Method for determining acetate content in compound sodium acetate and glucose injection

Abstract

The invention provides a method for determining the content of acetate in a compound sodium acetate-glucose injection, and belongs to the technical field of medicine component analysis, the content of acetate is determined by adopting a high performance liquid chromatography (HPLC) technology, high-sensitivity and high-resolution separation and detection can be realized, and the detection accuracy is high. According to the method, chromatographic conditions (such as chromatographic column temperature, mobile phase composition, flow velocity, detection wavelength and the like) are accurately set, operation steps are standardized, a reference substance solution is reasonably selected, a hydrogen-type sulfonated cross-linked styrene-divinyl benzene copolymer is adopted as a chromatographic column filling agent, and ultrasonic degassing treatment is introduced; the accuracy and the reliability of determination are improved, so that the method is more accurate, higher in efficiency and better in stability when being used for detecting the content of acetate.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical component analysis technology, specifically a method for determining the acetate content in compound sodium acetate glucose injection. Background Art

[0002] Compound sodium acetate glucose injection is a complex preparation containing glucose and various electrolytes (such as sodium, potassium, and magnesium ions). This preparation is primarily used in medical practice to maintain the body's water and electrolyte balance while providing essential energy. Acetate, as a component of this complex preparation, plays a crucial role in regulating the body's acid-base balance. Accurate concentration determination ensures the effectiveness and safety of the preparation in restoring and maintaining electrolyte and acid-base balance, avoiding adverse reactions caused by excessively high or low concentrations. During production, accurate determination of acetate content is critical for monitoring and quality control. This helps to promptly identify deviations and problems in the production process, allowing for appropriate adjustments to ensure the quality of the final product.

[0003] The determination of acetate in the section on synthetic polypeptides in Part IV of the Chinese Pharmacopoeia is mentioned. This method uses a C18 column, an acidic mobile phase, and a UV detector. Although this method can provide accurate results in some cases, it has significant limitations. The results of chromatographic analysis using this method are shown in Table 14 below:

[0004] concentration 0.16mg / ml 0.4mg / ml 1.6mg / ml Injection volume 10μl 100 μl 10μl Peak shape normal Crack peak Crack peak Advantages and Disadvantages The peak area is too small Generate crack peaks Generate crack peaks

[0005] Table 14

[0006] From the results in the table, we can see that: Figure 5 As shown, the main peak shape is normal only when the sample concentration is 0.16 mg / ml, but at this point the peak area is too small, the response is too low, and accurate quantification is impossible. Figure 6 and Figure 7 As shown, when the concentration increased to 0.4 mg / ml and 1.6 mg / ml, even when the injection volume increased to 100 μl at 0.4 mg / ml, peak splitting occurred in both cases, which interferes with the accuracy of peak resolution and quantification. Peak splitting suggests that while increasing concentration helps improve peak area, it may also introduce analytical errors.

[0007] Application No. 202311694644.0 discloses an HPLC method for determining the content of citrate, gluconate, and acetate in compound sodium acetate Ringer's injection, which involves the detection of acetate content. After comparison with the solution of this invention, the differences are as follows:

[0008] Column temperature setting: The operating temperature of the chromatographic column in this invention is set to 55-65℃, which is lower than the 75-85℃ in the comparative document. The lower column temperature helps protect the chromatographic column and improve the stability of the analyte, while also helping to improve the peak shape and resolution of acetate and reduce analytical errors caused by thermal degradation.

[0009] Mobile phase pH and flow rate: The mobile phase used in this invention is a 0.045-0.055 mol / L sulfuric acid solution with a low pH, which helps enhance the ionization of acetate ions and improves their adsorption and elution on the chromatographic column. Furthermore, the flow rate is set at 0.7-0.9 mL / min, slightly higher than the 0.4-0.6 mL / min in the comparative document, which reduces analysis time and improves detection efficiency.

[0010] Precise preparation of test solutions and reference solutions: The preparation of test solutions and reference solutions is described in detail in this invention, especially the precise preparation of reference solutions and the degassing treatment of test solutions. This helps to reduce fluctuations and errors in experiments and ensure the repeatability and accuracy of experimental results.

[0011] Detection Wavelength: The detection wavelength range selected in this invention is 210±2nm, while the comparative document uses 210nm. In the near-ultraviolet region, 210nm is a wavelength at which many organic compounds, including acetate, exhibit strong absorption characteristics. Therefore, selecting the vicinity of 210nm for measurement allows the spectrometer to utilize its high sensitivity at this wavelength. Providing a wavelength range (e.g., 210±2nm) allows experiments to be adjusted around the optimal sensitivity point, ensuring that absorption peaks are accurately captured, which is crucial for improving the accuracy of the measurement. Wavelength settings may have slight deviations, especially with frequent use or long calibration intervals. By setting a wavelength range rather than a fixed wavelength, greater flexibility is provided to the operator to adapt to the actual performance of the instrument, ensuring reliable data even with small wavelength shifts.

[0012] These two HPLC methods are similar in implementation, but they differ in specific operating parameters such as column temperature, flow rate, pH control, and detection wavelength.

[0013] In view of the above problems, it is necessary to develop a special method for determining the acetate content in compound sodium acetate glucose injection, so as to improve the accuracy and reliability of the determination, making it more precise, efficient and stable in detecting acetate content. Summary of the Invention

[0014] The purpose of this invention is to address the aforementioned problems in existing technologies by proposing a method for determining the acetate content in compound sodium acetate glucose injection.

[0015] The objective of this invention can be achieved through the following technical solution: A method for determining the acetate content in compound sodium acetate glucose injection, wherein the acetate content in compound sodium acetate glucose injection is determined by high performance liquid chromatography (HPLC), and the specific determination steps are as follows:

[0016] S1 solution preparation: The test sample of compound sodium acetate glucose injection is degassed and used as the test solution;

[0017] Reference solution: A sodium acetate solution of 1.64 mg / mL was prepared using deionized water as the solvent;

[0018] A sodium acetate solution of 1.64 mg / mL was chosen for the following reasons: To ensure the accuracy and precision of the determination: For any analytical method, a standard substance of known concentration is needed as a reference to verify the accuracy of the analysis. In liquid chromatography, the concentration of the analyte can be inferred by comparing the response (e.g., peak area) of the sample to a standard of known concentration. Therefore, a sodium acetate solution of 1.64 mg / mL, with its precisely calculated concentration, provides a reliable benchmark for calculating the acetate content in the sample.

[0019] Choosing an appropriate concentration range: The concentration of 1.64 mg / mL was selected considering both the detection sensitivity of liquid chromatography and the convenience of sample preparation. This concentration is high enough to produce clear chromatographic peaks in the column, while also avoiding excessive concentration or dilution that could affect the analytical results.

[0020] Compatibility with chemical reactions: During the dissolution process, the chemical stability and solubility of sodium acetate must meet the requirements of chromatographic analysis. A concentration of 1.64 mg / mL has been experimentally verified as suitable as a reference standard for the external standard method, providing stable and reproducible analytical results.

[0021] In summary, a 1.64 mg / mL sodium acetate solution was selected as the reference standard to ensure the accuracy and reliability of the method for determining the acetate content in compound sodium acetate glucose injection. As a standard, it helps determine the acetate concentration in the test sample by comparison with the test sample, thereby assessing its quality and specifications.

[0022] S2 Chromatographic conditions are set as follows: In a high performance liquid chromatograph, a strong cation hydrogen exchange column with hydrogen-form sulfonated crosslinked styrene-divinylbenzene copolymer as the packing material is selected as the chromatographic column.

[0023] The column operating temperature is set to 55-65℃;

[0024] A 0.045-0.55 mol / L sulfuric acid solution was selected as the mobile phase.

[0025] The flow rate of the mobile phase was set to 0.7-0.9 mL / min;

[0026] The injection volume of both the test solution and the reference solution was set to 20 μl.

[0027] The parameters of the hydrogen-form sulfonated crosslinked styrene-divinylbenzene copolymer are as follows: crosslinking degree 8%; spherical shape; particle size 8-10 μm; pore size... to

[0028] The detection wavelength was set to 210±2nm;

[0029] The choice of a wavelength of 210±2 nm for high-performance liquid chromatography (HPLC) analysis is based on several key factors, specifically for the detection and measurement of acetate or its salts (such as sodium acetate). The reasons for choosing this particular wavelength are as follows:

[0030] Absorption characteristics: Acetate and its salts have specific absorption peaks in the ultraviolet region. Although the absorption of acetate itself is not very strong in the visible and ultraviolet regions, its absorption peak usually appears in the wavelength range below 230 nm. Choosing 210±2 nm can effectively capture the absorption of acetate at this wavelength, thereby ensuring sensitive and specific detection of acetate content in the sample.

[0031] Chemical sensitivity: The wavelength of 210±2 nm is also within the absorption region of many organic acids and their salts, making it a suitable choice for analyzing such compounds. This wavelength is sensitive to a variety of organic acid compounds dissolved in injection solutions, thus providing a good analytical window in complex matrices.

[0032] Standardization of equipment and methods: Using a wavelength of 210±2nm for detection also helps to standardize the analytical process, which is beneficial for method replication and cross-laboratory comparison, because using standardized operating conditions can reduce the complexity of method development and the variability of experimental results.

[0033] Optimized sensitivity and accuracy: Detection at 210±2 nm typically offers superior sensitivity and accuracy compared to other wavelengths, especially for organic acids and their salts. This is particularly important for ensuring quality control of pharmaceutical products such as compound sodium acetate glucose injection.

[0034] In summary, the selection of a wavelength of 210±2 nm for HPLC analysis of acetate content is based on a comprehensive consideration of its chemical absorption characteristics, improved detection specificity, reduced interference, method standardization, and optimized detection sensitivity and accuracy. These factors work together to ensure the effectiveness and reliability of this method.

[0035] The reasons for choosing hydrogen-form sulfonated crosslinked styrene-divinylbenzene copolymers and the parameter requirements are as follows:

[0036] Ion exchange capacity: After sulfonation treatment, the styrene-divinylbenzene copolymer exhibits good ion exchange capacity. For the negatively charged acetate ions in compound sodium acetate glucose injection, this filler can effectively undergo ion exchange reaction with them, so as to achieve the separation and determination of the target analyte.

[0037] Chemical stability and high temperature resistance: In high performance liquid chromatography analysis, columns need to be able to withstand high temperatures and chemically unstable samples. Sulfonated crosslinked styrene-divinylbenzene copolymer has good chemical stability and can maintain structural integrity at an operating temperature of 55-65℃, ensuring the stability and reliability of the analysis process.

[0038] A crosslinking degree of 8% ensures both the physical strength and chemical stability of the column, while providing sufficient exchange sites to enhance the efficiency and selectivity of ion exchange.

[0039] Spherical shape and particle size of 8-10 μm: Spherical particles facilitate uniform column packing, ensuring uniform fluid flow within the column, thereby improving analytical reproducibility and accuracy. The choice of particle size aims to provide sufficient surface area while maintaining good separation capability and peak symmetry.

[0040] Aperture size ( to This pore size range can balance the retention and separation of sample molecules, making it particularly suitable for the analysis of small molecule ions such as acetate, ensuring the sensitivity and resolution of the analysis.

[0041] In summary, sulfonated crosslinked styrene-divinylbenzene copolymer, as a chromatographic column packing material, can effectively meet the requirements for determining the acetate content in compound sodium acetate glucose injection, providing superior ion exchange performance and stable analytical conditions, thus ensuring the accuracy and reliability of the analytical results.

[0042] The reasons for choosing 0.045-0.055 mol / L sulfuric acid solution as the mobile phase are as follows: Excellent ion exchange performance: Sulfuric acid solution can effectively undergo ion exchange reactions with negatively charged ions (such as acetate ions). In this method, the chromatographic column uses hydrogen-form sulfonated cross-linked styrene-divinylbenzene copolymer as the packing material. This type of column has good cation exchange capacity and can effectively separate acetate ions from compound sodium acetate glucose injection.

[0043] Stable chromatographic conditions: 0.045–0.055 mol / L sulfuric acid solution provides a stable acidic environment in chromatographic analysis, helping to maintain analytical consistency and reproducibility. This is crucial for ensuring the accuracy of analytical results between test solutions and reference solutions.

[0044] Compatibility: The physical and chemical properties of sulfuric acid solution during chromatographic separation will not damage the column, maintaining the stability and lifespan of the packing material, while not interfering with the analysis and detection of the target substance.

[0045] Flowability and separation efficiency: 0.045-0.055 mol / L sulfuric acid solution can effectively propel the sample through the chromatographic column at an appropriate flow rate (0.7-0.9 mL / min), ensuring analytical efficiency. In addition, the selected column operating temperature is 55-65℃, which helps to improve the column's separation efficiency and the sample analysis speed.

[0046] In summary, the selection of 0.045-0.055 mol / L sulfuric acid solution as the mobile phase is based on its superior ion exchange performance, stable chromatographic conditions, and compatibility with the column and analytical process. This selection ensures the accurate determination of acetate content in compound sodium acetate glucose injection, meeting analytical requirements and standards.

[0047] The mobile phase flow rate was set at 0.7-0.9 mL / min in the liquid chromatography method for determining the acetate content in compound sodium acetate glucose injection based on the following considerations:

[0048] Balancing Separation Efficiency and Analysis Speed: The choice of flow rate directly affects both the chromatographic separation efficiency and the analysis speed. Lower flow rates generally improve separation efficiency, allowing the target substance to interact more fully with the packing material, thereby enhancing separation capability. For the analysis of acetate ions, sufficient resolution is required to avoid interference with other components; therefore, a moderate flow rate of 0.7-0.9 mL / min was selected.

[0049] Column design and performance: In this method, a strong cation exchange column using hydrogen-form sulfonated crosslinked styrene-divinylbenzene copolymer as the packing material was employed. This column exhibits selectivity and efficiency in the retention and separation of acetate ions, and operation at a flow rate of 0.7–0.9 mL / min ensures analytical accuracy and reproducibility.

[0050] Instrument and column compatibility is crucial: HPLC systems and columns are typically designed to operate within a specific flow rate range to ensure uniform mobile phase distribution within the column, preventing premature column failure or inaccurate analytical results. Choosing a flow rate of 0.7–0.9 mL / min takes these factors into account, aiming to optimize separation and analytical speed while ensuring stable operation of the column and instrument for accurate and reproducible analytical results.

[0051] Trade-offs between detection sensitivity and analysis speed: The choice of flow rate also takes into account the actual needs of the analysis, including the optimization of detection sensitivity and analysis time. Higher flow rates may reduce analysis time, but sometimes sacrifice separation efficiency or sensitivity. 0.7-0.9 mL / min is a reasonable trade-off point to ensure separation efficiency while minimizing analysis time.

[0052] Therefore, setting the mobile phase flow rate to 0.7-0.9 mL / min was a choice made after balancing multiple considerations of separation effect, analysis speed and instrument performance in chromatographic analysis.

[0053] The selection of a column operating temperature of 55-65℃ for the determination of acetate content in compound sodium acetate glucose injection was based on comprehensive considerations.

[0054] Optimal separation effect: Temperature has a significant impact on the separation effect of the chromatographic column. At a temperature of 55-65℃, the intermolecular spatial activity of the packing material (hydrogen-form sulfonated crosslinked styrene-divinylbenzene copolymer) is enhanced, making it easier for solvent and sample molecules to enter and interact with the packing material. This ensures that acetate ions are fully separated from other present ions or impurities, thereby accurately determining their content.

[0055] Chromatographic peak resolution and shape: When the column operates at 55-65℃, chromatographic peaks are typically sharper and more symmetrical. This is due to the increased diffusion rate of sample molecules within the pores of the packing material, which in turn improves resolution and peak shape symmetry. This is crucial for ensuring the accuracy and reproducibility of the measurement results.

[0056] Sample stability: Samples used in chromatographic analysis typically dissolve better at relatively high temperatures, ensuring that insufficient solubility or precipitation does not occur during the analysis. A temperature of 55-65℃ ensures sufficient sample dissolution without causing decomposition or reaction, thus guaranteeing the accuracy and stability of the analytical results.

[0057] Column performance: The packing material of a chromatographic column is typically designed with optimal operating temperature within a certain range in mind. For columns using strong cation exchange media, 55-65℃ helps maintain stability and separation efficiency, ensuring reliability and repeatability over long periods of operation.

[0058] Method validation and standardization: Using an operating temperature of 55-65℃ helps to meet previous validation data and method standardization requirements. This consistency ensures the comparability and consistency of analytical results between different laboratories and operators, thereby improving the reliability and applicability of the assay method.

[0059] In summary, setting the column operating temperature to 55-65℃ is to maximize the separation effect, sample solubility and stability, and to ensure the consistency and reliability of analytical conditions, thereby ensuring the accurate determination of acetate content in compound sodium acetate glucose injection and the successful application and effective implementation of the determination method.

[0060] In the determination of acetate content in compound sodium acetate glucose injection by high performance liquid chromatography (HPLC), setting the injection volume of both the test solution and the reference solution to 20 μl has multiple considerations and advantages: It ensures measurement precision and accuracy: the injection volume directly affects the formation and peak area in the chromatogram. An injection volume of 20 μl allows sufficient time for acetate to separate and adsorb in the chromatographic column, forming a clear peak, while maintaining analytical precision. This helps to accurately measure the acetate content, avoid peak overlap or misalignment, and ensure the reliability and repeatability of the measurement results.

[0061] Maximizing column utilization efficiency: The chromatographic column is a crucial separation device in the assay process. By using a reasonable injection volume, the column's separation capacity and resolution can be fully utilized, improving analytical efficiency and detection sensitivity. An injection volume of 20 μl satisfies the requirements for chromatographic resolution while allowing the mobile phase to pass through the sample and control concentration changes.

[0062] Reducing the likelihood of measurement errors: Injection volumes that are too large or too small can lead to poor chromatographic separation, thus affecting the accuracy of measurement results. A 20 μl injection volume balances the relationship between sample concentration and analysis time to a certain extent, effectively reducing systematic and random errors caused by injection and ensuring the reliability of measurement data.

[0063] Laboratory standardization and comparison: In analytical laboratories, using an injection volume of 20 μl makes experimental results easier to reproduce and compare. This standardized injection volume promotes the dissemination of experimental methods and the sharing of results, contributing to the standardization process and the uniformity of experimental methods.

[0064] In summary, setting the injection volume of the detection solution and the reference solution to 20 μl is to maximize the accuracy, repeatability, and efficiency of the analysis during the determination of acetate content in compound sodium acetate glucose injection by high performance liquid chromatography, thereby obtaining reliable and practical measurement results.

[0065] S3 Obtaining Chromatograms: The test solution and the reference solution are detected by liquid chromatography to obtain chromatograms of the test solution and the reference solution, respectively;

[0066] S4 Chromatographic Analysis: The content of acetate in the test solution was calculated using the external standard method. Specifically, the peak area of ​​acetate in the chromatogram of the test solution was compared with the peak area of ​​acetate in the chromatogram of the reference solution. The content of acetate in the test solution was then calculated using the following formula:

[0067] f = C 对 / A 对 ;

[0068] Acetate content = f × A1 ÷ 82.03 × 1000;

[0069] Among them, C 对 A represents the concentration of sodium acetate in the standard solution; 对 A1 represents the peak area of ​​acetate in the chromatogram of the reference solution; A2 represents the peak area of ​​acetate in the chromatogram of the detection solution; 82.03 represents the molecular weight of sodium acetate, and the unit of acetate content is mmol / L.

[0070] Preferably, in step S1, the degassing treatment of the test solution is ultrasonic treatment, and the ultrasonic treatment process is as follows: set the ultrasonic processor parameters: power: 100-200W; frequency: 40kHz; treatment time: 5-10 minutes; temperature: 25-30°C;

[0071] Operating steps: Place the compound sodium acetate glucose injection sample to be processed in the ultrasonic cleaner; start the ultrasonic equipment and process it according to the ultrasonic processor parameters.

[0072] Results check: After the treatment is completed, check the surface of the solution for any residual bubbles.

[0073] The ultrasonic processor parameters (power: 100-200W; frequency: 40kHz; processing time: 5-10 minutes; temperature: 25-30℃) are set to effectively degas the solution and ensure that the compound sodium acetate glucose injection solution is free of any interfering air bubbles before entering high-performance liquid chromatography (HPLC) analysis. These parameters are set for the following reasons:

[0074] Power (100-200W): Setting the power within this range provides sufficient energy to generate tiny bubbles, which helps to rapidly release dissolved gases from the solution. Excessive power may cause overheating of the sample or generate shear forces, which could affect the chemical stability of the solution. Therefore, selecting a moderate power range helps to balance efficiency and sample protection.

[0075] Frequency (40kHz): The key to choosing the right frequency is to provide sufficient vibration to promote bubble formation and aggregation, which in turn facilitates bubble rise and removal. 40kHz has been proven to effectively remove bubbles from solution without damaging the chemical composition.

[0076] Processing time (5-10 minutes): This time range is sufficient for most of the dissolved gases to escape from the solution. Too short a time may not be enough for complete degassing, while too long a time may unnecessarily consume energy or cause thermal degradation of the sample.

[0077] Temperature (25-30℃): Maintaining the temperature within this range can prevent changes in sample properties or solubility issues caused by excessively high or low temperatures. Keeping the temperature near room temperature helps maintain the stability of chemical substances and avoids thermal decomposition, while also facilitating more efficient bubble escape.

[0078] These parameters are considered holistically to ensure the sample is in optimal physical condition before HPLC analysis, while avoiding sample deterioration or deviations in analytical results due to improper operation. This setup ensures experimental repeatability and accuracy, which is particularly crucial for analytical quality control requiring stringent procedures.

[0079] Preferably, in step S1, the preparation process of the reference solution is as follows: Sodium acetate of the required purity (HPLC grade) is dried, accurately weighed, and then added to deionized water and stirred until completely dissolved. Further deionized water is added to adjust the concentration to 1.64 mg / mL. Subsequently, the solution is degassed to avoid interference from air bubbles in subsequent steps. The degassed treatment is ultrasonic treatment, and the drying temperature is 120°C.

[0080] Preferably, in step S2, the chromatographic column parameters are as follows: inner diameter of 7.8 mm and length of 300 mm.

[0081] The selection of the column's inner diameter and length (7.8 mm and 300 mm) is an important comprehensive consideration in the method of determining the acetate content in compound sodium acetate glucose injection by liquid chromatography, mainly reflected in the following aspects:

[0082] Separation efficiency and resolution: The size of the chromatographic column directly affects the separation efficiency and resolution. A longer column (300 mm in length) provides more separation paths, facilitating the effective separation of the target compound (acetate ion) from other components in the sample. This is particularly important for complex sample systems such as compound sodium acetate glucose injection, ensuring the accuracy and reliability of the analytical results.

[0083] Sample adsorption and retention time: The 7.8 mm inner diameter column provides sufficient surface area and internal space for the mobile phase to pass through, which facilitates the adsorption and desorption of sample components on the packing surface, thereby controlling the retention time for analysis. This design balances the speed of sample separation and analysis, ensuring peak formation and separation efficiency.

[0084] Economy and performance optimization of the mobile phase: The selection of inner diameter and length is also related to the selection and performance optimization of the mobile phase. Under the conditions of a mobile phase of 0.045-0.055 mol / L sulfuric acid solution, a column with an inner diameter of 7.8 mm can maintain the homogeneity and stability of the mobile phase at a flow rate of 0.7-0.9 mL / min, thereby ensuring the accuracy and repeatability of the analysis.

[0085] Experimental cost and efficiency considerations: The selection of inner diameter and length also involves a balance between experimental cost and efficiency. An inner diameter of 7.8 mm ensures analytical performance while controlling experimental costs, because a wider column may require more mobile phase and sample, increasing the cost of experimental operation and the use of consumables.

[0086] Therefore, considering factors such as chromatographic separation effect, analysis speed, mobile phase stability and experimental cost, a chromatographic column with an inner diameter of 7.8 mm and a length of 300 mm was selected as the analytical tool to achieve the best analytical effect and experimental economy when determining the acetate content in compound sodium acetate glucose injection by liquid chromatography.

[0087] Preferably, in step S3, the specific process of obtaining the chromatogram of the detection solution is as follows: start the high-performance liquid chromatograph, allow the detection solution to be separated in the chromatographic column according to the chromatographic conditions, use the detector of the high-performance liquid chromatograph to continuously monitor the components flowing out of the chromatographic column at the detection wavelength, and record the results to generate the chromatogram of the detection solution.

[0088] Preferably, the detector type is an ultraviolet detector.

[0089] Compared with existing technologies, this method for determining the acetate content in compound sodium acetate glucose injection has the following advantages:

[0090] 1. Improved accuracy and reliability of determination: Application of high-performance liquid chromatography (HPLC) technology: This invention uses high-performance liquid chromatography (HPLC) technology to determine acetate content, achieving high sensitivity and high resolution separation and detection, significantly improving the accuracy of the determination. HPLC technology can effectively separate various components in the sample, ensuring accurate determination of acetate.

[0091] 2. Standardized chromatographic conditions: By precisely setting chromatographic conditions (such as column temperature, mobile phase composition, flow rate, and detection wavelength), this invention optimizes the determination process, reduces analytical errors, and improves the reliability of the results.

[0092] 3. Improved Detection Efficiency: Optimized Column Selection: Using hydrogen-form sulfonated crosslinked styrene-divinylbenzene copolymer as the column packing material, combined with specific column parameters (such as inner diameter and length), ensures high-efficiency separation performance, shortens detection time, and improves overall detection efficiency. The structural design of hydrogen-form sulfonated crosslinked styrene-divinylbenzene copolymer (i.e., a strong cation exchange resin) endows it with excellent selectivity. Its strong cation exchange properties can effectively separate positively charged analytes (such as acetate ions) from other components, improving chromatographic resolution. For the complex ionic components in compound sodium acetate glucose injection, this high selectivity ensures accurate determination of acetate ions without interference from other ions.

[0093] 4. Precise mobile phase and injection volume: The precise preparation of the mobile phase and the standardized setting of the injection volume make the determination process more stable and efficient, reducing the analysis time and the need for repeatability tests.

[0094] 5. Enhanced method stability: Ultrasonic degassing treatment: In step S1, the test solution and the reference solution are subjected to ultrasonic degassing treatment to remove air bubbles from the solution, avoiding interference from air bubbles with the detection results. This treatment method ensures the stability of the solution and enhances the accuracy and consistency of the measurement results.

[0095] 6. Precise control of experimental conditions: By setting experimental conditions, such as the power, frequency and processing time of ultrasonic treatment, the stability of the degassing process is ensured, further improving the reliability and repeatability of the measurement method.

[0096] 7. The detection process is simplified and standardized: Clear operating steps: This invention specifies in detail the preparation, degassing treatment, chromatographic condition setting and calculation methods of the detection solution and reference solution, so that the entire detection process has a high level of standardization and reduces human operation error.

[0097] 8. External standard method: The external standard method is used to calculate the acetate content, which provides a simple and effective quantitative analysis method, reduces the interference of other components in the sample on the results, and improves the accuracy of the analysis results.

[0098] 9. Wavelength selection and detector type: The detection wavelength was set to 210±2nm and an ultraviolet detector was used, which can detect acetate ions with high sensitivity and meet the strict detection requirements for acetate ion content in compound sodium acetate glucose injection.

[0099] In summary, this invention provides an efficient, accurate, and stable method for determining acetate content in compound sodium acetate glucose injection. By optimizing chromatographic conditions, standardizing operating procedures, and introducing ultrasonic degassing, the accuracy, efficiency, and stability of the determination are significantly improved, providing reliable technical support for quality control and analysis in related fields. Attached Figure Description

[0100] Figure 1 This is the chromatogram of the blank solvent in Specific Example 2.

[0101] Figure 2 This is a chromatogram of the negative preparation solution in Specific Example 2.

[0102] Figure 3 This is a chromatogram of the reference solution in Specific Example 2.

[0103] Figure 4 This is a chromatogram of the sample detection solution in Specific Example 2.

[0104] Figure 5 The chromatogram is for a test sample with a concentration of 0.16 mg / ml in the background art.

[0105] Figure 6 The chromatogram of the test sample with a concentration of 0.4 mg / ml in the background art is shown.

[0106] Figure 7 The chromatogram is for a test sample with a concentration of 1.6 mg / ml in the background art.

[0107] Figure 8 This is the standard curve for each linear sampling solution in Table 5. DETAILED DESCRIPTION

[0108] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments. Specific Implementation Example 1:

[0110] A method for determining the acetate content in compound sodium acetate glucose injection is disclosed. The method utilizes high-performance liquid chromatography (HPLC) to determine the acetate content in the compound sodium acetate glucose injection. The specific determination steps are as follows:

[0111] S1 solution preparation: The test solution is degassed and then used as the sample detection solution;

[0112] Reference solution: A sodium acetate solution of 1.64 mg / mL was prepared using deionized water as the solvent;

[0113] The degassing treatment of the test solution was performed using ultrasonic treatment, and the ultrasonic treatment process is as follows:

[0114] Set the ultrasonic processor parameters: Power: 100W; Frequency: 40kHz; Processing time: 5-10 minutes; Temperature: 25-30℃.

[0115] Operating steps: Place the compound sodium acetate glucose injection sample to be processed in the ultrasonic cleaner; start the ultrasonic equipment and process it according to the ultrasonic processor parameters.

[0116] Results check: After the treatment is completed, check the surface of the solution for any residual bubbles.

[0117] The preparation process of the reference solution is as follows: HPLC-grade sodium acetate was dried at 120℃ for 2 hours. After accurate weighing, it was placed in a container, deionized water was added and stirred until completely dissolved. Further deionized water was added to adjust the concentration to 1.64 mg / mL. Subsequently, the solution was degassed to avoid interference from air bubbles in subsequent steps. The degassed treatment was performed using ultrasound.

[0118] S2 Chromatographic conditions were set as follows: A strong cation exchange column with hydrogen-form sulfonated crosslinked styrene-divinylbenzene copolymer as the packing material was selected as the chromatographic column in a high-performance liquid chromatograph (HPLC); the inner diameter was 7.8 mm; the length was 300 mm.

[0119] The column operating temperature is set to 60℃.

[0120] 0.05 mol / L sulfuric acid solution was selected as the mobile phase.

[0121] The flow rate of the mobile phase was set to 0.8 mL / min;

[0122] The injection volume for both the sample solution and the reference solution was set to 20 μl.

[0123] The parameters of the hydrogen-form sulfonated crosslinked styrene-divinylbenzene copolymer are as follows: crosslinking degree 8%; spherical shape; particle size 8-10 μm; pore size... to

[0124] S3. Obtaining Chromatograms: The sample test solution and the reference solution are detected by liquid chromatography to obtain chromatograms of the sample solution and the reference solution, respectively.

[0125] The specific process for obtaining the chromatogram of the sample solution is as follows: Start the high-performance liquid chromatograph (HPLC), allow the sample solution to be separated in the column according to the chromatographic conditions, use the detector of the HPLC to continuously monitor the components flowing out of the column at a wavelength of 210 nm, and record the results to generate the chromatogram of the sample solution.

[0126] The detector type is an ultraviolet detector.

[0127] S4 Chromatographic Analysis: The content of acetate in the sample solution was calculated using the external standard method. Specifically, the peak area of ​​acetate in the chromatogram of the sample solution was compared with the peak area of ​​acetate in the chromatogram of the reference solution. The content of acetate in the sample solution was then calculated using the following formula:

[0128] f = C 对 / A 对 ;

[0129] Acetate content = f × A1 ÷ 82.03 × 1000;

[0130] Among them, C 对 A represents the concentration of sodium acetate in the standard solution; 对 A1 represents the peak area of ​​acetate in the chromatogram of the reference solution; A2 represents the peak area of ​​acetate in the chromatogram of the sample solution; 82.03 represents the molecular weight of sodium acetate, and the unit of acetate content is mmol / L.

[0131] Specific example two:

[0132] Specific Example 2 is a system suitability test for a method for determining acetate content in compound sodium acetate glucose injection mentioned in Specific Example 1. System suitability testing is the process of verifying that an analytical method can be performed stably and reliably under given conditions. In high-performance liquid chromatography (HPLC) analysis, system suitability testing is mainly used to ensure the suitability of the chromatographic system, method procedures, and analytical operations. This test typically includes checking several key parameters, such as retention time, theoretical plate number, and tailing factor, to evaluate the performance of the chromatographic system and the repeatability of the method. Specific Example 2 is as follows:

[0133] 1) Solution preparation:

[0134] Blank solvent: water;

[0135] Reference solution: Accurately weigh about 41 mg of sodium acetate reference standard (dried at 120℃ for 2 h) and place it in a 25 ml volumetric flask. Add water to dissolve and dilute to the mark, shake well, and then degas.

[0136] Sample test solution: The test solution after degassing;

[0137] Negative reagent stock solution: Accurately weigh 730 mg sodium chloride, 260 mg potassium chloride, 255 mg magnesium chloride, 680 mg potassium dihydrogen phosphate, 25 g anhydrous glucose, and an appropriate amount of hydrochloric acid into a 100 ml volumetric flask. Dissolve and dilute to the mark with water, and shake well. This is called the negative reagent stock solution. Accurately measure 20 ml of the negative reagent stock solution into a 100 ml volumetric flask, dilute to the mark with water, and shake well.

[0138] (2) Results: Inject each of the above solutions separately, record the chromatograms, and the results are as follows: Figure 1 、 Figure 2 、 Figure 3 and Figure 4 As shown:

[0139] (3) Conclusion: Regarding the above Figure 1 、 Figure 2 、 Figure 3 and Figure 4 The analysis was conducted, and the specific results are shown in Table 1, the results of the system suitability test.

[0140] Solution name Retention time (min) Theoretical number of plates Tail factor blank solvent N / A N / A N / A Negative preparation solution N / A N / A N / A Reference solution 10.143 19013 1.33 Test solution 10.144 18894 1.36

[0141] Table 1

[0142] As shown in Table 1, the theoretical plate number of the acetate peak in the reference solution is 19013 and the tailing factor is 1.33; the theoretical plate number of the acetate peak in the sample detection solution is 18894 and the tailing factor is 1.36, which meets the system suitability requirements. Specific Implementation Example 3:

[0144] Specific Embodiment Three is a specificity test of the method for determining acetate content in compound sodium acetate glucose injection mentioned in Specific Embodiment One. Specificity refers to the ability of an analytical method to accurately determine the target analyte without interference from other components in the sample. Specificity is an important parameter for evaluating the reliability of an analytical method, especially when determining in complex samples such as biological samples or mixtures containing multiple compounds. A series of forced degradation experiments (also known as stability tests) were conducted, including acid degradation, alkali degradation, oxidative degradation, high temperature degradation, and light degradation, to test the specificity of the analytical method under various extreme conditions. Through these tests, the stability and reliability of the analytical method under various storage and handling conditions that the sample may undergo can be evaluated. The content of Specific Embodiment Three is as follows:

[0145] 1. Solution preparation:

[0146] Blank solvent: water;

[0147] Negative reagent stock solution: Accurately weigh 730 mg sodium chloride, 260 mg potassium chloride, 255 mg magnesium chloride, 680 mg potassium dihydrogen phosphate, 25 g anhydrous glucose, and an appropriate amount of hydrochloric acid into the same 100 ml volumetric flask, dissolve and dilute with water to the mark, and shake well.

[0148] Negative reagent solution: Accurately measure 20 ml of negative reagent stock solution into a 100 ml volumetric flask, dilute with water to the mark, and shake well.

[0149] 0.01 mol / L azobisisobutyronitrile solution: Weigh 16.4 mg of azobisisobutyronitrile into a 10 ml volumetric flask, add 3 ml of ethanol to dissolve, add water to make up to volume, and shake well.

[0150] Acid-base blank solution: Accurately measure 1 ml of 1 mol / L hydrochloric acid solution into a 10 ml volumetric flask, add 1 ml of 1 mol / L sodium hydroxide solution, dilute with water to the mark, and shake well.

[0151] Oxidation blank solution: Accurately measure 1 ml of 0.01 mol / L azobisisobutyronitrile solution into a 10 ml volumetric flask, dilute with water to the mark, and shake well.

[0152] Undamaged sample solution: Accurately measure 7 ml of the injection solution into a 10 ml volumetric flask, dilute with water to the mark, and shake well.

[0153] Sample acid destruction solution: Accurately measure 7 ml of the injection solution into a 10 ml volumetric flask, add 1 ml of 1 mol / L hydrochloric acid solution, let stand at room temperature for 2 h, then add 1 ml of 1 mol / L sodium hydroxide solution, dilute with water to the mark, and shake well.

[0154] Sample alkaline destruction solution: Accurately measure 7 ml of the injection solution into a 10 ml volumetric flask, add 1 ml of 1 mol / L sodium hydroxide solution, let stand at room temperature for 2 h, then add 1 ml of 1 mol / L hydrochloric acid solution, dilute with water to the mark, and shake well.

[0155] Sample oxidation damage solution: Accurately measure 7 ml of injection solution into a 10 ml volumetric flask, add 1 ml of 0.01 mol / L azobisisobutyronitrile solution, let stand at room temperature for 2 h, then dilute with water to the mark and shake well.

[0156] High-temperature destruction solution for samples: Take 7 ml of the injection solution into a 10 ml volumetric flask, heat it in an 80℃ water bath for 2 hours, cool it to room temperature, dilute it with water to the mark, and shake well.

[0157] Sample light damage solution: Take 7 ml of the injection solution into a 10 ml volumetric flask, place it in a light exposure chamber (4500 lx ± 500 lx) for 48 h, dilute with water to the mark, and shake well.

[0158] Undamaged negative reagent solution: Accurately measure 7 ml of negative reagent solution into a 10 ml volumetric flask, dilute with water to the mark, and shake well.

[0159] Acid-destroying solution for anion-positive preparations: Accurately measure 7 ml of anion-positive preparation solution into a 10 ml volumetric flask, add 1 ml of 1 mol / L hydrochloric acid solution, let stand at room temperature for 2 h, then add 1 ml of 1 mol / L sodium hydroxide solution, dilute with water to the mark, and shake well.

[0160] Negative reagent base destruction solution: Accurately measure 7 ml of the negative reagent into a 10 ml volumetric flask, add 1 ml of 1 mol / L sodium hydroxide solution, let stand at room temperature for 2 h, then add 1 ml of 1 mol / L hydrochloric acid solution, dilute with water to the mark, and shake well.

[0161] Oxidative agent destruction solution: Accurately measure 7 ml of the anion agent into a 10 ml volumetric flask, add 1 ml of 0.01 mol / L azobisisobutyronitrile solution, let stand at room temperature for 2 h, then dilute with water to the mark and shake well.

[0162] High-temperature destruction solution for negative reagent: Accurately measure 7 ml of negative reagent into a 10 ml volumetric flask, heat in an 80°C water bath for 2 hours, cool to room temperature, dilute with water to the mark, and shake well.

[0163] Negative reagent light-destroying solution: Accurately measure 7 ml of negative reagent into a 10 ml volumetric flask, place it in a light test chamber (4500 lx ± 500 lx) for 48 h, dilute with water to the mark, and shake well.

[0164] 2. Results: The above solutions were injected separately, and the chromatograms were recorded and analyzed. The specific results are shown in Table 2 below (specificity experiment results, forced degradation experiment).

[0165] Degradation conditions Main peak purity Separation between the main peak and adjacent peaks Sample (undamaged) 999.9 2.520 Sample acid destruction 999.9 2.628 Sample alkali destruction 999.9 2.690 Sample oxidation damage 999.9 2.263 High-temperature damage to samples 999.9 2.109 Sample light damage 999.9 2.216 Negative reagent (undamaged) N / A N / A Acid destruction of negative preparations N / A N / A Negative agents are destroyed by alkalinity. N / A N / A Oxidative damage of negative agents N / A N / A Negative agents are destroyed by high temperature N / A N / A Negative agents are destroyed by light. N / A N / A

[0166] Table 2

[0167] 3. Conclusion: Blank solution does not interfere with the determination of the main peak.

[0168] The negative reagent did not produce any impurity peaks after being destroyed by acid, high temperature, oxidation, and light. An impurity peak was produced under alkaline destruction conditions, with a retention time of 9.399 min, which did not affect the determination of acetate.

[0169] After the sample solution was subjected to acid, alkali, oxidation, high temperature, and light irradiation, the separation degree between the main peak and adjacent peaks was greater than 1.5, and the peak purity was 999.9%, which did not affect the detection of acetate. This method has good specificity. Specific Implementation Example 4:

[0171] Specific Implementation Four involves the linear validation of a method for determining acetate content in compound sodium acetate glucose injection mentioned in Specific Implementation One. Linear validation is a verification process in analytical chemistry used to ensure that an analytical method has a good linear response within a certain concentration range. This is an important part of analytical method validation, aiming to prove that the method can accurately determine the concentration of the analyte within a specified concentration range, and that the determination result is proportional to the actual concentration of the analyte in the sample. The steps of linear validation are as follows: Solution preparation: First, prepare a series of standard solutions of known concentrations, covering the required concentration range from low to high; Sample injection and determination: Using the same experimental conditions, perform multiple tests on each concentration of standard solution to ensure the reliability of the data. Specific details are as follows:

[0172] 1. Solution preparation:

[0173] Reference stock solution: Accurately weigh approximately 164 mg of sodium acetate reference standard (dried at 120℃ for 2 hours) and place it in a 25 ml volumetric flask. Dissolve and dilute to the mark with water, and shake well to obtain the reference stock solution (6.56 mg / ml). Prepare according to Table 3 below:

[0174]

[0175]

[0176] Table 32. Injection sequences, as shown in Table 4:

[0177] Sample Name Number of injection needles L_20％ 2 L_50％ 2 L_80％ 2 L_100％ 2 L_120％ 2 L_200％ 2

[0178] Table 4

[0179] 3. Result Processing:

[0180] Perform linear regression on the concentration of each linear solution (X) and the peak area (Y) to create a standard curve. The correlation coefficient (r) should be no less than 0.999.

[0181] The Y-axis intercept should be within 2% of the 100% response value.

[0182] The relative standard deviation of the response factor should not exceed 2%.

[0183] 4. Results: Each linear solution was injected separately, and the chromatograms were recorded. The analytical results are shown in Table 5.

[0184]

[0185] Table 5

[0186] 5. Conclusion: As shown in Table 5, sodium acetate exhibits good linearity in the concentration range of 0.3283 mg / ml to 3.2834 mg / ml, with the linear equation being y = 594.7921x + 9.1705 and a correlation coefficient r of 0.9999. The response factor RSD is 0.61%, and the |Y-intercept| / 100% concentration response value (%) is 0.93%. The method demonstrates good linearity. Specific Implementation Example 5:

[0188] Specific Implementation Five is a validation of the injection precision of a method for determining acetate content in compound sodium acetate glucose injection mentioned in Specific Implementation One. Injection precision, also known as injection accuracy or repeatability, is an indicator used in analytical chemistry to evaluate the consistency and reliability of an analytical method when testing samples under repeated experimental conditions. It is mainly used to measure the degree of variation in test results (such as chromatographic peak area, retention time, etc.) when the same sample is tested multiple times consecutively under the same operating conditions. Importance of injection precision: Quality control: Ensuring the reliability and consistency of analytical results is particularly important in fields such as pharmaceuticals, food safety, and environmental monitoring. Method validation: It is a necessary step to verify the applicability of newly developed or modified analytical methods. How to evaluate injection precision: Measurement indicators: Usually evaluated by measuring the retention time and peak area of ​​the chromatographic peak obtained after multiple injections of the same sample. Statistical processing: Calculate the mean and standard deviation (SD) of these measurements, and from this, calculate the relative standard deviation (RSD), also known as percentage deviation (%RSD). Specific details are as follows:

[0189] 1. Solution preparation: Select the reference solution and sample detection solution from Example 2, and inject them continuously for 6 injections.

[0190] 2. Results: Each of the above solutions was injected separately, and the chromatograms were recorded. The analytical results are shown in Table 6, which shows the injection precision results.

[0191]

[0192] Table 6

[0193] 3. Conclusion: As shown in Table 6, when the reference solution was injected 6 times consecutively, the RSD of the retention time of the main peak was 0.02% and the RSD of the peak area of ​​the main peak was 0.11%; when the sample detection solution was injected 6 times consecutively, the RSD of the retention time of the main peak was 0.01% and the RSD of the peak area of ​​the main peak was 0.08%, indicating that the method has good injection precision. Specific Implementation Example Six:

[0195] Specific Example Six is ​​a repeatability verification of the method for determining acetate content in compound sodium acetate glucose injection mentioned in Specific Example One. Repeatability is a key term in experimental science, used to describe the consistency of results when the same sample is measured multiple times under the same experimental conditions. This is an important indicator of the stability and reliability of experimental methods, especially important in analytical chemistry and quality control. The core elements of repeatability include: identical operating conditions: the experiment should be conducted under exactly the same operating conditions, including using the same instruments, the same experimental procedures, and the same sample processing methods. Identical samples: multiple parts of the same batch or the same sample are being tested. Identical time and place: the experiment should generally be completed within a short period of time in the same laboratory environment. The specific details are as follows:

[0196] 1. Solution preparation: Select 2 parts of the reference solution and 6 parts of the sample solution from the specific example 2.

[0197] result

[0198] 2. Inject each of the above solutions separately, record the chromatograms, and perform analysis. The results are shown in Table 7 for repeatability.

[0199]

[0200] Table 7

[0201] 3. Conclusion: The results above show that the RSD of acetate content in the 6 test solutions was 0.13%, indicating good method repeatability. Specific Implementation Example 7:

[0203] Specific embodiment seven is an intermediate precision validation of the method for determining acetate content in compound sodium acetate glucose injection mentioned in specific embodiment one. Intermediate precision validation is a means of assessing the variability of an analytical method under routine experimental conditions. Unlike repeatability testing, which mainly focuses on the consistency of measurements under the same conditions, intermediate precision extends to a wider range of experimental conditions, including different times, different operators, and different instruments. This test is mainly used to simulate the reliability and stability of experiments under normally varying conditions. The core elements of intermediate precision validation include: different operators: the experiment is performed by different personnel to assess the impact of human factors on the experimental results. different times: the experiment is conducted on different dates or times to examine the impact of time variation on the experimental results. different instruments: the same experimental procedures are performed using different equipment or instruments to assess the impact of instrument differences on the results.

[0204] Intermediate precision measurement and evaluation: Measurement: This is performed by repeatedly measuring a specific property of the same sample under varying experimental conditions. Result presentation: Similar to repeatability, results are typically expressed as relative standard deviation (RSD), used to evaluate the consistency of the method under routine laboratory conditions.

[0205] Evaluation criteria: Generally, it is acceptable for the RSD value of intermediate precision to be slightly higher than that of repeatability, as it contains more sources of variation.

[0206] The specific acceptance criteria for RSD depend on the type of analysis and the required precision, but generally in chemical and pharmaceutical analysis, a method is considered to have good intermediate precision if the RSD value is below 5%.

[0207] The specific contents are as follows:

[0208] 1. Different personnel, at different times, using different instruments, perform the same operation as under the "Reproducibility Validation" section.

[0209] result

[0210] 2. Inject each of the above solutions separately, record the chromatograms, and perform analysis. The specific results are shown in Table 8, which shows the intermediate precision results.

[0211]

[0212] Table 8

[0213] The results above show that the RSD value of the 12 test sample solutions was 0.17%, indicating good method precision. Specific Implementation Example 8:

[0215] Specific Example 8 is an accurate verification of the method for determining acetate content in compound sodium acetate glucose injection mentioned in Specific Example 1. Accuracy verification is a core verification parameter in analytical chemistry, used to evaluate how close the measured value of an analytical method is to the true value, that is, its ability to correctly determine the analyte. In laboratory analysis, accuracy is usually verified by evaluating the recovery rate of the method, especially in quantitative analysis. The core concepts and steps of accuracy verification are: solution preparation and standard preparation.

[0216] The samples were prepared into different concentration levels through precise weighing and dilution to simulate possible variations in real samples.

[0217] Spiked recovery experiment: Add an additional analyte (spiked) to a sample of known concentration, and then determine the total amount by analytical methods.

[0218] Recovery rate is calculated as (measured amount / added amount) × 100% and is used to evaluate the accuracy of the method.

[0219] A recovery rate close to 100% usually indicates that the analytical method has high accuracy.

[0220] Recovery rates are typically expected to be between 98% and 102%, which means that the method can reflect the true sample content very closely.

[0221] The specific contents are as follows:

[0222] 1. Solution preparation:

[0223] Sodium acetate stock solution: Weigh 1360.8 mg of sodium acetate (raw material, trihydrate) into a 50 ml volumetric flask, add water to make up to volume, shake well (containing 1.64 mg / ml of sodium acetate), and degas.

[0224] Sodium acetate reference solution: see “Reproducibility Validation”.

[0225] Negative reagent stock solution: See "System Suitability Validation".

[0226] Recovery solution: According to Table 9 below, take an appropriate amount of sodium acetate stock solution, add 5.0 ml of negative reagent stock solution, and finally dilute with water to the mark to prepare solutions of three concentration levels: 80%, 100%, and 120%. Prepare three parallel aliquots for each concentration.

[0227] Limit concentration level Transfer sodium acetate stock solution (ml) Transfer the negative test preparation stock solution (ml) Adjust the volume to (ml) 80％ 2.0 5.0 25 100％ 2.5 5.0 25 120％ 3.0 5.0 25

[0228] Table 92, Calculation Formula:

[0229]

[0230] 3. Results: Inject each of the above solutions separately, record the chromatograms, and perform analysis. The specific results are shown in Table 10, which shows the accuracy results.

[0231]

[0232] Table 10

[0233] 4. Conclusion: As shown in Table 10, at the limit concentration levels of 80%, 100%, and 120%, the recovery rate was in the range of 98% to 102%, with an average recovery rate of 100.5% and an RSD of 0.6%; the method has good accuracy. Specific Implementation Example Nine:

[0235] Specific Example 9 is a solution stability verification of the method for determining acetate content in compound sodium acetate glucose injection mentioned in Specific Example 1. Solution stability verification is a process in analytical chemistry that evaluates whether the composition and properties of a solution remain unchanged after a period of time under specified conditions. This verification is to ensure the accuracy and reliability of the analytical method during the experimental process, especially in long-term analysis or multi-batch sample processing. The importance of solution stability verification: Ensuring data consistency: Stability verification ensures that the properties of the solution remain consistent throughout the entire analytical cycle, thereby ensuring that the obtained data are comparable. Extending the shelf life of samples and standards: Through verification, the shelf life of the solution under specific storage conditions can be determined, reducing waste. Specific details are as follows:

[0236] 1. Solution preparation: Blank solution: water; Negative reagent: same as under "System Suitability Validation"; Reference solution: same as under "System Suitability Validation"; Sample test solution: same as under "System Suitability Validation";

[0237] 2. Results: The above solutions were injected separately, the chromatograms were recorded, and the results were analyzed. The specific results are shown in Table 11 (Stability Results of Reference Solution) and Table 12 (Stability Results of Sample Solution).

[0238]

[0239] Table 11

[0240]

[0241] Table 12

[0242] 3. Conclusion: The experimental results show that the blank solution and negative control solution showed no changes and no impurity peaks were generated. The peak areas of the main peaks in the reference solution and test solution did not change significantly after 140 hours at room temperature, and the ratio of the peak area to the peak area at 0 hours was between 0.98 and 1.02, all meeting the requirements. Therefore, the reference solution and test solution showed good stability at room temperature for at least 140 hours. Specific Implementation Example 10:

[0244] Specific Example 10 is a robustness verification of the method for determining acetate content in compound sodium acetate glucose injection mentioned in Specific Example 1. Robustness verification is an important experimental design in analytical chemistry, used to evaluate the reliability and stability of analytical methods under slightly varying conditions. This verification mainly focuses on whether the analytical method can still provide accurate and consistent results under some non-ideal or slightly deviating standard operating conditions, to ensure the robustness and applicability of the method in practical applications.

[0245] Key components of robustness validation include: Variation of conditions: Adjusting several key parameters that may affect analytical results, such as: Column temperature: Particularly important for chromatographic analysis, as temperature changes affect retention time and separation efficiency. Flow rate: Affects sample flow time in the column, thus impacting separation efficiency and analysis time. Detection wavelength: In spectroscopic analysis, small changes in wavelength can affect detection sensitivity and specificity. Mobile phase composition: Changing the sulfuric acid concentration in the mobile phase will affect sample solubility and peak shape. Experimental design: Typically, tests are conducted under both standard and modified conditions to compare the consistency and accuracy of results.

[0246] Data analysis: Observe the changes in retention time, peak area, and concentration calculation of the main peak under different conditions, as well as the repeatability of the results (e.g., RSD, relative standard deviation). Specific details are as follows:

[0247] 1. Solution preparation: Sample solution: Same as under "System Suitability Validation"; Standard conditions are: column temperature 60℃; flow rate 0.8 ml / min; absorption wavelength 210 nm; mobile phase concentration 0.05 mol / L;

[0248] 2. Change the conditions: column temperature (±5℃), flow rate (±0.1ml / min), detection wavelength (±2nm), and sulfuric acid concentration of the mobile phase (0.045 and 0.055mol / L).

[0249] 3. Results: The above solutions were injected separately, chromatograms were recorded, and analyses were performed. Specific results are shown in Table 13, Robustness Results.

[0250]

[0251] Table 13

[0252] 4. Conclusion: The results above show that when the chromatographic parameters were examined for column temperature variation ±5℃, flow rate variation ±0.1ml / min, detection wavelength variation ±2nm, and mobile phase sulfuric acid concentrations of 0.045 and 0.055mol / L, no impurity peaks were generated in the negative reagent, which did not interfere with the determination of the main peak; the RSD of the content was 0.13%; minor changes in the chromatographic conditions of this method did not affect the determination of acetate content, and the method has good robustness.

[0253] The specific embodiments described herein are merely illustrative of the spirit of the present invention. Persons skilled in the art may make various modifications, additions, or substitutions to the described specific embodiments without departing from the spirit of the present invention or exceeding the scope of the appended claims.

Claims

1. A method for determining the content of acetate in compound sodium acetate glucose injection, characterized in that: The acetate content in compound sodium acetate glucose injection was determined by liquid chromatography using a high performance liquid chromatograph. The specific determination steps are as follows: Preparation of S1 solution: Degas the test sample of compound sodium acetate glucose injection and use it as the test solution; Reference solution: Prepare a sodium acetate solution with a concentration of 1.64 mg / mL using deionized water as solvent; S2 setting chromatographic conditions: in a high performance liquid chromatograph, a strong cation hydrogen exchange column with hydrogen-type sulfonated cross-linked styrene divinylbenzene copolymer as a filler is selected as a chromatographic column; The column operating temperature was set at 55-65°C; Select 0.045-0.055 mol / L sulfuric acid solution as the mobile phase. The flow rate of the mobile phase was set at 0.7-0.9 mL / min; The injection volume of the test solution and the reference solution was set to 20 μl; The detection wavelength was set to 210 ± 2 nm; The parameters of hydrogen-type sulfonated cross-linked styrene divinyl benzene copolymer are as follows: cross-linking degree is 8%; shape is spherical; particle size is 8-10μm; pore size is to S3 obtaining a chromatogram: using liquid chromatography to detect the test solution and the reference solution respectively, to obtain a chromatogram of the test solution and a chromatogram of the reference solution; S4 Chromatographic analysis: Use the external standard method to calculate the content of acetate in the test solution. The specific operation is to obtain the acetate peak area in the chromatogram of the test solution and the acetate peak area in the chromatogram of the reference solution; calculate the content of acetate in the test solution. The specific calculation formula is as follows: f=C 对 / A 对 ; Acetate content = f × A1 ÷ 82.03 × 1000; Among them, C 对 A is the concentration of sodium acetate in the reference solution; 对 is the acetate peak area in the chromatogram of the reference solution; A1 is the acetate peak area in the chromatogram of the test solution; 82.03 is the molecular weight of sodium acetate, and the unit of acetate content is mmol / L.

2. The method for determining the acetate content in the compound sodium acetate glucose injection according to claim 1, characterized in that: In step S1, the degassing treatment of the test sample of compound sodium acetate glucose injection is ultrasonic treatment, and the ultrasonic treatment process is as follows: Set the ultrasonic processor parameters: Power: 100-200W; Frequency: 40kHz; Processing time: 5-10 minutes; Temperature: 25-30℃; Operation steps: Place the compound sodium acetate glucose injection sample to be processed in an ultrasonic cleaner; start the ultrasonic equipment and process according to the ultrasonic processor parameters; Effect inspection: After the treatment is completed, check whether there are any residual bubbles on the surface of the solution.

3. The method for determining the acetate content in the compound sodium acetate glucose injection according to claim 1, characterized in that: In step S1, the preparation process of the reference solution is as follows: drying the sodium acetate of the required purity of HPLC grade, accurately weighing it, adding deionized water and stirring it until it is completely dissolved, further adding deionized water and adjusting the concentration to 1.64 mg / mL; then, degassing the solution to avoid interference of bubbles with subsequent steps; the degassing treatment is ultrasonic treatment, and in the drying operation, the drying temperature is 120°C.

4. The method for determining the acetate content in the compound sodium acetate glucose injection according to claim 1, characterized in that: In step S2, the parameters of the chromatographic column are as follows: inner diameter is 7.8 mm; length is 300 mm.

5. The method for determining the acetate content in the compound sodium acetate glucose injection according to claim 1, characterized in that: In step S3, the specific process of obtaining the chromatogram of the detection solution is as follows: start the high performance liquid chromatograph, according to the set chromatographic conditions, inject the detection solution and pass it through the chromatographic column under the action of the mobile phase, the detection solution is separated in the chromatographic column, and the detector of the high performance liquid chromatograph is used to continuously monitor the components flowing out of the chromatographic column at the detection wavelength, and record the results to generate a chromatogram of the detection solution.

6. The method for determining the acetate content in compound sodium acetate glucose injection according to claim 5, characterized in that: The detector type is UV detector.

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