Method for determining in-vitro dissolution rate of acetaminophen and ibuprofen suspension medicine

Abstract

The invention provides a method for measuring the in-vitro dissolution rate of an acetaminophen and ibuprofen suspension medicine, belongs to the technical field of medicine analysis, and solves the problems that the in-batch difference of experimental results measured by a conventional dissolution instrument is large, and the single-point dissolution rate difference is large. By adopting a closed-loop flow cell mode, the in-vivo dissolution environment of the medicine can be more accurately simulated, the dissolution rate is effectively controlled, the in-batch difference is reduced, and the accuracy and the stability of dissolution rate measurement are improved. Through multi-step filtration and dilution treatment, the purity and accuracy of a dissolution solution sample are ensured, and the dissolution rate is accurately measured by using a high performance liquid chromatography (HPLC) technology, so that more reliable in-vitro dissolution rate data are obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of drug analysis, and specifically relates to a method for determining the in vitro dissolution rate of a drug, in particular to a method for determining the in vitro dissolution rate of acetaminophen and ibuprofen suspension drugs. Background Art

[0002] Acetaminophen and ibuprofen suspension is a comprehensive drug that can enhance efficacy in many aspects, including analgesia, antipyretic and anti-inflammatory. The combined use of the two can not only more effectively relieve the discomfort caused by fever and mild to moderate pain, but also reduce the side effects of a single drug. The dissolution determination of acetaminophen and ibuprofen suspension is a crucial part of drug development and production, especially in drug quality control, efficacy verification and safety assessment. Evaluate the release characteristics of the drug: Dissolution is the rate and extent of drug release from the preparation under specific conditions, reflecting the bioavailability of the drug. The in vitro dissolution process of the drug components (such as acetaminophen and ibuprofen) in acetaminophen and ibuprofen suspension directly affects its absorption rate and efficacy in vivo. Ensure the consistency of drug quality: Acetaminophen and ibuprofen suspension drugs are compound preparations. In order to ensure the quality and efficacy of each batch of drugs, dissolution testing must be performed to ensure that the dissolution behavior of each batch of products is consistent.

[0003] The following problems exist in the drug dissolution test of acetaminophen and ibuprofen suspension using conventional dissolution apparatus: 1. Inconsistency between dissolution rate and in vivo absorption rate: Tests conducted using conventional dissolution apparatus often cannot accurately simulate the release and absorption behavior of drugs in the human body. It usually shows a faster dissolution rate than the actual absorption in the human body, which leads to a significant deviation between the test results and the actual bioavailability of the drug. 2. Reliability issues in dissolution determination: Conventional dissolution test methods often have large inter-batch and intra-batch differences in dissolution determination, which affects the reliability and consistency of the test data. This inconsistency complicates the evaluation of drug quality and also poses challenges to the quality control of drug preparations.

[0004] In response to this problem, the closed-loop flow cell method, as a new dissolution testing technology, has gradually attracted widespread attention in recent years. However, when using the closed-loop flow cell method for determination, for acetaminophen and ibuprofen suspensions, due to their large particles and low drug solubility, in practical applications, how to choose a flow environment and absorption process that simulates more physiological conditions, how to prepare higher purity dissolution samples, and how to perform more accurate cumulative dissolution tests on solution samples are the difficulties of current drug testing technology.

[0005] Therefore, an improved in vitro drug dissolution determination method is urgently needed to improve the accuracy, reproducibility and reliability of acetaminophen and ibuprofen suspension dissolution testing. Summary of the invention

[0006] The purpose of the present invention is to solve the above problems in the prior art and to provide a method for determining the in vitro dissolution rate of acetaminophen and ibuprofen suspension drugs.

[0007] The purpose of the present invention can be achieved by the following technical scheme: A method for determining the in vitro dissolution rate of acetaminophen and ibuprofen suspension drugs, comprising the following:

[0008] S1 obtains a sample of a suspension drug dissolution liquid: adopts a closed-loop circulation pool method to perform dissolution sampling operations on n suspension drug samples respectively to obtain a dissolution liquid sample set; during the dissolution sampling operation, a phosphate solution with a pH value of 5.5 is used as a dissolution medium. When sampling, samples are taken from a dissolution medium container according to a preset sampling time point. After each sampling, a dissolution medium equal to the volume of the liquid taken is added;

[0009] In the method using the closed-loop circulation pool mode, the dissolution sampling system used includes a circulation pool dissolution device, a solvent delivery workstation and an automatic sampling workstation. The circulation pool dissolution device is designed as follows: a polypropylene filter membrane and a polycarbonate filter membrane are placed in the filter chamber in sequence according to the direction of liquid flow during filtration, a ruby ​​is placed at the lower end of the inverted cone of the sample pool; glass beads are placed inside the sample pool;

[0010] The design of the solvent delivery workstation is as follows: a circulation pump is provided; the dissolution medium storage container has a stirring function.

[0011] As a new dissolution test method, the circulation cell method has been included in the 2020 edition of the "Chinese Pharmacopoeia". Under this method, the dissolution medium preheated to a suitable temperature is continuously passed through the circulation cell at a certain flow rate by a constant flow pump, contacting with the drug in the circulation cell and releasing it. The dissolution rate can be adjusted by changing the dissolution mode, adjusting the flow rate, changing the inner diameter of the circulation cell, adjusting the amount of glass beads, etc., so that the dissolution method can better simulate the in vivo environment. Especially in the closed-loop mode, the sample is always exposed to fresh dissolution medium, and the appropriate sink conditions are always maintained. It is more suitable for in vitro dissolution studies of poorly soluble drugs, and shows better discrimination in in vitro dissolution studies of some drugs.

[0012] The reasons for using a closed-loop mode for acetaminophen and ibuprofen suspension drugs are as follows: 1. To control the dissolution environment: Through a closed-loop system, the conditions in the dissolution process, such as temperature, flow rate, and pH, can be more accurately controlled. This control ensures the repeatability and comparability of the test. 2. Reduce drug sedimentation: In the test of suspension drugs, drug particles are easy to settle. The closed-loop flow cell can help maintain the suspension state and reduce particle sedimentation through continuous circulation, thereby more realistically reflecting the release of the drug in the body. 3. Better sample representativeness: Because the dissolution medium is constantly circulating and contacts the drug from all directions, it can better simulate the environment of the drug in the body, and a more representative sample can be obtained when sampling from the dissolution medium. 4. Realize dynamic sampling: Dynamic sampling at different time points without stopping the system operation or interfering with the overall dissolution process. This method can more accurately capture the dynamic changes of drug release.

[0013] The use of polypropylene and polycarbonate filters, as well as the placement of the polycarbonate filter at the inlet of the filtration chamber, is not only to ensure the purity and accuracy of sampling, but also closely related to the evaluation of drug dissolution effect. Specifically, such a configuration helps to achieve the following key advantages in dissolution effect evaluation: Filtration efficiency and selectivity: Polycarbonate filter: This filter has good chemical stability and high mechanical strength, and is suitable for use in high-pressure environments. The pore size of the polycarbonate filter can be precisely controlled and can effectively intercept particles of a certain size. Therefore, it can be placed at the inlet of the filtration chamber as the first filtration barrier to intercept larger undissolved particles or impurities. Polypropylene filter: The polypropylene filter membrane has good chemical and thermal stability and is used for the filtration of biological preparations and drugs. In this system, it can be used as a second-stage filter to process the dissolution medium after passing through the polycarbonate filter membrane to further remove smaller particles and potential dissolved impurities. Prevent clogging and maintain flow rate: During the filtration process, larger particles are first intercepted by the polycarbonate filter membrane, reducing the risk of clogging of the polypropylene filter membrane. This hierarchical filtration method can maintain a stable flow rate and pressure during the dissolution test and ensure the continuous flow of the dissolution medium. Keep the dissolution medium clean and stable: By first passing through a polycarbonate filter membrane to intercept larger particles and then filtering more finely through a polypropylene filter membrane, impurities and tiny particles that may cause deviations in test results can be effectively removed. This double-layer filtration ensures the cleanliness and stability of the dissolution medium, making the dissolution test of the drug more reliable. Improve dissolution kinetics evaluation: The dissolution rate of a drug is an important indicator for evaluating the drug release behavior. Accurate dissolution evaluation requires ensuring that there is no unnecessary physical and chemical interference in the test environment. Through double filtration, the release behavior of the drug under physiological conditions can be more accurately simulated and evaluated, which is crucial to understanding the actual performance of the drug in vivo. Optimize the accuracy of subsequent analysis: It is very important to ensure the purity of the sample before liquid chromatography analysis. Through effective filtration settings, problems that may be encountered in high-performance liquid chromatography (HPLC) analysis, such as column blockage and baseline noise, can be minimized, thereby improving the accuracy and reproducibility of analytical results. In summary, such a filtration setup not only ensures the purity and accuracy of sampling by providing clean and stable dissolution media, but also provides a reliable basis for evaluating the dissolution behavior and kinetics of drugs, which helps to make accurate decisions during drug development and quality control.

[0014] The selection of a phosphate solution with a pH of 5.5 can better simulate the dissolution behavior of drugs in the small intestine environment after eating, which is very important for understanding the behavior of drugs under such physiological conditions.

[0015] The practice of adding a volume of dissolution medium equal to the volume of liquid removed after each sampling can be compared to maintaining the constancy of the human body's internal environment. There are several important reasons for this practice: Simulating the stability of the human body's internal environment: In the human body, the volume and concentration of liquid in the organism are maintained at a relatively stable state. By adding the same volume of dissolution medium in the experiment, the stability of this biological internal environment can be simulated to ensure that the drug dissolution environment is similar to that in the human body, so that the experimental results are closer to the actual situation. Prevent rapid changes in drug concentration in the dissolution system: In the human body, the volume of blood and body fluids remains basically constant during the absorption and distribution of drugs, which helps the drug achieve the desired therapeutic effect in the body. Adding the same volume of dissolution medium in the experiment helps avoid unexpected increases in drug concentrations due to a reduction in the volume of the dissolution medium, thereby maintaining the consistency and controllability of drug release.

[0016] S2 prepares the sample solution: each sample in the dissolution sample set is subjected to preliminary filtration, filtrate sampling, filtration sample dilution and re-filtration operations to obtain the sample solution set. In the preliminary filtration operation, a microporous filter membrane of PES material is used for filtration; in the re-filtration operation, a nylon filter head is used for filtration; in the dilution operation, a methanol-water solution is used as a diluent for dilution according to a preset dilution ratio. In the methanol-water solution, the volume ratio of methanol: water is 1:1;

[0017] The main reason for using PES (polyethersulfone) microporous membrane filtration is that it is suitable for the preliminary filtration operation of drug samples. The specific reasons include: good chemical compatibility and biological inertness: PES material has excellent chemical stability and biological inertness, will not produce unexpected chemical reactions or contamination to drug samples, and ensure the purity of samples and the accuracy of analysis. Microporous structure and precise separation ability: PES microporous membrane usually has a uniform pore size distribution and precise separation ability, which can effectively remove solid particles, microorganisms and macromolecules in samples, and ensure the clarity and stability of solutions. Efficient particle capture ability: Due to its microporous structure design, PES membrane can efficiently capture particulate matter in samples, avoiding these particulate matter from affecting the accuracy and sensitivity of subsequent chromatographic analysis or other detection methods. Convenient operation and stability: PES membrane usually has high physical strength and stability, is not easy to break or deform during use, is suitable for high-pressure filtration operations, and can ensure the stability and reproducibility of filtration operations. In summary, the selection of PES microporous membrane as a tool for preliminary filtration is to ensure the safety, accuracy and efficiency of sample processing.

[0018] The nylon filter head has little adsorption to the target drug components (acetaminophen and ibuprofen) in the experiment. In the dissolution experiment, if the filter head material adsorbs more drug components, it will lead to a low drug concentration in the sample, affecting the accuracy of the analysis. The nylon filter head can minimize the drug adsorption effect while ensuring the filtration effect. In the multi-step filtration of the dissolution experiment, filter membranes of different materials (such as PES microporous filter membranes and nylon filter heads) are used at different stages. The purpose is to fully remove impurities and particles while maintaining the stability of the drug components. The selection of nylon filter heads meets the requirements of precise control and secondary purification during the experiment.

[0019] Mixing methanol and water in a 1:1 volume ratio can effectively adjust the polarity of the solution. This medium-polarity solution helps dissolve the two drugs, acetaminophen and ibuprofen.

[0020] S3 prepare reference solution samples: prepare at least two reference solution samples, and the specific preparation process is as follows: dissolve the acetaminophen standard and the ibuprofen standard in a methanol-water solution to obtain an initial reference solution, in which the concentration of acetaminophen is the same as the concentration of acetaminophen in the suspension drug sample, and the concentration of ibuprofen is the same as the concentration of ibuprofen in the suspension drug sample;

[0021] Then, the initial control solution is diluted with methanol aqueous solution to obtain a reference substance solution sample; in the reference substance solution sample, the concentration of acetaminophen is set to be the same as the concentration of acetaminophen in a comprehensive solution formed by diluting a portion of the suspension drug sample in the dissolution medium contained in the dissolution medium container; the concentration of ibuprofen is set to be the same as the concentration of ibuprofen in a comprehensive solution formed by diluting a portion of the suspension drug sample in the dissolution medium contained in the dissolution medium container;

[0022] The core role of the reference solution in the experiment is to provide a standardized reference benchmark for the concentration determination of the test solution. The standard curve is established using a standard solution (reference solution) of known concentration to calculate the concentration of the unknown sample. The standard curve is drawn based on the high performance liquid chromatography (HPLC) analysis results of the reference solution sample. When using the external standard method for dissolution calculation, the accuracy of the standard curve has a direct impact on the final dissolution calculation. By performing high performance liquid chromatography measurements on all reference solution samples, taking the average value based on the measurement results, the prepared standard curve is more accurate. In this way, the sample dissolution data obtained based on the standard curve will also be more accurate, avoiding deviations from the true value due to accidental operational errors or instrument abnormalities (such as transient interference, light scattering caused by solution particles, etc.).

[0023] The purpose of dilution in step S2 and step S3 is: directly using the undiluted solution for measurement may cause the measurement result to exceed the detection range of the instrument or cause the chromatogram to be too crowded (i.e., signal saturation). Therefore, it is necessary to dilute the final solution so that the concentration falls within the linear detection range of the HPLC system to ensure the reliability of the data. Avoid instrument overload: The HPLC system has a certain detection limit and linear range. If the sample concentration is too high, it may cause signal saturation or peak distortion, affecting the accuracy and analysis of the data. By dilution, it can be ensured that the concentration of the sample is within the effective detection range of the HPLC system, so as to obtain an accurate concentration value. Sample complexity: The concentrations of acetaminophen and ibuprofen are relatively high, and direct liquid chromatography analysis may cause peak overlap or interference. Dilution can not only reduce the concentration, making the chromatographic peaks of each compound clearer, but also help to reduce the influence of possible interfering substances, thereby improving the accuracy of the measurement.

[0024] S4 Liquid chromatography determination: Use a high performance liquid chromatography system to perform liquid chromatography determination on each sample in the reference solution sample set and the test solution sample set by gradient elution. After the liquid chromatography determination is completed, the cumulative dissolution of acetaminophen and the cumulative dissolution of ibuprofen in the suspension drug are calculated based on the determination results by using the external standard method and the rehydration method. Based on the calculated cumulative dissolution values, the cumulative dissolution curves of acetaminophen and ibuprofen are drawn.

[0025] The specific calculation process is as follows: according to the results of the liquid chromatography determination operation, the external standard method is used for calculation, and the n single-point solubility values ​​of acetaminophen and the n single-point solubility values ​​of ibuprofen are calculated at each preset sampling time point, and then, for each preset sampling time point, the n single-point solubility values ​​of acetaminophen are averaged as the acetaminophen solubility value at the preset sampling time point, and the n single-point solubility values ​​of ibuprofen are averaged as the ibuprofen solubility value at the preset sampling time point; finally, the rehydration method is used for calculation to obtain the cumulative solubility value of acetaminophen and the cumulative solubility value of ibuprofen at each preset sampling time point; according to the cumulative solubility values, the cumulative dissolution curves of acetaminophen and the cumulative dissolution curves of ibuprofen are drawn respectively; the external standard method is a well-known method in the art and will not be introduced in detail here.

[0026] In the liquid chromatography measurement operation, the following settings were used: detection wavelength 222 nm, mobile phase: mobile phase A: 0.1% (V / V) trifluoroacetic acid aqueous solution, mobile phase B: methanol.

[0027] Detection wavelength 222nm: The selection of detection wavelength is based on the ultraviolet absorption characteristics of the substance to be tested. This wavelength can provide better sensitivity and selectivity, making the detection of target compounds more accurate and sensitive.

[0028] Mobile phase composition: Mobile phase A: 0.1% (V / V) trifluoroacetic acid aqueous solution: Trifluoroacetic acid is a mobile phase additive that can improve the shape of chromatographic peaks and increase the selectivity and sensitivity of chromatographic separation. Its addition can help suppress matrix effects in samples and reduce peak tailing. Mobile phase B: Methanol: Methanol is used to adjust the polarity of the mobile phase and helps improve the elution of non-polar to medium-polar components. In gradient elution, the proportion of methanol is gradually increased, which can effectively elute various components on the chromatographic column.

[0029] Preferably, in step S1, the sample pool uses a standard flow cell with an inner diameter of 22.6 mm, the diameter of the ruby ​​is 5 mm, the diameter of the glass beads is 1 mm, and the dissolution sampling operation parameters are set as follows: maintain the fluid temperature at 37°C, the fluid flow rate is 8 mL / min, and the volume ratio of the suspension drug to the dissolution medium is set to 3:400; the volume of each sampling is 1 / 400 of the volume of the dissolution medium.

[0030] The above parameter settings are to ensure the accuracy and repeatability of the drug dissolution experiment. Each parameter setting here plays a key role in the accuracy and consistency of the experimental results. The reasons for these parameter settings: Sample cell size and ruby ​​diameter: The standard flow cell with an inner diameter of 22.6mm and the ruby ​​with a diameter of 5mm are selected to ensure the flow characteristics of the dissolution medium in the flow cell and the uniform contact of the sample. The role of ruby ​​is to prevent the medium in the sample cell from flowing back into the pipeline. The use of ruby ​​is related to its hardness and chemical stability. It can remain stable in high-speed flowing liquids, so as not to affect the composition of the dissolution medium. Glass bead diameter: Glass beads with a diameter of 1mm increase the turbulence in the sample cell, which helps to break the interface layer around the sample, thereby improving the dissolution rate and uniformity of the drug. Temperature and flow rate control: Maintaining the fluid temperature at 37℃ and the flow rate at 8mL / min helps to simulate the drug release environment under human conditions and ensure the biological relevance of the experimental results. 37℃ is the normal body temperature of the human body, and the appropriate flow rate can simulate the flow of blood or body fluids in the in vivo environment. The volume ratio of the suspension drug to the dissolution medium is set to 5.5:400, which means that the ratio of the suspension to the dissolution medium is moderate, which not only ensures that the drug particles can fully contact the dissolution medium, but also avoids nonlinear dissolution behavior caused by oversaturation of the dissolution medium or excessive drug concentration. Sampling volume ratio: The sampling volume of each time is 1 / 400 of the volume of the dissolution medium. This setting ensures that each sampling has little impact on the overall dissolution pool, thereby avoiding changes in the drug concentration and dissolution kinetics in the dissolution pool during continuous sampling.

[0031] Together, these settings ensure the control and reproducibility of the experiment, ensuring that the data obtained from the experiment can effectively reflect the dissolution behavior of the drug and have physiological relevance, which is crucial for evaluating the in vitro release characteristics of the drug and predicting its behavior in vivo.

[0032] Preferably, in step S1, the preset sampling time points are 10, 15, 30, 45, 60, 90, 120, 180, 240 and 360 minutes respectively. In order to ensure that the drug is completely dissolved, the sampling time points need to cover the entire release process, including the rapid release stage, the sustained release stage and the complete release stage, until the dissolution curve enters the plateau phase; through reasonable time point settings, a complete dissolution curve from rapid release to sustained release to complete dissolution can be drawn. This is very critical for evaluating drug release behavior, dosage form design and dissolution mechanism. Physicochemical properties of drugs: solubility differences between acetaminophen and ibuprofen: acetaminophen is a highly hydrophilic drug with a faster dissolution rate, while ibuprofen is a hydrophobic drug with a relatively slow dissolution rate. Therefore, the selection of sampling time points needs to take into account the dissolution kinetics of the two drugs: the drug in the suspension exists in the form of suspended particles, and the release is affected by the surface area, particle size and solubility of the particles, and has a multiphase dissolution characteristic. By sampling at different time points, the release process of the drug can be fully reflected. In the dissolution test, the closed-loop circulation cell method is used to ensure the dynamic circulation of the dissolution medium and the uniformity of the sample, which puts forward requirements for the selection of time points: at the early time points, the sampling frequency of the samples is high to avoid missing the rapid release stage. At the later time points, the concentration gradually approaches the plateau value, and the sampling frequency is appropriately reduced to reduce the risk of operational errors and drug degradation. The selection of these sampling time points is based on: the rapid release, sustained release and plateau dissolution characteristics of the drug. The physical and chemical properties and solubility differences of acetaminophen and ibuprofen. Ensure the integrity, accuracy and consistency of the experimental data. The need for dynamic sampling and rehydration operations. By setting multiple reasonable time points, the dissolution curve can be comprehensively drawn to provide reliable data support for drug development and quality evaluation.

[0033] Preferably, the pore size of the polypropylene filter membrane is 20 μm, the pore size of the polycarbonate filter membrane is 20 μm; the pore size of the microporous filter membrane of PES material is 0.45 μm, and the pore size of the filter membrane in the nylon filter head is 0.22 μm.

[0034] The pore size of the polypropylene filter membrane and the polycarbonate filter membrane is set to 20 microns: these two filter membranes are used for coarse preliminary filtration. The pore size of 20 microns can prevent larger particles from entering the subsequent processing links. Such a pore size is sufficient to prevent larger solid particles or undissolved drug agglomerates that are not fully dispersed in the dissolution medium from passing through, reducing the blockage or interference that these particles may cause to downstream analysis.

[0035] The pore size of the PES microporous filter membrane is set to 0.45 microns: PES (polyethersulfone) filter membrane has good chemical stability and low protein adsorption, which is suitable for filtering biological samples. The 0.45 micron pore size is used for further fine filtration to remove fine particles and other possible tiny impurities. The use of this filter membrane in dissolution testing helps to ensure the cleanliness of the sample and the accuracy of the analysis.

[0036] The pore size of the filter membrane in the nylon filter head is 0.22 microns: The pore size of 0.22 microns provides extremely high filtration accuracy for the final filtration of the solution, ensuring that the solution does not block the chromatographic column before entering the HPLC analysis. This is especially important because HPLC has extremely high requirements for sample purity, and any unfiltered tiny particles may cause clogging of the chromatographic column or errors in the analysis results.

[0037] Through this multi-stage filtration strategy, accurate and reliable analysis results can be obtained. In addition, the selection of each filter material and pore size takes into account its physical and chemical properties to adapt to different experimental conditions and requirements, ensuring the overall efficiency and effect of the dissolution test.

[0038] Preferably, in the dilution operation of step S2, the volume ratio of the diluted object: the diluted solution is 1:50. In the dissolution test, the initial concentration of the drug is often high and may exceed the effective detection range of the liquid chromatograph (HPLC). By diluting the sample, the risk of clogging the chromatographic column by the high-viscosity water-soluble auxiliary materials in the suspension can be greatly reduced, the fluctuation of the chromatographic peak elution time can be reduced, and the probability of the chromatographic peak tailing and the front can be reduced. Thereby improving the accuracy of the measurement.

[0039] Preferably, in step S4, the configuration of the high performance liquid chromatography system is as follows: detector: ultraviolet detector; chromatographic column: C18 chromatographic column, the inner diameter of the chromatographic column is 4.6 mm, the length of the chromatographic column is 150 mm, and the diameter of the filler particles is 3.5 μm; guard column: the inner diameter of the guard column is 4.6 mm, the length of the guard column is 10 mm, and the diameter of the filler particles is 5 μm.

[0040] Preferably, in step S4, during the gradient elution process using the mobile phase, the volume ratio of mobile phase A to mobile phase B in the mobile phase is set as follows: 0-3 minutes, the ratio of mobile phase A and mobile phase B is uniformly adjusted from the initial 75:25 to 5:95; 3-3.1 minutes, the ratio of mobile phase A and mobile phase B is uniformly adjusted from 5:95 to 75:25; 3.1-8.5 minutes, the ratio of mobile phase A and mobile phase B is maintained at 75:25.

[0041] The purpose of gradient elution is to optimize chromatographic separation and improve analytical efficiency and resolution. By changing the ratio of mobile phase components (i.e., the volume ratio of mobile phase A to mobile phase B), a better separation effect can be achieved. The following are specific considerations and reasons: Initial high-polarity mobile phase: Using a higher proportion of mobile phase A (0.1% trifluoroacetic acid aqueous solution, with higher polarity) at the beginning of the analysis helps to quickly elute analytes with higher polarity. This helps prevent excessive retention of polar analytes on the chromatographic column, allowing them to pass through the chromatographic column quickly and reduce analysis time. Gradually increase non-polar mobile phase B: As the ratio of mobile phase A to B is adjusted from 75:25 to 5:95, that is, gradually increasing the proportion of methanol (non-polar mobile phase), it is beneficial for non-polar analytes to be eluted from the chromatographic column. This is because non-polar analytes interact more strongly with non-polar solvents and are therefore more easily eluted. Rapid recovery of initial conditions: Near the end of sample analysis, the mobile phase ratio is quickly adjusted from 5:95 back to 75:25 and maintained at this ratio for a period of time. This is to quickly clean the column and restore the initial state to prepare for the next sample analysis. This helps to maintain the stability of the chromatographic system and the repeatability of the analysis. Overall process optimization: The entire mobile phase adjustment process is to ensure that each component can be well separated on the chromatographic column during analysis. This rapid mobile phase adjustment not only optimizes the elution time of each analyte, but also improves the throughput and efficiency of the analysis. Through this gradient elution design, effective separation of suspension drugs such as acetaminophen and ibuprofen can be achieved, ensuring that each component can be analyzed under the most suitable conditions, thereby obtaining accurate dissolution data.

[0042] Compared with the prior art, the method for determining the in vitro dissolution of acetaminophen and ibuprofen suspension drugs has the following beneficial effects:

[0043] 1. Introduction of closed-loop circulation cell method: By adopting the closed-loop circulation cell method, the present invention solves the problem that conventional dissolution instruments cannot accurately simulate the behavior of drugs in vivo due to the too fast dissolution rate. By simulating a flow environment closer to physiological conditions, the in vivo and in vitro correlation of the dissolution test is improved, thereby being able to more accurately predict the behavior of drugs in the human body. By introducing the circulation cell method, the present invention significantly reduces the test differences between batches and within batches. This improvement increases the consistency and reliability of the dissolution test results, and provides strong technical support for the quality control and standardization of drugs.

[0044] 2. Improved dissolution test accuracy: This method can more accurately simulate the drug release environment in the human body by using a closed-loop flow cell mode and precisely controlling the pH value of the dissolution medium, thereby providing drug release data that is closer to reality.

[0045] 3. Enhanced reproducibility: By using standardized equipment configuration and operating procedures, such as standard flow cells and precise sampling time point settings, the present invention ensures the reproducibility and reliability of experimental results, which is particularly important for drug development and quality control.

[0046] 4. Optimized sample processing: The multi-step sample processing process in this method (including preliminary filtration, dilution and re-filtration operations) ensures the purification and stability of the sample, reduces possible measurement errors and improves the accuracy of the analysis.

[0047] 5. Efficient analytical technology: Using high performance liquid chromatography (HPLC) technology, combined with gradient elution and precise detection wavelength setting, this method can effectively separate and accurately determine the two drug components in the suspension, making drug analysis more accurate and efficient.

[0048] In summary, the present invention provides a new in vitro dissolution determination method for acetaminophen and ibuprofen suspension drugs. By adopting the method of closed-loop circulation cell, the present invention can more accurately simulate the dissolution environment of the drug in vivo, can effectively control the dissolution rate, reduce the intra-batch difference, and improve the accuracy and stability of dissolution determination. In addition, by multi-step filtration and dilution treatment, the purity and accuracy of the dissolution solution sample are ensured, and the dissolution is accurately determined using high performance liquid chromatography (HPLC) technology, thereby obtaining more reliable in vitro dissolution data. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Schematic diagram of the dissolution sampling system.

[0050] Figure 2 This is the dissolution curve of acetaminophen in closed-loop flow cell mode.

[0051] Figure 3 This is the dissolution curve of acetaminophen under conventional dissolution apparatus conditions.

[0052] Figure 4 This is the dissolution curve of ibuprofen in the closed-loop flow cell mode.

[0053] Figure 5 This is the dissolution curve of ibuprofen under conventional dissolution apparatus conditions.

[0054] Figure 6 Typical chromatograms for acetaminophen and ibuprofen. DETAILED DESCRIPTION

[0055] The following are specific embodiments of the present invention and the accompanying drawings to further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0056] This specific embodiment

[0057] A method for determining the in vitro dissolution of acetaminophen and ibuprofen suspension drugs specifically comprises the following steps:

[0058] S1 Obtaining suspension drug dissolution sample: Using the closed-loop circulation cell method, 6 suspension drug samples are respectively subjected to dissolution sampling operations to obtain a dissolution sample set; the specification of the suspension drug is 3.2g of acetaminophen and 0.96g of ibuprofen per 100ml. The saturated solubility of acetaminophen under the medium condition is 15mg / ml (satisfying the sink condition) and that of ibuprofen is 0.16mg / ml (not satisfying the sink condition).

[0059] like Figure 1 As shown, in the method using a closed-loop circulation cell, the dissolution sampling system used includes a circulation cell dissolution device, a solvent delivery workstation and an automatic sampling workstation.

[0060] The operation for each suspension drug is as follows: select a standard circulation pool with an inner diameter of 22.6mm, put a ruby ​​with a diameter of 5mm at the lower end of the inverted cone to prevent the medium in the sample pool from flowing back into the pipeline, use a syringe to draw 3mL of suspension drug and accurately weigh it, add it to the ruby ​​at the lower end of the cone of the sample pool, fill the sample pool with 5g of glass beads first, then inject 2.5ml of suspension drug with a syringe, and then add 15g of glass beads with a diameter of 1mm to ensure that the glass beads cover the drug solution; add polypropylene into the filter chamber Epoxy filter (pore size 20 μm) and polycarbonate membrane (pore size 20 μm) were used as filter membranes, and Teflon gaskets were installed. After the filter chamber and sample pool were assembled, they were installed in the dissolution instrument in closed-loop mode with the following parameters: temperature 37 °C, flow rate 8 mL / min, sampling at 10, 15, 30, 45, 60, 90, 120, 180, 240 and 360 min, respectively, phosphate solution with a pH value of 5.5 was used as the dissolution medium, the amount of the dissolution medium was 400 ml, and 1 ml was sampled each time;

[0061] S2 prepares the sample of the test solution: each sample in the suspension drug dissolution sample set is subjected to preliminary filtration, filtrate sampling, filtrate sampling dilution and re-filtration operations to obtain the sample set of the test solution. In the preliminary filtration operation, a microporous filter membrane of PES material is used for filtration; in the re-filtration operation, a nylon filter head is used for filtration, and the 2 ml liquid initially passed through the nylon filter head is discarded; in the dilution operation, a methanol-water solution is used as a diluent for dilution according to a preset dilution multiple, and in the methanol-water solution, the volume ratio of methanol: water is 1:1; in the dilution operation, the volume ratio of the diluted object: the diluted solution is 1:50;

[0062] S3 prepares a reference solution sample: dissolves the acetaminophen standard and the ibuprofen standard in a methanol-water solution to prepare an initial reference solution; in the initial reference solution, the concentration of acetaminophen is the same as the concentration of acetaminophen in the suspension drug, and the concentration of ibuprofen is the same as the concentration of ibuprofen in the suspension drug;

[0063] In the reference solution sample, the concentration of acetaminophen was set to be the same as the concentration of acetaminophen in the mixed solution formed by diluting 5.5 mL of the suspension drug in 400 mL of the dissolution medium; the concentration of ibuprofen was set to be the same as the concentration of ibuprofen in the mixed solution formed by diluting 5.5 mL of the suspension drug in 400 mL of the dissolution medium;

[0064] S4 Liquid chromatography determination and calculation: Figure 6 As shown, in Figure 6 In the figure, the first peak is for acetaminophen, and the second peak is for ibuprofen. The two reference solution samples and each sample in the test solution sample set are subjected to liquid chromatography determination by gradient elution using a high performance liquid chromatography system. After the liquid chromatography determination is completed, the cumulative dissolution of acetaminophen and the cumulative dissolution of ibuprofen in the acetaminophen and ibuprofen suspension drugs are calculated based on the determination results using the external standard method and the rehydration method. Based on the calculated cumulative dissolutions, the cumulative dissolution curves of acetaminophen and the cumulative dissolution curves of ibuprofen are drawn respectively.

[0065] The calculation formula using the fluid replacement method is as follows: Where Xi is the dissolution value of the i-th sampling, V1 is the total volume of the dissolution medium; V2 is the volume added after each sampling;

[0066] The liquid chromatography condition parameters are shown in Table 1:

[0067]

[0068]

[0069] Table 1

[0070] The gradient elution parameters are shown in Table 2:

[0071] Time / min Mobile phase A Mobile phase B 0 75 25 3 5 95 3.1 75 25 8.5 75 25

[0072] Table 2

[0073] The results of the determination of acetaminophen in the closed-loop flow cell mode are shown in Table 3, and the corresponding curve graph is shown in Figure 2 As shown:

[0074]

[0075]

[0076] Table 3

[0077] The results of the determination of acetaminophen under conventional dissolution conditions are shown in Table 4, and the corresponding curve diagram is shown in Figure 3 As shown,

[0078]

[0079]

[0080] Table 4

[0081] Experimental conclusion: The dissolution rate of acetaminophen under conventional dissolution apparatus conditions is fast, has no discriminatory power, and the intra-batch differences of samples are slightly large. However, the intra-batch differences of samples under flow cell conditions are smaller and more discriminatory. And the 6 curves are more similar. 1. The consistency of dissolution is higher (RSD is smaller): RSD (relative standard deviation) is an indicator to measure the consistency of dissolution test results. The smaller the RSD, the more stable and reliable the test results. The RSD of the closed-loop flow cell method gradually decreases, especially when the test time is longer (such as 120 minutes and above), the RSD has dropped to about 0.5%. For example, at 360 minutes, the RSD is only 0.58, showing a very high consistency. The RSD of the conventional dissolution apparatus method is relatively large throughout the entire measurement process, especially in the early time period (such as 10 minutes to 30 minutes), the RSD is higher, at 9.67% and 6.32% respectively. Even in a longer period of time (such as 360 minutes), the RSD is maintained at 0.72%. Although it has improved compared with the previous period, the overall consistency is still lower than the flow cell method. Analysis: The RSD of the flow cell method is lower, indicating that it has higher accuracy and repeatability, and is suitable for drug quality control requiring high consistency. 2. Stability and fidelity of the dissolution curve: From the table data, it can be seen that the dissolution rate of the flow cell method increases more steadily, especially in the later period (such as more than 120 minutes) when the cumulative dissolution rate reaches close to 100%, and the dissolution rate gradually tends to stabilize. This means that the dissolution process of the drug is relatively continuous and uniform. The data of the conventional dissolution instrument method showed a certain volatility, especially in the early period (such as between 45 minutes and 90 minutes), the dissolution rate showed a slight decline or unstable fluctuation (such as the dissolution fluctuation of sample 1 and sample 5). In addition, the dissolution rate did not stabilize at about 100%, and there was a slight fluctuation (such as at 240 minutes, the measured cumulative dissolution rate was 100.75%). Analysis: The dissolution process of the closed-loop flow cell method is more stable, consistent with the actual release pattern of the drug in the body, and can better simulate the release characteristics of the drug in the human body, avoiding the fluctuations or unevenness that may occur in the conventional dissolution instrument method. 3. The early dissolution rate is relatively stable: In the early stage (10-60 minutes), the dissolution rate of the flow cell method increases relatively slowly and consistently. For example, the dissolution rates of all samples at 15 minutes are relatively close, and the subsequent rate of increase remains relatively stable. This result usually indicates that the drug release is more in line with expectations and is particularly important for the development of sustained-release or sustained-release preparations. The dissolution rate of the conventional dissolution instrument method is relatively volatile in the early stage (10-60 minutes), and the differences between different samples are large, resulting in inconsistent measurement results, especially at 90 minutes, when the dissolution rate begins to show a larger gap. The dissolution rate of some samples is higher (such as sample 2 is 101.93%, sample 3 is 96.43%), while the dissolution rate of some samples is relatively low.Analysis: The flow cell method can better control the dissolution rate of the drug in the early stage, provide more consistent and controllable dissolution characteristics, and avoid the volatility existing in the conventional dissolution instrument method. 4. Controllability and simulation of drug release: The flow cell method can better simulate the dynamic dissolution process of the drug in the body through a closed-loop flow system. The drug solution in the flow cell circulates continuously, simulating the actual environment in the human body. This is of great significance for simulating the release behavior of drugs under different physiological environments. Conventional dissolution instrument methods usually use static dissolution systems. The drug release rate is limited by the fluidity of the solution, which may lead to incomplete or unstable dissolution processes. Especially when the volume is large or the drug release is slow, conventional dissolution instruments may not be able to effectively simulate the actual in vivo release. Analysis: The closed-loop flow cell method can simulate the in vivo environment more realistically, ensure that the dissolution of the drug is close to the physiological environment, and provide more accurate data support for the clinical application of the drug. 5. Reliability and repeatability of test data: During the measurement process, the results of the flow cell method showed higher stability and reliability, especially when repeated tests, the dissolution curves of different samples were relatively consistent. The conventional dissolution method showed large fluctuations in some samples, especially in the early time period. For example, the dissolution of sample 1 at 90 minutes was 85.83% by the flow cell method, while it was 99.50% by the conventional dissolution method. This difference may mean that the conventional method is not as stable as the flow cell method in controlling dissolution. Analysis: The test data provided by the flow cell method is more reliable and can better reflect the performance of the drug in actual use, which is suitable for more accurate control and evaluation of drug quality.

[0082] The results of ibuprofen determination in the closed-loop flow cell mode are shown in Table 5, and the corresponding curve graph is shown in Figure 4 As shown:

[0083]

[0084] Table 5

[0085] The results of ibuprofen measured under conventional dissolution apparatus conditions are shown in Table 6, and the corresponding dissolution curve is shown in Figure 5 As shown,

[0086]

[0087]

[0088] Table 6

[0089] Experimental conclusion: The dissolution rate of ibuprofen under conventional dissolution apparatus conditions is fast, with no discrimination, and the intra-batch differences of samples are slightly large. However, the intra-batch differences of samples under flow cell conditions are small, with more discrimination. And the 6 curves are more similar.

[0090] Compared with conventional technologies, the advantages of this method are as follows:

[0091] Improved dissolution consistency: smaller RSD, more stable data, suitable for high quality control.

[0092] The dissolution process is more stable and realistic: it avoids the volatility of conventional dissolution apparatus methods and simulates a more in vivo environment.

[0093] Better early dissolution control: The release rate is stable, which can ensure the ideal release of the drug in the body.

[0094] Provide more reliable test data: It can provide higher accuracy and repeatability, which is suitable for drug research and development and production.

[0095] The specific embodiments described herein are merely examples of the spirit of the present invention. A person skilled in the art of the present invention may make various modifications or supplements to the specific embodiments described or replace them in a similar manner, but will not deviate from the spirit of the present invention or exceed the defined scope. Although the present invention is described and described in detail in the drawings and the foregoing description, such descriptions and descriptions are considered to be illustrative or exemplary rather than restrictive. It should be understood that within the scope of the following claims, changes and modifications may be made by a person of ordinary skill. Specifically, the present invention encompasses additional embodiments having any combination of features from the above-mentioned different embodiments. With respect to the use of the expression "generally" or "substantially", this patent application should be understood to disclose that these features and values ​​are also fully satisfied, i.e., there is no aforementioned characterization as "generally" or "substantially".

Claims

1. A method for determining the in vitro dissolution rate of acetaminophen and ibuprofen suspension drugs, characterized in that: The method for determining the in vitro dissolution of acetaminophen and ibuprofen suspension drugs includes the following: S1 obtains the dissolution liquid sample: adopts the closed-loop circulation cell method to perform dissolution sampling operation on n portions of acetaminophen and ibuprofen suspension drug samples respectively to obtain a dissolution liquid sample set; during the dissolution sampling operation, a phosphate solution with a pH value of 5.5 is used as the dissolution medium. When sampling, samples are taken from the dissolution medium container according to the preset sampling time point. After each sampling, the dissolution medium is supplemented with a volume equal to the volume of the liquid taken away; The filter chamber and sample pool used in the closed-loop flow cell method are set as follows: a polypropylene filter membrane and a polycarbonate filter membrane are placed in the filter chamber in sequence according to the direction of liquid flow during filtration, a ruby ​​is placed at the lower end of the inverted cone of the sample pool; glass beads are placed inside the sample pool; S2 prepares the sample solution: each sample in the dissolution sample set is subjected to preliminary filtration, filtrate sampling, filtration sample dilution and re-filtration operations to obtain the sample solution set. In the preliminary filtration operation, a microporous filter membrane of PES material is used for filtration; in the re-filtration operation, a nylon filter head is used for filtration; in the dilution operation, a methanol-water solution is used as a diluent, and in the methanol-water solution, the volume ratio of methanol: water is 1:1; S3 prepare reference solution samples: prepare at least two reference solution samples, and the specific preparation process is as follows: dissolve the acetaminophen standard and the ibuprofen standard in a methanol-water solution to obtain an initial reference solution, in which the concentration of acetaminophen is the same as the concentration of acetaminophen in the suspension drug sample, and the concentration of ibuprofen is the same as the concentration of ibuprofen in the suspension drug sample; The initial control solution is diluted with methanol aqueous solution to obtain a reference substance solution sample; in the reference substance solution sample, the concentration of acetaminophen is set to be the same as the concentration of acetaminophen in a mixed solution formed by diluting a portion of the suspension drug sample in the dissolution medium contained in the dissolution medium container; the concentration of ibuprofen is set to be the same as the concentration of ibuprofen in a mixed solution formed by diluting a portion of the suspension drug sample in the dissolution medium contained in the dissolution medium container; S4 Liquid chromatography determination: Use a high performance liquid chromatography system to perform liquid chromatography determination on each sample in the reference solution sample and the test solution sample set by gradient elution. After the liquid chromatography determination is completed, the cumulative dissolution of acetaminophen and the cumulative dissolution of ibuprofen in the suspension drug are calculated based on the determination results by using the external standard method and the rehydration method. Based on the calculated cumulative dissolution, the cumulative dissolution curves of acetaminophen and the cumulative dissolution curves of ibuprofen are drawn respectively. In the liquid chromatography measurement operation, the following settings were used: detection wavelength 222 nm, mobile phase: mobile phase A: 0.1% (V / V) trifluoroacetic acid aqueous solution, mobile phase B: methanol.

2. The method for measuring the in vitro dissolution of acetaminophen and ibuprofen suspension as claimed in claim 1, characterized in that: In step S1, the sample cell uses a standard flow cell with an inner diameter of 22.6 mm, the diameter of the ruby ​​is 5 mm, and the diameter of the glass bead is 1 mm.

3. The method for measuring the in vitro dissolution of acetaminophen and ibuprofen suspension as claimed in claim 1, characterized in that: In step S1, the dissolution sampling operation parameters are set as follows: the dissolution medium temperature is maintained at 37°C, the flow rate is 8 mL / min, and the volume ratio of the suspension drug to the dissolution medium is set to 5.5:400; the volume of each sampling is 1 / 400 of the volume of the dissolution medium.

4. The method for measuring the in vitro dissolution of acetaminophen and ibuprofen suspension as claimed in claim 1, characterized in that: In step S1, n≥5, and the preset sampling time points are 10, 15, 30, 45, 60, 90, 120, 180, 240 and 360 minutes, respectively.

5. The method for measuring the in vitro dissolution of acetaminophen and ibuprofen suspension as claimed in claim 1, characterized in that: The pore size of the polypropylene filter membrane is 20μm, and the pore size of the polycarbonate filter membrane is 20μm; the pore size of the PES microporous filter membrane is 0.45μm, and the pore size of the filter membrane in the nylon filter head is 0.22μm.

6. The method for determining the in vitro dissolution of acetaminophen and ibuprofen suspension as claimed in claim 1, characterized in that: In the filtered sample dilution operation of step S2, the volume ratio of the diluted object to the diluted solution is 1:

50.

7. The method for determining the in vitro dissolution of acetaminophen and ibuprofen suspension as claimed in claim 1, characterized in that: In step S4, the configuration of the HPLC system is as follows: detector: UV detector; chromatographic column: C18 chromatographic column, the inner diameter of the chromatographic column is 4.6 mm, the length of the chromatographic column is 150 mm, and the diameter of the filler particles is 3.5 μm; guard column: the inner diameter of the guard column is 4.6 mm, the length of the guard column is 10 mm, and the diameter of the filler particles is 5 μm.

8. The method for determining the in vitro dissolution of acetaminophen and ibuprofen suspension as claimed in claim 1, characterized in that: In step S4, during the gradient elution process using the mobile phase, the volume ratio of mobile phase A to mobile phase B in the mobile phase is set as follows: 0-3 minutes, the ratio of mobile phase A to mobile phase B is uniformly adjusted from the initial 75:25 to 5:95; 3-3.1 minutes, the ratio of mobile phase A to mobile phase B is uniformly adjusted from 5:95 to 75:25; 3.1-8.5 minutes, the ratio of mobile phase A to mobile phase B is maintained at 75:25.

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