A method for detecting related substances in mecobalamin tablets

Abstract

This invention relates to the field of pharmaceutical analysis and detection technology, and proposes a method for detecting related substances in methylcobalamin tablets. The method employs reversed-phase high-performance liquid chromatography (RP-HPLC) with an ultraviolet (UV) detector. The chromatographic conditions of the RHPLC-UV detector method include: mobile phases A and B; mobile phase A is a 92:8 mixture of potassium dihydrogen phosphate solution and acetonitrile, and mobile phase B is a 92:8 mixture of acetonitrile and water; gradient elution is used. This technical solution solves the problem of inaccurate detection of related substances in methylcobalamin tablets in related technologies.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical analysis and detection technology, specifically to a method for detecting related substances in methylcobalamin tablets. Background Technology

[0002] Methylcobalamin, full name Coα-[α-(5,6-dimethylbenzimidazolyl)]-Coβ-methylcobalamin, is a vitamin B12 compound. 12 A member of the vitamin family, and the only vitamin in the human body containing a metallic element. Its main indication is peripheral neuropathy. Methylcobalamin is an endogenous form of vitamin B. 12 It exists in blood and bone marrow, along with vitamin B. 12 In comparison, it has a good effect on improving neuronal conduction. It can promote nucleic acid-protein-lipid metabolism through methyl conversion reaction. As a coenzyme of methionine synthase, it can convert homocysteine ​​into methionine, participate in the process of deoxynucleosides synthesizing thymine, promote nucleic acid and protein synthesis, promote intraaxonal transport and axonal regeneration and myelin formation, prevent axonal degeneration, and repair damaged nerve tissue.

[0003]

[0004] The figure shows the structural formula of mecobalamin. Mecobalamin is available in tablet, capsule, and injection forms, with tablets (0.5mg) being the most widely sold. As a photosensitizing drug, mecobalamin tablets are typically film-coated or sugar-coated. Excipients mainly include lactose, corn starch, microcrystalline cellulose, magnesium (calcium) stearate, and film-coating premixes.

[0005] There are currently nine quality standards for mecobalamin tablets, namely the 2025 edition of the Chinese Pharmacopoeia, Part II (hereinafter referred to as the Pharmacopoeia Method), the 18th edition of the Japanese Pharmacopoeia, the original imported registration standard, and six enterprise registration standards. The quality standard for mecobalamin capsules is only the 2025 edition of the Chinese Pharmacopoeia, Part II. Except for the Japanese Pharmacopoeia, all other standards include a "related substances" test item, and the methods are basically consistent with the Pharmacopoeia methods.

[0006] Based on the manufacturing process of methylcobalamin tablets, it is known that the relevant substances in methylcobalamin tablets should include at least the known impurity vitamin B. 12 Photodegradation products such as hydroxycobalamin and other unknown impurities are detected, but existing detection methods have shortcomings: on the one hand, they do not detect vitamin B12. 12 On the one hand, under these chromatographic conditions, the retention time of the hydroxycobalamin peak is relatively short, making it difficult to achieve baseline separation from the excipient and solvent peaks. This interferes with accurate quantification, leading to deviations in the results and hindering the safety control of methylcobalamin tablets.

[0007] Therefore, it is necessary to propose a detection method that can more comprehensively and accurately assess the related substance safety of methylcobalamin tablets. Summary of the Invention

[0008] This invention proposes a method for detecting related substances in methylcobalamin tablets, which solves the problem of inaccurate detection of related substances in methylcobalamin tablets in related technologies.

[0009] The technical solution of this invention is as follows: This invention proposes a method for detecting related substances in methylcobalamin tablets, using reversed-phase high-performance liquid chromatography-ultraviolet detection (RP-HPLC-UV detector) to detect related substances in methylcobalamin tablets; the chromatographic conditions of the R-HPLC-UV detector method include: The mobile phase includes mobile phase A and mobile phase B; mobile phase A is a potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 92:8, and mobile phase B is acetonitrile and water in a volume ratio of 92:8. The elution process uses gradient elution.

[0010] As a further technical solution, the gradient elution procedure is as follows: From 0 min to 8 min, the volume percentage of mobile phase A changed from 100% to 92%, and the volume percentage of mobile phase B changed from 0% to 8%. From 8 min to 30 min, the volume percentage of mobile phase A was maintained at 92%, and the volume percentage of mobile phase B was maintained at 8%. Over 30-31 minutes, the volume percentage of mobile phase A changed from 92% to 100%, and the volume percentage of mobile phase B changed from 8% to 0%.

[0011] As a further technical solution, the concentration of the potassium dihydrogen phosphate solution is 0.025~0.034 mol / L, preferably 0.03 mol / L, and the pH value of the potassium dihydrogen phosphate solution is 4.4~4.6, preferably 4.5.

[0012] As a further technical solution, the chromatographic conditions also include: the packing material is octadecylsilane-bonded silica gel, the detection wavelength is 340~344nm, preferably 342nm, the column temperature is 30~40℃, preferably 35℃, the injection volume is 5~50μL, preferably 20μL, and the flow rate is 0.8~1.2mL / min, preferably 0.9~1mL / min, and more preferably 1mL / min.

[0013] As a further technical solution, the method for detecting related substances in the methylcobalamin tablets includes the following steps: Preparation of test solution: Weigh the fine powder of methylcobalamin tablets, add it to mobile phase A to dissolve and dilute, filter, and obtain the test solution; Control solution: Measure the test solution and dilute it with mobile phase A to obtain the control solution; Systemic adaptability solution: Weigh out hydroxycobalamin and vitamin B1, respectively. 12 The reference standard and methylcobalamin reference standard were dissolved and diluted in mobile phase A to obtain a system suitability solution. The test solution, the control solution, and the system suitability solution were subjected to reversed-phase liquid chromatography-ultraviolet detection to obtain chromatograms.

[0014] As a further technical solution, in the test sample solution, the mass-to-volume ratio of methylcobalamin in the methylcobalamin tablet to the mobile phase A is 0.5 mg: 1 mL.

[0015] As a further technical solution, the volume concentration of the test solution in the control solution is 1%.

[0016] As a further technical solution, in the system suitability solution, the mass-to-volume ratio of hydroxycobalamin to mobile phase A is 0.5 μg:1 mL, and vitamin B... 12 The mass-to-volume ratio of the reference standard to mobile phase A was 0.5 μg:1 mL, and the mass-to-volume ratio of the methylcobalamin reference standard to mobile phase A was 0.5 mg:1 mL.

[0017] As a further technical solution, the pH adjuster of the potassium dihydrogen phosphate solution includes sodium hydroxide solution or phosphoric acid solution, wherein the concentration of sodium hydroxide solution is 0.15~0.24 mol / L or 0.2 mol / L, and the concentration of phosphoric acid solution is 0.15~0.24 mol / L or 0.2 mol / L.

[0018] The working principle and beneficial effects of this invention are as follows: In the detection method for related substances in methylcobalamin tablets of this invention, a gradient elution method is employed. Initially, the proportion of acetonitrile in mobile phase A is relatively low, which prolongs the retention of hydroxycobalamin, separating the excipient peak, water peak, and solvent peak. This improves the specificity of the detection method and facilitates accurate quantification of hydroxycobalamin. It effectively solves the problems of short retention time of hydroxycobalamin and difficulty in separating excipient and solvent peaks in existing detection processes, leading to result deviations. Attached Figure Description

[0019] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0020] Figure 1 This is a chromatogram of the test solution of Example 1 of the present invention; Figure 2 This is a chromatogram of the control solution of Example 1 of the present invention; Figure 3This is a chromatogram of the system-adaptive solution of Example 1 of the present invention; Figure 4 This is the detection chromatogram of the test solution of Comparative Example 1 of the present invention; Figure 5 This is the detection chromatogram of the test solution of Comparative Example 2 of the present invention; Figure 6 This is the detection chromatogram of the test solution of Comparative Example 3 of the present invention; Figure 7 This is the detection chromatogram of the test solution of Comparative Example 4 of the present invention; Figure 8 This is the detection chromatogram of the test solution of Comparative Example 5 of the present invention; Figure 9 This is the detection chromatogram of the test solution of Comparative Example 6 of the present invention; Figure 10 This is a chromatogram of the photodamage specificity test of the present invention; Figure 11 This is a chromatogram of the alkali degradation specificity test of the present invention; Figure 12 This is a chromatogram of the acid degradation specificity test of the present invention; Figure 13 This is a chromatogram of the oxidative damage specificity test of the present invention; Figure 14 This is a chromatogram of the high-temperature damage specificity test of the present invention. Detailed Implementation

[0021] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0022] In the following examples and comparative examples: The instruments used were: a U3000 high-performance liquid chromatograph equipped with an ultraviolet detector; and an XS-105 electronic balance. Mecobalamin tablets: 0.5mg, from Eisai (China) Pharmaceutical Co., Ltd.; Hydroxycobalamin: USP reference standard; Vitamin B 12 Reference standard: Reference standard from the National Institutes for Food and Drug Control (NIFDC); Methylcobalamin reference standard: National Institutes for Food and Drug Control (NIFDC) reference standard; Potassium dihydrogen phosphate: from Tianjin Kemeo Reagent Co., Ltd.; Acetonitrile: from Merck Reagents GmbH, Germany; Water: Homemade water from a Merck Millpore IQ7000 ultrapure water system.

[0023] Example 1 1. Chromatographic conditions Column: The packing material is octadecylsilane-bonded silica gel; Mobile phase A consists of potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 92:8, and mobile phase B consists of acetonitrile and water in a volume ratio of 92:8. The concentration of potassium dihydrogen phosphate solution is 0.03 mol / L, and the pH is adjusted to 4.5 using 0.2 mol / L sodium hydroxide solution or phosphoric acid solution. The gradient elution procedure is as follows:

[0024] The detection wavelength was 342 nm, the column temperature was 35 ℃, the injection volume was 20 μL, and the flow rate was 1 mL / min. 2. Preparation of the test solution Weigh out the methylcobalamin tablets, grind them into a fine powder, weigh an appropriate amount of the fine powder, add it to mobile phase A to dissolve and dilute, filter, and obtain the test solution. The mass-volume ratio of methylcobalamin in the methylcobalamin tablets to mobile phase A in the test solution is 0.5 mg: 1 mL. 3. Preparation of control solution Measure out the test solution, dilute it with mobile phase A to obtain a control solution, the volume concentration of the test solution in the control solution being 1%; 4. Preparation of system-adaptive solutions Weigh out hydroxycobalamin and vitamin B separately. 12 The reference standard and methylcobalamin reference standard were dissolved and diluted in mobile phase A to obtain a system suitability solution, wherein the mass-to-volume ratio of hydroxycobalamin to mobile phase A was 0.5 μg:1 mL, and vitamin B... 12 The mass-to-volume ratio of the reference standard to mobile phase A was 0.5 μg:1 mL, and the mass-to-volume ratio of the methylcobalamin reference standard to mobile phase A was 0.5 mg:1 mL. The test solution, control solution, and system suitability solution were subjected to reversed-phase liquid chromatography-ultraviolet detection to obtain chromatograms, among which... Figure 1 This is the chromatogram of the test solution from Example 1. Figure 2 This is the chromatogram of the control solution from Example 1. Figure 3 This is a chromatogram of the system suitability solution from Example 1.

[0025] Comparative Example 1 Compared to Example 1, Comparative Example 1 differs in that the mobile phase in this comparative example is a fixed ratio of 84:16 potassium dihydrogen phosphate solution-acetonitrile, wherein the pH of the potassium dihydrogen phosphate solution is adjusted to 4.5 with 0.2 mol / L sodium hydroxide solution or phosphoric acid, and isocratic elution is used. The system suitability solution does not contain vitamin B. 12 And hydroxycobalamin.

[0026] Figure 4 The chromatogram is for the test solution of Comparative Example 1.

[0027] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the mobile phase in this comparative example is only mobile phase A; Figure 5 The chromatogram is for the detection of the test solution in Comparative Example 2.

[0028] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that the mobile phase in this comparative example is only mobile phase B; Figure 6 The chromatogram is for the test solution of Comparative Example 3.

[0029] Comparative Example 4 Compared with Example 1, Comparative Example 4 differs in that, in this comparative example, mobile phase A is a potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 85:15, and mobile phase B is acetonitrile and water in a volume ratio of 92:8. Figure 7 The chromatogram is for the test solution of Comparative Example 4.

[0030] Comparative Example 5 Compared with Example 1, Comparative Example 5 differs in that, in this comparative example, mobile phase A is a potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 92:8, and mobile phase B is acetonitrile and water in a volume ratio of 85:15. Figure 8 The chromatogram is for the test solution of Comparative Example 5.

[0031] Comparative Example 6 Compared with Example 1, Comparative Example 6 differs in that, in this comparative example, mobile phase A is a potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 85:15, and mobile phase B is acetonitrile and water in a volume ratio of 85:15. Figure 9 The chromatogram is for the test solution of Comparative Example 6.

[0032] A comparison of Example 1 and Comparative Example 1 shows that the detection method of the present invention has good specificity, not only solving the problems in the original method, but also more accurately detecting the content of hydroxycobalamin, and simultaneously detecting the impurity vitamin B.12 This, to a certain extent, better ensures the safety of mecobalamin tablets and provides better technical support for the safety of medication use by the public.

[0033] Compared with Comparative Examples 2 and 3, Example 1 shows that the solvent / excipient peak in Comparative Example 2 interferes with the determination of the known impurity hydroxycobalamin. In Comparative Example 3, the content of acetonitrile in the organic phase is relatively high, and the elution ability is strong, so the detection purpose cannot be achieved.

[0034] Comparing Example 1 with Comparative Examples 4, 5, and 6, it can be seen that in Comparative Example 4, increasing the acetonitrile content in mobile phase A significantly affects the peak time of methylcobalamin, which is delayed from 2 minutes to 5 minutes. In Comparative Example 5, the delayed peak time of methylcobalamin makes it difficult to elute some less polar impurities, resulting in a lower number of detected impurities and hindering the detection of related substances. In Comparative Example 6, the peak shape of methylcobalamin is poor, failing to achieve the detection objective.

[0035] 5. Methodological Validation 5.1 Specificity Test 20 μL each of the methylcobalamin reference solution and negative control solution, after being subjected to strong acid, strong alkali, high temperature, light, and oxidation degradation, were injected into the liquid chromatograph to investigate their specificity. The various degradation conditions are shown in Table 1. Table 1. Specific Forced Degradation Conditions

[0036] Depend on Figures 10-14 It can be seen that, using the chromatographic conditions of Example 1 of this scheme, the impurities produced by methylcobalamin after being subjected to strong acid, strong alkali, oxidation and light damage can all be separated from the main peak at the baseline, meeting the relevant requirements of the 2025 edition of the Chinese Pharmacopoeia.

[0037] Figure 10 This is a chromatogram of the light-damaged components of the present invention; Figure 11 This is the chromatogram of the alkali-degraded material in this invention; Figure 12 This is a chromatogram of the acid-degraded components of the present invention; Figure 13 This is a chromatogram of the oxidative damage of the present invention; Figure 14 This is a chromatogram of the high-temperature destruction of the present invention.

[0038] 5.2 Examination of Linear Relationships Linear regression was performed with the mass concentration of methylcobalamin as the x-axis and the peak area as the y-axis. The regression equation was y = 0.1911x + 0.1926, with a correlation coefficient of 1.0000. This indicates a good linear relationship within the mass concentration range of 1.09 μg / ml to 109 μg / ml.

[0039] 5.3 Precision Test One portion of the test solution was injected six times consecutively to examine the relative standard deviation of the content of each impurity. The results showed that the RSD6 for hydroxycobalamin was 1%, and for vitamin B... 12 The RSD6 is 0.2%, the RSD6 of a single impurity is 0.3%, and the RSD6 of the total impurities is 0.

[0040] 5.4 Repeatability Test Six parallel test solutions were prepared from a batch of methylcobalamin tablets. Related substances were detected according to the method described in the examples, and the RSD of the results was examined. The RSD of hydroxycobalamin content was 1%, and vitamin B... 12 The RSD6 is 0.3%, and the RSD6 of the total impurities is 0.

[0041] 5.5 Recovery Rate Examination Prepare 18 portions of negative control solution (weigh 1g of a mixture of all excipients used in the tablet preparation, accurately add 10ml of mobile phase A to dissolve, and use this as the negative control solution), each approximately 1g, and add hydroxycobalamin reference solution and vitamin B. 12 The reference solution and mobile phase A were used to prepare cobalt amine reference standard and vitamin B, respectively. 12 The reference standards were solutions at concentrations of 80%, 100%, and 120% of the limit level, which were used as solutions for recovery determination. The recovery rates of the two known impurities were determined according to the detection method in Example 1.

[0042] The results are shown in Table 2: Table 2 Results of Method Recovery Determination

[0043] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for detecting related substances in methylcobalamin tablets, characterized in that, The reversed-phase high-performance liquid chromatography-ultraviolet detection method was used to detect related substances in methylcobalamin tablets; the chromatographic conditions of the reversed-phase high-performance liquid chromatography-ultraviolet detection method included: The mobile phase includes mobile phase A and mobile phase B; mobile phase A is a potassium dihydrogen phosphate solution and acetonitrile in a volume ratio of 92:8, and mobile phase B is acetonitrile and water in a volume ratio of 92:

8. The elution process uses gradient elution.

2. The method for detecting related substances in methylcobalamin tablets according to claim 1, characterized in that, The gradient elution procedure is as follows: From 0 min to 8 min, the volume percentage of mobile phase A changed from 100% to 92%, and the volume percentage of mobile phase B changed from 0% to 8%. From 8 min to 30 min, the volume percentage of mobile phase A was maintained at 92%, and the volume percentage of mobile phase B was maintained at 8%. Over 30-31 minutes, the volume percentage of mobile phase A changed from 92% to 100%, and the volume percentage of mobile phase B changed from 8% to 0%.

3. The method for detecting related substances in methylcobalamin tablets according to claim 1, characterized in that, The concentration of the potassium dihydrogen phosphate solution is 0.025~0.034 mol / L, and the pH value of the potassium dihydrogen phosphate solution is 4.4~4.

6.

4. The method for detecting related substances in methylcobalamin tablets according to claim 1, characterized in that, The chromatographic conditions also include: the packing material is octadecylsilane-bonded silica gel, the detection wavelength is 340~344nm, the column temperature is 30~40℃, the injection volume is 5~50μL, and the flow rate is 0.8~1.2mL / min.

5. The method for detecting related substances in methylcobalamin tablets according to claim 1, characterized in that, Includes the following steps: Preparation of test solution: Weigh out fine powder of methylcobalamin tablets, add mobile phase A to dissolve and dilute, filter, and obtain test solution; Control solution: Measure the test solution and dilute it with mobile phase A to obtain the control solution; Systemic adaptability solution: Weigh out hydroxycobalamin and vitamin B1, respectively. 12 The reference standard and methylcobalamin reference standard were dissolved and diluted in mobile phase A to obtain a system suitability solution. The test solution, the control solution, and the system suitability solution were subjected to reversed-phase high-performance liquid chromatography-ultraviolet detection to obtain chromatograms.

6. The method for detecting related substances in methylcobalamin tablets according to claim 5, characterized in that, In the test solution, the mass-to-volume ratio of methylcobalamin in the methylcobalamin tablet to the mobile phase A is 0.5 mg: 1 mL.

7. The method for detecting related substances in methylcobalamin tablets according to claim 5, characterized in that, In the control solution, the volume concentration of the test solution is 1%.

8. The method for detecting related substances in methylcobalamin tablets according to claim 5, characterized in that, In the system suitability solution, the mass-to-volume ratio of hydroxycobalamin to mobile phase A is 0.5 μg:1 mL, and vitamin B... 12 The mass-to-volume ratio of the reference standard to mobile phase A was 0.5 μg:1 mL, and the mass-to-volume ratio of the methylcobalamin reference standard to mobile phase A was 0.5 mg:1 mL.

9. The method for detecting related substances in methylcobalamin tablets according to claim 3, characterized in that, The pH adjuster of the potassium dihydrogen phosphate solution includes sodium hydroxide solution or phosphoric acid solution, wherein the concentration of sodium hydroxide solution is 0.15~0.24 mol / L and the concentration of phosphoric acid solution is 0.15~0.24 mol / L.

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