Method for detecting related substances in cobamamide capsule

Abstract

The invention discloses a method for detecting related substances in cobamamide capsules, which adopts high performance liquid chromatography for detection, and the chromatographic conditions are as follows: a chromatographic column takes octadecylsilane chemically bonded silica with the particle size of 3-5 microns as a filler; the column length is 150-250 mm, and the column inner diameter is 3-4.6 mm; a mobile phase: a mobile phase A is a monopotassium phosphate solution with the concentration of 0.045-0.055 mol / L and the pH value of 4.8-5.2; the mobile phase B is methanol; and gradient elution. The detection method is easy to operate, high in sensitivity, good in specificity and high in accuracy, the content of specific impurities (adenosine, hydroxycobalamin, cyanocobalamin and mecobalamin) and non-specific impurities in the cobalamin capsule can be accurately controlled, the quality of the cobalamin capsule is effectively controlled, and the detection method has important significance on quality control of the cobalamin capsule.

Description

Technical Field

[0001] This application belongs to the technical field of pharmaceutical analysis and detection, and specifically relates to a method for detecting related substances in adenosylcobalamin capsules. Background Art

[0002] Adenosylcobalamin capsules are vitamins, mainly used for the prevention and treatment of vitamin B12 deficiency, megaloblastic anemia, and the treatment of neurological diseases related to pernicious anemia. The original research of adenosylcobalamin capsules was by Eisai Co., Ltd., which was approved for marketing in Japan on January 19, 1967. The marketed specifications were 0.25 mg (this specification was withdrawn from the market on April 1, 1995) and 0.5 mg, with the trade name Hycobal, and it passed the re-evaluation in Japan in April 1976. The Japanese Orange Book determined the 0.5 mg specification of adenosylcobalamin capsules of Eisai Co., Ltd. as the reference preparation. In March 1974, Tsuruha Pharmaceutical Co., Ltd. was approved for marketing a 0.25 mg specification of adenosylcobalamin capsules in Japan with a generic drug application.

[0003] Currently, the quality standards of adenosylcobalamin raw materials are included in both ChP2020 and USP-NF2024. ChP2020 includes the quality standards of adenosylcobalamin tablets, but only controls hydroxocobalamin (UV method) and does not include the related substances inspection item. In addition, the detection methods for related substances in adenosylcobalamin injections and adenosylcobalamin tablets have also been publicly reported in the existing technical literature. Currently, the quality standards of adenosylcobalamin capsules have not been included or reported in the pharmacopoeias of various countries and the existing technical literature.

[0004] Adenosylcobalamin capsules are small-specification preparations, and the main drug adenosylcobalamin accounts for only 0.4%. The components of 99.6% are the fillers corn starch and microcrystalline cellulose. Therefore, compared with tablets or injections, there are more excipients in adenosylcobalamin capsules, which requires higher separation requirements and greater difficulty for the extraction of test samples and detection methods. Therefore, the chromatographic conditions under the related substances item in the standards of the raw materials included in ChP2020 and EP11.0, as well as the detection conditions for injections and tablets in the existing technical literature, cannot be directly applied to the analysis and detection of adenosylcobalamin capsules.

[0005] Therefore, there is an urgent need to develop an analytical and detection method for related substances in adenosylcobalamin capsules. Summary of the Invention

[0006] In view of this, the primary objective of this application is to provide a method for detecting related substances in adenosylcobalamin capsules. This detection method uses high-performance liquid chromatography, which is simple to operate, has high sensitivity, good specificity, and high accuracy. It can accurately control the content of specific impurities (adenosine, hydroxocobalamin, cyanocobalamin, methylcobalamin) and non-specific impurities in this product, and can effectively control the quality of adenosylcobalamin capsules, which is of great significance for the quality control of adenosylcobalamin capsules.

[0007] To achieve the above object, the present application adopts the following technical solutions:

[0008] The present application discloses a method for detecting related substances in adenosylcobalamin capsules, which is detected by high performance liquid chromatography. The chromatographic conditions of the high performance liquid chromatography are as follows:

[0009] Chromatographic column: Octadecylsilane chemically bonded silica gel is used as the packing material, and the particle size of the packing material is 3 - 5μm; the length of the chromatographic column is 150 - 250mm, and the inner diameter is 3 - 4.6mm;

[0010] Mobile phase: Mobile phase A is a potassium dihydrogen phosphate solution with a concentration of 0.045 - 0.055mol / L and a pH of 4.8 - 5.2; mobile phase B is methanol; gradient elution;

[0011] Sample injection volume: 50 - 100μl; flow rate: 0.8 - 1.2ml / min; detection wavelength: 258 - 262nm; column temperature: 35 - 42°C; injector temperature: 4 - 25°C.

[0012] Further, the chromatographic conditions of the high performance liquid chromatography are as follows:

[0013] Chromatographic column: Octadecylsilane chemically bonded silica gel is used as the packing material, and the particle size of the packing material is 5μm; the length of the chromatographic column is 250mm, and the inner diameter is 4.6mm;

[0014] Mobile phase: Mobile phase A is a potassium dihydrogen phosphate solution with a concentration of 0.05mol / L and a pH of 5.0; mobile phase B is methanol; gradient elution;

[0015] Sample injection volume: 100μl; flow rate: 1.0ml / min; detection wavelength: 260nm; column temperature: 40°C; injector temperature: 4°C.

[0016] Further, the elution program of the gradient elution is one of the following gradients (1) - (3):

[0017] Gradient (1):

[0018]

[0019] Gradient (2):

[0020]

[0021]

[0022] Gradient (3):

[0023]

[0024] In some specific embodiments of the present application, the elution program for gradient elution is gradient (2).

[0025] In a further aspect, the detection method further includes a step of preparing a solution, and the preparation of the solution includes:

[0026] Preparation of the test solution: Weigh this product precisely, protect from light, dissolve with a diluent and quantitatively dilute to the mark, shake well to prepare a solution with a cobamamide concentration of 0.1 - 0.2 mg / ml, filter, and take the subsequent filtrate;

[0027] Preparation of the control solution: Precisely measure the test solution, protect from light, quantitatively dilute with a diluent to prepare a control solution with a concentration of 1 - 2 μg / ml;

[0028] Excipient solution: Weigh corn starch precisely, protect from light, dissolve with a diluent and quantitatively dilute to the mark, shake well to prepare a solution with a concentration of 12 - 26 mg / ml, filter, and take the subsequent filtrate;

[0029] System suitability solution: Weigh adenosine, hydroxocobalamin, cyanocobalamin, methylcobalamin and cobamamide precisely, dissolve and dilute with a diluent to prepare a mixed solution containing 0.1 - 0.2 mg of cobamamide and 1 - 2 μg of each of adenosine, hydroxocobalamin, cyanocobalamin and methylcobalamin per 1 ml;

[0030] Among them, the diluent is a calcium chloride or magnesium chloride solution with a mass concentration of 1% - 2%;

[0031] Preferably, the diluent is a calcium chloride solution with a mass concentration of 1%.

[0032] In some specific embodiments of the present application, the concentration of the test solution is 0.1 mg / ml;

[0033] The concentration of the control solution is 1 μg / ml;

[0034] The concentration of the excipient solution is 12 mg / ml;

[0035] In the system suitability solution, each 1 ml contains 0.1 mg of cobamamide and 1 μg of each of adenosine, hydroxocobalamin, cyanocobalamin and methylcobalamin.

[0036] It can be understood that the protection from light can be achieved by well-known brown volumetric containers in the art, such as volumetric flasks or measuring flasks, without special limitations.

[0037] In the present application, in the chromatogram of the system suitability solution, the elution order is adenosine, hydroxocobalamin, cyanocobalamin, cobamamide, methylcobalamin in sequence; among them, the resolution between the cobamamide peak and the cyanocobalamin peak is not less than 5.0.

[0038] In this application, the specific impurities, the content of other single impurities and the total impurities are calculated according to the following methods:

[0039]

[0040] Total impurities = ∑(content of specific impurities + content of other single impurities);

[0041] Among them, the specific impurities are adenosine, hydroxocobalamin, cyanocobalamin and methylcobalamin;

[0042] In the formula, A x is the peak area of the specific impurity in the test solution;

[0043] A 单杂 is the peak area of other single impurities in the test solution;

[0044] f n is the correction factor for each specific impurity. Among them, the correction factors for adenosine, hydroxocobalamin, cyanocobalamin and methylcobalamin are 0.41, 2.0, 2.1 and 1.7 respectively;

[0045] A 1％对照 is the peak area of the 1% reference solution.

[0046] During specific calculation, in the chromatogram of the test solution, the peak areas of adenosine, hydroxocobalamin, cyanocobalamin and methylcobalamin shall not be greater than the main peak area of the 1% reference solution after being multiplied by the correction factors 0.41, 2.0, 2.1 and 1.7 respectively; the area of other single impurities shall not exceed 0.5 times (0.5%) of the main peak area of the reference solution, and the sum of the peak areas of each impurity calculated according to the corrected peak area shall not be greater than 2 times (0.2%) of the main peak area of the reference solution.

[0047] Advantages of this application:

[0048] The method for detecting related substances in the cobamamide capsules provided by this application is based on high performance liquid chromatography for detection. It has simple operation, high sensitivity, good specificity, high accuracy and good durability. Under the chromatographic conditions of this application, the contents of specific impurities adenosine, hydroxocobalamin, cyanocobalamin, methylcobalamin and other single impurities in the cobamamide capsules can be accurately determined. Moreover, the separation between each impurity peak, between the main peak and the adjacent impurity peak is good, and the impurities can be effectively separated from the solvent and excipients, achieving full separation of each impurity, effectively controlling the product quality of the cobamamide capsules, and having important significance for the quality control of the cobamamide capsules. Description of the drawings

[0049] Figure 1 It is the chromatogram of the system suitability solution in Example 1.

[0050] Figure 2It is the chromatogram overlay of the blank solvent, system suitability solution, blank excipient solution, test solution, control solution and each component localization solution in Example 2.

[0051] Figure 3 It is the chromatogram overlay detected after changing the chromatographic conditions in the robustness experiment of Example 3.

[0052] Figure 4 It is the chromatogram of the spiked test solution with the concentration of each impurity being 1.0 μg / ml in Example 4.

[0053] Figure 5 It is the chromatogram overlay of multiple batches of test solutions in Example 8.

[0054] Figure 6 It is the overlay of the localization of each component of the related substances in the comparative example. Detailed implementation manners

[0055] The implementation manners of the present application will be clearly and completely described below. The technical solutions in the following described implementation manners are exemplary and only possible technical implementations of the present application, not all possible implementations. Those skilled in the art can completely combine the implementation manners of the present application and obtain other implementation manners without creative labor, and these implementation manners are also within the protection scope of the present application.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs. The terms used in the specification of this application are only for the purpose of describing specific implementation manners and are not intended to limit this application.

[0057] In addition, unless otherwise specified, the methods without specific conditions or steps recorded are all conventional methods, and the reagents and materials used can be obtained from commercial channels.

[0058] The sample information involved in the following examples is specifically as follows:

[0059]

[0060]

[0061] Establishment of the detection method for related substances in cobamamide capsules in Example 1

[0062] A detection method for related substances in cobamamide capsules is provided in this example. The specific method is as follows:

[0063] (1) High performance liquid chromatograph: Shimadzu LC20A.

[0064] (2) High performance liquid chromatography conditions:

[0065] Chromatographic column: Agilent TC-C18(2), 4.6×250 mm, 5 μm;

[0066] Mobile phase: Potassium dihydrogen phosphate solution with a concentration of 0.05 mol / L (adjust the pH value to 5.0 with sodium hydroxide test solution) was used as mobile phase A; methanol was used as mobile phase B, and gradient elution was carried out according to the gradient (2) shown in the following table:

[0067]

[0068] Flow rate 1.0 ml / min, column temperature 40 °C, detection wavelength 260 nm, injection volume 100 μl, injector temperature 4 °C.

[0069] (3) Solution preparation:

[0070] Diluent: Take 1 g of anhydrous calcium chloride, add water to dilute to 100 ml, and shake well.

[0071] System suitability solution: Weigh accurately reference substances adenosine, hydroxocobalamin, cyanocobalamin, methylcobalamin and cobamamide, dissolve and dilute with the diluent to prepare a mixed solution containing about 0.1 mg of cobamamide and about 1 μg of each of adenosine, hydroxocobalamin, cyanocobalamin and methylcobalamin in 1 ml.

[0072] (4) Sample determination:

[0073] After the system is stable, inject the system suitability solution into the high performance liquid chromatography, and record the chromatogram according to the chromatographic conditions in this example.

[0074] Figure 1 The chromatogram of the system suitability solution is shown. It can be seen that the elution order is adenosine, hydroxocobalamin, cyanocobalamin, cobamamide, methylcobalamin in turn; among them, the resolution between the cobamamide peak and the cyanocobalamin peak is not less than 5.0.

[0075] Example 2 Method specificity

[0076] 1. Solution preparation

[0077] Diluent: Take 1 g of anhydrous calcium chloride, add water to dilute to 100 ml, and shake well.

[0078] Test solution: Weigh accurately about 0.65 g of the content of this cobamamide capsule (equivalent to 2.5 mg of cobamamide), place it in a 25 ml brown volumetric flask, dissolve it with the diluent, dilute to the mark, shake well, prepare a solution with a concentration of 0.1 mg / ml, filter, and take the subsequent filtrate.

[0079] Control solution: Accurately measure the test solution, place it in a brown volumetric flask, dilute it quantitatively with the diluent to prepare a control solution with a concentration of 1 μg / ml.

[0080] Blank excipient solution: Weigh accurately 0.65 g of blank excipient (a mixture of corn starch and microcrystalline cellulose in a mass ratio of 6:7), place it in a 25-ml brown volumetric flask, dissolve it with diluent, dilute to the mark, shake well, filter, and take the subsequent filtrate.

[0081] System suitability solution: Weigh accurately the reference substances adenosine, hydroxocobalamin, cyanocobalamin, methylcobalamin, and cobamamide, dissolve and dilute them with diluent to prepare a mixed solution containing about 0.1 mg of cobamamide and about 1 μg of each of adenosine, hydroxocobalamin, cyanocobalamin, and methylcobalamin per 1 ml.

[0082] Adenosine localization solution: Weigh accurately the reference substance adenosine, dissolve and dilute it quantitatively with diluent to prepare an adenosine localization solution of 1 μg / ml.

[0083] Hydroxocobalamin localization solution: Weigh accurately the reference substance hydroxocobalamin hydrochloride, dissolve and dilute it quantitatively with diluent to prepare a hydroxocobalamin localization solution of 1 μg / ml.

[0084] Cyanocobalamin localization solution: Weigh accurately the reference substance cyanocobalamin, dissolve and dilute it quantitatively with diluent to prepare a cyanocobalamin localization solution of 1 μg / ml.

[0085] Methylcobalamin localization solution: Weigh accurately the reference substance methylcobalamin, dissolve and dilute it quantitatively with diluent to prepare a methylcobalamin localization solution of 1 μg / ml.

[0086] 2. Adopt the chromatographic conditions established in Example 1.

[0087] 3. Sample determination:

[0088] After the system is stable, take the above solutions and inject them into the high-performance liquid chromatograph respectively, record the chromatogram, and examine the specificity of the related substances detection method. The chromatogram is shown in Figure 2 , and the detection and analysis results are shown in Table 1.

[0089] Table 1 Specificity results of the related substances detection method

[0090]

[0091]

[0092] Through Figure 2From the test results in Table 1, it can be concluded that the diluent does not interfere with the determination of impurities and the main component. In the chromatogram of the system suitability solution, the minimum resolution between the known impurity and the adjacent impurity peak is 2.280 (>1.0), and the minimum resolution between the main peak and the adjacent impurity peak is 7.179 (>1.5). In the chromatogram of the test solution, the adenosine peak coincides with the excipient peak. Adenosine widely exists in plant cells. In the blank excipient, this peak is introduced by the excipient corn starch. For strict control, it is all counted as the adenosine peak. The blank excipient solution peak does not interfere with the determination of other impurities and the main component.

[0093] Method Durability of Example 3

[0094] In this example, by changing conditions such as column temperature, flow rate, pH value of buffer solution A in the mobile phase, salt concentration of mobile phase A, and using different chromatographic columns in the chromatographic conditions, with the system suitability solution in Example 2 as the sample, the durability of the detection method was investigated. The results are shown in Figure 3 and Table 2.

[0095] Table 2 Transformation Conditions and Detection Results of the Durability of the Detection Method

[0096]

[0097] Note: The transformed chromatographic conditions in Table 2 are all single variables. Except for the transformed chromatographic conditions, other detection conditions are the same as those in Example 1.

[0098] From the results in Table 2, it can be seen that under the transformed chromatographic conditions, the minimum resolution between adjacent impurity peaks (adenosine and hydroxocobalamin) is 5.482, and the minimum resolution between the main peak and the cyanocobalamin peak is 21.787. Combining with Figure 3 the chromatographic overlay map, it can be seen that all substances in the detection method provided by this application can be effectively separated, and the peak shapes are good, indicating that the durability of this method is good.

[0099] Method Accuracy of Example 4

[0100] 1. Diluent Selection

[0101] (1) Diluent: 1% calcium chloride solution;

[0102] Spiked test solution: Weigh accurately the reference substances adenosine, hydroxocobalamin, cyanocobalamin, methylcobalamin and the capsule content of cobamamide, dissolve and dilute quantitatively with the solvent to prepare a mixed solution containing about 0.1 mg of cobamamide and about 1 μg of each of adenosine, hydroxocobalamin, cyanocobalamin and methylcobalamin per 1 ml.

[0103] At the same time, set water as the solvent as the control, and prepare a spiked test solution with the same concentration.

[0104] (2) Using the same chromatographic conditions as in Example 1, spiked test solutions prepared with different diluents were separately injected into the high-performance liquid chromatograph, and the experimental results were recorded. The results are shown in Table 3.

[0105] Table 3 Spiked recovery rate

[0106]

[0107] From the test results in Table 3, it can be seen that when 1% calcium chloride is used as the diluent, the recovery rates of each impurity are all within the range of 90% - 110%, and the contents of each impurity in the sample can be accurately determined. When water is used as the diluent, the recovery rate of hydroxocobalamin is about 60%. Therefore, 1% calcium chloride solution is selected as the diluent in this application.

[0108] 2. Accuracy experiment

[0109] Diluent: 1% calcium chloride solution.

[0110] Spiked test solution: Weigh accurately the contents of adenosine, hydroxocobalamin, cyanocobalamin, methylcobalamin and cobamamide capsules, dissolve and dilute quantitatively with the diluent to prepare a solution containing about 0.1 mg of cobamamide and about 0.5 μg of each of adenosine, hydroxocobalamin, cyanocobalamin and methylcobalamin per 1 ml;

[0111] Refer to the above method to prepare spiked test solutions containing 0.1 mg of cobamamide and 1.0 μg and 1.5 μg of each of adenosine, hydroxocobalamin, cyanocobalamin and methylcobalamin per 1 ml respectively.

[0112] Three portions were prepared for each of the above solutions at each concentration.

[0113] Using the same chromatographic conditions as in Example 1 to investigate the accuracy of the detection method. Among them, Figure 4 is the chromatogram of the spiked test solution with the concentration of each impurity being 1.0 μg / ml. All the detection and analysis results are shown in Table 4.

[0114] Table 4 Accuracy of the detection method

[0115]

[0116]

[0117] From the results in Table 4, it can be seen that the recovery rates of each impurity are all within the range of 90% - 110%, indicating that the accuracy of the detection method in this application is good.

[0118] Detection limit, quantitation limit and linearity of the method in Example 5

[0119] 1. Detection limit and quantitation limit

[0120] Appropriately take reference substances of adenosine, hydroxocobalamin, cyanocobalamin, methylcobalamin, and cobamamide, and after successive dilution with a diluent (1% calcium chloride solution), inject 100 μl (chromatographic conditions are the same as in Example 1), record the chromatogram, take about 3 as the detection limit with the signal-to-noise ratio (S / N), and take about 10 as the quantification limit with the signal-to-noise ratio (S / N). The results are shown in Table 5.

[0121] Table 5 Results of the detection limit and quantification limit of the detection method

[0122]

[0123] Note ★ : Calculate the percentage of each impurity equivalent to the test substance based on the concentration of the test substance solution being 0.1 mg / ml.

[0124] It can be seen from the results in Table 5 that the sensitivity of the present invention is good, it can effectively detect related substances with relatively low contents, eliminate potential medication risks, and ensure the effectiveness and safety of the drug quality.

[0125] 2. Linearity

[0126] Diluent: 1% calcium chloride solution.

[0127] Appropriately take reference substances of adenosine, hydroxocobalamin, cyanocobalamin, methylcobalamin, and cobamamide, dissolve them separately with the diluent and quantitatively dilute to prepare solutions containing about 100 μg per 1 ml as respective stock solutions.

[0128] Quantification limit solution: Appropriately take the above respective stock solutions, add the diluent to prepare solutions with a signal-to-noise ratio (S / N) of 10 as the quantification limit solutions.

[0129] Linearity solutions 2 - 7: Accurately measure 1 ml, 1 ml, 2 ml, 2 ml, 3 ml, and 5 ml of the above respective stock solutions, place them in volumetric flasks of 250 ml, 50 ml, 50 ml, 20 ml, 20 ml, and 20 ml respectively, add the diluent to dilute to the scale, shake well, as linearity solutions 2 - 7. The specific concentrations of the linearity solutions are shown in Table 6:

[0130] Table 6 Composition of the linearity solutions

[0131]

[0132] Prepare the linearity solutions in the above manner by different experimenters on different days. Inject the quantification limit solution and linearity solutions 2 - 7 into another high-performance liquid chromatograph of a different model (chromatographic conditions are the same as in Example 1), and record the chromatogram.

[0133] Using the reference substance concentration as the abscissa (X) and the peak area as the ordinate (Y), perform linear regression analysis to investigate the correction factors of specific impurities (adenosine, hydroxocobalamin, cyanocobalamin, methylcobalamin) (correction factor = slope of the main component / slope of the impurity). The results are shown in Table 7.

[0134] Table 7 Linear Results of the Detection Method

[0135]

[0136] From the results in Table 7, it can be seen that using the detection method of Example 1, the linear relationships of each impurity in the range of the limit of quantitation to about 2.5 μg / ml and adenosylcobalamin in the range of the limit of quantitation to about 2.5 μg / ml are good, indicating that the linear relationship of this application is good and the contents of each impurity can be quantitatively detected.

[0137] Repeatability and Precision of the Method in Example 6

[0138] In this example, 6 test solution samples (taking 0.65 g of the contents of 9353230701 batch of adenosylcobalamin capsules → 25 ml, and the specific preparation is the same as in Example 2) were prepared by different analysts on different dates and detected on different instruments. The chromatographic conditions were the same as in Example 1, and the test results are shown in Table 8.

[0139] Table 8 Results of Repeatability and Precision

[0140]

[0141] From the test results in Table 8, it can be seen that among the 6 test solution samples, the average content of adenosine is 0.053%, the RSD is 1.1%, which is less than 20%. The average contents of hydroxocobalamin and the maximum unknown single impurity are 0.383% and 0.110% respectively, and the RSDs are 2.9% and 1.0% respectively, both of which are less than 10%. The average content of total impurities is 0.794%, and the RSD is 1.6%, which is less than 5%, indicating that the repeatability of this method is good.

[0142] Among the 12 test solution samples, the absolute differences in the average contents of adenosine, hydroxocobalamin and the maximum unknown single impurity are 0.036%, 0.050% and 0.008% respectively, all of which are less than 0.1%. The absolute difference in the average content of total impurities is 0.094%, which is less than 0.2%, indicating that the precision of this method is good.

[0143] Solution Stability in Example 7

[0144] In this example, take the test solution (taking 0.65 g of the contents of 9353230701 batch of adenosylcobalamin capsules → 25 ml), place it at room temperature (25 °C) and 4 °C, sample and measure at different time points to investigate the stability of the solution. The solution preparation and chromatographic conditions are the same as in Example 1, and the results are shown in Tables 9 - 10.

[0145] Table 9 Solution Stability - 25°C

[0146]

[0147]

[0148] Table 10 Solution Stability - 4°C

[0149]

[0150] From the experimental results in Table 9, it can be seen that when the test solution was placed at room temperature (25°C) for 12 hours, the absolute differences were all less than 0.10%, and no new impurities greater than 0.1% were generated, indicating that the test solution was stable within 12 hours at room temperature (25°C); from the experimental results in Table 10, it can be seen that when the test solution was placed at 4°C for 24 hours, the absolute differences were all less than 0.10%, and no new impurities greater than 0.1% were generated, indicating that the test solution was stable within 24 hours at 4°C.

[0151] Example 8 Detection of Related Substances in Cyanocobalamin Capsules

[0152] The related substance tests were carried out on 4 batches of samples (batch numbers: 9353230701, 353230801, 353230802, 353230803, source: Chengdu Tongde Pharmaceutical Co., Ltd.) according to the related substance test method provided in Example 2. The method is as follows:

[0153] 1. Solution Preparation:

[0154] Diluent: 1% calcium chloride solution.

[0155] Test Solution: Take an appropriate amount of the contents of this product (equivalent to 2.5 mg of cyanocobalamin), weigh accurately, place it in a 25 ml brown volumetric flask, add an appropriate amount of diluent, shake to dissolve and dilute to the mark, shake well, filter, and take the subsequent filtrate.

[0156] Reference Solution: Accurately measure 1 ml of the test solution, place it in a 100 ml brown volumetric flask, and dilute to the mark with the diluent, shake well.

[0157] Excipient Solution: Take about 0.3 g of corn starch, weigh accurately, place it in a 25 ml brown volumetric flask, add an appropriate amount of diluent, shake to dissolve and dilute to the mark, shake well, filter, and take the subsequent filtrate.

[0158] System Suitability Solution: Take appropriate amounts of adenosine, hydroxocobalamin, cyanocobalamin, methylcobalamin, and cyanocobalamin, dissolve and dilute with the diluent to prepare a mixed solution containing about 0.1 mg of cyanocobalamin and about 1 μg of adenosine, hydroxocobalamin, cyanocobalamin, and methylcobalamin per 1 ml.

[0159] 2. The chromatographic conditions refer to Example 1.

[0160] 3. System suitability:

[0161] (1) Under the above chromatographic conditions, accurately pipette 100 μl of the excipient solution, test solution, and reference solution respectively, inject them into the liquid chromatograph, and record the chromatogram.

[0162] (2) In the chromatogram of the system suitability solution, the elution order is adenosine, hydroxocobalamin, cyanocobalamin, cobamamide, and methylcobalamin in sequence; the resolution between the cobamamide peak and the cyanocobalamin peak shall not be less than 5.0.

[0163] 4. Calculation formula:

[0164]

[0165] Total impurities = ∑(specific impurity content + other single impurity content)

[0166] Where:

[0167] A x : Peak area of specific impurities in the test solution;

[0168] A 单杂 : Peak area of other single impurities in the test solution;

[0169] f n : Correction factor for each impurity (the correction factors for adenosine, hydroxocobalamin, cyanocobalamin, and methylcobalamin are 0.41, 2.0, 2.1, and 1.7 respectively);

[0170] A 1％对照 : Peak area of the 1% reference solution.

[0171] 5. Limit:

[0172] If there are impurity peaks in the chromatogram of the test solution, after multiplying the peak areas of adenosine, hydroxocobalamin, cyanocobalamin, and methylcobalamin by the correction factors 0.41, 2.0, 2.1, and 1.7 respectively, they shall not be greater than the main peak area of the reference solution (1.0%); other individual impurities shall not exceed 0.5 times the main peak area of the reference solution (0.5%), and the sum of the peak areas of each impurity calculated according to the corrected peak area shall not be greater than 2 times the main peak area of the reference solution (2.0%).

[0173] For the overlay map of the chromatograms of multiple batches of test solutions, see Figure 5 , and the test analysis results of multiple batches of samples are shown in Table 11.

[0174] Table 11 Test Results of Multiple Batches of Cobamamide Capsules

[0175]

[0176] By Figure 5 and the test results in Table 11, it can be seen that the test results of related substances in 4 batches of samples all meet the relevant regulations.

[0177] Comparative Example

[0178] The 2020 edition of the Chinese Pharmacopoeia (ChP2020) includes the quality standard for cobamamide tablets, and only ultraviolet-visible spectrophotometry is used to check the absorbance of hydroxocobalamin. To demonstrate the advantages of the detection method provided in this application, in this comparative example, the chromatographic conditions under the related substances item of the cobamamide raw material in ChP2020 were used to investigate the related substances of cobamamide capsules.

[0179] 1. Chromatographic conditions:

[0180] Chromatographic column: Agilent TC-C18(2), 4.6×250mm, 5μm;

[0181] Mobile phase: 0.05mol / L potassium dihydrogen phosphate solution (adjusted to pH 3.2 with phosphoric acid)-acetonitrile (85:15);

[0182] Column temperature 35°C; detection wavelength 260nm; injection volume 100μl, flow rate 1.0ml / min.

[0183] 2. Solution preparation

[0184] System suitability solution: Weigh appropriate amounts of adenosine, hydroxocobalamin, cyanocobalamin, methylcobalamin, and cobamamide accurately, dissolve with water and dilute to make a mixed solution containing 0.1mg of cobamamide and 1μg of each of adenosine, hydroxocobalamin, cyanocobalamin, and methylcobalamin per 1ml.

[0185] Test solution: Weigh an appropriate amount of the reference substance cobamamide accurately, dissolve with water and quantitatively dilute to make a solution containing 1mg per 1ml.

[0186] Each localization solution: Weigh appropriate amounts of adenosine, hydroxocobalamin, cyanocobalamin, methylcobalamin, and cobamamide accurately, dissolve with water and dilute to make a solution containing approximately 100μg of each of adenosine, hydroxocobalamin, cyanocobalamin, and methylcobalamin and approximately 10μg of cobamamide per 1ml.

[0187] After filtering each solution, take the subsequent filtrate and inject it into the high-performance liquid chromatograph. The chromatogram is shown in Figure 6 .

[0188] The results show that when the chromatographic conditions under the related substances item of the cobamamide raw material in the 2020 edition of the Chinese Pharmacopoeia (ChP2020) are used to detect cobamamide capsules, the solvent peak interferes with the detection of hydroxocobalamin; it can be seen from the localization solution that the elution order is adenosine, hydroxocobalamin, cyanocobalamin, cobamamide in sequence, and methylcobalamin fails to elute and peak under these conditions.

[0189] Through the above examples and comparative examples, it can be concluded that the existing methods for adenosylcobalamin raw materials are not applicable to the analysis and detection of related substances in adenosylcobalamin capsules. The detection method provided by this application can accurately control the contents of specific impurities (adenosine, hydroxocobalamin, cyanocobalamin, methylcobalamin) and non-specific impurities in this product. Each peak is well separated, with high sensitivity, good specificity, and high accuracy. It can effectively control the quality of adenosylcobalamin capsule products and is of great significance.

[0190] It should be noted that this application is not limited to the above embodiments. The above embodiments are only examples, and embodiments with the same structure and the same effect as the technical idea within the technical solution scope of this application are included in the technical scope of this application. In addition, within the scope of not departing from the main idea of this application, various modifications that those skilled in the art can think of to the embodiments, as well as other ways constructed by combining some constituent elements in the embodiments, are also included in the scope of this application.

Claims

1. A method for detecting related substances in adenosylcobalamin capsules, characterized in that: The HPLC method was used for detection, and the chromatographic conditions of the HPLC method were as follows: Chromatographic column: Octadecylsilane bonded silica gel is used as filler, and the filler particle size is 3-5μm; the length of the chromatographic column is 150-250mm, and the inner diameter is 3-4.6mm; Mobile phase: Mobile phase A is potassium dihydrogen phosphate solution with a concentration of 0.045-0.055 mol / L and a pH of 4.8-5.2; mobile phase B is methanol; gradient elution; Injection volume 50-100 μl; flow rate 0.8-1.2 ml / min; detection wavelength 258-262 nm; column temperature 35-42°C; injector temperature 4-25°C.

2. The method for detecting related substances in adenosylcobalamin capsules according to claim 1, characterized in that: In the chromatographic conditions of the high performance liquid chromatography, the chromatographic column uses octadecylsilane bonded silica gel as a filler, and the filler particle size is 5 μm; the length of the chromatographic column is 250 mm, and the inner diameter is 4.6 mm.

3. The method for detecting related substances in adenosylcobalamin capsules according to claim 1, characterized in that: In the chromatographic conditions of the high performance liquid chromatography, the mobile phase A is a potassium dihydrogen phosphate solution with a concentration of 0.05 mol / L and a pH of 5.0; the mobile phase B is methanol; and the elution is performed by gradient elution.

4. The method for detecting related substances in adenosylcobalamin capsules according to claim 1, characterized in that: The chromatographic conditions of the high performance liquid chromatography method are as follows: injection volume 100 μl; flow rate 1.0 ml / min; detection wavelength 260 nm; column temperature 40° C.; and injector temperature 4° C.

5. The method for detecting related substances in adenosylcobalamin capsules according to claim 1, characterized in that: The elution procedure of the gradient elution is one of the following gradients (1) to (3): Gradient (1): Gradient (2): Gradient (3): Preferably, the elution procedure of the gradient elution is gradient (2).

6. The method for detecting related substances in adenosylcobalamin capsules according to claim 1, characterized in that: The detection method further comprises the following steps: Preparation of test solution: accurately weigh the contents of adenosylcobalamin capsules, protect from light, add diluent to dissolve and quantitatively dilute to scale, shake well to make a solution with a concentration of 0.1-0.2 mg / ml adenosylcobalamin, filter and take the filtrate; Preparation of control solution: Accurately measure the test solution, protect from light, and quantitatively dilute with diluent to prepare a control solution with a concentration of 1-2 μg / ml; Excipient solution: accurately weigh corn starch, protect from light, add diluent to dissolve and quantitatively dilute to scale, shake well to make a solution with a concentration of 12-26 mg / ml, filter and take the filtrate; System suitability solution: accurately weigh the reference substances adenosine, hydroxocobalamin, cyanocobalamin, methylcobalamin and adenosylcobalamin, and use dilute A mixed solution of 1 to 2 μg each; Wherein, the diluent is a calcium chloride or magnesium chloride solution with a mass concentration of 1% to 2%; Preferably, the diluent is a calcium chloride solution with a mass concentration of 1%.

7. The method for detecting related substances in adenosylcobalamin capsules according to claim 6, characterized in that: The concentration of the test solution is 0.1 mg / ml; and / or, the concentration of the control solution is 1 μg / ml; and / or, the concentration of the excipient solution is 12 mg / ml; And / or, each 1 ml of the system suitability solution contains 0.1 mg of adenosylcobalamin and 1 μg each of adenosine, hydroxocobalamin, cyanocobalamin and methylcobalamin.

8. The method for detecting related substances in adenosylcobalamin capsules according to claim 6, characterized in that: In the chromatogram of the system suitability solution, the order of peaks is adenosine, hydroxocobalamin, cyanocobalamin, adenosylcobalamin, and methylcobalamin; wherein the separation degree between the adenosylcobalamin peak and the cyanocobalamin peak is not less than 5.

0.

9. The method for detecting related substances in adenosylcobalamin capsules according to claim 6, characterized in that: Specific impurities, other single impurities and total impurities are calculated as follows: Total impurities = ∑ (specific impurity content + other single impurity content); Among them, the specific impurities are adenosine, hydroxocobalamin, cyanocobalamin, and methylcobalamin; In the formula, A x is the peak area of ​​specific impurities in the test solution; A 单杂 is the area of ​​other single impurity peaks in the test solution; f n is the correction factor for each specific impurity, wherein the correction factors for adenosine, hydroxocobalamin, cyanocobalamin, and methylcobalamin are 0.41, 2.0, 2.1, and 1.7, respectively; A 1％对照 It is the peak area of ​​1% control solution.

10. The method for detecting related substances in adenosylcobalamin capsules according to claim 6, characterized in that: In the chromatogram of the test solution, the peak areas of adenosine, hydroxocobalamin, cyanocobalamin and methylcobalamin shall not be greater than the main peak area of ​​1% control solution after multiplying by correction factors of 0.41, 2.0, 2.1 and 1.7 respectively; other individual impurities shall not exceed 0.5 times the main peak area of ​​1% control solution, and the sum of the peak areas of each impurity calculated according to the corrected peak area shall not be greater than 2 times the main peak area of ​​1% control solution.

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