Method for detecting trace folic acid impurity A in multivitamin preparation

Abstract

The invention discloses a method for detecting trace folic acid impurity A in a multivitamin preparation, and belongs to the field of pharmaceutical analytical chemistry. According to the method, a high performance liquid chromatography is adopted, a mobile phase A and a mobile phase B are prepared from phosphate, a tetrabutylammonium hydrogen sulfate solution, methanol and isopropanol, and the content of the trace folic acid impurity A with potential genetic toxicity in the multivitamin preparation can be effectively detected through gradient elution. The detection method provided by the invention is high in sensitivity, good in separation degree and good in repeatability, can accurately and quantitatively detect the trace folic acid impurity A in the multivitamin preparation, and ensures the safety of multivitamin preparation products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical analysis chemistry, and particularly relates to a detection method for detecting trace folic acid impurity A

(2S)-2-[(4-aminobenzoyl)amino] pentanedioic acid

[0002] Vitamins are essential micronutrients for maintaining normal physiological functions of the human body, and play an irreplaceable role in key processes such as energy metabolism, immune function, and cell repair. According to their solubility, vitamins are divided into water-soluble vitamins and fat-soluble vitamins. Water-soluble vitamins are a class of nutrients that the human body must have but cannot synthesize itself, and need to be obtained through food and supplements. Water-soluble vitamins mainly include: vitamin C, dexpanthenol, riboflavin sodium phosphate, vitamin B6, vitamin B1, tetrahydrate coenzyme, folic acid, and nicotinamide. They are mainly involved in energy metabolism, nervous system regulation, and immune enhancement, and cannot be stored for a long time because they are easily soluble in water and need to be supplemented daily. Fat-soluble vitamins include vitamin D3, vitamin A, and vitamin E, which need to be dissolved in fat for absorption and are mainly involved in bone health, antioxidant, and coagulation physiological processes. Both water-soluble and fat-soluble vitamins need to be supplemented through diet or supplements, and excessive intake may cause accumulation and poisoning.

[0003] To ensure the nutritional needs of the market, research on various vitamins is carried out. At present, various vitamin injections being produced or developed include injection of various vitamins (12), injection of 12 kinds of complex vitamins, injection of water-soluble vitamins, various vitamin injection (13), injection of various vitamins (13), pediatric various vitamin injection (13), pediatric injection of various vitamins (13), and injection of water-soluble vitamins, etc. Taking injection of various vitamins (12) as an example, this product is the only injection of lyophilized powder for injection containing water-soluble vitamins and fat-soluble vitamins at home and abroad. Its greatest feature is that fat-soluble vitamins and water-soluble vitamins can be dissolved in the same container. The specification is 5mL / branch, the prescription contains 12 main components and 6-7 auxiliary components, and the content of each component is different. The content is trace, for example, the specification of folic acid is 414μg / 5mL, i.e. 82.8μg / mL.

[0004] Vitamins are unstable and easily degraded by light and heat to produce impurities, which directly affects the safety of clinical use of drugs, especially when multiple vitamins are supplemented intravenously. Therefore, the control of impurities is a key project for product development and quality control.

[0005] Folic acid is extremely unstable, easily destroyed in acidic conditions, easily oxidized under light and heat, and even oxidized at room temperature. It was found during the experiment that folic acid would also degrade when it encountered metal ions, resulting in a large number of impurities in folic acid, which would pose a certain harm to the human body, especially for pregnant women taking the drug, which should be treated more carefully. Therefore, it is necessary to study the degradation impurities of each component and control the impurity content to ensure that it is within a certain safe and effective range. Folic acid and its preparations have been included in the pharmacopoeias of many countries. In the EP / BP pharmacopoeia, the limit of impurity A in folic acid raw material is 0.5%, and in the BP pharmacopoeia, the limit of impurity A in folic acid injection is 2.0%.

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

[0007]

[0008] The warning structure of genetic toxic impurities has been listed in the "Warning Structure of Genetic Toxicity Impurities" published by the National Center for Drug Evaluation of the State Food and Drug Administration at the New Drug Research and Development Forum ([1] Ma Lei, Ma Yulan, Chen Zhen, et al. Warning structure of genetic toxicity impurities [J]. China New Drug Journal, 2014, 23(18): 2106-2111.), in which the first level of arylamine or the group capable of generating arylamine; aryl-substituted hydroxylamine and its derived ester are all warning structures. Folic acid impurity A contains arylamine groups in its structure, which has a potential genetic toxicity warning structure. Therefore, a high-sensitivity detection method is particularly important for the accuracy of folic acid impurity A content control. Most of the pharmacopoeias and literature at home and abroad are methods for controlling a single raw material or methods for controlling individual impurities in three or four vitamin preparations. There are few reports on effective control of impurities in 12 or more vitamin preparations.

[0009] The components of injectable multivitamins are complex, the content of each component is low, the content of impurities is lower, and the limit of genetic toxic impurities is lower. The detection methods mostly use LC-MS or GC-MS for detection; since LC-MS or GC-MS instruments are expensive, most QC departments of pharmaceutical companies do not have the detection conditions for LC-MS or GC-MS, so it is more practical to develop a conventional liquid chromatography method to detect genetic toxic impurities. The sensitivity of the conventional detection method in the prior art does not meet the requirements, or the mobile phase uses a buffer close to pure salt, which faces the problem of storage and use time limit.

[0010] In the prior art, CN 115047083 A discloses a detection method for folic acid and eight impurities thereof. The method uses octadecylsilane-bonded silica gel as the filler, methanol-ammonium acetate solution (12:88) as the mobile phase, and the column temperature is 25°C. The detection wavelength is 280 nm for folic acid and eight impurities. The detection results show that the peak time of folic acid impurity A is about 5.0 min, and the peak time of folic acid is about 10-15 min. However, the method does not disclose the limit of quantification and the detection limit, and is not suitable for accurate quantitative detection of folic acid impurity A in multiple vitamin preparations.

[0011] CN 11507852 B discloses a method for separating and detecting 10 kinds of folic acid impurities by Waters ultra-high performance liquid chromatography. The method uses an octadecylsilane-bonded silica gel column as the filler, and the mobile phase is ammonium acetate solution and methanol with a volume ratio of 90:10 as mobile phase A, and ammonium acetate solution and methanol with a volume ratio of 70:30 as mobile phase B for gradient elution. The detection limit of folic acid impurity A in the method is 0.09 μg / mL, and the limit of quantification is 0.17 μg / mL. The specification appendix Figure 1 shows that the peak time of folic acid impurity A is 2-3 min, which is easily interfered by other strong polar components and impurities in the preparation, and the method has poor specificity.

[0012] Li Xuelian et al. discloses a method for determining the content of related substances in folic acid tablets by high performance liquid chromatography. The method uses a Besil C18-B chromatographic column (4.6 mm x 250 mm, 3.5 μm) or a chromatographic column with equivalent performance; phosphate buffer (take potassium dihydrogen phosphate 10.0 g and dipotassium hydrogen phosphate 5.0 g, dissolve and dilute to 1000 mL with water, and adjust the pH value to 5.5 with phosphoric acid) as mobile phase A, methanol as mobile phase B, gradient elution; column temperature is 35°C; flow rate is 0.8 mL / min; ultraviolet detector (other related substances except impurity K), detection wavelength is 280 nm, impurity K is determined by fluorescence detector, excitation wavelength is 280 nm; emission wavelength is 450 nm; injection volume is 5 μL. The detection chart shows that 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, which is prone to bacterial growth, leading to abnormal peak, and is not easy to store for a long time (Li Xuelian, Xiao Xueju, Cui Delin, Han Yahui, Yi Bin. Determination of the content of related substances in folic acid tablets by high performance liquid chromatography [J]. Central South Pharmaceutical Journal, 2024, 22(9): 2429-2432).

[0013] Panzhaohui et al. used a chromatographic column: Kromasil C18 (250 mm x 4.6 mm x 5 μm); mobile phase: 0.05 M potassium dihydrogen phosphate solution (pH value was adjusted to 5.5 ± 0.03 with 5 M sodium hydroxide); flow rate: 1.5 mL / min; column temperature: room temperature; detection wavelength: 269 nm; injection volume: 20 μL. The mobile phase is still a high-pH pure salt buffer, which is not easy to store for a long time. The impurity A peak is still not well retained (Panzhaohui, Zhang Lihong, Xiao Hongming. Determination of folic acid hydrolysis product in folic acid ferrous fumarate tablets by high performance liquid chromatography [J]. North Pharmaceutical, 2014, 11 (4): 5-5).

[0014] CN 109239230 B discloses a method for analyzing folic acid impurity A, folic acid impurity D, p-aminobenzoic acid and 3-aminopropanol in a multi-vitamin preparation. The chromatographic conditions are as follows: column switching method is used, with octadecylsilane bonded silica gel chromatographic column (Merck, Lichrospher, 125 mm x 4 mm, 5 μm) as the pretreatment column and octadecylsilane bonded silica gel chromatographic column (250 mm x 4 mm, 5 μm) as the analysis column; acetic acid salt solution (weigh 60 g of anhydrous sodium acetate, dissolve and dilute to 1000 mL with water, add glacial acetic acid to adjust the pH to 6.0, add 1.5 mL of tetrahydrofuran, mix well, and filter with a 0.22 um filter membrane) is used as the mobile phase A, methanol is used as the mobile phase B, gradient elution is used, the flow rate is 1.5 mL / min, the detection wavelength is 380 nm, and the column temperature is 40℃. This method uses column switching method, and the test sample solution and the control sample solution need to be derivatized, which is complicated to operate. Moreover, the acetic acid sodium buffer salt concentration in the mobile phase is as high as 0.7 mL / L, which is not easy to store for a long time due to the high pH value. The retention time of folic acid impurity A in the spectrum is only 13 min, which is close to the adjacent peak. Considering the durability, there is a risk of poor separation degree. In addition, under this chromatographic condition, the limit of quantification of folic acid impurity A is only 10 ng, i.e. the limit of quantification concentration is 1 ng / μL, and the sensitivity is low.

[0015] CN113092618A discloses a detection method for the separation and control of six impurities in 13 kinds of compound vitamins for injection, namely 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 (150 mm x 4.6 mm, 3 μm) is used as the chromatographic column; acetonitrile-ethanol-0.01% tetrahydrofuran water (60:10:30) is used as the mobile phase A, and methanol-acetonitrile-ethanol (6:3:1) is used as the mobile phase B. The detection of fat-soluble components is affected by the high proportion of organic phase in the mobile phase, and the folic acid impurity A has strong polarity, which cannot be retained on the chromatographic column, so it cannot be used for quantitative detection of folic acid impurity A.

[0016] CN 118501298 B provides a method for detecting 2,3-diketone-L-gulonic acid in various vitamin preparations. The method uses a high-performance liquid chromatograph, and the chromatographic conditions are as follows: an octadecylsilane-bonded silica gel chromatographic column is used, a phosphate buffer solution containing tetrabutylammonium bromide-acetonitrile is used as the mobile phase A, acetonitrile-water is used as the mobile phase B, the mobile phases A and B are subjected to gradient programming, the column temperature is 40-50°C, the flow rate is 1.0 mL / min, the detection wavelength is 200-250 nm, and the injection amount is 20 μL. This method is not suitable for detecting folic acid impurity A in various vitamin preparations, and may cause peak broadening or tailing, resulting in inaccurate detection results.

[0017] CN115561371A provides a method for detecting the content of folic acid in health food and its application. The chromatographic conditions are isocratic method. However, the components of various vitamin preparations are complex, there are nearly 20 components of raw materials and excipients, and it takes a long time to flush the fat-soluble components, so gradient elution must be used. On the other hand, according to the literature spectrum of "Determination of Related Substances in Folic Acid Tablets by High Performance Liquid Chromatography", the retention time of impurity A is 6.2 min, and the retention time of folic acid is 34.1 min. It can be seen that the polarity of folic acid impurity A is quite different from that of folic acid. In the chromatographic conditions of CN115561371A patent, the retention time of folic acid peak is 31.904 min, and impurity A should elute before 10 min. Because there are many polar components in 12 vitamins, the dosage of water-soluble components such as VC, dexpanthenol, and nicotinamide in the prescription is large, and the polarity is large, which is difficult to retain on the chromatographic column and is easily eluted, which interferes with the determination of impurity A. Therefore, this method is not suitable.

[0018] In summary, there is an urgent need for an analysis method with good separation degree, high sensitivity, low detection limit, and accurate recovery rate to detect trace amounts of folic acid impurity A in various vitamin preparations. SUMMARY

[0019] To solve the above problems, the purpose of the present application is to provide a method for detecting trace amounts of folic acid impurity A in various vitamin preparations, which can effectively realize the determination and limit control of folic acid impurity A. The detection method has high sensitivity, strong specificity, good accuracy and simple operation.

[0020] To achieve the above purpose, the technical scheme of the present application is as follows:

[0021] A method for detecting trace amounts of folic acid impurity A in various vitamin preparations, which uses a high performance liquid chromatograph for determination, and the chromatographic conditions are as follows:

[0022] Chromatographic column: octadecylsilane bonded silica gel chromatographic column; preferably ChromaNik Sunniest C18 column, 250mm x 4.6mm, 5um;

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

[0024] Preferably, mobile phase A: 0.04mol / L phosphate buffer (take anhydrous dibasic potassium phosphate 6.97g, add tetrabutylammonium hydrogen sulfate 0.5g, add water 1000mL to dissolve, adjust pH 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 the gradient program;

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

[0028] Column temperature: 30-37℃, 43-45℃; preferably the injection temperature is 35℃;

[0029] Detection wavelength: 273nm;

[0030] Injection volume: 20uL;

[0031] The injection gradient program is as follows:

[0032] Time / min 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 sample solution: take an appropriate amount of contents of injection multiple vitamins (12) (about equivalent to 414ug of folic acid), accurately weigh and dissolve in water, and quantitatively dilute to prepare a solution containing about 80ug of folic acid per 1mL;

[0035] Impurity control stock solution preparation: Take a certain amount of folic acid impurity A control sample, accurately weigh, add 28.6 mg / mL sodium carbonate solution to dissolve and dilute with water to prepare a solution containing about 5 μg per 1 mL;

[0036] Limit of quantification solution preparation: accurately take the impurity control stock solution with a concentration of 5 μg / mL, dilute with water as solvent to the concentration of signal-to-noise ratio S / N=10 by stages, as the limit of quantification solution;

[0037] Limit of detection solution preparation: accurately take the impurity control stock solution with a concentration of 5 μg / mL, dilute with water as solvent to the concentration of signal-to-noise ratio S / N=3 by stages, as the limit of detection solution;

[0038] Recovery solution preparation: take two injections of multi-vitamins (12) (about equivalent to 828 μg of folic acid), accurately weigh, place in a 10 mL volumetric flask, respectively accurately add 0.5 mL, 1 mL, 2 mL, 3 mL of control stock solution, dissolve and dilute to the mark with water, shake well, respectively as 25%, 50%, 100%, 150% group accuracy solution, each concentration level is prepared in parallel for 3 times.

[0039] The method can be used for detecting the content of trace folic acid impurity A in multi-vitamin preparations, so as to study the stability, degradability and other properties of injections.

[0040] The beneficial effects of the present application are:

[0041] (1) The limit of quantification of the present application is 0.0746 μg / mL, i.e. 1.49 ng; the limit of detection is 0.0224 μg / mL, i.e. 0.45 ng; it is shown that the method has high detection sensitivity, and meets the qualitative and quantitative detection of trace folic acid impurity A in multi-vitamin preparations.

[0042] (2) By adding four different concentrations of folic acid impurity A standard to the sample with known concentration, the average recovery rate of 12 accuracy solutions is 99.70%, and the relative standard deviation is 0.77%, which meets the analysis requirements.

[0043] (3) The linear regression equation of the present application in the concentration range of 0.0746 μg / mL to 7.4650 μg / mL is A=73808C-662.42, and the correlation coefficient r is 1.0000, wherein C is in units of μg / mL, and A is the peak area. The technical solution can detect the content of folic acid impurity A in a small range, provides a strict quality control means for multi-vitamin products, and ensures the safety of the products.

[0044] (4) The preparation operation of the sample solution of the present method is simple, and does not need to be derived, which greatly improves the work efficiency.

[0045] (5) The present application adds isopropanol in mobile phase B, and uses methanol-isopropanol-water with a volume ratio of 30:50:20 as mobile phase B, which greatly improves the rapid elution ability of other components and impurities.

[0046] (6) The present application can quickly elute the ion pair (tetrabutylammonium hydrogen sulfate) peak (as shown in the accompanying drawings, the retention time is about 94 min) while improving the detection sensitivity, avoids the inaccurate detection results caused by continuous sample injection, and ensures the sample injection repeatability and the service life of the chromatographic column. Figure 3

[0047] By using the ultraviolet detector and adding the ion pair of tetrabutylammonium hydrogen sulfate in the mobile phase, the water phase solubility can be enhanced, the water / organic phase distribution ratio can be balanced, and it is more stable under acidic conditions, thereby solving the problem of poor separation degree of trace component folic acid impurity A in various vitamin preparations due to large polarity, and obtaining a method for accurately detecting trace folic acid impurity A in various vitamin preparations with high sensitivity and good repeatability. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 It is a chromatogram of the limit of quantification of the folic acid impurity A reference solution in Example 1.

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

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

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

[0052] Figure 5 It is a chromatogram of the injection multiple vitamins (12) with a column temperature of 40 DEG C in Example 6.

[0053] Figure 6 It is a chromatogram of the injection multiple vitamins (12) high temperature 60 DEG C influence factor 10 days test sample solution in Example 10.

[0054] Figure 7 It is a chromatogram of the injection multiple vitamins (12) high temperature 60 DEG C influence factor 10 days test sample solution in Comparative Example 1.

[0055] Figure 8 It is a chromatogram of the ion pair peak not eluted out by continuous 4 times sample injection in Comparative Example 2.

[0056] Figure 9 It is a chromatogram of the ion pair peak completely eluted out by continuous 4 times sample injection in Comparative Example 2. DETAILED DESCRIPTION​

[0057] The application will be further described in connection with specific examples. It should be understood that the specific examples described herein are intended to be illustrative only and are not intended to limit the scope of the application.

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

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

[0060] The source of the injection multivitamin (12) is: batch number YF09411111, North China Pharmaceutical Co., Ltd.; batch number LE20C076, Baxter S.A.

[0061] The source of the folic acid impurity A control sample is: batch number 8-RTU-33-1, TRC.

[0062] Example 1, detection of the limit of quantification and the limit of detection

[0063] (1) Chromatographic conditions:

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

[0065] Flow rate: 1.0 mL / min;

[0066] Wavelength: 273 nm;

[0067] Column temperature: 35°C;

[0068] Injection volume: 20 μL;

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

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

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

[0072] The mobile phases A and B are carried out according to the gradient program:

[0073] The gradient program is as follows:

[0074] Time / min 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] Stock solution of reference substance: 12.96 mg of folic acid impurity A reference substance (96% content) was precisely weighed into a 50 mL volumetric flask, 1 mL of 28.6 mg / mL sodium carbonate solution was added to dissolve, and water was added to dilute to the mark, and shaken well.

[0077] Limit of quantification solution: 5 mL of the stock solution of reference substance was precisely measured into a 50 mL volumetric flask, diluted to the mark with water, shaken well, 5 mL was precisely measured into a 50 mL volumetric flask, diluted to the mark with water, shaken well, 3 mL was precisely measured into a 100 mL volumetric flask, diluted to the mark with water, shaken well, and obtained.

[0078] Limit of detection solution: 3 mL of the limit of quantification solution was precisely measured into a 10 mL volumetric flask, diluted to the mark with water, shaken well, and obtained.

[0079] (3) Test results:

[0080] Limit of quantification: 20 μL of the limit of quantification solution was precisely measured and injected into the liquid chromatograph, the signal-to-noise ratio was S / N=10:1, and the limit of quantification concentration was 0.0746 μg / mL.

[0081] Limit of detection: 20 μL of the limit of detection solution was precisely measured and injected into the liquid chromatograph, the signal-to-noise ratio was S / N=3:1, and the limit of detection concentration was 0.0224 μg / mL.

[0082] Conclusion: The limit of quantification concentration of trace folic acid impurity A in the preparation of multiple vitamins (12) for injection detected by the method was 0.0746 μg / mL (0.07 ng / μL), the limit of quantification was 1.49 ng, and the limit of detection concentration was 0.0224 μg / mL, i.e. 0.45 ng. The sensitivity of the method was much higher than the limit of quantification concentration (1 ng / μL) in the patent technology CN 109239230A.

[0083] Example 2, determination of linear relationship range

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

[0085] (2) Solution preparation:

[0086] Stock solution for linear relationship: 12.96 mg of folic acid impurity A reference substance (96% content) was precisely weighed into a 50 mL volumetric flask, 1 mL of 28.6 mg / mL sodium carbonate solution was added to dissolve, and water was added to dilute to the mark, shaken well, 5 mL was precisely measured into a 50 mL volumetric flask, diluted to the mark with water, shaken well, and used as the stock solution for linear relationship.

[0087] Linear relationship solution: precisely pipette 0.5 mL, 1 mL, 2 mL, 2.4 mL, 3 mL of the linear relationship stock solution, respectively, into a 10 mL volumetric flask, dilute to the mark with water, shake well, and use as linear solution 1, linear solution 2, linear solution 3, linear solution 4, and linear solution 5, respectively.

[0088] (3) Test results:

[0089] Precisely pipette 20 μL of each of the linear solutions 1-5 in step (2) into the liquid chromatograph, respectively, take the concentration as the abscissa, and the peak area as the ordinate, perform linear regression, the linear equation is A = 73808C - 662.42, C is in the unit of μg / mL, and A is the peak area; the correlation coefficient r = 1.0000, and the folic acid impurity A has a good linear relationship 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] Control solution: take 12.96 mg of the folic acid impurity A control (96% in content), place into a 50 mL volumetric flask, add 1 mL of 28.6 mg / mL sodium carbonate solution to dissolve, dilute to the mark with water, shake well, precisely pipette 2 mL, place into a 100 mL volumetric flask, dilute to the mark with water, shake well, and obtain.

[0094] Control stock solution: take 12.96 mg of the folic acid impurity A control (96% in content), place into a 50 mL volumetric flask, add 1 mL of 28.6 mg / mL sodium carbonate solution to dissolve, dilute to the mark with water, shake well, precisely pipette 5 mL, place into a 50 mL volumetric flask, dilute to the mark with water, shake well, and obtain.

[0095] Test solution: take 2 bottles of the multivitamin for injection (12), precisely weigh, place into a 10 mL volumetric flask, dissolve and dilute to the mark with water, shake well, and obtain.

[0096] Recovery rate solution: take 2 bottles of the multivitamin for injection (12), precisely weigh, place into a 10 mL volumetric flask, precisely add 0.5 mL, 1 mL, 2 mL, 3 mL of the control stock solution, respectively, dissolve and dilute to the mark with water, shake well, and use as 25%, 50%, 100%, and 150% group accuracy solutions, respectively, and prepare 3 parallel samples at each concentration level.

[0097] (3) Test results:

[0098] Precisely pipet 20 μL of the above solution into the liquid chromatograph, record the chromatogram, and calculate the recovery rate and relative standard deviation according to the difference between the measured amount and the original amount and the input amount. The results are shown in the following table:

[0099]

[0100] Test Conclusion: The recovery rate of the 12 accuracy solutions is between 98.02% and 101.0%, with an average recovery rate of 99.70%, and the RSD of the 12 recovery rate results is 0.77%, indicating that the method has good accuracy.

[0101] Example 4, Determination of Folic Acid Impurity A in 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 substance (96% content) and place it in a 50 mL volumetric flask. Add 1 mL of 28.6 mg / mL sodium carbonate solution to dissolve it, and then dilute to the mark with water, shake well, and take 2 mL accurately, place it in a 100 mL volumetric flask, dilute to the mark with water, shake well, and use it as the reference solution.

[0105] Test solution: Take an appropriate amount of the contents of multivitamin (12) for injection (equivalent to about 414 μg of folic acid), accurately weigh, place it in a 5 mL volumetric flask, dissolve and dilute to the mark with water, shake well, and use it as the test solution. Prepare 6 samples in the same way.

[0106] (3) Test results:

[0107] Precisely pipet 20 μL of the above solution into the liquid chromatograph, record the chromatogram, and calculate the recovery rate and relative standard deviation according to the difference between the measured amount and the original amount and the input amount. The results are shown in the following table:

[0108] Number 1 2 3 4 5 6 Mean RSD(%) Mean 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 of the 6 test solutions is 1.92%, and the average deviation is 0.03%, indicating that the method has good repeatability.

[0110] In addition, the blank solvent does not interfere with the determination of folic acid impurity A in the test solution; the peak purity and separation degree of the folic acid impurity A peak in the test solution meet the requirements, and the method has good specificity.

[0111] Example 5, Determination of Folic Acid Impurity A in Multivitamin (12) for Injection - Investigation of Mobile Phase A

[0112] (1) Chromatographic conditions: Except for the mobile phase A, the other chromatographic conditions were the same as those in Example 1.

[0113] Mobile phase A: 0.04 mol / L phosphate buffer (take 6.97 g of anhydrous dibasic sodium phosphate, 0.5 g of tetrabutylammonium bisulfate, add 1000 mL of water to dissolve, and adjust the pH value 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 substance (96% content) and place it in a 50 mL volumetric flask, add 1 mL of 28.6 mg / mL sodium carbonate solution to dissolve, dilute to the mark with water, shake well, and take 2 mL accurately, place it in a 100 mL volumetric flask, dilute to the mark with water, shake well, and use it as the reference solution.

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

[0117] (3) Test results:

[0118] Accurately take 20 μL of each of the above solutions, inject them into the liquid chromatograph, record the chromatogram, and analyze them according to the above conditions, as shown in the following table:

[0119]

[0120] The test showed that under the above mobile phase proportions, the separation degree and theoretical plate number of folic acid impurity A and the adjacent peaks were high, and the detection content results were basically consistent.

[0121] Example 6, Determination of Trace Components Folic Acid Impurity A in Multivitamin Injection (12) - Column Temperature Investigation

[0122] (1) Chromatographic conditions:

[0123] Except for setting multiple column temperatures, the rest were the same as in Example 1; the column temperature was set to 30°C and 40°C, respectively.

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

[0125] (3) Test results:

[0126] Accurately take 20 μL of each of the above solutions, inject them into the liquid chromatograph, record the chromatogram, and analyze them according to the above conditions, as shown in the following table:

[0127]

[0128] Conclusion: When the column temperature is 40°C, the adjacent peaks are more sensitive to the column temperature, and folic acid impurity A is wrapped. Therefore, the column temperature is increased to 37°C, 43°C and 45°C, and under the conditions of 37°C and 43°C, folic acid impurity A and the adjacent peaks are just separated from the baseline. The later method should control the column temperature in the range of 30°C to 37°C or 43°C to 45°C.

[0129] Example 7, Determination of Folic Acid Impurity A in Multivitamin (12) Micro-ingredients for Injection - pH Investigation of Mobile Phase A

[0130] (1) Chromatographic conditions: except for the investigation of multiple pH values of mobile phase A, the rest are the same as in Example 1;

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

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

[0133] (3) Test results:

[0134] Precisely take 20 μL of the above solution, inject it into the liquid chromatograph, record the chromatogram, and analyze it under the above conditions, as shown in the following table:

[0135]

[0136] Conclusion: When the pH is 6.2, folic acid impurity A and the adjacent peaks are not separated from the baseline (separation degree is 1.43), so the pH is increased to 6.25. Under the condition of pH 6.25, the separation degree is 2.21, so the later method should control the pH of the mobile phase in the range of 6.25-6.4.

[0137] Example 8, Determination of Folic Acid Impurity A in Multivitamin (12) Micro-ingredients for Injection - Different Flow Rate Investigation

[0138] (1) Chromatographic conditions: except for setting the flow rate to 0.9-1.1 mL / min, the rest are the same as in Example 1.

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

[0140] (3) Test results:

[0141] Precisely take 20 μL of the above solution, inject it into the liquid chromatograph, record the chromatogram, and analyze it under the above conditions, as shown in the following table:

[0142]

[0143] Example 9, Determination of Folic Acid Impurity A in Multivitamin (12) for Injection - Investigation of Different Chromatographic Columns

[0144] (1) Chromatographic conditions: same as Example 1 except that different brand instruments, different brand or batch chromatographic columns and the following gradient settings were changed.

[0145] Time / min 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 Example 5.

[0147] (3) Test results: precisely take 20 μL of the above solution, inject into the liquid chromatograph, record the chromatogram, analyze according to the above conditions, and combine the results of Examples 5-8, the analysis is shown in the following table:

[0148]

[0149] Conclusion: the theoretical plate number of folic acid impurity A peak under the above durability conditions is greater than 2000, the separation degree of folic acid impurity A peak and adjacent peaks in the test solution meets the requirements; the RSD of the content determination result is 3.36%, the average deviation is 0.05%, the method has good durability, and the method is suitable for the detection of folic acid impurity A in multivitamin preparations.

[0150] Example 10, Determination of Folic Acid Impurity A in Multivitamin (12) for Injection

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

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

[0153] Test solution: prepare two batches of multivitamin (12) for injection influence factor test sample - high temperature 60 degrees influence factor 10 days solution.

[0154] (3) Test results:

[0155] Precisely take 20 μL of the above solution, inject into the liquid chromatograph, record the chromatogram, analyze according to the above conditions, and combine the results of Examples 5-8, the analysis is shown in the following table:

[0156] Name Retention time T(min) Folic acid impurity A(%) Multivitamin for injection (12) - batch number: YF09411111 - 60 degrees for 10 days 44.558 2.15 Multivitamin for injection (12) - batch number: LE20C076 - 60 degrees for 10 days 44.195 2.79

[0157] Conclusion: from the chromatogram of the test solution of multivitamin (12) for injection under the influence of high temperature 60 degrees for 10 days, the retention time of folic acid impurity A peak is about 44 min, and the separation degree from the adjacent peaks is good. See the following figure for details: Figure 6 .

[0158] Comparative Example 1, Determination of Folic Acid Impurity A in Multivitamin (12) for Injection

[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 / min 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 is to be explained that the above embodiments are only used to illustrate the technical solutions of the present application in detail but not to limit the present application, and the skilled in the art can modify or equivalently replace the technical solutions of the present application without departing from the purpose and scope of the present application, which should be covered in the scope of the claims of the present application.

Claims

1. A method of detecting trace amounts of folic acid impurity A in a plurality of vitamin preparations, characterized by, 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 the pH adjusted to 6.25~6.4 by phosphoric acid; Mobile phase B: Methanol-isopropanol-water; Mobile phases A and B are eluted using a gradient program, and the elution program is as follows: The percentages of mobile phases A and B are volume percentages.

2. The method according to claim 1, characterized in that, The chromatographic column is either ChromaNik Sunniest, 250mm × 4.6mm, 5µm or YMC-Triart C18, 250mm × 4.6mm, 5µm.

3. The method according to claim 1, characterized in that, The volume ratio of the dipotassium hydrogen phosphate buffer solution containing tetrabutylammonium hydrogen phosphate to methanol in the mobile phase A is 89~91:11~9.

4. 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.

5. The method according to claim 1, characterized in that, The mobile phase B is a mixed solution of methanol, isopropanol, and water, with a volume ratio of methanol-isopropanol-water of 30:50:

20.

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

7. 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.

8. 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.

9. 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.

10. The method according to claim 1, characterized in that, 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).

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