Three impurities in vitamin B12 injection and preparation method and application thereof

Abstract

The invention discloses three impurities in B12 injection as well as a preparation method and application thereof. The structures of three impurities in the vitamin B12 injection are respectively shown as an impurity 1, an impurity 2 and an impurity 3. According to the specific embodiment of the invention, the contents of three impurities in the vitamin B12 injection can be gradually increased in the storage period of the vitamin B12 injection, so that the contents exceed the limit of the impurities in the vitamin B12 injection, and the quality of the vitamin B12 injection is possibly influenced. Therefore, the inventor researches and characterizes the impurities, so as to monitor the impurities in the vitamin B12 injection.

Description

Vitamin B 12 Three impurities in injection solutions, their preparation methods and applications Technical Field

[0001] This invention relates to the technical field of analytical chemistry, specifically to vitamin B. 12 Three impurities in the injection solution, their preparation methods, and applications. Background Technology

[0002] In a cool, dry place, vitamin B 12 The shelf life of injectable solutions can reach 2-3 years. However, in environments with relatively poor light protection, impurities are easily generated, affecting the long-term storage of vitamin B12. 12 Three impurities were found in the injection solution, and their size gradually increased with prolonged storage, ultimately affecting vitamin B1. 12 The quality of the injection solution. Specific phenomena are as follows: Vitamin B... 12 During the high-temperature (60°C) test, the injection solution degraded to generate impurities 1, 2, and 3.

[0003] During normal (room temperature) storage, all impurities increased:

[0004] As can be seen, the levels increased during both high-temperature testing and room-temperature storage, exceeding the identification limit of this product. Therefore, impurities were prepared. Based on this, we conducted further investigations into the aforementioned impurities to ensure the quality of Vitamin B1. 12 The quality of the injection solution. Summary of the Invention

[0005] According to an embodiment of the first aspect of the present invention, the present invention provides a vitamin B 12 The structures of the three impurities in the injection solution were clearly characterized, which facilitates their application in subsequent preparation or detection.

[0006] Vitamin B according to specific embodiments of the present invention 12 The structures of the three impurities in the injection solution are shown as impurity 1, impurity 2, and impurity 3, respectively: .

[0007] Vitamin B according to specific embodiments of the present invention 12 The three impurities in the injection solution are in vitamin B. 12 During storage, the content of the injection solution will gradually increase, eventually exceeding that of vitamin B1. 12 The limits of impurities in the injection solution may affect vitamin B12. 12 The quality of the injection solution. Therefore, the inventors studied and characterized the aforementioned impurities in order to improve the quality of vitamin B12.12 Impurities in the injection solution are monitored. Specific characterization methods include proton NMR, carbon NMR, and DEPT spectroscopy. 1 H- 1 HCOSY, HSQC, HMBC, and NOESY spectra were used to characterize impurities 1, 2, and 3. Typical spectra of the characterization results are shown in Figures 1-21.

[0008] According to an embodiment of a second aspect of the present invention, the present invention provides a vitamin B according to an embodiment of a first aspect of the present invention. 12 The preparation method for the three impurities in the injection solution includes the following steps: 1. [The following text appears to be a separate, unrelated section:] Vitamin B... 12 The injection solution was stored at high temperature, and impurities 1, 2, and 3 were obtained, as shown in the following reaction: ; 2. Regarding the aforementioned vitamin B 12 Impurities 1, 2, and 3 generated in the injection solution were separated and enriched: III. Impurities 1, 2, and 3 were prepared using HPLC. The HPLC conditions included: an Agilent ZORBAX Eclipse XDB-C184.6×250mm 5μm column or a column with equivalent performance, using octadecylsilane-bonded silica gel as the packing material; and the following mobile phases: 0.01mol / L ammonium acetate-acetonitrile (90:10 volume) as mobile phase A and acetonitrile as mobile phase B, with gradient elution performed according to the table below.

[0009] The flow rate was 1.0 ml per minute; the column temperature was 30℃; the detection wavelength was 361 nm; the injection volume was 20 μl; and materials with relative retention times of approximately 2.6, 2.9, and 3.6 were collected, corresponding to impurity 1, impurity 2, and impurity 3, respectively.

[0010] Optionally, the high-temperature preservation temperature is not lower than 60°C; the high-temperature preservation duration is 10 days.

[0011] Optionally, the pH value of the 0.01 mol / L ammonium acetate is 3.3.

[0012] Optionally, the pH of the 0.01 mol / L ammonium acetate may be adjusted using acetic acid.

[0013] According to an embodiment of a second aspect of the present invention, the present invention provides a vitamin B according to an embodiment of a first aspect of the present invention. 12 The application of the three impurities in the injection solution, or the three impurities prepared by the preparation method according to the second aspect of the present invention, and the application of the impurities as impurity standards.

[0014] Optionally, the vitamin B... 12Three impurities in the injection solution in vitamin B 12 It is used as an impurity standard in the production or quality control of injection solutions.

[0015] Optionally, the application includes the following steps: preparing a blank solution and a test solution, and preparing a reference solution from the three impurities; detecting the blank solution, test solution, and reference solution using liquid chromatography (LC); the LC conditions are as follows: Column: Agilent ZORBAX Eclipse XDB-C184.6×250mm 5μm column or equivalent column with octadecylsilane-bonded silica gel as the packing material; Mobile phase: 0.05mol / L potassium dihydrogen phosphate-acetonitrile (90:10 v / v) as mobile phase A, acetonitrile as mobile phase B; column temperature: 30℃; detection wavelength: 361nm; injection volume: 20μl; gradient elution is performed according to the table below. .

[0016] Optionally, the preparation method of the reference solution includes the following steps: accurately weigh 2 mg of each of the three impurities, place them in a 20 ml volumetric flask, dissolve and dilute to the mark with 10% acetonitrile, shake well, then accurately measure 1 ml, place it in a 10 ml volumetric flask, dilute to the mark with 10% acetonitrile, shake well, and the solution is obtained.

[0017] Optionally, the pH of the 0.05 mol / L potassium dihydrogen phosphate may be adjusted to 3.3 using phosphoric acid. Attached Figure Description

[0018] Figure 1 shows the 1H NMR spectrum of impurity 1. 1 Figure 2 shows the carbon NMR spectrum of impurity 1 (H-NMR); 13 Figure 3 shows the DEPT spectrum of impurity 1; Figure 4 shows the DEPT spectrum of impurity 1. 1 H- 1 Figure 5 shows the HSQC spectrum of impurity 1; Figure 6 shows the HMBC spectrum of impurity 1; Figure 7 shows the NOESY spectrum of impurity 1; Figure 8 shows the 1H NMR spectrum of impurity 2. 1 H-NMR); Figure 9 shows the carbon NMR spectrum of impurity 2 (H-NMR); 13 (C-NMR); Figure 10 shows the DEPT spectrum of impurity 2; Figure 11 shows the spectrum of impurity 2. 1 H- 1 Figure 12 shows the HSQC spectrum of impurity 2; Figure 13 shows the HMBC spectrum of impurity 2; Figure 14 shows the NOESY spectrum of impurity 2; Figure 15 shows the 1H NMR spectrum of impurity 3. 1 H-NMR); Figure 16 shows the carbon NMR spectrum of impurity 3 (H-NMR); 13 (C-NMR); Figure 17 shows the DEPT spectrum of impurity 3; Figure 18 shows the spectrum of impurity 3. 1 H-1 Figure 19 shows the HSQC spectrum of impurity 3; Figure 20 shows the HMBC spectrum of impurity 3; Figure 21 shows the NOESY spectrum of impurity 3; Figure 22 shows the liquid chromatogram of the blank control of Example 1 of the present invention; Figure 23 shows the chromatogram of the mixed reference solution; Figure 24 shows the chromatogram of the test sample solution of the present invention; Figure 25 shows the linearity of impurity 1 of the present invention; Figure 26 shows the linearity of impurity 2 of the present invention; Figure 27 shows the linearity of impurity 3 of the present invention; Figure 28 shows the linearity of the main component of the present invention. Detailed Implementation

[0019] The following specific embodiments describe vitamin B of the present invention. 12 Three impurities in the injection solution.

[0020] Vitamin B according to specific embodiments of the present invention 12 The structures of the three impurities in the injection solution are shown as impurity 1, impurity 2, and impurity 3, respectively: .

[0021] Vitamin B according to specific embodiments of the present invention 12 The structures of the three impurities in the injection solution are shown as impurity 1, impurity 2, and impurity 3, respectively: .

[0022] After clear characterization, the impurity structures are clear, making them convenient to use as reference standards in production and quality control. Specifically, the characterization of impurities 1, 2, and 3 includes confirmation of their molecular structures and conformations, clearly defining their molecular structures, including their stereoconformities. The specific characterization is as follows: Figure 1 shows the 1H NMR spectrum of impurity 1 (…). 1 ¹H-NMR clearly characterizes the number and type of hydrogen atoms in impurity 1; Figure 2 shows the carbon NMR spectrum of impurity 1. 13 Figure 3 shows the DEPT spectrum of impurity 1, which clearly characterizes the number and types of carbon atoms in impurity 1. Figure 4 shows the DEPT spectrum of impurity 1, used to distinguish different types of carbon (primary carbon, secondary carbon, tertiary carbon, quaternary carbon) in organic molecules, thus supplementing conventional methods. 13 CNMR only provides chemical shifts and cannot directly determine the number of hydrogen atoms bonded to carbon; Figure 4 shows impurity 1. 1 H- 1H COSY spectroscopy reveals the bonding connections between hydrogen nuclei, enabling rapid identification of cross-peaks and construction of spin systems. It can efficiently deduce molecular skeletons and, combined with other two-dimensional spectra, can solve complex structural problems. Figure 5 shows the HSQC spectrum of impurity 1, a two-dimensional heteronuclear correlation NMR spectrum. Its core function is to establish the correspondence between directly linked ¹H and ¹³C. Figure 6 shows the HMBC spectrum of impurity 1. HMBC (Heteronuclear Multiple Bond Correlation) spectroscopy is a two-dimensional heteronuclear NMR spectrum based on long-range ¹H-¹³C coupling. Its core function is to reveal the correlation between ¹H and ¹³C separated by 2-3 chemical bonds. In some cases, it can even detect 4-bond coupling, making it a key tool for resolving complex molecular structures, especially suitable for solving quaternary carbon, heteroatom connection sites, and ring skeleton splicing. Figure 7 shows the NOESY spectrum of impurity 1. NOESY (Nuclear Overhauser Effect Spectroscopy) Effect correlation spectroscopy (ERS) is a two-dimensional nuclear magnetic resonance spectrum based on dipole-dipole interactions between spatially adjacent protons. Its core function is to reveal ¹H-¹H spatial correlations within a distance of less than 5 Å in molecules. It complements ¹H-¹HCOSY, which depends on chemical bond coupling, and is a key tool for resolving molecular stereochemistry, conformation, and intermolecular interactions. The characterization of impurities 2 and 3 is similar to that of other impurities, as shown in Figures 8-21.

[0023] According to an embodiment of the second aspect of the present invention, the present invention provides a method for preparing three impurities in a vitamin B12 injection solution according to an embodiment of the first aspect of the present invention, comprising the following steps: 1. [The method involves] preparing the vitamin B12 injection solution... 12 The injection solution was stored in a high-temperature environment, resulting in impurity 1, impurity 2, and impurity 3, as shown in the following reaction: With this setup, impurity 1, impurity 2 and impurity 3 can be easily prepared by placing them at a higher temperature.

[0024] II. Regarding Vitamin B 12 Impurities 1, 2, and 3 generated in the injection solution are separated and enriched: Impurities 1, 2, and 3 generated in step one are separated and enriched to facilitate the purification and extraction of impurities.

[0025] III. Impurity 1, Impurity 2, and Impurity 3 were prepared using HPLC. HPLC can achieve precise preparation of impurities 1, 2, and 3.

[0026] The HPLC conditions include: an Agilent ZORBAX Eclipse XDB-C18 4.6×250mm 5um column or a column with equivalent performance, using octadecylsilane-bonded silica gel as the packing material; and the following mobile phases: mobile phase A is 0.01mol / L ammonium acetate-acetonitrile at a volume ratio of 90:10, and mobile phase B is acetonitrile, with gradient elution performed according to the table below.

[0027] The flow rate was 1.0 ml / min; the column temperature was 30℃; the detection wavelength was 361 nm; the injection volume was 20 μl; impurities with relative retention times of approximately 2.6, 2.9, and 3.6 were collected, respectively. This setup, by employing appropriate high-performance liquid chromatography (HPLC) conditions, enabled the effective separation and preparation of the three impurities, avoiding cross-contamination or other contamination, and achieving the one-step preparation of three high-purity impurities.

[0028] According to some embodiments of the present invention, the high-temperature storage temperature is not lower than 60°C; the high-temperature storage period is 10 days. This setting, allowing for storage at a relatively high temperature for a period of time, is beneficial for vitamin B. 12 The formation of three impurities in the injection solution.

[0029] According to some embodiments of the present invention, the pH value of 0.01 mol / L ammonium acetate is 3.3. With this setup, using ammonium acetate-acetonitrile as mobile phase A at pH 3.3 allows for more efficient separation of vitamin B. 12 Raw materials and vitamin B 12 It can eliminate three impurities in the injection solution and maintain the stability of the impurity structure.

[0030] According to some embodiments of the present invention, acetic acid is used to adjust the pH value of 0.01 mol / L ammonium acetate. This configuration allows for effective pH adjustment of the sodium acetate solution while maintaining the uniformity of the anions in the sodium acetate solution.

[0031] According to an embodiment of a third aspect of the present invention, the present invention provides a vitamin B according to an embodiment of a first aspect of the present invention. 12 The application of the three impurities in the injection solution, or the three impurities prepared according to the preparation method of the second aspect of the present invention, and the application of the three impurities as impurity standards. Impurities in a drug are closely related to its safety and efficacy stability. Accurate identification of the impurity structure enables scientific analysis and evaluation of the impurities, which is the basis for establishing reliable impurity testing methods and a prerequisite for ensuring comprehensive and effective control of impurities in pharmaceuticals.

[0032] According to some embodiments of the present invention, in vitamin B12 In the production or quality control of injectable solutions, these impurities are used as impurity standards. Studies have found that the above three impurities gradually increase during the stability period. This finding is relevant to vitamin B12. 12 It plays an important role in the production and quality control of vitamin B1, and can provide vitamin B12. 12 This provides a reference standard for the quality control of injectable solutions and the safety testing of clinical medication, thereby ensuring the safety and reliability of clinical medication. Furthermore, it can be used to analyze impurity content and determine reasonable control strategies, which is of great significance for clinical medication safety testing.

[0033] According to some embodiments of the present invention, the steps include: preparing a blank solution, a test solution, and preparing a reference solution for the three impurities; and detecting the blank solution, the test solution, and the reference solution by liquid chromatography.

[0034] The liquid chromatography conditions are as follows: Column: Agilent ZORBAX Eclipse XDB-C18 4.6×250mm 5μm column or equivalent performance column with octadecylsilane-bonded silica gel as the packing material; Mobile phase: 0.05mol / L potassium dihydrogen phosphate-acetonitrile (90:10 v / v) as mobile phase A, acetonitrile as mobile phase B; column temperature: 30℃; detection wavelength: 361nm; injection volume: 20μl; gradient elution is performed according to the table below. .

[0035] According to some embodiments of the present invention, the preparation method of the reference solution includes the following steps: accurately weigh 2 mg of each of the three impurities, place them in separate 20 ml volumetric flasks, dissolve and dilute to the mark with 10% acetonitrile, shake well, then accurately measure 1 ml of each and place them in separate 10 ml volumetric flasks, dilute to the mark with 10% acetonitrile, shake well, and the solution is obtained. This setup allows for the precise preparation of reference solutions for impurities 1, 2, and 3, facilitating the detection and monitoring of these three impurities in vitamin injection solutions.

[0036] According to some embodiments of the present invention, phosphoric acid is used to adjust the pH of 0.05 mol / L potassium dihydrogen phosphate to 3.3. This setting, by using phosphoric acid to adjust the pH of 0.05 mol / L potassium dihydrogen phosphate, can effectively adjust the pH of potassium dihydrogen phosphate while ensuring the uniformity of the anions in the potassium dihydrogen phosphate solution.

[0037] Optionally, the blank solution is 10% acetonitrile. This configuration allows for the elimination of interference from the blank solution in the detection method of this invention by detecting the blank solution.

[0038] The present invention will be further described in detail below with reference to specific exemplary embodiments, performance testing experiments and other details.

[0039] The raw materials, reagents, solvents, etc. used in this invention can all be obtained commercially.

[0040] Example 1 Example 1 provides a vitamin B 12 The structures of the three impurities in the injection solution are shown as impurity 1, impurity 2, and impurity 3, respectively: .

[0041] Its preparation method includes the following steps: 1. Vitamin B 12 The injection solution was stored at high temperature, and impurities 1, 2, and 3 were obtained, as shown in the following reaction: The high-temperature storage temperature should not be lower than 60℃; the high-temperature storage period is 10 days; II. Regarding Vitamin B... 12 Impurities 1, 2, and 3 generated in the injection solution were separated and enriched: III. Impurities 1, 2, and 3 were prepared using HPLC. The HPLC conditions included: an Agilent ZORBAX Eclipse XDB-C184.6×250mm 5μm column or a column with equivalent performance, using octadecylsilane-bonded silica gel as the packing material; the mobile phase was as follows: mobile phase A was 0.01 mol / L ammonium acetate-acetonitrile at a volume ratio of 90:10, and mobile phase B was acetonitrile, with gradient elution performed according to the table below; the pH of the 0.01 mol / L ammonium acetate was 3.3; the pH of the 0.01 mol / L ammonium acetate was adjusted with acetic acid.

[0042] The flow rate was 1.0 ml per minute; the column temperature was 30℃; the detection wavelength was 361 nm; the injection volume was 20 μl; and impurities with relative retention times of approximately 2.6, 2.9, and 3.6 were collected respectively to obtain the final product.

[0043] Example 2 Example 2 provides a vitamin B 12 Three impurities in the injection solution in vitamin B 12 In the production or quality control of the injection solution, it is used as an impurity standard, including the following steps: preparing a blank solution, a test solution, and preparing reference solutions for the three impurities; detecting the blank solution, test solution, and reference solution using liquid chromatography (LC); the LC conditions are as follows: Column: Agilent ZORBAX Eclipse XDB-C184.6×250mm 5μm column or equivalent column with octadecylsilane-bonded silica gel as the packing material; Mobile phase: 0.05mol / L potassium dihydrogen phosphate-acetonitrile (90:10 v / v) as mobile phase A, acetonitrile as mobile phase B; column temperature: 30℃; detection wavelength: 361nm; injection volume: 20μl; flow rate: 1.0ml / min; gradient elution is performed according to the table below. The preparation method of the reference solution includes the following steps: accurately weigh 2 mg of each of the three impurities, place them in 20 ml volumetric flasks, dissolve and dilute to the mark with 10% acetonitrile, shake well, then accurately measure 1 ml of each and place them in 10 ml volumetric flasks, dilute to the mark with 10% acetonitrile, shake well, and the solution is obtained; adjust the pH of 0.05 mol / L potassium dihydrogen phosphate to 3.3 with phosphoric acid; the blank solution is 10% acetonitrile.

[0044] The blank solution is 10% acetonitrile.

[0045] Test solution: Vitamin B1 stored for 3 months 12 Injection solution.

[0046] Example 3 is basically the same as Example 2, except that the pH value is 3.0.

[0047] Example 4 is basically the same as Example 2, except that the pH value is 3.5.

[0048] Example 5 is basically the same as Example 2, except that the column temperature is 28°C.

[0049] Example 6 is basically the same as Example 2, except that the column temperature is 32°C.

[0050] Comparative Example 1 is basically the same as Example 2, except that the flow rate is 0.8 ml.

[0051] Comparative Example 2 is basically the same as Example 2, except that the flow rate is 1.2 ml.

[0052] Comparative Example 3 is basically the same as Example 2, except that mobile phase A is 10 mmol / L ammonium acetate aqueous solution and mobile phase B is methanol.

[0053] The impurity detection levels for each embodiment and comparative example are shown in Table 1 below.

[0054]

[0055] Based on Examples 2-6 and their test results, it can be concluded that the vitamin B provided by this invention... 12 Three impurities in the injection solution in vitamin B 12 In the production or quality control of injectable solutions, its application as an impurity standard can accurately monitor vitamin B1. 12 Three types of impurities in the production or quality control of injectable solutions. High specificity and reliability.

[0056] As shown in Comparative Examples 1-3 and their detection results, the specificity of the method decreases when the detection parameters are changed. For example, in Comparative Examples 1 and 2, when the flow rate of the detection process is changed, the resolution between impurity 2 peak and impurity 1 peak is 1.32 and 1.08, respectively, which is less than the standard resolution (1.5). When the mobile phase is changed, for example in Comparative Example 3, mobile phase A is 10 mmol / L ammonium acetate aqueous solution; mobile phase B is methanol, and the resolution between impurity 2 peak and impurity 1 peak is 0.8, which is less than 1.5.

[0057] Methodology Validation

[0058]

[0059]

[0060]

[0061] 4.1 Specificity assessment of the method's resolution requires that the blank solution and blank excipients do not interfere with the related substances test of this product; in the chromatogram of the test solution, the theoretical plate number is calculated according to vitamin B... 12 Peak calculation not less than 2000, Vitamin B 12 The resolution between the main component peak and adjacent impurity peaks should be greater than 1.5; in the chromatogram of the mixed reference solution, the resolution between the main component peak and adjacent impurity peaks, as well as the resolution between each impurity peak, should meet the requirements.

[0062] Blank solution: 10% acetonitrile.

[0063] Blank excipient solution: Take blank excipient solution.

[0064] Impurity A stock solution: Accurately weigh approximately 1 mg of impurity A reference standard, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with 10% acetonitrile, and shake well. (100 μg / ml) Prepare stock solutions for impurities B, C, D, E, G, H, and impurities 1, 2, and 3 using the same method as the impurity A stock solution.

[0065] Impurity A positioning solution: Accurately measure 1 ml of impurity A stock solution and place it in a 10 ml volumetric flask. Dilute to the mark with 10% acetonitrile and shake well. (10 μg / ml) Prepare positioning solutions for impurities B, C, D, E, G, H, and impurities 1, 2, and 3 using the same method as the impurity A positioning solution.

[0066] Mixed reference solution: Take vitamin B 12Weigh approximately 10 mg of the reference standard accurately and place it in a 10 ml volumetric flask. Add 1 ml each of impurity A, B, C, D, E, G, H and impurity 1, 2, 3 stock solutions. Dissolve and dilute to the mark with 10% acetonitrile and shake well.

[0067] Reference solution: Take vitamin B 12 Accurately weigh approximately 2 mg of the reference standard and place it in a 20 ml volumetric flask. Dissolve and dilute to the mark with 10% acetonitrile, and shake well. Then accurately measure 1 ml of the solution and place it in a 10 ml volumetric flask. Dilute to the mark with 10% acetonitrile and shake well. (10 μg / ml) Test solution: Take this product.

[0068] Accurately measure blank solution, blank excipient solution, reference solution, sensitivity solution, mixed reference solution, each positioning solution, and each test solution, and inject them into the liquid chromatograph separately, and record the chromatograms.

[0069]

[0070] Conclusion: Neither the blank solution (see Figure 22) nor the blank excipient interfered with the related substances test of this product; in the chromatogram of the test solution (see Figure 24), the theoretical plate number was determined according to vitamin B... 12 The peak value is calculated to be 7322, which is greater than 2000, indicating vitamin B. 12 The resolution between each peak and adjacent impurity peaks was greater than 1.5 (minimum value 2.63); in the chromatogram of the mixed reference solution (see Figure 23), the resolution between each impurity peak and between the main component and adjacent peaks was greater than 1.5 (minimum value 1.75), which meets the requirements. Therefore, the method specificity of related substances of this product is good.

[0071] 4.2 Limit of Quantitation (LOQ) and Limit of Detection (LOD) are defined as the concentration at a signal-to-noise ratio (SNR) of approximately 10 and the concentration at a SNR of approximately 3.

[0072] Take vitamin B 12 In addition, appropriate amounts of impurity 1, 2, and 3 reference standards were quantitatively diluted to prepare limit of quantitation solutions and limit of detection solutions.

[0073] Accurately measure the limit of quantitation solution and limit of detection solution, inject them into the liquid chromatograph, and record the chromatogram.

[0074]

[0075] Conclusion: The limits of quantitation and detection of impurities 1, 2, 3 and the main component all meet the requirements, and the sensitivity is good, satisfying the detection needs.

[0076] 4.3 Linearity and Range, Correction Factor: Examine the linearity of each impurity content within the LOQ range of 2% (equivalent to the limit concentration percentage, see the table below); linear regression with concentration as the x-axis and peak area as the y-axis is required, with a correlation coefficient r ≥ 0.995 and the y-intercept within 15% of the 100% limit response value.

[0077]

[0078] Limit of Quantification Solution: Take the limit of quantification solution for each impurity from the "Limit of Quantification and Limit of Detection" list.

[0079] Impurity 1 Stock Solution: Accurately weigh approximately 10 mg of Impurity 1 reference standard, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with 10% acetonitrile, and shake well. (1000 μg / ml) Prepare Impurity 2, 3, and Vitamin B using the same method as the Impurity 1 stock solution. 12 Stock solution.

[0080] Linear stock solution: Accurately measure impurities 1, 2, 3, and vitamin B. 12 Add 1 ml of each stock solution to a 20 ml volumetric flask, dissolve and dilute to the mark with 10% acetonitrile, and mix well. (50 μg / ml)

[0081] Accurately measure each linear solution, inject it into the liquid chromatograph, and record the chromatogram (see Figures 25-28).

[0082]

[0083] 4.7 Accuracy test: When the limits of each impurity are approximately 25%, 50%, 100%, and 150%, the average recovery rate should be between 80% and 120%, and the RSD should be ≤20% (n=12).

[0084] Blank solution: 10% acetonitrile. Blank excipient solution: Take blank excipient.

[0085] Reference solution: Take vitamin B 12 Weigh approximately 2 mg of the reference standard accurately and place it in a 20 ml volumetric flask. Dissolve and dilute to the mark with 10% acetonitrile, and shake well. Then accurately measure 1 ml of the reference standard and place it in a 10 ml volumetric flask. Dilute to the mark with 10% acetonitrile and shake well.

[0086] Impurity 1 stock solution: Accurately weigh approximately 10 mg of impurity 1 reference standard, place it in a 10 ml volumetric flask, dissolve and dilute to the mark with 10% acetonitrile, and shake well. (1000 μg / ml) Prepare impurity 2 and 3 stock solutions using the same method as impurity 1 stock solution.

[0087] Impurity Mixture Stock Solution: Accurately measure 5 ml each of impurity 2 and 3 stock solutions, place them in the same 50 ml volumetric flask, dilute to the mark with 10% acetonitrile, and mix well. (Impurity concentration: 100 μg / ml) Impurity Mixture Solution: Accurately measure 1 ml of the impurity mixture stock solution, place it in a 10 ml volumetric flask, dilute to the mark with 10% acetonitrile, and mix well. (Impurity concentration: 10 μg / ml) Background Solution: Take this product; it is ready.

[0088] 50% Accuracy Solution: Accurately measure 1 ml of the impurity mixture stock solution and place it in a 20 ml volumetric flask. Dilute to the mark with this product and mix well. (Impurity concentration: 5 μg / ml) Prepare three parallel aliquots of 100% Accuracy Solution: Accurately measure 2 ml of the impurity mixture stock solution and place it in a 20 ml volumetric flask. Dilute to the mark with this product and mix well. (Impurity concentration: 10 μg / ml) Prepare three parallel aliquots of 150% Accuracy Solution: Accurately measure 3 ml of the impurity mixture stock solution and place it in a 20 ml volumetric flask. Dilute to the mark with this product and mix well. (Impurity concentration: 15 μg / ml) Prepare three parallel aliquots of the blank solution, blank excipient solution, reference solution, sensitivity solution, impurity mixture solution, and each accuracy solution. Inject each aliquot into the liquid chromatograph and record the chromatogram.

[0089]

[0090]

[0091]

[0092] Conclusion: Using the principal component external standard method with correction factor, the average recoveries of impurities 1, 2, and 3, approximately 0.5%, 1.0%, and 1.5% (approximately equivalent to 50%, 100%, and 150% limits), were all within the range of 80%–120%, with RSDs all less than 20% (maximum value 0.98%), meeting the requirements. Therefore, the related substances method for this product has good accuracy.

[0093] Sample test results

[0094]

[0095]

[0096]

[0097] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. Vitamin B 12 The three impurities in the injection solution are characterized by, The structures of impurities 1, 2, and 3 are shown below: 。 2. The vitamin B according to claim 1 12 A method for preparing three impurities in an injection solution, characterized in that, The steps include:

1. Adding the vitamin B... 12 The injection solution was stored at high temperature, and impurities 1, 2, and 3 were obtained, as shown in the following reaction: ; 2. Regarding the aforementioned vitamin B 12 Impurities 1, 2, and 3 generated in the injection solution were separated and enriched: III. Impurities 1, 2, and 3 were prepared using HPLC. The HPLC conditions included: an Agilent ZORBAX Eclipse XDB-C18 4.6×250mm 5μm column or a column with equivalent performance, using octadecylsilane-bonded silica gel as the packing material; and the following mobile phases: 0.01mol / L ammonium acetate-acetonitrile (90:10 volume) as mobile phase A and acetonitrile as mobile phase B, with gradient elution performed according to the table below: The flow rate was 1.0 ml per minute; the column temperature was 28℃~32℃; the detection wavelength was 361 nm; the injection volume was 20 μl; materials with relative retention times of approximately 2.6, 2.9, and 3.6 were collected, corresponding to impurity 1, impurity 2, and impurity 3, respectively.

3. The preparation method according to claim 2, characterized in that, The high-temperature preservation temperature is not lower than 60°C; the high-temperature preservation time is 10 days.

4. The preparation method according to claim 2, characterized in that, The pH value of the 0.01 mol / L ammonium acetate is 3.0~3.

5.

5. The preparation method according to claim 4, characterized in that, The pH value of the 0.01 mol / L ammonium acetate was adjusted using acetic acid.

6. The vitamin B according to claim 1 12 The application of the three impurities in the injection solution or the three impurities prepared by the method according to any one of claims 2-5, characterized in that, The three impurities are used as impurity standards.

7. The application according to claim 6, characterized in that, The vitamin B mentioned 12 Three impurities in the injection solution in vitamin B 12 It is used as an impurity standard in the production or quality control of injection solutions.

8. The application according to claim 7, characterized in that, The procedure includes the following steps: preparing a blank solution and a test solution, and preparing a reference solution for the three impurities; detecting the blank solution, test solution, and reference solution using liquid chromatography (LC). The LC conditions are as follows: Column: Agilent ZORBAX Eclipse XDB-C18 4.6×250mm 5μm column or a column with equivalent performance, with octadecylsilane-bonded silica gel as the packing material; Mobile phase: 0.05mol / L potassium dihydrogen phosphate-acetonitrile (90:10 v / v) as mobile phase A and acetonitrile as mobile phase B; column temperature: 28℃~32℃; detection wavelength: 361nm; injection volume: 20μl; flow rate: 1.0ml / min; gradient elution is performed according to the table below. 。 9. The application according to claim 8, characterized in that, The preparation method of the reference solution includes the following steps: Take 2 mg of each of the three impurities, accurately weigh them, place them in 20 ml volumetric flasks, add 10% acetonitrile to dissolve and dilute to the mark, shake well, then accurately measure 1 ml of each and place them in 10 ml volumetric flasks, dilute to the mark with 10% acetonitrile, shake well, and the solution is obtained.

10. The application according to claim 8, characterized in that, The pH of the 0.05 mol / L potassium dihydrogen phosphate was adjusted to 3.0-3.5 using phosphoric acid.

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