Liquid chromatography method for measuring aspirin content in carbaspirin calcium mother liquor prepared by calcium chloride method

Abstract

The invention belongs to the technical field of pharmaceutical analysis, and relates to a liquid chromatography method for measuring aspirin content in carbaspirin calcium mother liquor prepared by a calcium chloride method. A high performance liquid chromatography external standard method is adopted, and a high-water-phase-resistant reversed-phase chromatographic column is used; the mobile phase A is acetonitrile, and the mobile phase B is a 0.05-0.2% phosphoric acid aqueous solution; the detection wavelength is 225-235 nm, the flow velocity is 0.8-1.2 mL / min, the column temperature is 25-40 DEG C, and the sample size is 5-20 [mu] L. The method can effectively separate aspirin, salicylic acid and other components in the mother liquor matrix, and is high in specificity, high in accuracy, good in reproducibility and simple and convenient to operate. The method is suitable for rapidly and accurately monitoring the residual amount of aspirin in the mother liquor in the technological process of preparing carbaspirin calcium through a calcium chloride method, and has important significance on optimizing the production technology, controlling the content of impurities such as aspirin and salicylic acid in a final product and improving the product quality.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical analysis technology, specifically relating to a liquid chromatography method for determining the aspirin content in the mother liquor of carbaspirin calcium prepared by the calcium chloride method. Background Technology

[0002] Carbaspirin calcium, a highly water-soluble aspirin prodrug, is widely used in livestock farming for antipyretic and analgesic purposes. One of its production methods is the calcium chloride process, which involves the reaction of aspirin with a calcium source under specific conditions. In this process, the mother liquor is complex, typically containing high concentrations of calcium chloride, unreacted aspirin, potentially generated salicylic acid (a hydrolyzed impurity of aspirin), and other process-related impurities. Accurate monitoring of the residual aspirin content in the mother liquor is crucial for process control: excessively high aspirin content may indicate incomplete reaction, affecting yield and cost; excessively low content may indicate over-reaction or undesirable hydrolysis of aspirin, affecting the purity and stability of the final product. One of the main application drawbacks of carbaspirin calcium is related to its impurity control, particularly excessively high levels of free salicylic acid, which may lead to increased gastrointestinal irritation in animals. Therefore, monitoring the content of aspirin and its hydrolyzed impurity, salicylic acid, at the source is of great significance for ensuring the quality of the final product.

[0003] Currently, there are relevant standards for the quality analysis of carbaspirin calcium. However, for reaction mother liquors with high salt content, high concentration, and rapidly changing composition, conventional titration or ultraviolet spectrophotometry lacks specificity and cannot distinguish aspirin from salicylic acid and other impurities, making it difficult to accurately reflect the real-time state of the process. Upstream process impurities such as acetylsalicylic anhydride, polyacetylated salicylic acid derivatives, and degradation products (in addition to salicylic acid, there may also be phenyl acetylsalicylate, etc.) may not be completely separated from the main peak under poor chromatographic conditions, hiding in the tail or shoulder of the main peak, leading to missed detection or inaccurate quantification of impurities.

[0004] Therefore, there is an urgent need to develop an HPLC method specifically for the determination of aspirin content in carbaspirin calcium mother liquor using the calcium chloride method, in order to meet the process requirements. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a high-performance liquid chromatography external standard method that is highly specific, accurate, reproducible, easy to operate, and applicable to the determination of aspirin content in carbaspirin calcium mother liquor using the calcium chloride method.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A high-performance liquid chromatography (HPLC) method for determining the aspirin content in the mother liquor of carbaspirin calcium prepared by the calcium chloride method is disclosed. The method employs an external standard method and the chromatographic conditions are as follows: a reversed-phase column resistant to high aqueous phase, preferably an octadecylsilane-bonded silica (C18) column, with a column length of 150-250 mm, an inner diameter of 4.6 mm, and a particle size of 3-5 μm. Gradient elution is performed using a mixed mobile phase A (acetonitrile) and mobile phase B (0.05%-0.2% phosphoric acid aqueous solution); the detection wavelength is 225-235 nm, preferably 230 nm; the flow rate is 0.8-1.2 mL / min; the column temperature is 25-40℃; and the injection volume is 5-20 μL.

[0008] During the gradient elution process, the initial ratio of mobile phase A to mobile phase B is 45:55; within 0-8 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from the initial ratio to 65:35 at a uniform rate; within 8-10 minutes, the volume ratio of mobile phase A to mobile phase B remains constant at 65:35; within 10-12 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 65:35 to 45:55 at a uniform rate; and within 12-15 minutes, the volume ratio of mobile phase A to mobile phase B remains constant at 45:55.

[0009] The liquid chromatography method for determining the aspirin content in the mother liquor for preparing carbaspirin calcium by the calcium chloride method according to the present invention further includes the following steps:

[0010] S1. Preparation of the test solution: Accurately measure a certain volume of carbaspirin calcium to prepare the mother liquor, adjust the pH to 5.5-7.5 with phosphoric acid, dilute and dissolve with a diluent to ensure that the main component of carbaspirin calcium dissolves without significant acid hydrolysis, quantitatively dilute with a diluent to a suitable concentration, filter through a 0.45 μm microporous membrane, and take the filtrate as the test solution.

[0011] S2. Preparation of reference solutions: Accurately weigh an appropriate amount of reference standard, dissolve and quantitatively dilute it with the diluent to prepare a series of reference solutions of known concentrations.

[0012] S3. Determination: Under the chromatographic conditions described above, accurately pipette the reference solution and the test solution, respectively, and inject them into the liquid chromatograph, recording the chromatograms. Calculate the concentration of aspirin in the test solution based on the peak area using the external standard method, and then convert the concentration to the aspirin content in the mother liquor according to the dilution factor.

[0013] The formula for calculating aspirin content is:

[0014] Aspirin content (mg / mL) = (A1 × C) 1s × D1) / (A 1s )

[0015] Wherein, A1: the peak area of ​​aspirin in the test solution;

[0016] A 1s Peak area of ​​aspirin in the reference solution;

[0017] C 1s Concentration of the reference solution (mg / mL);

[0018] D1: Total dilution factor of the test solution.

[0019] The formula for calculating the free salicylic acid content is:

[0020] Free salicylic acid content (mg / mL) = (A2 × C) 2s × D2) / (A 2s )

[0021] Where, A2: peak area of ​​salicylic acid in the test solution;

[0022] A 2s Peak area of ​​salicylic acid in the reference solution;

[0023] C 2s Concentration of the reference solution (mg / mL);

[0024] D2: Total dilution factor of the reference solution.

[0025] The method of this invention can be applied to the process control of preparing carbaspirin calcium by the calcium chloride method, to monitor the residual amount of aspirin in the reaction mother liquor, and can also be used to evaluate the content of salicylic acid impurities in the carbaspirin calcium mother liquor.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] (1) High specificity: This method can effectively separate aspirin, salicylic acid (the main process impurities and degradation products) and other unknown impurities in the mother liquor, ensuring that the aspirin peak is not disturbed and the quantification is accurate.

[0028] (2) Strong anti-interference ability: Through optimized chromatographic conditions (especially mobile phase composition), it can withstand the matrix effect of high concentration of calcium chloride in the mother liquor, avoid salt precipitation in the chromatographic system, protect the chromatographic column and ensure good peak shape.

[0029] (3) High accuracy and good reproducibility: The external standard method is adopted. Through strict system suitability control and linear range investigation, the method precision (RSD) is good, the recovery test results are ideal, and it fully meets the requirements of quantitative analysis.

[0030] (4) Fast and simple: The gradient elution procedure shortens the single-needle analysis time and facilitates rapid monitoring of the process. Only dilution and filtration are required, making it suitable for online or near-line sampling and analysis.

[0031] (5) Significant application value: The method of this invention can be directly used to determine the critical control point reaction endpoint in the preparation of carbaspirin calcium by the calcium chloride method. By monitoring the aspirin residue and optimizing process parameters, the generation of salicylic acid impurity can be reduced from the source, which has important guiding significance for improving the quality and stability of the final product and controlling its gastrointestinal irritation. At the same time, this method can also be used for the quantitative evaluation of salicylic acid in the mother liquor. Attached Figure Description

[0032] Figure 1 The image shows the liquid chromatogram of the aspirin reference solution in Example 1. Detailed Implementation

[0033] The present invention will be further described in detail below with reference to the embodiments, but the scope of protection of the present invention is not limited thereto.

[0034] Example 1

[0035] (a) Chromatographic conditions and system suitability test:

[0036] Chromatographic column: Agilent ZORBAX SB-C18 column (4.6 mm × 150 mm, 5 μm); mobile phase: mobile phase A is acetonitrile, mobile phase B is 0.1% phosphoric acid aqueous solution, gradient elution; column temperature: 30℃; detection wavelength: 230 nm; injection volume: 10 μL; flow rate: 1.0 mL / min; the theoretical plate number of the aspirin peak is not less than 2500, and the resolution between the aspirin peak and the salicylic acid peak is greater than 2.0.

[0037] During the gradient elution process, the initial ratio of mobile phase A to mobile phase B is 45:55; within 0-8 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from the initial ratio to 65:35 at a uniform rate; within 8-10 minutes, the volume ratio of mobile phase A to mobile phase B remains constant at 65:35; within 10-12 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 65:35 to 45:55 at a uniform rate; and within 12-15 minutes, the volume ratio of mobile phase A to mobile phase B remains constant at 45:55.

[0038] (II) Solution preparation:

[0039] Diluent: Methanol-Water (50:50, v / v)

[0040] Aspirin reference solution: Accurately weigh approximately 25 mg of aspirin reference standard, place it in a 50 mL volumetric flask, dissolve and dilute to the mark with diluent, and shake well to prepare the stock solution. Accurately measure an appropriate amount and dilute stepwise with diluent to a concentration of 20 μg / mL, then filter through a 0.45 μm filter membrane.

[0041] Salicylic acid reference solution: Prepare a 5 μg / mL salicylic acid reference solution using the same method.

[0042] Mixed solution: Take an appropriate amount of the above aspirin and salicylic acid stock solution and dilute it with a diluent to prepare a mixed solution containing 20 μg of aspirin and 5 μg of salicylic acid per 1 mL.

[0043] Test solution: Accurately measure 0.5 mL of carbaspirin calcium reaction stock solution and place it in a 100 mL volumetric flask. Adjust the pH to 5.5-7.5 with phosphoric acid solution, quantitatively dilute with diluent to the appropriate concentration, shake well, and filter through a 0.45 μm filter membrane.

[0044] Determination: Accurately pipette 10 μL each of the aspirin reference solution and the test solution into the liquid chromatograph and record the chromatograms. (Chemistry of the test solution) Figure 1 The results showed that the carbaspirin calcium peak (retention time approximately 6.362 min), salicylic acid peak (retention time approximately 10.021 min), aspirin peak (retention time approximately 11.417 min), and other impurity peaks (retention time approximately 19.319 min) were all baseline separated, indicating that the method has good specificity.

[0045] Example 2

[0046] Accurately weigh an appropriate amount of aspirin reference standard and prepare a stock solution with a concentration of approximately 100 μg / mL using diluent. Accurately measure an appropriate amount of this stock solution and dilute it with diluent to prepare a series of reference standard solutions with concentrations of approximately 5, 10, 20, 40, 60, and 80 μg / mL. Inject the solutions sequentially under the chromatographic conditions of Example 1. Perform linear regression with concentration (C μg / mL) as the abscissa and peak area (A) as the ordinate. The regression equation is: A = 35624C - 1852, with a correlation coefficient r = 0.9999. The results indicate that aspirin exhibits good linearity in the concentration range of 5-80 μg / mL.

[0047] Example 3

[0048] Take the same sample solution (prepared according to Example 1), inject it six times consecutively under the chromatographic conditions of Example 1, and record the peak area of ​​aspirin. The relative standard deviation (RSD) is calculated to be 0.32%, indicating that the method has good precision.

[0049] Example 4

[0050] The spiking recovery method was employed. Nine 0.25 mL aspirin stock solutions of known low-concentration aspirin were precisely measured and placed in separate 50 mL volumetric flasks, divided into three groups. For each group, three concentration levels of known-concentration aspirin reference standard stock solution (low, medium, and high) were precisely added. The solutions were then diluted to the mark with diluent and analyzed according to the method in Example 1. The calculated average recovery rate was 99.8%, with an RSD of 0.95% (n=9), indicating that the method accuracy met the requirements.

[0051] Example 5

[0052] Based on the chromatographic conditions of Example 1, minor variations were made to the organic phase ratio in the mobile phase (±5%), flow rate (±0.1 mL / min), column temperature (±5℃), and a different brand of the same type of C18 column. The effects on system suitability (mainly the resolution between aspirin and salicylic acid) and the peak area of ​​aspirin were investigated. The results showed that under these minor variations, the resolution was greater than 2.0 and the peak area RSD was less than 2.0%, indicating good robustness of the method.

[0053] Example 6

[0054] Three batches of carbaspirin calcium preparation mother liquor from different reaction time points using the calcium chloride method were collected. Test solutions were prepared and determined according to the method in Example 1. The aspirin content in the mother liquor was calculated using the external standard method. The results can be used to monitor the reaction progress and assist in determining the reaction endpoint. The aspirin residue in batch 1 (mid-reaction) was 15.2 g / L, in batch 2 (near the endpoint) it was 2.1 g / L, and in batch 3 (at the end of the reaction) it was 0.5 g / L. This method provides accurate data support for process optimization.

[0055] While the specific embodiments of the present invention have been described above, they are not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

Claims

1. A liquid chromatography method for determining the aspirin content in the mother liquor from the calcium chloride method for preparing carbaspirin calcium, characterized in that, The external standard method of high performance liquid chromatography (HPLC) was used. The HPLC conditions included: using a reversed-phase column resistant to high aqueous phase; gradient elution using a mixture of mobile phases A and B, wherein mobile phase A is acetonitrile and mobile phase B is a 0.05%-0.2% aqueous phosphoric acid solution; detection wavelength of 225-235 nm; flow rate of 0.8-1.2 mL / min; column temperature of 25-40℃; and injection volume of 5-20 μL.

2. The liquid chromatography method for determining the aspirin content in the mother liquor for preparing carbaspirin calcium by the calcium chloride method according to claim 1, characterized in that, The chromatographic column is an octadecylsilane-bonded silica gel column.

3. A liquid chromatography method for determining the aspirin content in the mother liquor for preparing carbaspirin calcium by the calcium chloride method according to claim 1 or 2, characterized in that, During the gradient elution process, the initial ratio of mobile phase A to mobile phase B is 45:55; within 0-8 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from the initial ratio to 65:35 at a uniform rate; within 8-10 minutes, the volume ratio of mobile phase A to mobile phase B remains constant at 65:35; within 10-12 minutes, the volume ratio of mobile phase A to mobile phase B gradually changes from 65:35 to 45:55 at a uniform rate; and within 12-15 minutes, the volume ratio of mobile phase A to mobile phase B remains constant at 45:

55.

4. The liquid chromatography method for determining the aspirin content in the mother liquor for preparing carbaspirin calcium by the calcium chloride method according to claim 1, characterized in that, Includes the following steps: S1. Preparation of the test solution: Adjust the pH of the carbaspirin calcium preparation mother liquor, dilute with diluent, filter, and obtain the test solution; S2. Preparation of reference solution: Dissolve the reference standard with a diluent to obtain a reference solution of known concentration; S3. Determination: Inject the reference solution and the test solution separately, and calculate the aspirin content in the carbaspirin calcium preparation mother liquor by peak area using the external standard method.

5. The liquid chromatography method for determining the aspirin content in the mother liquor for preparing carbaspirin calcium by the calcium chloride method according to claim 4, characterized in that, In step S1, the pH of the carbaspirin calcium preparation mother liquor is adjusted to 5.5-7.5 using phosphate.

6. The liquid chromatography method for determining the aspirin content in the mother liquor for preparing carbaspirin calcium by the calcium chloride method according to claim 4, characterized in that, The diluent is a mixed solution of methanol and water; by volume ratio, methanol:water = 1:

1.

7. The application of the liquid chromatography method for determining the aspirin content in the mother liquor of calcium carbaspirin prepared by the calcium chloride method as described in any one of claims 1-6 in the process control of calcium carbaspirin prepared by the calcium chloride method.

8. The liquid chromatography method for determining the aspirin content in the mother liquor of carbaspirin calcium prepared by the calcium chloride method as described in any one of claims 1-6 is used to evaluate the salicylic acid impurity content in the carbaspirin calcium mother liquor.

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