Method for detecting vitamin B6 related substances in multivitamin preparation

Abstract

The invention provides a method for detecting vitamin B6 related substances in a multivitamin preparation, and belongs to the field of analytical chemistry. According to the method, a high performance liquid chromatograph is adopted, and chromatographic conditions are as follows: an octadecyl silane bonded silica gel chromatographic column is adopted, a mobile phase is a phosphate buffered solution (containing sodium hexanesulfonate)-acetonitrile, and a mobile phase A and a mobile phase B are performed according to a gradient procedure; the flow velocity is 0.8 to 1.2 mL / min; the column temperature is 28-40 DEG C, the detection wavelength is 320-340nm, and the sample size is 20-50mu L. The detection method has the advantages of high sensitivity and good linear relationship, and is suitable for analysis and content determination of vitamin B6 related substances in various vitamin preparations.

Description

Technical Field

[0001] The invention belongs to the field of analytical chemistry, and in particular relates to a method for detecting vitamin B6-related substances in a multivitamin preparation. The method is used for analyzing and determining vitamin B6-related substances in the multivitamin preparation. Background Art

[0002] Vitamin B6 is found in high concentrations in yeast, liver, cereal grains, meat, fish, eggs, beans, and peanuts. It serves as a coenzyme for transaminases and amino acid decarboxylases in the human body and is particularly involved in numerous metabolic reactions, particularly amino acid metabolism. Vitamin B6 preparations are clinically used to prevent and treat vomiting during pregnancy and radiation sickness. Vitamin B6 primarily acts on the blood, muscles, nerves, and skin. Its functions include antibody synthesis, gastric acid production in the digestive system, fat and protein utilization (especially recommended for weight loss), and maintaining sodium / potassium balance (stabilizing the nervous system). Common symptoms of vitamin B6 deficiency include loss of appetite, poor food utilization, weight loss, vomiting, and diarrhea. Severe vitamin B6 deficiency can lead to acne, anemia, arthritis, seizures in children, depression, headaches, hair loss, inflammation, learning disabilities, and weakness.

[0003] Vitamin B6, also known as pyridoxine, includes pyridoxine, pyridoxal, and pyridoxamine. It exists in the body in the form of phosphate esters and is a water-soluble vitamin. It is easily destroyed by light or alkali and is not resistant to high temperatures. Pyridoxine is stable in acidic solutions but easily destroyed in alkaline solutions. Its heat resistance is superior to that of its counterparts, pyridoxal and pyridoxamine. Many multi-component vitamin preparations that have been marketed and contain vitamin B6, such as water-soluble vitamins for injection containing 9 water-soluble vitamins, multivitamins for injection containing 12 vitamins (12), pediatric multivitamin injection containing 13 vitamins (13), multivitamin injection (13), etc., contain pyridoxine as the active ingredient of vitamin B6.

[0004] Pyridoxine, also known as vitamin B6 or anti-dermatitis factor, has a molecular formula of C8H 11 NO3·HCl has a molecular weight of 205.64 and is readily soluble in water and ethanol. It was first isolated by A. von Szent-Györgyi in 1935, named vitamin B6 in 1936, and synthesized artificially in 1939. The structural formula of pyridoxine is as follows:

[0005]

[0006] Pyridoxine is unstable and sensitive to light and heat, and can oxidize and degrade to produce impurities. If improperly stored during transportation, it can easily oxidize and degrade to produce various impurities, including Impurity C (pyridoxal) and Impurity F, thereby reducing or even eliminating the efficacy of vitamin B6.

[0007] The structural formula of impurity C (i.e. pyridoxal) is as follows:

[0008]

[0009] Vitamin B6 impurity F is a vitamin B6 dimer with the molecular formula C 16 H 20 N2O5, molecular weight is 320.35, and its structural formula is:

[0010]

[0011] Among vitamin B6 products and preparations, pyridoxine is more stable than pyridoxal and pyridoxamine, and has higher average bioavailability than pyridoxal and pyridoxamine (Yaman M, Mızrak ÖF. Determination and evaluation of in vitro bioaccessibility of the pyridoxal, pyridoxine, and pyridoxamine forms of vitamin B6 in cereal-based baby foods. Food Chem. 2019 Nov 15;298:125042.). Therefore, determining the content of impurity C (pyridoxal) in vitamin B6 is of great significance for the research of multivitamin drugs containing vitamin B6.

[0012] CN 109239230B relates to a method for detecting impurities in multivitamin injections. This method can effectively detect folic acid impurity A, folic acid impurity D, p-aminobenzoic acid, and 3-aminopropanol in multivitamin preparations. CN 110715995A reports a method for detecting impurities in multivitamin injections, primarily involving the detection of oxalic acid, 3-aminopropanol, retinol, α-tocopherol, menadione, and the cis-isomer of vitamin K1. Neither invention addresses the detection of vitamin B6 degradation impurities, and the method is not applicable to vitamin B6 impurities C (pyridoxal) and impurity F.

[0013] CN 114280198B is a method for detecting vitamin B6 and its related substances in vitamin B6 injection using octadecylsilane bonded silica gel as filler and sodium heptanesulfonate solution and methanol as mobile phases for gradient elution. This method is suitable for the detection of vitamin B6 and its related substances in single-ingredient vitamin B6 injection, but is not suitable for the detection of compound preparations containing vitamin B6. The specificity does not meet the requirements.

[0014] In the non-patent literature, Liu Yan et al. studied an HPLC method for determining the content of vitamin B6 and related substances in vinblastine eyewash (Liu Yan, Li Rui, Li Yongchun, et al. HPLC determination of the content of vitamin B6 and related substances in vinblastine eyewash [J]. Food and Drug, 2009, 11(09): 38-40.). The literature involved the detection of vitamin B6 related substances. The high performance liquid chromatography method was used in the literature, and the chromatographic column was Zorbax SB-C 18 The column was filled with a 4.6 mm × 150 mm column, 5 μm column. The mobile phase consisted of a sodium hexanesulfonate buffer (1.3 g sodium hexanesulfonate, 0.57 g anhydrous sodium acetate, dissolved in water, 22 mL glacial acetic acid, diluted to 1000 mL with water, and adjusted to pH 3.0 with 6 mol / L sodium hydroxide) and methanol (92:8 ratio). The flow rate was 1.0 mL / min. The column temperature was 35°C. The detection wavelengths were 291 nm for content analysis and 275 nm for related substances. This method is suitable for the determination of vitamin B6 degradation impurities in vitamin B6 potassium vinblastine eyewash. The reported vitamin B6 degradation impurities are primarily photolysis products, which is inconsistent with the degradation pathway of vitamin B6 impurity F. Furthermore, detailed studies of the degradation impurities were not reported, making it unsuitable for the detection of vitamin B6 impurity F in multi-component vitamins.

[0015] Liu Guiguo et al. used high-performance liquid chromatography to detect vitamin B6 in fortified foods (infant formula, infant cereal complementary foods, and functional beverages). The chromatographic column was Eclipse Plus C18 (5 μm, 4.6 mm×250 mm, Agilent Technologies, Inc., USA); the mobile phase was V (methanol) (A): V (0.2 g / L sodium octane sulfonate, 0.25% triethylamine solution, pH = 3.2±0.1 adjusted with acetic acid) (B) = 21:79, the flow rate was 1 mL / min, the injection volume was 10 μL, and the column temperature was 30 ℃; the detection wavelength was: excitation wavelength 293 nm, emission wavelength 395 nm. This method requires sample treatment before detection, which is complicated to operate. The test results show that the limit of quantification of pyridoxal is 0.03 mg / 100 g, but the peak time is around 5.5-6 min, which is completely unsuitable for the detection of pyridoxal in multivitamin preparations. The separation effect is poor, and the triethylamine in the mobile phase has irreversible damage to the chromatographic column (Liu Guiguo, Wang Haomiao, Li Jiahui, et al. Determination of vitamin B6 content in fortified foods by high performance liquid chromatography [J]. Journal of Food Safety and Quality, 2019, 10(21): 7349-7353.).

[0016] Zhan Yuecheng et al. separated and determined taurine, vitamin B1, vitamin B2, niacin, niacinamide, pyridoxine, pyridoxal, pyridoxamine, pantothenic acid, folic acid ...12 The content of 12 water-soluble vitamins such as vitamin C, biotin, etc. (Zhan Yuecheng, He Bin, Liu Mengting, et al. Research on the method of rapid detection of multiple water-soluble vitamins in food [J]. Agricultural Products Processing (Second Half of the Month), 2019(8):49-52,56.). The recovery rate of this method at three mass concentration levels was 78.5%-89.4%, the relative standard deviation (RSD) was 2.00%-4.99%, the detection limit of pyridoxal was 5.0μg / kg, and the quantification limit was 20.0μg / kg. However, this method uses liquid chromatography and mass spectrometry in series for detection, the instrument price is high, and the analysis cost is high.

[0017] Vitamin ingredients have complex physical and chemical properties, are often unstable, and are sensitive to light, heat, oxygen, metal ions, acidity, and other factors. Multivitamin compound preparations have variable ingredients and complex reaction mechanisms. Compared to single vitamin preparations, multi-component vitamin preparations are difficult to detect and have significant mutual interference. Furthermore, the content of each vitamin in vitamin preparations is often trace, especially in compound vitamin preparations for injection, which contain even lower levels of degradation impurities. Therefore, quality control testing of compound vitamins, especially impurity detection, is a difficult aspect of the development of such preparations. Currently, there is a lack of analytical methods for the detection of vitamin B6-related substances in multi-component vitamin preparations. Summary of the Invention

[0018] The purpose of the present invention is to solve the above-mentioned problems existing in the prior art and to provide a high performance liquid chromatography method with good specificity, high sensitivity, low detection limit, simplicity and high efficiency for separating and analyzing vitamin B6-related substances in multivitamin preparations, thereby achieving quality control of vitamin B6 in multivitamin preparations.

[0019] The technical solutions of the present invention are as follows:

[0020] A method for detecting vitamin B6-related substances in a multivitamin preparation, the method using high performance liquid chromatography, the chromatographic conditions of which are:

[0021] Chromatographic column: octadecylsilane bonded silica gel column; the preferred chromatographic column is Sunniest C18, 250 mm × 4.6 mm, 5 μm or YMC Triart C18, 250 mm × 4.6 mm, 5 μm;

[0022] Mobile phase A: sodium hexanesulfonate potassium dihydrogen phosphate buffer solution-acetonitrile, adjust the pH value to 4.0-5.5 with phosphoric acid, preferably 5.4-5.5;

[0023] Mobile phase B: sodium hexanesulfonate potassium dihydrogen phosphate buffer solution-acetonitrile, adjust the pH value to 4.0-5.5 with phosphoric acid, preferably 5.4-5.5;

[0024] Flow rate: 0.8-1.2 mL / min; preferably 1.0 mL / min;

[0025] Column temperature: 28-40°C; the preferred injection temperature is 35°C;

[0026] Ultraviolet detector, preferably detection wavelength: 320-340nm; more preferably detection wavelength: 330nm;

[0027] Injection volume: 20μL-50μL;

[0028] Mobile phases A and B were prepared according to the following gradient program:

[0029] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0~12 95 5 12~25 95~90 5~10 25~50 90~85 10~15 50~59 85 15 59~60 85~50 15~50 60~70 50 50 70~71 50~95 50~5 71~100 95 5

[0030] The preferred mobile phase A is a 0.01 mol / L potassium dihydrogen phosphate buffer solution containing 0.2% sodium hexanesulfonate; the buffer solution is adjusted to a pH of 5.4 to 5.5 with phosphoric acid, and the preferred pH is 5.5;

[0031] The preferred volume ratio of potassium dihydrogen phosphate buffer solution (containing ammonium hexanesulfonate): acetonitrile is 80-95:20-5, more preferably 85:15.

[0032] The preferred mobile phase B is a 0.01 mol / L potassium dihydrogen phosphate buffer solution containing 0.2% sodium hexane sulfonate, the pH value of the buffer solution is adjusted to 5.5 with phosphoric acid, and the volume ratio of potassium dihydrogen phosphate buffer solution (containing sodium hexane sulfonate): acetonitrile is 25~50:50~75, more preferably 30:70.

[0033] The preferred gradient program is:

[0034] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 12 95 5 25 90 10 50 85 15 59 85 15 60 50 50 70 50 50 71 95 5 100 95 5

[0035] This method can be used to detect the content of vitamin B6-related substances in various vitamin preparations, so as to study the stability and degradation performance of vitamin B6 in the preparations.

[0036] Beneficial effects of the present invention:

[0037] 1) The detection method of the present invention can not only detect vitamin B6 and impurity C qualitatively and quantitatively, but also detect impurity F qualitatively and quantitatively.

[0038] 2) After testing, the quantification limit of impurity C of the present invention was 6.40 ng, and the detection limit was 2.00 ng; the quantification limit of impurity F was 6.60 ng, and the detection limit was 2.00 ng, indicating that the detection sensitivity of this method is high and meets the requirements for the qualitative and quantitative detection of vitamin B6-related substances in multivitamin preparations.

[0039] 3) By selecting a specific gradient elution procedure, using a conventional C18 chromatographic column, and adding the ion-pairing reagent sodium hexanesulfonate to the mobile phase, the detection of the vitamin B6 impurity F macromolecular dimer in multi-component vitamin preparations was achieved. This avoided the problems of poor separation between the dimer and the main component and inaccurate detection results, and obtained an analytical method with high sensitivity, good repeatability, and accurate quantitative detection of vitamin B6-related substances in multiple vitamin preparations.

[0040] 4) Vitamin B6 impurities C and F were added to the samples, and the recoveries were calculated. The average recovery of impurity C was 87.97%, and the average recovery of impurity F was 100.0%, both meeting the acceptance criteria and indicating that the method has good accuracy.

[0041] 5) For vitamin B6, within the concentration range of 0.33 μg / mL to 27.44 μg / mL, the linear regression equation was A = 18178C + 144.24, with a correlation coefficient r of 1.0000, indicating a good linear relationship. For impurity C, within the concentration range of 0.32 μg / mL to 26.83 μg / mL, the linear regression equation was A = 17066C - 422.22, with a correlation coefficient of 0.9999, indicating a good linear relationship. The correction factor for vitamin B6 was 1.07. For vitamin B6 impurity F, within the concentration range of 0.33 μg / mL to 27.25 μg / mL, the linear regression equation was A = 23052C - 2921.4, with a correlation coefficient of 1.0000, indicating a good linear relationship. The correction factor for vitamin B6 was 0.8. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the standard curve of vitamin B6 in Example 1.

[0043] Figure 2 This is a standard curve diagram of vitamin B6 impurity C in Example 1.

[0044] Figure 3 This is a standard curve diagram of vitamin B6 impurity F in Example 1.

[0045] Figure 4 This is the liquid chromatogram of the 100% accuracy solution in Example 3.

[0046] Figure 5 It is the implementation of 9 accelerated 6 months - upright -40 ℃ sample chromatogram.

[0047] Figure 6 It is the liquid chromatogram of Comparative Example 1. DETAILED DESCRIPTION

[0048] The present invention will be further described below in conjunction with specific embodiments. The following description is only for the purpose of explaining the present invention. The scope of protection of the present invention is not limited to these embodiments. In order to enable those skilled in the art to better understand the present invention, equivalent replacements or corresponding improvements made to the contents of the present invention still fall within the scope of protection of the present invention.

[0049] Example 1 Determination of standard curve

[0050] (1) Chromatographic conditions: Column: Sunniest C18, 250 mm × 4.6 mm, 5 μm;

[0051] Flow rate: 1.0 mL / min;

[0052] Column temperature: 35°C;

[0053] UV detector detection wavelength: 330nm;

[0054] Injection volume: 20 μL;

[0055] Mobile phase A: potassium dihydrogen phosphate buffer solution (containing sodium hexane sulfonate)-acetonitrile in a volume ratio of (85:15); 0.01 mol / L phosphate buffer solution containing 0.2% sodium hexane sulfonate, the buffer solution was adjusted to pH 5.5 with phosphoric acid;

[0056] Mobile phase B: potassium dihydrogen phosphate buffer solution (containing sodium hexane sulfonate)-acetonitrile in a volume ratio of (30:70); 0.01 mol / L phosphate buffer solution containing 0.2% sodium hexane sulfonate, and the pH value of the buffer solution was adjusted to 5.5 with phosphoric acid.

[0057] Mobile phases A and B were prepared according to the gradient program:

[0058] The gradient program is:

[0059] Time (minutes) Mobile phase A (%) Mobile phase B (%) 0 95 5 12 95 5 25 90 10 50 85 15 59 85 15 60 50 50 70 50 50 71 95 5 100 95 5

[0060] (2) Solution preparation

[0061] Step (1) Reference substance stock solution: Take approximately 27 mg of vitamin B6 reference substance, accurately weigh it, place it in a 100 mL volumetric flask, add water to dissolve it and dilute it to the mark, shake it well, and use it as vitamin B6 stock solution. Take approximately 27 mg of impurity C reference substance, accurately weigh it, place it in a 100 mL volumetric flask, add water to dissolve it and dilute it to the mark, shake it well, and use it as impurity C stock solution. Take approximately 27 mg of impurity F reference substance, accurately weigh it, place it in a 100 mL volumetric flask, add water to dissolve it and dilute it to the mark, shake it well, and use it as impurity F stock solution.

[0062] Step (2) Linear stock solution: Take 5 mL each of the vitamin B6 stock solution, impurity C stock solution, and impurity F stock solution prepared in step (1), place them in the same 50 mL volumetric flask, dilute to the mark with water, and shake well to prepare the linear stock solution.

[0063] Step (3) Linear solution: Take 1 mL, 2 mL, 5 mL, 8 mL, and 10 mL of the linear stock solution of step (2), place them in a 10 mL volumetric flask, add water to dilute to the scale, and shake well to prepare 20%, 40%, 100%, 160%, and 200% linear solutions of vitamin B6, and 120%, 240%, 600%, 960%, and 1200% linear solutions of impurity C and impurity F.

[0064] (3) Measurement results

[0065] Accurately measure 20 μL of each linear solution 1-5 and inject it into the liquid chromatograph. Including the limit of quantification, use the concentration C (μg / mL) as the horizontal axis and the peak area A as the vertical axis to perform linear regression. See the attached manual for details. Figure 1 、 Figure 2 The linear regression equation of vitamin B6 is A=18178C+144.24, and the correlation coefficient r is 1.0000; the linear regression equation of impurity C is A=17066C-422.22, and the correlation coefficient is 0.9999; the linear regression equation of impurity F is 23052C-2921.4, and the correlation coefficient is 1.0000.

[0066] Example 2 Detection Limit and Quantification Limit

[0067] Chromatographic conditions: same as in Example 1.

[0068] Solution preparation:

[0069] (1) Accurately measure 1 mL of the linear solution 1 prepared in step (3) of Example 1, place it in a 10 mL volumetric flask, dilute to the mark with water, and shake well; this is the quantitative limit solution;

[0070] (2) Accurately measure 3 mL of the solution in step (1), place it in a 10 mL volumetric flask, dilute to the mark with water, and shake well; this is the detection limit solution;

[0071] Measurement results:

[0072] Limit of quantitation: Accurately measure 20 μL of the limit of quantitation solution and inject it into the liquid chromatograph. The signal-to-noise ratio (S / N) is 10:1. According to the test results, the limit of quantitation concentration of impurity C is 0.32 μg / mL; the limit of quantitation concentration of impurity F is 0.33 μg / mL.

[0073] Detection limit: Accurately measure 20 μL of the detection limit solution and inject it into the liquid chromatograph. The signal-to-noise ratio S / N is 3:1. According to the test results, the detection limit concentration of impurity C is 0.10 μg / mL; the detection limit concentration of impurity F is 0.10 μg / mL.

[0074] Conclusion: The detection limit of vitamin B6 impurity C by this detection method is 0.32 μg / mL, and the limit of quantification is 6.40 ng; the limit of quantification of impurity F is 0.33 μg / mL, and the limit of quantification is 6.60 ng; the detection limit of vitamin B6 impurity C is 2.00 ng, and the detection limit concentration is 0.10 μg / mL; the detection limit of impurity F is 2.00 ng, and the detection limit concentration is 0.10 μg / mL, indicating that the detection method has high sensitivity.

[0075] Example 3 Determination of the Recovery Rate of Vitamin B6 Related Substances in Multivitamin Preparations

[0076] (1) Chromatographic conditions: Same as in Example 1.

[0077] (2) Solution preparation

[0078] System suitability solution: Take 50 mg of vitamin B6 and place it in a 25 mL volumetric flask, add water to dissolve and dilute to the scale, shake well, and destroy it at 80°C for 72 hours. Cool to room temperature; accurately measure 2 mL and place it in a 10 mL volumetric flask, add water to dilute to the scale, and shake well.

[0079] Preparation of test solution: Take an appropriate amount of the contents of multivitamin for injection (12) (approximately equivalent to 5.5 mg of vitamin B6), accurately weigh it, place it in a 25 mL volumetric flask, add water to dissolve it and dilute it to the scale, and shake it well;

[0080] Preparation of reference solution: Take about 22 mg of vitamin B6 reference substance, accurately weigh it, place it in a 100 mL volumetric flask, add water to dissolve and dilute to the scale, accurately measure 2 mL, place it in a 200 mL volumetric flask, add water to dilute to the scale, shake well, and use it as the reference solution.

[0081] Recovery rate stock solution: (1) Take about 22 mg of impurity C reference substance, accurately weigh it, place it in a 100 mL volumetric flask, add water to dissolve it and dilute it to the mark, shake it well, and use it as the impurity C reference substance stock solution; take about 22 mg of impurity F reference substance, accurately weigh it, place it in a 100 mL volumetric flask, add water to dissolve it and dilute it to the mark, shake it well, and use it as the impurity F reference substance stock solution;

[0082] (2) Accurately measure 5 mL of the reference stock solution of impurity C and impurity F, place it in a 100 mL volumetric flask, dilute to the mark with water, and shake well to use as the recovery stock solution;

[0083] Prepare the spiked test solution: Accurately weigh approximately 750 mg of multivitamins for injection into a 25 mL volumetric flask. Accurately add 5 mL of the recovery stock solution, dissolve in water, and dilute to the mark. Shake well. This is the 100% accuracy test solution. Prepare 6 more samples in the same manner.

[0084] (3) Measurement results

[0085] Accurately measure 20 μL of each of the above solutions and inject them into the liquid chromatograph. According to the main component external standard method, the content of impurity C and impurity F in the test sample is calculated by peak area. The recovery rate is calculated by the ratio of the difference between the measured amount and the original amount to the added amount. The results of the accuracy test of the impurity C content determination are as follows:

[0086]

[0087] The results of the accuracy test of impurity F content determination are as follows:

[0088]

[0089] Conclusion: The recovery rate of vitamin B6 impurity C was 85.71%-90.31%, with an average recovery rate of 87.97%; the recovery rate of impurity F was 97.35%-102.2%, with an average recovery rate of 100.0%, indicating that the method has good accuracy.

[0090] Example 4 Repeatability Determination of Vitamin B6 Related Substances in Multivitamin Preparations

[0091] (1) Preparation of solution.

[0092] Blank solvent: water;

[0093] Preparation of system suitability solution: same as in implementation 3;

[0094] Preparation of reference solution: Take an appropriate amount of vitamin B6 reference substance, accurately weigh it, dissolve it in water and quantitatively dilute it to a solution containing approximately 2.2 μg of vitamin B6 per 1 mL;

[0095] Preparation of test solution: Take an appropriate amount of the contents of multivitamins for injection (12) (approximately equivalent to 5.5 mg of vitamin B6) (accelerated 6-month sample at 40°C), accurately weigh, place in a 25 mL volumetric flask, dissolve in water and dilute to the mark, shake well, and prepare 6 portions in the same way;

[0096] (2) Liquid chromatography conditions were the same as in Example 1;

[0097] Accurately measure 20 μL of each of the above solutions and inject them into the liquid chromatograph. According to the main component external standard method, the content of impurity C and impurity F in the test sample is calculated by peak area to calculate the repeatability. The results are as follows:

[0098]

[0099] The results showed that in the 6 test solutions, the average content of vitamin B6 impurity C was 0.52%, with an average deviation of 0.10%; the average content of impurity F was 0.44%, with an average deviation of 0.01%, indicating that this detection method has good reproducibility.

[0100] Example 5: Durability of the Determination Method for Vitamin B6-Related Substances in Multivitamin Preparations - Investigation of pH Value

[0101] (1) The solution preparation is the same as in Example 4;

[0102] (2) Chromatographic conditions

[0103] Except for the change in pH value of potassium dihydrogen phosphate buffer in the mobile phase (5.5±0.1), everything else was the same as in Example 1;

[0104] pH value: 5.4, 5.5, 5.6.

[0105] (3) Sampling and result analysis

[0106] Accurately measure 20 μL of the system suitability solution and the test solution, inject them into the liquid chromatograph, and record the chromatogram.

[0107] (4) The experimental results show that the data in the chromatogram recorded at pH values ​​of 5.4, 5.5, and 5.6.

[0108] The results of the system suitability solution test are shown in the table below:

[0109]

[0110] The test results of the test solution are shown in the following table:

[0111]

[0112] Results: In the system suitability solution chromatogram, at a mobile phase pH of 5.4, the resolution between impurity F and adjacent chromatographic peaks met the requirements. At a pH of 5.6, the resolution between vitamin B6 and impurity F did not meet the requirements. This indicates that the standard method is robust at standard pH and slightly lower. When the mobile phase pH is higher than the standard, the resolution between vitamin B6 and impurity F decreases or even fails to meet the requirements. The pH robustness range for impurity F is somewhat narrow; increasing the pH value can affect resolution, so this should be considered.

[0113] Example 6: Durability of the Determination of Vitamin B6-Related Substances in Multivitamin Preparations - Investigation of Different Column Temperatures

[0114] (1) The solution preparation is the same as in Example 4;

[0115] (2) Chromatographic conditions

[0116] Except for the different column temperatures, other chromatographic conditions were the same as in Example 1;

[0117] Column temperature: 30℃, 35℃, 40℃;

[0118] (3) Sampling and result analysis

[0119] Accurately measure 20 μL of the system suitability solution and the test solution, and record the chromatograms at column temperatures of 30°C, 35°C, and 40°C. The system suitability solution test results are shown in the table below:

[0120]

[0121] The test results of the test solution are shown in the following table:

[0122]

[0123] The results show that the separation between the peaks of vitamin B6 impurity F and adjacent peaks of the system suitability solution and the test solution at column temperatures of 30℃, 35℃ and 40℃ all meet the requirements, and the column temperature durability under this condition is good.

[0124] Example 7 Development of a method for determining vitamin B6-related substances in multivitamin preparations - Investigation of different chromatographic columns

[0125] (1) The solution preparation is the same as in Example 4;

[0126] (2) Chromatographic conditions

[0127] Except for the change of different chromatographic columns, everything else is the same as Example 1;

[0128] Chromatographic columns: chromatographic columns of the same brand but different batches (Sunniest C18, 250 mm × 4.6 mm, 5 μm); chromatographic columns of different brands (YMC Triart C18, 250 mm × 4.6 mm, 5 μm).

[0129] (3) Sampling and result analysis

[0130] After replacing different chromatographic columns and fully equilibrating, the system suitability solution and the test solution were injected and the chromatograms were recorded. When replacing the chromatographic columns of the same brand but different batches (Sunniest C18, 250mm×4.6mm, 5μm) and the chromatographic columns of different brands (YMC Triart C18, 250mm×4.6mm, 5μm), the chromatograms recorded for the system suitability solution and the test solution did not change significantly. The separation between vitamin B6, impurity C, impurity D, impurity E, and impurity F and adjacent peaks in the test solution met the requirements. The test results of the test solution are shown in the table:

[0131]

[0132] The experimental results show that the separation degree of each impurity peak and adjacent peak of vitamin B6 in the test samples of multiple chromatographic columns in this technical solution meets the requirements.

[0133] Example 8 Development of a method for determining vitamin B6-related substances in multivitamin preparations - Investigation of different organic phase ratios in mobile phase A

[0134] (1) The solution preparation is the same as in Example 4;

[0135] (2) Chromatographic conditions.

[0136] Except for the change in the ratio of mobile phase A to organic phase, everything else was the same as in Example 1;

[0137] Mobile phase A organic phase ratio: 84:16, 85:15, 86:14;

[0138] (3) Sampling and result analysis.

[0139] Inject the system suitability solution and the test solution separately and record the chromatogram. The system suitability solution test results are shown in the table below:

[0140]

[0141] The test results of the test solution are shown in the following table:

[0142]

[0143] The experimental results show that, under a mobile phase A / organic phase ratio of 84:16, the separation of impurity F in the system suitability solution chromatogram does not meet the requirements. This indicates that the system is more robust at slightly lower organic phase ratios. When the organic phase ratio of mobile phase A is higher than the standard conditions, the separation of impurity F decreases or even fails to meet the requirements. Increasing the organic phase ratio in mobile phase A used in the detection of impurity F will affect the separation, so this should be considered.

[0144] Example 9 Determination of Vitamin B6 Related Substances in Multivitamins for Injection (12)

[0145] (1) Chromatographic conditions: Same as in Example 1.

[0146] (2) Solution preparation

[0147] Blank solvent: water;

[0148] Preparation of reference solution: Take an appropriate amount of vitamin B6 reference substance, weigh accurately, dissolve in water and quantitatively dilute to a solution containing approximately 2.2 μg of vitamin B6 per 1 mL.

[0149] Preparation of test solution: Take an appropriate amount of the contents of the stable multivitamin for injection (12) (approximately equivalent to 5.5 mg of vitamin B6), accurately weigh it, place it in a 25 mL volumetric flask, add water to dissolve it and dilute it to the scale, shake it well;

[0150] The manufacturer of the test samples in this embodiment, batches 1, 2, and 3, is Shandong Lusheng Pharmaceutical Co., Ltd. (trade name: Wanxiwei), and the manufacturer of batches 4, 5, and 6 is Baxter SA (trade name: Shiniweita).

[0151] (3) Determination results: 20 μL of each of the above solutions was accurately measured and injected into the liquid chromatograph respectively. The chromatogram was recorded and the results of the vitamin B6 related substances in the test solution were calculated by the main component external standard method based on the peak area. The results are as follows:

[0152] Test sample batch Impurity C content (%) Impurity F content (%) Batch 1 (long term 6 months - upright -25℃) Not detected Not detected Batch 2 (long term 6 months - upright -25℃) Not detected Not detected Batch 3 (long term 6 months - upright -25℃) Not detected Not detected Batch 4 (long term 6 months at -25°C) 0.10 Not detected Batch 5 (long term -25℃ for 6 months) 0.08 Not detected Batch 6 (long term -25℃ for 6 months) 0.06 Not detected Batch 1 (mid-June - upright -30°C) Not detected Not detected Batch 2 (mid-June - upright -30°C) Not detected Not detected Batch 3 (mid-June - upright -30°C) Not detected Not detected Batch 4 (mid-June -30°C) 0.13 Not detected Batch 5 (mid-June -30°C) 0.13 Not detected Batch 6 (mid-June -30°C) 0.08 Not detected Batch 1 (accelerated 6 months - upright -40°C) 0.67 0.45 Batch 2 (accelerated 6 months - upright -40°C) 0.79 0.43 Batch 3 (accelerated 6 months - upright -40°C) 0.53 0.47 Batch 4 (accelerated 6 months -40℃) 1.18 0.47

[0153] Comparative Example 1

[0154] (1) The solution preparation is the same as in Example 4;

[0155] (2) Chromatographic conditions

[0156] Except for the change of gradient elution procedure, everything else is the same as Example 1;

[0157] The gradient elution program was:

[0158] Time / min Mobile phase A / % Mobile phase B / % 0-45 100 0 45-50 0 100 50-70 0 100 71-100 100 0

[0159] (3) Sampling and result analysis

[0160] Add the test solution and record the chromatogram.

[0161] The experimental results show that: due to the complex composition of multivitamins, the mobile phase ratio and gradient suitable for detecting vitamin B6-related substances in multivitamin preparations are relatively specific. In mobile phase A: potassium dihydrogen phosphate buffer solution (containing sodium hexane sulfonate)-acetonitrile volume ratio is (85:15); mobile phase B: potassium dihydrogen phosphate buffer solution (containing sodium hexane sulfonate)-acetonitrile volume ratio is (30:70); 0.01 mol / L phosphate buffer solution, containing sodium hexane sulfonate at a concentration of 0.2%, the buffer solution is adjusted to pH 5.5 with phosphoric acid, and the gradient program is changed, such as Figure 3 The liquid chromatogram of the test solution shown in the figure shows that the separation degree between impurity F and vitamin B6 is 0.7. The test result is inaccurate, does not meet the requirements, and is not suitable for the detection of vitamin B6-related substances.

Claims

1. A method for detecting vitamin B6-related substances in a multivitamin preparation, characterized in that: The vitamin B6 related substances are vitamin B6 impurities C and F. The structural formula of impurity C is: ； The structural formula of impurity F is: ； The method adopts high performance liquid chromatography to determine the content of the product, and the chromatographic conditions are as follows: The chromatographic column is an octadecylsilane bonded silica gel column or a column with equivalent performance; The flow rate was 1.0 mL / min; Column temperature is 28-40°C; The detection wavelength is 320-340nm; The injection volume is 20μL-50μL; UV detector; Mobile phase A: potassium dihydrogen phosphate buffer solution containing sodium hexanesulfonate-acetonitrile, the potassium dihydrogen phosphate buffer solution was adjusted to pH 5.4-5.5 with phosphoric acid; Mobile phase B: potassium dihydrogen phosphate buffer solution containing sodium hexanesulfonate-acetonitrile, the potassium dihydrogen phosphate buffer solution was adjusted to pH 5.4-5.5 with phosphoric acid; Mobile phases A and B were prepared according to the gradient program: The gradient program is: The percentages of mobile phase A and mobile phase B are by volume.

2. The method according to claim 1, characterized in that The chromatographic column is any one of Sunniest C18, 250 mm×4.6 mm, 5 μm and YMC Triart C18, 250 mm×4.6 mm, 5 μm.

3. The method according to claim 1, characterized in that The gradient program is: The percentages of mobile phase A and mobile phase B are by volume.

4. The method according to claim 1, characterized in that The concentration of sodium hexanesulfonate in the mobile phase A is 0.2%, the concentration of potassium dihydrogen phosphate buffer solution is 0.01 mol / L, and the pH is 5.5; The concentration of sodium hexanesulfonate in the mobile phase B is 0.2%, the concentration of potassium dihydrogen phosphate buffer solution is 0.01 mol / L, and the pH is 5.

5.

5. The method according to claim 1, characterized in that: The volume ratio of the mobile phase A: potassium dihydrogen phosphate buffer solution of sodium hexanesulfonate to acetonitrile is 84-86:16-14; The volume ratio of the mobile phase B: potassium dihydrogen phosphate buffer solution of sodium hexanesulfonate-acetonitrile is 30:

70.

6. The method according to claim 1, characterized in that The volume ratio of the mobile phase A: potassium dihydrogen phosphate buffer solution of sodium hexanesulfonate-acetonitrile is 85:

15.

7. The method according to claim 1, characterized in that: The column temperature was 35°C.

8. The method according to claim 1, characterized in that: The detection wavelength of the ultraviolet detector is 330 nm.

9. The method according to claim 1, characterized in that The limit of quantification of the impurity C is 6.40 ng, and the limit of detection is 2.00 ng; the limit of quantification of the impurity F is 6.60 ng, and the limit of detection is 2.00 ng.

10. The method according to claim 1, characterized in that: The multivitamin preparation includes any one of multivitamins for injection (12), 12 kinds of compound vitamins for injection, water-soluble vitamins for injection, multivitamin injection (13), multivitamins for injection (13), pediatric multivitamin injection (13), and pediatric multivitamin injection (13).

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