Method for detecting purity of benzoyl galanthamine
The purity of benzoylgalantamine was determined by high performance liquid chromatography, which solves the problem of inaccuracy in existing detection methods and enables rapid and accurate purity detection, supporting drug development and clinical applications.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHANG JIA GANG VINSCE BIO PHARM
- Filing Date
- 2026-02-13
- Publication Date
- 2026-05-01
AI Technical Summary
The lack of effective methods in the current technology to accurately detect the purity of benzoylgalantamine affects its drug development, pharmacological activity evaluation and future clinical application.
The purity of benzoylgalantamine was determined by high performance liquid chromatography (HPLC). A C18 column, phosphate-methanol buffer, and acetonitrile were used as the mobile phase, with gradient elution. Qualitative and quantitative analysis were performed at 230 nm using a UV-Vis absorption detector.
It enables rapid and accurate detection of benzoylgalantamine purity, exhibiting good specificity, operability, linearity, precision, and robustness, and is suitable for the production and application of benzoylgalantamine.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of pharmaceutical analysis technology, and in particular to a method for detecting the purity of benzoylgalantamine. Background Technology
[0002] Galantamine is a natural alkaloid, initially extracted from plants in the Amaryllidaceae family (such as snowdrops and daffodils), and later also synthesized artificially. It belongs to the second-generation competitive cholinesterase inhibitors. By inhibiting acetylcholinesterase, it increases the concentration of acetylcholine in the brain and is primarily used to treat Alzheimer's disease (AD). Cholinesterase inhibitors play a crucial role in current AD treatment, and galantamine remains an important option for symptomatic treatment. Further research into its derivatives is expected to drive the development of next-generation drugs that combine symptom improvement and disease modification. Therefore, research on galantamine or its derivatives is of significant scientific importance and will greatly contribute to the fight against Alzheimer's disease (AD).
[0003] Benzoylgalantamine is a benzoylated derivative of galantamine, and its structural modifications may endow it with unique pharmacological properties. Benzoylgalantamine is currently still in the theoretical or early research stages, and the study of its purity detection methods is crucial for drug development, pharmacological activity evaluation, and future clinical applications. Purity detection not only directly affects the reliability and reproducibility of the compound, but may also determine its pharmacological activity, toxicological properties, and druggability. Summary of the Invention
[0004] In view of this, the technical problem to be solved by the present invention is to provide a method for detecting the purity of benzoyl galantamine, and the method provided by the present invention is accurate and reliable.
[0005] This invention provides a method for detecting the purity of benzoyl galantamine, characterized by comprising the following steps:
[0006] A) The benzoyl galantamine sample was dissolved in a solvent to obtain the test solution;
[0007] Benzoyl galantamine reference standard was dissolved in a solvent to obtain a reference standard solution;
[0008] B) The test solution and the reference solution are determined by high performance liquid chromatography to obtain the chromatograms of the test solution and the reference solution; based on the chromatogram of the reference solution, the benzoyl galantamine in the chromatogram of the test solution is qualitatively and quantitatively determined, and the purity of benzoyl galantamine is calculated.
[0009] The chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column; mobile phase A is phosphate methanol buffer solution, mobile phase B is acetonitrile, and gradient elution is used.
[0010] The structural formula of benzoylgalantamine of this invention is as follows:
[0011]
[0012] The method for detecting the purity of benzoyl galantamine provided by the present invention firstly involves dissolving the benzoyl galantamine sample in a solvent to obtain a test solution.
[0013] The benzoyl galantamine test sample described in this invention includes benzoyl galantamine raw material.
[0014] All the raw materials mentioned above can be subjected to quality control and qualitative and quantitative detection by the method of the present invention.
[0015] The solvent described in this invention is methanol;
[0016] In one specific embodiment, the mass-to-volume ratio (mg / mL) of the benzoylgalantamine test sample and the solvent is 1 mg: 1 mL.
[0017] Benzoyl galantamine reference standard was dissolved in a solvent to obtain a reference standard solution;
[0018] In one specific embodiment, the solvent is methanol; the concentration of the reference solution is 1 mg / mL.
[0019] Within the concentration range of benzoylgalantamine (451.20 μg / mL to 1353.60 μg / mL), the linear equation for the concentration-peak area relationship was y = 6753.6x + 71096, r = 0.9998. This indicates that the method exhibits good linearity.
[0020] The test solution and the reference solution are determined by high performance liquid chromatography to obtain the chromatograms of the test sample and the reference solution. Based on the chromatogram of the reference solution, the benzoylgalantamine in the chromatogram of the test sample is qualitatively and quantitatively determined, and the purity of benzoylgalantamine is calculated. The calculation method described in this invention is preferably the peak area normalization method.
[0021] According to the present invention, the chromatographic conditions for the high performance liquid chromatography method are as follows: the chromatographic column is a C18 column;
[0022] The chromatographic column has dimensions of 4.6 mm * 150 mm and a diameter of 4 μm; the column temperature is room temperature, preferably 30 °C; and the theoretical plate number, calculated based on the benzoyl galantamine peak, is not less than 2000.
[0023] In one specific embodiment, the chromatographic column is an Agilent Proshell 120EC C18 4.6mm*150mm 4μm column.
[0024] Mobile phase A is a phosphate-methanol buffer solution, wherein the concentration of disodium hydrogen phosphate dihydrate is 4.22 mmol / L, the concentration of anhydrous disodium hydrogen phosphate is 19.48 mmol / L, and the volume fraction of methanol is 5%.
[0025] In one specific embodiment of the present invention
[0026] The specific preparation method for phosphate-methanol buffer solution is as follows: Weigh 0.79g of disodium hydrogen phosphate dihydrate and 2.46g of anhydrous sodium dihydrogen phosphate, dissolve them in 1L of ultrapure water to prepare the phosphate buffer solution, then measure 950mL of the phosphate buffer solution and add 50mL of methanol, mix well, and the solution is ready.
[0027] Mobile phase B is acetonitrile, and gradient elution is used.
[0028] According to the present invention, the elution gradient is:
[0029] 0–20 min, Phase A: 70%, Phase B: 30%;
[0030] 20–21 min, Phase A: 70% → 5%, Phase B: 30% → 95%;
[0031] 21–26 min, Phase A: 5%, Phase B: 95%;
[0032] 26–26.1 min, Phase A: 5% → 70%, Phase B: 95% → 30%;
[0033] 26.1–35 min, Phase A: 70%, Phase B: 30%.
[0034] The present invention exhibits good baseline separation, good peak separation, and stable baseline under the above-mentioned elution gradient.
[0035] The flow rate of the mobile phase described in this invention is 0.8-1.2 mL / min; more preferably 1.0 mL / min.
[0036] The detector is an ultraviolet-visible absorption detector, and the detection wavelength of this invention is 230nm.
[0037] The inventors discovered that the chromatographic peak at 230 nm contains more information, the chromatographic baseline is more stable, and the peak area is larger.
[0038] The injection volume of this invention is 3~5μL.
[0039] This invention provides a method for detecting the purity of benzoylgalantamine, comprising the following steps: A) Dissolving the benzoylgalantamine sample in a solvent to obtain a test solution; dissolving the benzoylgalantamine reference standard in a solvent to obtain a reference solution; B) Determining the purity of the test solution and the reference solution using high-performance liquid chromatography (HPLC) to obtain chromatograms of the test solution and the reference standard; qualitatively and quantitatively determining the benzoylgalantamine in the chromatogram of the test solution based on the chromatogram of the reference standard, and calculating the purity of benzoylgalantamine; the HPLC conditions are as follows: a C18 column; mobile phase A is phosphate-methanol buffer solution, mobile phase B is acetonitrile, and gradient elution is used. This invention provides for the first time a method for detecting the purity of benzoyl galantamine by liquid chromatography. The method of this invention has high specificity, good operability, and good accuracy, linearity, precision and robustness. It can realize the rapid determination of the purity of benzoyl galantamine, which is of great significance to the production and application of benzoyl galantamine. Attached Figure Description
[0040] Figure 1 The results are for linearity and range tests;
[0041] Figure 2 The chromatogram is of benzoyl galantamine reference solution;
[0042] Figure 3 Chromatogram of benzoylgalantamine test solution;
[0043] Figure 4 The chromatogram of benzoyl galantamine solution under mobile phase ratio 1 is shown.
[0044] Figure 5 The chromatogram of benzoylgalantamine solution under mobile phase ratio 2 is shown. Detailed Implementation
[0045] This invention provides a method for detecting the purity of benzoylgalantamine. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of protection of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can obviously make modifications or appropriate alterations and combinations to the method and application described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.
[0046] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.
[0047] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.
[0048] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.
[0049] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.
[0050] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.
[0051] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.
[0052] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.
[0053] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.
[0054] The embodiments and comparative examples of this invention describe some examples, in which the embodiments illustrate certain implementations of the invention. However, this does not mean that the effects of the invention can only be achieved in these examples.
[0055] The main instruments, equipment, and reagents used in the following examples are as follows:
[0056] Liquid Chromatograph 1: Agilent 1260; UV detector; serial number MYYF030016;
[0057] Electronic balance: Sartorius Scientific Instruments (Beijing) Co., Ltd., Model BCE124-1CCN; Serial number MYYF03002;
[0058] CNC ultrasonic cleaner: Shanghai Jingqi Instrument Co., Ltd.; Model JQ-040SF-2; Serial number YF-0103;
[0059] Methanol: Xilong Scientific; Batch No. 250325B1; Chromatographic grade;
[0060] Acetonitrile: Xilong Scientific; Batch No. 250515A1; Chromatographic grade;
[0061] Disodium hydrogen phosphate dihydrate: Pyrizoline; Batch No. KB57JEDO-WKT; Analytical grade;
[0062] Anhydrous sodium dihydrogen phosphate: Rakugen; batch number Le062377193; analytical grade;
[0063] Benzoylgalantamine reference standard: Zhangjiagang Weisheng Pharmaceutical Co., Ltd.; batch number 20250218; content 99.0%.
[0064] Benzoylgalantamine samples: Zhangjiagang Weisheng Pharmaceutical Co., Ltd.; batch numbers 20250310, 20250311, 20250312.
[0065] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a method for detecting the purity of benzoyl galantamine provided by the present invention.
[0066] Example 1
[0067] A method for determining the purity of benzoylgalantamine includes the following steps:
[0068] S1. Preparation of reference solution:
[0069] Accurately weigh 250 mg of benzoylgalantamine reference standard, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with methanol, shake well, and use it as the reference standard stock solution. Then accurately transfer 5 mL of the stock solution to a 25 mL volumetric flask, dilute to the mark with methanol, shake well, and use it as the reference standard solution.
[0070] S2. Preparation of the test solution:
[0071] Accurately weigh 25 mg of benzoylgalantamine sample (20250310), place it in a 25 mL volumetric flask, dissolve and dilute with methanol to the mark, shake well, and use it as the test solution.
[0072] S3. Accurately pipette 3 μL each of the reference solution and the test solution into the liquid chromatograph, determine the chromatogram, and obtain the chromatogram; calculate the purity of benzoyl galantamine by peak area normalization method.
[0073] The chromatographic conditions of the liquid chromatograph are as follows:
[0074] Column: Agilent Proshell 120EC C18 4.6mm*150mm 4μm;
[0075] Detector: Ultraviolet-Vis absorption detector; Wavelength: 230nm;
[0076] Mobile phase: Mobile phase A: Phosphate-methanol buffer solution (weigh 0.79g of disodium hydrogen phosphate dihydrate and 2.46g of anhydrous sodium dihydrogen phosphate, dissolve in 1L of ultrapure water to prepare the phosphate buffer solution, then measure 950mL of the phosphate buffer solution and add 50mL of methanol, mix well); Mobile phase B: Acetonitrile;
[0077] Gradient elution:
[0078]
[0079] Column temperature: 30℃;
[0080] Injection volume: 3 μL.
[0081] Example 2 Specificity Test
[0082] (I) Solution Preparation
[0083] Reference solution: Accurately weigh 45.1 mg of benzoyl galantamine reference standard, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well to obtain the reference solution;
[0084] Preparation of the test solution: Accurately weigh 55.8 mg of benzoylgalantamine sample, place it in a 50 mL volumetric flask, dissolve and dilute with methanol to the mark, shake well, and use as the test solution.
[0085] Blank solvent: methanol.
[0086] (II) Measurement Method
[0087] Accurately pipette 3 μL each of blank solvent, reference solution and test solution into the liquid chromatograph and determine.
[0088] (III) Results of Specificity Tests
[0089] The blank solvent did not interfere with the elution position of benzoyl galantamine; the theoretical plate number of the benzoyl galantamine peak in the reference solution was 37313, which is greater than 2000; the resolution between benzoyl galantamine and the adjacent peak in the test solution was 2.805, which is greater than 1.5.
[0090] As an example, refer to Figure 2 The above chromatogram is of the benzoyl galantamine reference solution. Figure 3 This is the chromatogram of the test solution.
[0091] (IV) Conclusion
[0092] Experimental results show that this method has good specificity.
[0093] Example 3 System Precision Test
[0094] (I) Solution Preparation
[0095] The reference solution is the same as the reference solution in Example 2.
[0096] (II) Measurement Method
[0097] Accurately pipette 3 μL of the reference solution and inject it into the liquid chromatograph for determination.
[0098] (III) Results and Data: The results are shown in Table 1.
[0099] Table 1. Injection precision results
[0100]
[0101] (IV) Conclusion
[0102] Experimental results show that:
[0103] Injection precision: The relative standard deviation of the peak area of benzoyl galantamine was 0.10% after six repeated injections of the reference solution (the repeatability (RSD) of 1.0 g / g shall not exceed 1%), which meets the requirements of the Chinese Pharmacopoeia. The method has good system precision.
[0104] Example 4: Linearity and Range Test
[0105] (I) Solution Preparation
[0106] Benzoylgalantamine reference stock solution: Accurately weigh 1128.4 mg of benzoylgalantamine reference standard, place it in a 250 mL volumetric flask, dissolve and dilute to the mark with methanol, shake well, and use as the reference stock solution.
[0107] Benzoylgalantamine linear solution:
[0108] 50% linear solution: Accurately measure 5 mL of benzoylgalantamine reference stock solution, place it in a 50 mL volumetric flask, dilute to the mark with methanol, shake well, filter, and collect the filtrate.
[0109] 80% linear solution: Accurately measure 8 mL of benzoylgalantamine reference stock solution, place it in a 50 mL volumetric flask, dilute to the mark with methanol, shake well, filter, and collect the filtrate.
[0110] 100% linear solution: Accurately measure 10 mL of benzoylgalantamine reference stock solution, place it in a 50 mL volumetric flask, dilute to the mark with methanol, shake well, filter, and collect the filtrate.
[0111] 120% linear solution: Accurately measure 12 mL of benzoylgalantamine reference stock solution, place it in a 50 mL volumetric flask, dilute to the mark with methanol, shake well, filter, and collect the filtrate.
[0112] 150% linear solution: Accurately measure 15 mL of benzoylgalantamine reference stock solution, place it in a 50 mL volumetric flask, dilute to the mark with methanol, shake well, filter, and collect the filtrate.
[0113] (II) Measurement Method
[0114] Precisely measure 3 μL of each linear solution at each concentration level, inject it into the liquid chromatograph, and measure the concentration. Perform linear regression with concentration on the x-axis and peak area on the y-axis, and calculate the linear correlation coefficient.
[0115] Linear regression is performed using the peak area as the ordinate, and the linear equation is calculated.
[0116] (III) Results
[0117] The data results are shown in Table 2. Figure 1
[0118] Table 2. Results of Linearity and Range Tests
[0119]
[0120] Figure 1 Linearity and range test results
[0121] (IV) Conclusion
[0122] Experimental results show that within the range of 451.20 μg / mL to 1353.60 μg / mL, the linear relationship between concentration and peak area of benzoylgalantamine is y = 6753.6x + 71096, r = 0.9998, indicating good linearity of the method.
[0123] Example 5 Recovery Rate Test
[0124] (I) Solution Preparation
[0125] Benzoylgalantamine reference stock solution: Accurately weigh 1128.4 mg of benzoylgalantamine reference standard, place it in a 250 mL volumetric flask, dissolve and dilute to the mark with methanol, shake well, and use as the reference stock solution.
[0126] Reference solution: Accurately measure 5 mL of benzoyl galantamine reference stock solution, place it in a 25 mL volumetric flask, dilute to the mark with ethanol, shake well, filter, and collect the filtrate.
[0127] Test solution: Accurately weigh 22.0 mg of benzoylgalantamine sample, place it in a 25 mL volumetric flask, add ethanol to dissolve and dilute to the mark, shake well, filter, and take the filtrate.
[0128] Preparation of 50% recovery solution: Accurately weigh 22.1 mg of benzoylgalantamine sample and place it in a 25 mL volumetric flask. Add 2.5 mL of benzoylgalantamine reference stock solution, dissolve and dilute to the mark with ethanol, shake well, filter, and collect the filtrate. Prepare 3 aliquots using the same method.
[0129] Preparation of 100% recovery solution: Accurately weigh 22.6 mg of benzoylgalantamine sample and place it in a 25 mL volumetric flask. Add 5 mL of benzoylgalantamine reference stock solution, dissolve and dilute to the mark with ethanol, shake well, filter, and collect the filtrate. Prepare 3 aliquots using the same method.
[0130] Preparation of 150% recovery solution: Accurately weigh 22.3 mg of benzoylgalantamine sample and place it in a 25 mL volumetric flask. Add 7.5 mL of benzoylgalantamine reference stock solution, dissolve and dilute to the mark with ethanol, shake well, filter, and collect the filtrate. Prepare 3 aliquots using the same method.
[0131] Recovery rate = (CA) / B × 100%
[0132] In the formula, A represents the amount of the analyte contained in the test sample.
[0133] B represents the amount of reference standard added.
[0134] C represents the measured value;
[0135] (II) Measurement Method
[0136] Accurately pipette 3 μL each of the reference solution, the test solution, and the recovery solutions of different concentrations of the test solution into the liquid chromatograph and determine the concentration. Calculate the recovery rate of benzoylgalantamine using the external standard method. The recovery rate should be between 98% and 102%, and its relative standard deviation (RSD) should not exceed 2%.
[0137] (III) Results
[0138] The data results are shown in Table 3.
[0139] Table 3 Results of the recovery rate test
[0140]
[0141] (IV) Conclusion
[0142] Experimental results showed that the recovery rate of benzoylgalantamine calculated using the external standard method was between 98% and 102%, with an RSD of 0.45%, less than 2%, which meets the requirements of the Chinese Pharmacopoeia. The method demonstrated good accuracy.
[0143] Example 6 Precision Test
[0144] I. Repetitiveness
[0145] (I) Solution Preparation
[0146] Preparation of the test solution: Accurately weigh 55.8 mg of benzoylgalantamine sample, place it in a 50 mL volumetric flask, dissolve and dilute with methanol to the mark, shake well, and use as the test solution.
[0147] (II) Measurement Method
[0148] Accurately pipette 3 μl each of the reference solution and the test solution into the liquid chromatograph and determine the content. Calculate the content of this product using the peak area normalization method. The relative standard deviation (RSD) should not exceed 1%.
[0149] (III) Results
[0150] The data results are shown in Table 4.
[0151] Table 4 Results of Repeatability Tests
[0152]
[0153] (IV) Conclusion
[0154] The experimental results show that the average content of the repeatable test sample is 99.582%, the RSD is 0.01%, which is less than 1% and meets the requirements.
[0155] II. Intermediate Precision
[0156] (I) Solution Preparation
[0157] Prepare the solution according to the solution preparation method under the repeatability section.
[0158] (II) Measurement Method
[0159] Two testers performed the repeatability test at different times, accurately injecting 3 μL each of the reference solution and the test solution into the liquid chromatograph for determination. The content of the product was calculated using the peak area normalization method. The content of the product and its relative standard deviation were calculated using the peak area normalization method, and the relative standard deviations of the results from the two testers and 12 determinations were also calculated; all relative standard deviations should not exceed 6.0%.
[0160] (III) Results
[0161] The data results are shown in Table 5.
[0162] Table 5 Results of intermediate precision test
[0163]
[0164] (IV) Conclusion
[0165] The experimental results showed that the average content of the 12 intermediate precision test samples was 99.583%, the RSD% was 0.02%, which was less than 6.0%, and met the requirements.
[0166] Example 7 Solution Stability Test
[0167] (I) Solution Preparation
[0168] Preparation of the reference solution for the test: Prepare the reference solution according to the method for repeatability under the test and place it in a liquid chromatography vial.
[0169] Preparation of the test solution: Prepare the test solution according to the method for repeatability under the test solution preparation section and place it in a liquid chromatography vial.
[0170] (II) Measurement Method
[0171] Take the above-mentioned reference solution and test solution, and place them at room temperature for 0, 4, 8, 12, 18, 24, 36, and 48 hours. Accurately pipette 3 μL of each solution and inject it into the liquid chromatograph for determination. Record the peak area of the chromatogram; the ratio of the main peak area to the zero point at each time point should be between 98% and 102%.
[0172] (III) Results
[0173] The data results are shown in Tables 6 and 7.
[0174] Table 6 Results of stability test of reference solution
[0175]
[0176] Table 7 Results of the stability test of the test sample solution
[0177]
[0178] (IV) Conclusion
[0179] Experimental results showed that, under room temperature conditions, the ratio of the peak area to the zero point for the benzoyl galantamine peak measured in the reference solution within 48 hours was 98%–102%, with no new impurities generated; similarly, the ratio of the peak area to the zero point for the benzoyl galantamine peak measured in the test solution within 48 hours was also 98%–102%, with no new impurities generated. These results indicate that both the reference solution and the test solution are relatively stable at room temperature for 48 hours.
[0180] Example 8 Durability Test
[0181] (I) Solution Preparation
[0182] Reference solution: Accurately weigh 46.8 mg of benzoyl galantamine reference standard, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with methanol, and shake well to obtain the reference solution;
[0183] Preparation of the test solution: Accurately weigh 54.2 mg of benzoylgalantamine sample, place it in a 50 mL volumetric flask, dissolve and dilute with methanol to the mark, shake well, and use as the test solution.
[0184] (II) Measurement Method
[0185] Adjust the chromatographic conditions according to the table below. Accurately pipette 3 μL each of the reference solution and the test solution and inject them into the liquid chromatograph. Measure the results. Record the peak areas of the chromatograms. The relative standard deviation of the peak areas should not exceed 2%.
[0186] The purity of this product and its relative standard deviation were calculated and compared using the peak area normalization method.
[0187] Table 8 Chromatographic conditions
[0188]
[0189] (III) Results
[0190] The data results are shown in Tables 9 and 10;
[0191] Table 9. Durability test results of benzoylgalantamine reference standard.
[0192]
[0193] Table 10 Results of the durability test of benzoylgalantamine test sample
[0194]
[0195] (IV) Conclusion
[0196] Experimental results show that under different chromatographic column and column temperature conditions for the purity determination of benzoyl galantamine, the average content is 99.540%, the RSD value is 0.01%, which is less than 2%, and meets the requirements.
[0197] Example 9: Determination of the purity of benzoylgalantamine in the sample
[0198] Three batches of test samples (Zhangjiagang Weisheng Pharmaceutical Co., Ltd., batch numbers 20250310, 20250311, and 20250312) were prepared according to the solution preparation method under the repeatability section and determined under the above chromatographic conditions. The test results are shown in Table 11.
[0199] Table 11 Results of purity determination of benzoylgalantamine
[0200]
[0201] After testing the above three batches of samples, the purity results of benzoylgalantamine were found to be parallel and stable.
[0202] Example 10 Comparative Test
[0203] (I) Solution Preparation
[0204] Preparation of test solution: Weigh 52.0 mg of benzoylgalantamine sample, place it in a 50 mL volumetric flask, dissolve and dilute with methanol to the mark, shake well, and use as test solution.
[0205] (II) Measurement Method
[0206] Adjust the chromatographic conditions according to the table below, accurately pipette 3 μL each of the reference solution and the test solution, inject them into the liquid chromatograph, and determine the result.
[0207] Table 12 Comparison Conditions
[0208]
[0209] (III) Results
[0210] Experimental results show that:
[0211] Under mobile phase ratio 1, the retention time of the main component in the benzoyl galantamine purity determination was 29.908 min, and the tailing factor was 4.8, which is greater than 1.2, indicating severe peak tailing; this condition was unsuitable. Under mobile phase ratio 2, the retention time was 1.914 min, and the main component was not retained; this condition was also unsuitable. Under the original conditions, the retention time of the main component was 10.166 min, and the tailing factor was 1.04, which is less than 1.2, indicating strong specificity and good accuracy, linearity, precision, and robustness.
[0212] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for detecting the purity of benzoylgalantamine, characterized in that, Includes the following steps: A) The benzoylgalantamine sample was dissolved in a solvent to obtain the test solution; Benzoyl galantamine reference standard was dissolved in a solvent to obtain a reference standard solution; B) The test solution and the reference solution are determined by high performance liquid chromatography to obtain the chromatograms of the test solution and the reference solution; based on the chromatogram of the reference solution, the benzoyl galantamine in the chromatogram of the test solution is qualitatively and quantitatively determined, and the purity of benzoyl galantamine is calculated. The chromatographic conditions for the high performance liquid chromatography method are as follows: the column is a C18 column; mobile phase A is phosphate methanol buffer solution, mobile phase B is acetonitrile, and gradient elution is used.
2. The detection method according to claim 1, characterized in that, The solvent is methanol; the concentration of the reference solution is 1 mg / mL.
3. The detection method according to claim 1, characterized in that, The concentration of disodium hydrogen phosphate dihydrate in the phosphate-methanol buffer solution is 4.22 mmol / L, the concentration of anhydrous disodium hydrogen phosphate is 19.48 mmol / L, and the volume fraction of methanol is 5%.
4. The detection method according to claim 1, characterized in that, The elution gradient is: 0–20 min, Phase A: 70%, Phase B: 30%; 20–21 min, Phase A: 70% → 5%, Phase B: 30% → 95%; 21–26 min, Phase A: 5%, Phase B: 95%; 26–26.1 min, Phase A: 5% → 70%, Phase B: 95% → 30%; 26.1–35 min, Phase A: 70%, Phase B: 30%.
5. The detection method according to claim 1, characterized in that, The chromatographic column has dimensions of 4.6 mm * 150 mm and 4 μm; the column temperature is 30℃; and the theoretical plate number, calculated based on the benzoyl galantamine peak, is not less than 2000.
6. The detection method according to claim 1, characterized in that, The injection volume is 3~5μL.
7. The detection method according to claim 1, characterized in that, The detection wavelength is 230nm.
8. The detection method according to claim 1, characterized in that, The mobile phase flow rate was 1.0 mL / min.
9. The detection method according to claim 1, characterized in that, The mass-to-volume ratio (mg / mL) of the benzoylgalantamine test sample and solvent was 1 mg: 1 mL. The benzoyl galantamine test sample includes benzoyl galantamine active pharmaceutical ingredient.
10. The detection method according to claim 1, characterized in that, In the range of 451.20 μg / mL to 1353.60 μg / mL, the linear relationship between concentration and peak area for benzoylgalantamine is given by the equation y = 6753.6x + 71096, r = 0.9998.