in health foods and its application. The chromatographic conditions used are isocratic. However, multivitamin preparations have complex compositions, with nearly 20 components including raw materials and excipients. Washing away fat-soluble components requires a long time, necessitating gradient elution. Furthermore, according to the chromatogram in the literature "Determination of Related Substances in Folic Acid Tablets by High Performance Liquid Chromatography," impurity A has a retention time of 6.2 min, while folic acid has a retention time of 34.1 min. This indicates a significant difference in polarity between folic acid impurity A and folic acid. In the chromatographic conditions of CN115561371A, the folic acid peak retention time is 31.904 min, meaning impurity A should elute before 10 min. Because there are many polar components in the 12-dimensional vitamin C formulation, and the dosage of water-soluble components such as vitamin C, dextropanol, and nicotinamide is relatively large and highly polar, their retention on the chromatographic column is poor, making them easily eluted and interfering with the determination of impurity A. Therefore, this method is not applicable.

[0018] In summary, there is an urgent need for an analytical method with good separation, high sensitivity, low detection limit, and accurate recovery to detect trace amounts of folic acid impurity A in multivitamin preparations. Summary of the Invention

[0019] To address the aforementioned problems, the present invention aims to provide a method for detecting trace amounts of folic acid impurity A in multivitamin preparations, effectively achieving the determination and limit control of folic acid impurity A. The detection method is highly sensitive, specific, accurate, and easy to operate.

[0020] To achieve the above objectives, the technical solution of the present invention is as follows:

[0021] A method for detecting trace amounts of folic acid impurity A in multivitamin preparations, wherein the method uses high-performance liquid chromatography (HPLC) under the following chromatographic conditions:

[0022] Chromatographic column: Octadecylsilane-bonded silica gel column; preferably ChromaNik Sunniest C18 column, 250 mm × 4.6 mm, 5 μm;

[0023] Mobile phase A: phosphate buffer-methanol volume ratio of 89~91:11~9; pH 6.25~6.4;

[0024] Preferably, the mobile phase A is: 0.04 mol / L phosphate buffer (6.97 g of anhydrous dipotassium hydrogen phosphate, 0.5 g of tetrabutylammonium hydrogen sulfate, 1000 mL of water to dissolve, and pH adjusted to 6.3 with phosphoric acid) - methanol (90:10).

[0025] Mobile phase B: Methanol-isopropanol-water (30:50:20);

[0026] The above mobile phases A and B are eluted according to a gradient program;

[0027] Flow rate: 0.9~1.1 mL / min;

[0028] Column temperature: 30℃~37℃, 43℃~45℃; the preferred injection temperature is 35℃.

[0029] Detection wavelength: 273nm;

[0030] Injection volume: 20 μL;

[0031] The injection gradient procedure is as follows:

[0032] Time / minute Mobile phase A / % Mobile phase B / % 0~55 98~100 0~2 55~60 0~2 98~100 60~90 0~2 98~100 90~95 98~100 0~2 95~115 98~100 0~2

[0033] Solution preparation:

[0034] Preparation of test solution: Take an appropriate amount of the contents of multivitamins for injection (12) (approximately equivalent to 414 μg of folic acid), accurately weigh it, dissolve it in water and quantitatively dilute it to prepare a solution containing approximately 80 μg of folic acid per 1 mL;

[0035] Preparation of impurity reference standard stock solution: Take an appropriate amount of folic acid impurity A reference standard, accurately weigh it, add an appropriate amount of 28.6 mg / mL sodium carbonate solution to dissolve it, and dilute it quantitatively with water to prepare a solution containing about 5 μg per 1 mL;

[0036] Preparation of limit of quantitation solution: Accurately measure the impurity reference standard stock solution with a concentration of 5 μg / mL, and dilute it stepwise with water as solvent to a concentration with a signal-to-noise ratio (S / N) of 10 to obtain the limit of quantitation solution;

[0037] Preparation of detection limit solution: Accurately measure 5 μg / mL of impurity reference standard stock solution, and dilute stepwise with water to a signal-to-noise ratio (S / N) of 3 to obtain the detection limit solution;

[0038] Preparation of recovery rate solution: Take two vials of multivitamin (12) for injection (approximately equivalent to 828 μg of folic acid), weigh them accurately, place them in a 10 mL volumetric flask, and accurately add 0.5 mL, 1 mL, 2 mL, and 3 mL of reference stock solution respectively. Dissolve and dilute with water to the mark, shake well, and prepare 25%, 50%, 100%, and 150% accuracy solutions respectively. Prepare 3 parallel solutions for each concentration level.

[0039] This method can be used to detect the content of trace folic acid impurity A in various vitamin preparations, so as to study the stability, degradation and other properties of the injection.

[0040] The beneficial effects of this invention are:

[0041] (1) The limit of quantitation of the present invention is 0.0746 μg / mL, i.e. 1.49 ng; the limit of detection is 0.0224 μg / mL, i.e. 0.45 ng; indicating that the method has high detection sensitivity and meets the qualitative and quantitative detection requirements of folic acid impurity A in various vitamin preparations.

[0042] (2) By adding four different concentrations of folic acid impurity A standard to samples of known concentration, the recovery rate was calculated. The average recovery rate of 12 accuracy solutions was 99.70%, and the relative standard deviation was 0.77%, which met the analytical requirements.

[0043] (3) In the concentration range of 0.0746μg / mL to 7.4650μg / mL, the linear regression equation of this invention is A=73808C-662.42, and the correlation coefficient r is 1.0000, where C is in μg / mL and A is the peak area. This technical solution can detect the content of folic acid impurity A in a small range, providing a strict quality control method for various vitamin products and ensuring the safety of the products.

[0044] (4) The preparation of the test solution in this method is simple and does not require derivatization, which greatly improves the work efficiency.

[0045] (5) Isopropanol is added to the mobile phase B of the present invention, and methanol-isopropanol-water with a volume ratio of 30:50:20 is used as the mobile phase B, which greatly improves the rapid elution ability of other components and impurities.

[0046] (6) While improving detection sensitivity, this invention can also rapidly detect the ion pair (tetrabutylammonium bisulfate) peak (as shown in the attached diagram). Figure 3 As shown, the retention time is approximately 94 minutes. This elution process avoids inaccurate detection results that may occur with continuous injections, ensuring injection repeatability and extending the lifespan of the chromatographic column.

[0047] By employing an ultraviolet detector and adding tetrabutylammonium bisulfate ion pairs to the mobile phase, the solubility in the aqueous phase can be enhanced, the water / organic phase distribution ratio can be balanced, and the system is more stable under acidic conditions. This solves the problem of high polarity and poor separation of folic acid impurity A in multivitamin preparations, resulting in a highly sensitive, reproducible, and accurate quantitative detection method for folic acid impurity A in multivitamin preparations. Attached Figure Description

[0048] Figure 1 The chromatogram shows the limit of quantitation of the folic acid impurity A reference solution in Example 1.

[0049] Figure 2 The chromatogram shows the detection limit of the folic acid impurity A reference solution in Example 1.

[0050] Figure 3 This is the chromatogram of the blank solvent in Example 1.

[0051] Figure 4 The chromatogram shows the linear relationship of the folic acid impurity A reference solution in Example 2.

[0052] Figure 5 The chromatogram is of the injectable multivitamin (12) with a column temperature of 40°C in Example 6.

[0053] Figure 6 The chromatogram of the test solution of the injectable multivitamin (12) in Example 10, which was affected by high temperature (60 degrees Celsius) for 10 days.

[0054] Figure 7 The chromatogram of the test solution of injectable multivitamins (12) in Comparative Example 1 after 10 days of exposure to high temperature (60 degrees Celsius).

[0055] Figure 8 The chromatogram shows the ion pair peaks that were not eluted in four consecutive injections in Comparative Example 2.

[0056] Figure 9 The chromatogram shows the ion pair peaks completely eluted from four consecutive injections in Comparative Example 2. Detailed Implementation

[0057] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0058] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all reagents and consumables used in the following examples are commercially available products.

[0059] The sources of the test samples and control samples involved in the examples are as follows:

[0060] Injectable multivitamins (12) Source: Batch number YF09411111, North China Pharmaceutical Co., Ltd.; Batch number LE20C076, Baxter SA.

[0061] Folic acid impurity A reference standard source: batch number 8-RTU-33-1, TRC.

[0062] Example 1: Detection of Limit of Quantitation and Limit of Detection

[0063] (1) Chromatographic conditions:

[0064] Chromatographic column: ChromaNik Sunniest C18 column, 250mm × 4.6mm, 5μm.

[0065] Flow rate: 1.0 mL / min;

[0066] Wavelength: 273nm;

[0067] Column temperature: 35℃;

[0068] Injection volume: 20 μL;

[0069] Solvents: water, sodium carbonate solution at 28.6 mg / mL;

[0070] Mobile phase A: 0.04 mol / L phosphate buffer (dissolve 6.97 g of anhydrous dipotassium hydrogen phosphate and 0.5 g of tetrabutylammonium hydrogen sulfate in 1000 mL of water, and adjust the pH to 6.3 with phosphoric acid) - methanol (90:10);

[0071] Mobile phase B: Methanol-isopropanol-water (30:50:20);

[0072] Mobile phases A and B are processed according to a gradient program:

[0073] The gradient procedure is as follows:

[0074] Time / minute Mobile phase A / % Mobile phase B / % 0~55 100 0 55~60 0 100 60~90 0 100 90~95 100 0 95~115 100 0

[0075] (2) Solution preparation:

[0076] Reference stock solution: Accurately weigh 12.96 mg of folic acid impurity A reference standard (content 96%), place it in a 50 mL volumetric flask, add 1 mL of 28.6 mg / mL sodium carbonate solution to dissolve it, dilute with water to the mark, and shake well;

[0077] Limit of Quantitation Solution: Accurately measure 5 mL of the above reference stock solution and place it in a 50 mL volumetric flask. Dilute with water to the mark and shake well. Accurately measure 5 mL of the same solution and place it in a 50 mL volumetric flask. Dilute with water to the mark and shake well. Accurately measure 3 mL of the same solution and place it in a 100 mL volumetric flask. Dilute with water to the mark and shake well.

[0078] Detection limit solution: Accurately measure 3 mL of the quantitation limit solution, place it in a 10 mL volumetric flask, dilute with water to the mark, and shake well to obtain the solution.

[0079] (3) Experimental results:

[0080] Limit of quantitation: Accurately measure 20 μL of the limit of quantitation solution and inject it into the liquid chromatograph. With a signal-to-noise ratio of S / N = 10:1, the limit of quantitation concentration is 0.0746 μg / mL.

[0081] Limit of detection: Accurately measure 20 μL of the limit of detection solution and inject it into the liquid chromatograph. With a signal-to-noise ratio of S / N = 3:1, the limit of detection concentration is 0.0224 μg / mL.

[0082] Conclusion: The limit of quantitation (LOQ) of this method for detecting trace folic acid impurity A in injectable multivitamin (12) preparations is 0.0746 μg / mL (0.07 ng / μL), the limit of quantitation (LOQ) is 1.49 ng, and the limit of detection (LOD) is 0.0224 μg / mL, i.e. 0.45 ng. The sensitivity of this method is much higher than the limit of quantitation (1 ng / μL) in the patent technology of CN 109239230A.

[0083] Example 2: Determination of the range of linear relationship

[0084] (1) Chromatographic conditions: Same as in Example 1.

[0085] (2) Solution preparation:

[0086] Linearity stock solution: Accurately weigh 12.96 mg of folic acid impurity A reference standard (content 96%), place it in a 50 mL volumetric flask, add 1 mL of 28.6 mg / mL sodium carbonate solution to dissolve it, dilute with water to the mark, shake well, accurately measure 5 mL, place it in a 50 mL volumetric flask, dilute with water to the mark, shake well, and use it as the linearity stock solution.

[0087] Linearity solutions: Accurately measure 0.5 mL, 1 mL, 2 mL, 2.4 mL, and 3 mL of the linearity stock solution, place them in 10 mL volumetric flasks, dilute with water to the mark, and shake well. These are respectively used as linear solution 1, linear solution 2, linear solution 3, linear solution 4, and linear solution 5.

[0088] (3) Experimental results:

[0089] Accurately measure 20 μL each of linear solutions 1-5 from step (2) and inject them into the liquid chromatograph, including the limit of quantitation. Plot the concentration on the x-axis and the peak area on the y-axis for linear regression. The linear equation is A = 73808C - 662.42, where C is in μg / mL and A is the peak area. The correlation coefficient r = 1.0000, indicating that the folic acid impurity A shows good linearity within the concentration range of 0.0746 μg / mL to 7.4650 μg / mL.

[0090] Example 3: Determination of the recovery rate of folic acid impurity A

[0091] (1) Chromatographic conditions: Same as in Example 1.

[0092] (2) Solution preparation:

[0093] Reference solution: Take 12.96 mg of folic acid impurity A reference standard (content 96%), place it in a 50 mL volumetric flask, add 1 mL of 28.6 mg / mL sodium carbonate solution to dissolve it, dilute with water to the mark, shake well, accurately measure 2 mL, place it in a 100 mL volumetric flask, dilute with water to the mark, shake well, and the solution is ready.

[0094] Reference stock solution: Take 12.96 mg of folic acid impurity A reference standard (content 96%), place it in a 50 mL volumetric flask, add 1 mL of 28.6 mg / mL sodium carbonate solution to dissolve it, dilute with water to the mark, shake well, accurately measure 5 mL, place it in a 50 mL volumetric flask, dilute with water to the mark, shake well, and the solution is ready.

[0095] Test solution: Take two vials of multivitamins (12) for injection, weigh them accurately, place them in a 10mL volumetric flask, add water to dissolve and dilute to the mark, shake well, and the solution is ready.

[0096] Recovery solution: Take two vials of multivitamins (12) for injection, weigh them accurately, place them in a 10mL volumetric flask, and add 0.5mL, 1mL, 2mL and 3mL of the reference stock solution respectively. Dissolve and dilute with water to the mark, shake well, and prepare 25%, 50%, 100% and 150% accuracy solutions respectively. Prepare 3 parallel solutions for each concentration level.

[0097] (3) Experimental results:

[0098] Accurately measure 20 μL of each of the above solutions and inject them into the liquid chromatograph. Record the chromatograms. Calculate the recovery rate by the ratio of the difference between the measured amount and the original amount to the amount injected, and calculate the relative standard deviation. The results are shown in the table below:

[0099]

[0100] Experimental conclusions: The individual recovery rates of the 12 accuracy solutions ranged from 98.02% to 101.0%, with an average recovery rate of 99.70%. The RSD of the 12 recovery results was 0.77%, indicating good method accuracy.

[0101] Example 4: Determination of folic acid impurity A in a multivitamin (12) for injection

[0102] (1) Chromatographic conditions: Same as in Example 1.

[0103] (2) Solution preparation:

[0104] Reference solution: Take 12.96 mg of folic acid impurity A reference standard (content 96%), place it in a 50 mL volumetric flask, add 1 mL of 28.6 mg / mL sodium carbonate solution to dissolve it, dilute with water to the mark, shake well, accurately measure 2 mL, place it in a 100 mL volumetric flask, dilute with water to the mark, shake well, and use it as the reference solution.

[0105] Test solution: Accurately weigh an appropriate amount of the contents of multivitamins for injection (12) (approximately equivalent to 414 μg of folic acid), place it in a 5 mL volumetric flask, add water to dissolve and dilute to the mark, shake well, and use as the test solution. Prepare a total of 6 portions using the same method.

[0106] (3) Experimental results:

[0107] Accurately measure 20 μL of each of the above solutions and inject them into the liquid chromatograph, recording the chromatograms. Calculate the peak area using the external standard method. The experimental results are shown in the table below:

[0108] serial number 1 2 3 4 5 6 average value RSD (%) average deviation content(%) 1.67 1.69 1.67 1.60 1.68 1.66 1.66 1.92 0.03

[0109] Conclusion: The RSD of the folic acid impurity A content determination results in the 6 test sample solutions was 1.92%, the average deviation was 0.03%, and the method repeatability was good.

[0110] Furthermore, the blank solvent did not interfere with the determination of folic acid impurity A in the test solution; the peak purity and resolution of folic acid impurity A peak in the test solution met the requirements, indicating good method specificity.

[0111] Example 5: Determination of folic acid impurity A in injectable multivitamins (12) - Investigation of mobile phase A.

[0112] (1) Chromatographic conditions: Except for mobile phase A, which was used to investigate multiple proportions, the other chromatographic conditions were the same as in Example 1.

[0113] Mobile phase A: 0.04 mol / L phosphate buffer (dissolve 6.97 g of anhydrous dipotassium hydrogen phosphate and 0.5 g of tetrabutylammonium hydrogen sulfate in 1000 mL of water, and adjust the pH to 6.3 with phosphoric acid) - methanol (89:11, 91:9).

[0114] (2) Solution preparation:

[0115] Reference solution: Take 12.96 mg of folic acid impurity A reference standard (content 96%), place it in a 50 mL volumetric flask, add 1 mL of 28.6 mg / mL sodium carbonate solution to dissolve it, dilute with water to the mark, shake well, accurately measure 2 mL, place it in a 100 mL volumetric flask, dilute with water to the mark, shake well, and use it as the reference solution.

[0116] Test solution: Take an appropriate amount of the contents of multivitamins for injection (12) (approximately equivalent to 414 μg of folic acid), accurately weigh it, place it in a 5 mL volumetric flask, add water to dissolve and dilute to the mark, shake well, and use it as the test solution.

[0117] (3) Experimental results:

[0118] Accurately measure 20 μL of each of the above solutions, inject them into the liquid chromatograph, record the chromatograms, and analyze them under the conditions described above, as shown in the table below:

[0119]

[0120] Experiments show that under the above mobile phase ratios, the separation degree and theoretical plate number of folic acid impurity A from adjacent peaks are high, and the detection results are basically consistent.

[0121] Example 6: Determination of folic acid impurity A in a multivitamin (12) for injection - column temperature investigation

[0122] (1) Chromatographic conditions:

[0123] Except for setting multiple column temperatures, everything else is the same as in Example 1; the column temperatures are set to 30℃ and 40℃ respectively.

[0124] (2) Solution preparation: Same as in Example 5.

[0125] (3) Experimental results:

[0126] Accurately measure 20 μL of each of the above solutions, inject them into the liquid chromatograph, record the chromatograms, and analyze them under the conditions described above, as shown in the table below:

[0127]

[0128] Conclusion: At a column temperature of 40℃, folic acid impurity A was found to be encapsulated by adjacent peaks due to their greater sensitivity to temperature. Therefore, column temperatures of 37℃, 43℃, and 45℃ were further investigated. At 37℃ and 43℃, folic acid impurity A achieved baseline separation from adjacent peaks. Future methods should maintain column temperatures within the range of 30℃–37℃ or 43℃–45℃.

[0129] Example 7: Determination of folic acid impurity A in a multivitamin (12) for injection - mobile phase A - pH determination

[0130] (1) Chromatographic conditions: Except for mobile phase A, which was used to investigate multiple pH values, all other conditions were the same as in Example 1;

[0131] Mobile phase A: 0.04 mol / L phosphate buffer (dissolve 6.97 g of anhydrous dipotassium hydrogen phosphate and 0.5 g of tetrabutylammonium hydrogen sulfate in 1000 mL of water, and adjust the pH to 6.2-6.4 with phosphoric acid) - methanol (90:10).

[0132] (2) Solution preparation: Same as in Example 5.

[0133] (3) Experimental results:

[0134] Accurately measure 20 μL of each of the above solutions, inject them into the liquid chromatograph, record the chromatograms, and analyze them under the conditions described above, as shown in the table below:

[0135]

[0136] Conclusion: At pH 6.2, folic acid impurity A did not achieve baseline separation from adjacent peaks (resolution was 1.43). Therefore, an additional test at pH 6.25 was conducted. At pH 6.25, the resolution was 2.21. Therefore, subsequent methods must control the mobile phase pH within the range of 6.25–6.4.

[0137] Example 8: Determination of folic acid impurity A in a multivitamin (12) for injection - Investigation at different flow rates

[0138] (1) Chromatographic conditions: Except for setting the flow rate to 0.9~1.1 mL / min, all other conditions are the same as in Example 1.

[0139] (2) Solution preparation: Same as in Example 5.

[0140] (3) Experimental results:

[0141] Accurately measure 20 μL of each of the above solutions, inject them into the liquid chromatograph, record the chromatograms, and analyze them under the conditions described above, as shown in the table below:

[0142]

[0143] Example 9: Determination of folic acid impurity A in injectable multivitamins (12) – Investigation using different chromatographic columns

[0144] (1) Chromatographic conditions: Except for changing to different brands of instruments, different brands or batches of chromatographic columns and the following gradient settings, all other conditions are the same as in Example 1;

[0145] Time / minute Mobile phase A / % Mobile phase B / % 0 98 2 55 98 2 60 2 98 90 2 98 95 98 2 115 98 2

[0146] (2) Solution preparation: Same as in Example 5.

[0147] (3) Experimental results: 20 μL of each of the above solutions were accurately measured and injected into the liquid chromatograph. The chromatograms were recorded, and the analysis was performed under the above conditions. The results were combined with those of Examples 5 to 8 above, and the analysis is shown in the table below:

[0148]

[0149] Conclusion: Under all the above robustness conditions, the theoretical plate number of the folic acid impurity A peak is greater than 2000, and the resolution between the folic acid impurity A peak and adjacent peaks in the test solution meets the requirements. The RSD of the content determination results is 3.36%, and the average deviation is 0.05%. The method has good robustness and is suitable for the detection of folic acid impurity A in various vitamin preparations.

[0150] Example 10: Determination of folic acid impurity A in a trace component of multivitamin (12) for injection

[0151] (1) Chromatographic conditions: Same as in Example 1.

[0152] (2) Solution preparation: Same as in Example 5.

[0153] Test solution: Prepare two batches of test samples for the influence of multivitamins for injection (12) - high temperature 60 degrees Celsius influence factor 10 days solution.

[0154] (3) Experimental results:

[0155] Accurately measure 20 μL of each of the above solutions, inject them into the liquid chromatograph, record the chromatograms, and analyze them under the conditions described above. The results are shown in the table below:

[0156] name Retention time T (min) Folic acid impurity A (%) Multivitamins for Injection (12) - Batch No.: YF09411111 - 60 degrees Celsius for 10 days 44.558 2.15 Injectable Multivitamins (12) - Batch No.: LE20C076 - 60 degrees Celsius for 10 days 44.195 2.79

[0157] Conclusion: The chromatogram of the test solution of multivitamins for injection (12) at 60°C for 10 days shows that the retention time of peak A (folic acid impurity) is approximately 44 min, and the separation from adjacent peaks is good. See attached [document / details]. Figure 6 .

[0158] Comparative Example 1: Determination of Folic A Impurity A in Trace Components of Multivitamin for Injection (12)

[0159] (1) Chromatographic conditions: Except for mobile phase B, all other conditions are the same as in Example 1;

[0160] Mobile phase B: Acetonitrile-water (80:20).

[0161] (2) Solution preparation: Same as in Example 5;

[0162] Test solution: Prepared sample for the test of influencing factors of multivitamins for injection (12) (batch number: YF09411111) - high temperature 60 degrees Celsius influencing factor solution.

[0163] (3) Experimental results:

[0164] Accurately measure 20 μL of each of the above solutions, inject them into the liquid chromatograph, record the chromatograms, and analyze them under the conditions described above. The results are shown in the attached figure. Figure 7 As shown in the figure, the gradient peak elution time is between 64 and 94 minutes, while the attached peak... Figure 6 The gradient elution time is 60-80 min. When isopropanol is used as the mobile phase B component, the gradient elution of impurity peaks is significantly more than that of the acetonitrile-water system. This helps to effectively elute lipid-soluble components, avoids the residue of lipid-soluble components and less polar impurities in the chromatographic column, and ensures the accuracy of folic acid impurity A detection.

[0165] Comparative Example 2: Investigation of Ion Pair Peak Elution

[0166] (1) Chromatographic conditions: Same as in Example 1 except for the gradient program; the gradient elution program is as follows:

[0167] Time / minute Mobile phase A / % Mobile phase B / % 0 95 5 65 83 17 65~66 30 70 66~75 30 70 75~76 95 5 76~90 95 5 .

[0168] (2) Solution preparation: Same as the preparation of the reference solution in Example 5.

[0169] (3) Experimental results: Accurately measure 20 μL of each of the above solutions, inject them into the liquid chromatograph, record the chromatogram, and perform four consecutive injections. Analyze the results under the above conditions. The results are attached. Figure 8 As shown in the figure, the ion pair peaks of the reference solution were not eluted. After continuous injection, ion pair peaks appeared at 13 min starting from the second injection, which could easily be mistaken for folic acid impurity A peak, leading to incorrect peak labeling and inaccurate detection results. However, the results obtained by continuous injection four times according to the gradient program of Example 1 showed that... Figure 9 This means that the ion pair peaks in the reference solution (approximately 95 min) have been completely eluted, and no other chromatographic peaks were found in subsequent continuous injections except for the folic acid impurity A peak, ensuring the accuracy of the test results.

[0170] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention in detail and are not intended to limit it. Those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for detecting trace amounts of folic acid impurity A in a multivitamin preparation, characterized in that, The folic acid impurity A is (2S)-2-[(4-aminobenzoyl)amino]glutaric acid; The method is high-performance liquid chromatography, and the chromatographic conditions are as follows: Chromatographic column: Octadecylsilane-bonded silica gel column; Column temperature: 30℃~37℃ or 43℃~45℃; Flow rate: 0.9 mL / min~1.1 mL / min; Detection wavelength: 273nm; Injection volume: 20 μL; Mobile phase A: dipotassium hydrogen phosphate buffer solution containing tetrabutylammonium hydrogen sulfate - methanol, with a volume ratio of 89~91:11~9, and the pH is adjusted to 6.25~6.4 with phosphoric acid; Mobile phase B: methanol-isopropanol-water, volume ratio 30:50:20; Mobile phases A and B are eluted using a gradient program, and the elution program is as follows: Wherein, the percentages of mobile phases A and B are volume percentages; The multivitamin preparations include any one of the following: multivitamins for injection (12), 12-compound vitamins for injection, water-soluble vitamins for injection, multivitamin injection solution (13), multivitamins for injection (13), pediatric multivitamin injection solution (13), and pediatric multivitamins for injection (13).

2. The method according to claim 1, characterized in that, The chromatographic column can be either ChromaNik Sunniest C18 with a length of 250 mm, a diameter of 4.6 mm, and a packing particle size of 5 µm, or YMC-Triart C18 with a length of 250 mm, a diameter of 4.6 mm, and a packing particle size of 5 µm.

3. The method according to claim 1, characterized in that, The concentration of dipotassium hydrogen phosphate in the dipotassium hydrogen phosphate buffer solution of mobile phase A is 0.04 mol / L, and the concentration of tetrabutylammonium hydrogen sulfate in the dipotassium hydrogen phosphate buffer solution is 0.5 g / L; the pH of mobile phase A is 6.

3.

4. The method according to claim 1, characterized in that, The column temperature is 35°C.

5. The method according to claim 1, characterized in that, The elution procedure is as follows: The percentages of mobile phases A and B are volume percentages.

6. The method according to claim 1, characterized in that, The elution procedure is as follows: The percentages of mobile phases A and B are volume percentages.

7. The method according to claim 1, characterized in that, The detection range of folic acid impurity A is 0.0746 μg / mL to 7.4650 μg / mL; the limit of quantification of folic acid impurity A is 0.0746 μg / mL, and the limit of detection of folic acid impurity A is 0.0224 μg / mL.

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