A method for detecting and verifying a liposome related substance

Abstract

This application discloses a method for detecting and validating liposome-related substances, relating to the field of liposome analysis and detection technology. The method includes the following steps: diluting a liposome sample to obtain a sample to be tested; sequentially performing ultrafiltration separation, a first release treatment, and a second release treatment on the liposome sample to be tested; obtaining a first detection solution, a second detection solution, and a third detection solution respectively; determining the mass of free-state related substances, the mass of membrane-bound related substances, and the mass of intracapsule-loaded related substances; summing the masses of free-state related substances, membrane-bound related substances, and intracapsule-loaded related substances; and combining this sum with the mass of the liposome sample to obtain the mass percentage content of related substances in the liposome sample. This application achieves the distribution and localization of related substances in different structural regions of liposomes through a continuous gradient release method, which can reduce the impact of strong destructive treatments on the liposome structure and improve the reliability of detection results.

Description

Technical Field

[0001] This application relates to the field of liposome analysis and detection technology, specifically a method for detecting and validating liposome-related substances. Background Technology

[0002] Liposomes are nanoscale vesicle structures formed by a lipid bilayer, typically composed of phospholipids, cholesterol, and other lipid materials. During storage and use, the phospholipid components may undergo degradation reactions such as oxidation and hydrolysis, producing liposome-related substances, such as lysophospholipids, free fatty acids, and lipid oxidation products. Due to the presence of the lipid bilayer structure, the liposome system differs from ordinary homogeneous solution systems, exhibiting distinct spatial distribution characteristics among its internal components. Existing methods for detecting liposome-related substances typically employ direct and forced endosome disruption, such as treatment with high-concentration organic solvents, strong acids or bases, or ultrasonic destruction. However, these methods have the following drawbacks in practical applications:

[0003] It is impossible to distinguish the spatial distribution of relevant substances in liposomes, and strong destructive treatment (i.e., forced capsule disruption) may change the structural state of liposomes, increasing the risk of generating non-target peaks during detection and causing the detection results to be too high. Summary of the Invention

[0004] In view of the deficiencies in the existing technology, the technical problem to be solved by this application is: how to realize the detection and distribution localization of liposome-related substances.

[0005] To achieve the above objectives, embodiments of this application provide a method for detecting and verifying liposome-related substances, the method comprising the following steps:

[0006] S1. After diluting the liposome sample, obtain the liposome sample to be tested;

[0007] S2. Perform ultrafiltration separation on the liposome sample to be tested, collect the filtrate as the first detection solution, and the remaining part is the preliminary release treatment material;

[0008] S3. After adding surface release treatment liquid to the preliminary release treatment material for the first release treatment, perform ultrafiltration separation treatment, collect the filtrate as the second detection liquid, and the remaining part is the secondary release treatment material.

[0009] S4. After adding vesicle destruction treatment fluid to the secondary release treatment material for the second release treatment, separate and obtain the third detection fluid.

[0010] S5. Use chromatographic detection methods to detect the first detection solution, the second detection solution and the third detection solution respectively, and obtain the mass of free related substances, the mass of membrane-bound related substances and the mass of encapsulated related substances.

[0011] S6. After summing the masses of the free-state related substances, the membrane-bound related substances, and the encapsulated related substances, and combining this with the mass of the liposome sample, obtain the mass percentage of related substances in the liposome sample.

[0012] In one embodiment, the method for diluting the liposome sample is as follows: the liposome sample is added to an aqueous medium and mixed thoroughly; wherein the mass ratio of the liposome sample to the aqueous medium is 1:5-50.

[0013] In one embodiment, the ultrafiltration separation uses an ultrafiltration membrane of 50kDa-200kDa.

[0014] In one embodiment, the surface release treatment liquid comprises a nonionic surfactant and an aqueous medium, wherein the mass ratio of the nonionic surfactant to the aqueous medium is 1:100-5000.

[0015] In one embodiment, the method of adding a surface release treatment liquid to the initially released material for the first release treatment includes:

[0016] Add 1-10 times the mass of surface release treatment solution to the initial release treatment material, and shake at 50-200 rpm for 10-60 minutes at 2℃-15℃.

[0017] In one embodiment, the vesicle destruction treatment solution comprises a nonionic surfactant, an organic co-solvent, and an aqueous medium, wherein the mass ratio of the nonionic surfactant, the organic co-solvent, and the aqueous medium is 1:5-30:50-200.

[0018] The organic co-solvent is one or more of ethanol, isopropanol, and methanol.

[0019] In one embodiment, the method for separating and obtaining the third detection liquid after adding vesicle destruction treatment fluid to the secondary release treatment material for the second release treatment includes:

[0020] Add 2-10 times the mass of vesicle destruction solution to the secondary release treatment material, and treat it at 500rpm-1500rpm for 10min-60min in an environment of 25℃-45℃.

[0021] After centrifuging at 8000rpm-15000rpm for 5min-15min, collect the supernatant.

[0022] The supernatant was filtered through a 0.20μm-0.45μm filter membrane to obtain the third detection solution.

[0023] In one embodiment, the aqueous medium is one of deionized water, phosphate buffer, or physiological saline.

[0024] In one embodiment, the nonionic surfactant is one or more of Tween surfactants, poloxamer surfactants, and polyoxyethylene surfactants.

[0025] In one embodiment, the method of obtaining the mass of free related substances, membrane-bound related substances, and encapsulated related substances by detecting the first detection solution, the second detection solution, and the third detection solution respectively using chromatographic detection methods includes:

[0026] The first, second, and third detection solutions were detected by chromatographic detection methods to obtain the chromatographic response parameters of the corresponding detection solutions.

[0027] Based on the pre-established correspondence between chromatographic response parameters and related substance concentrations, the concentrations of related substances in the corresponding detection solutions are determined.

[0028] Calculate the product of the concentration of the relevant substance and the volume of the corresponding detection solution to obtain the mass of the relevant substance in free state, the mass of the relevant substance in membrane-bound state, and the mass of the relevant substance in the capsule-loaded state.

[0029] Compared with the prior art, the advantages of this application are:

[0030] By using a continuous gradient release method, the mass of related substances in the free state, membrane-bound state, and intracapsular loaded state can be obtained sequentially, enabling the distribution and localization of related substances in different structural regions of liposomes. The first and second release treatments are performed using a low-temperature and mild release method, which can reduce the impact of strong destructive treatment on the liposome structure and improve the reliability of the detection results. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a flowchart of the detection and verification method for liposome-related substances in the embodiments of this application. Detailed Implementation

[0033] Liposome-related substances refer to all impurities in liposome samples produced due to lipid degradation. These include lysophospholipids, free fatty acids, and lipid oxidation products.

[0034] To achieve the detection and verification of liposome-related substances, this application classifies related substances according to their location by continuously gradient-releasing them from the liposome structure: free related substances (i.e., related substances existing in the continuous phase outside the liposome, which are not bound to the liposome structure and exist directly in the dispersion medium), membrane-bound related substances (i.e., related substances bound to the liposome phospholipid bilayer structure or adsorbed in the lipid membrane region, which are restricted by the lipid membrane structure and cannot be directly detected by conventional separation methods), and intravesical loaded related substances (i.e., related substances located in the aqueous phase space inside the liposome or covered by the lipid bilayer, which require disruption of the liposome vesicle structure before they can be released).

[0035] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0036] The flowchart shown in the attached diagram is for illustrative purposes only and does not necessarily include all content and operations / steps, nor does it necessarily have to be performed in the order described. For example, some operations / steps can be broken down, combined, or partially merged, so the actual execution order may change depending on the actual situation.

[0037] To make the objectives, technical solutions, and advantages of this application clearer, the embodiments of this application will be described in further detail below with reference to the accompanying drawings.

[0038] This application provides a method for detecting and validating liposome-related substances, referring to... Figure 1 The method includes the following steps:

[0039] S1. After weighing the liposome sample, add the liposome sample to an aqueous medium and mix well. The mass ratio of the liposome sample to the aqueous medium is 1:5-50. After completing the dilution process of the liposome sample, obtain the liposome sample to be tested.

[0040] S2. Use a 50kDa-200kDa ultrafiltration membrane to perform ultrafiltration separation on the liposome sample to be tested. This process retains intact liposome particles and allows free related substances located in the continuous phase outside the liposomes to pass through the ultrafiltration membrane. Collect the filtrate as the first detection solution, and the remaining part is the preliminary release treatment material.

[0041] S3. After adding surface release treatment liquid to the preliminary release treatment material for the first release treatment, perform ultrafiltration separation treatment, collect the filtrate as the second detection liquid, and the remaining part is the secondary release treatment material; wherein the surface release treatment liquid includes nonionic surfactant and aqueous medium, and the mass ratio of nonionic surfactant to aqueous medium is 1:100-5000.

[0042] Nonionic surfactants are one or more of the following: Tween surfactants, poloxamer surfactants, and polyoxyethylene surfactants;

[0043] The aqueous medium is one of deionized water, phosphate buffer, or physiological saline.

[0044] The above-mentioned method of adding surface release treatment liquid to the preliminary release treatment material for the first release treatment includes:

[0045] Add 1-10 times the mass of surface release treatment solution to the initial release treatment material, and shake at 50-200 rpm for 10-60 minutes at 2℃-15℃.

[0046] Furthermore, an ultrafiltration membrane of 50kDa-200kDa was used to perform ultrafiltration separation on the initially released material after shaking to obtain a second detection solution and a secondary released material.

[0047] S4. After adding vesicle destruction treatment solution to the secondary release treatment material for the second release treatment, separate and obtain the third detection solution; wherein the vesicle destruction treatment solution includes nonionic surfactant, organic cosolvent and aqueous medium, and the mass ratio of nonionic surfactant, organic cosolvent and aqueous medium is 1:5-30:50-200.

[0048] Nonionic surfactants are one or more of the following: Tween surfactants, poloxamer surfactants, and polyoxyethylene surfactants;

[0049] The organic co-solvent is one or more of ethanol, isopropanol, and methanol;

[0050] The aqueous medium is one of deionized water, phosphate buffer, or physiological saline.

[0051] The method for separating and obtaining the third detection solution after adding vesicle destruction treatment solution to the secondary release treatment material for the second release treatment includes:

[0052] Add 2-10 times the mass of vesicle destruction solution to the secondary release treatment material, and treat it at 500rpm-1500rpm for 10min-60min in an environment of 25℃-45℃.

[0053] After centrifuging at 8000rpm-15000rpm for 5min-15min, collect the supernatant.

[0054] The supernatant was filtered through a 0.20μm-0.45μm filter membrane to obtain the third detection solution.

[0055] S5. Calculate the mass of the related substances in the free state. The procedure for obtaining the mass of the related substances in the free state is as follows:

[0056] The first detection solution is detected by a chromatographic method (one of high performance liquid chromatography, ultra-high performance liquid chromatography, or liquid chromatography-mass spectrometry) to obtain free chromatographic response parameters (one of peak area, peak height, and peak area integral value). Based on the pre-established correspondence between the chromatographic response parameters and the concentrations of related substances, the concentration of free related substances corresponding to the free chromatographic response parameters is determined. The concentration of free related substances is the sum of one or more of the following: lysophospholipid concentration, free fatty acid concentration, and lipid oxidation product concentration. The specific concentration is determined according to the target requirements. That is, if a certain related substance is determined, only that related substance is determined; if multiple related substances are determined, the concentrations are summed after each related substance is determined.

[0057] The product of the concentration of the free-state related substance and the volume of the first detection solution is calculated as the mass of the free-state related substance.

[0058] The process for calculating the mass of membrane-bound related substances is as follows:

[0059] The second detection solution is detected by chromatographic detection method to obtain membrane-bound chromatographic response parameters. Based on the pre-established correspondence between chromatographic response parameters and related substance concentrations, the concentration of membrane-bound related substances corresponding to the membrane-bound chromatographic response parameters is determined. The concentration of membrane-bound related substances is the sum of one or more of the following: lysophospholipid concentration, free fatty acid concentration, and lipid oxidation product concentration. The specific concentration is determined according to the target requirements. That is, if a certain related substance is determined, only that related substance is determined. If multiple related substances are determined, the concentrations are summed after each related substance is determined.

[0060] The product of the concentration of the membrane-bound related substance and the volume of the second detection solution is calculated as the mass of the membrane-bound related substance.

[0061] The process for calculating the mass of the relevant substances in the encapsulated state is as follows:

[0062] The third detection solution is detected by chromatographic detection method to obtain the chromatographic response parameters of the in-capsule loaded state. Based on the pre-established correspondence between the chromatographic response parameters and the concentrations of related substances, the concentration of related substances in the in-capsule loaded state corresponding to the chromatographic response parameters is determined. The concentration of related substances in the in-capsule loaded state is the sum of one or more of the following: lysophospholipid concentration, free fatty acid concentration, and lipid oxidation product concentration. The specific concentration is determined according to the target requirements. That is, if a certain related substance is determined, only that related substance is determined. If multiple related substances are determined, the concentrations are summed after each related substance is determined.

[0063] The product of the concentration of the related substances loaded in the capsule and the volume of the third detection solution is calculated as the mass of the related substances loaded in the capsule.

[0064] S6. After calculating the sum of the masses of the free-state related substances, the membrane-bound related substances, and the intracapsular related substances, and combining this with the mass of the liposome sample, the mass percentage of related substances in the liposome sample can be obtained.

[0065] Based on this, the mass of related substances in the free state, membrane-bound state and intracapsular state can be obtained sequentially through continuous gradient release, and the mass of related substances can be located at different positions of the liposomes. The first and second release treatments are carried out by low temperature and gentle release, which can reduce the impact of strong destructive treatment on the liposome structure and improve the reliability of the detection results.

[0066] Furthermore, by calculating the ratios of the mass of the free-state related substances, the mass of the membrane-bound related substances, and the mass of the encapsulated related substances to the mass of the liposome sample, the mass percentage of related substances at different locations in the liposome sample can be obtained.

[0067] This allows for precise identification of the source and content of relevant substances.

[0068] The method provided in this application will now be described in detail with reference to specific embodiments.

[0069] Example 1

[0070] Liposome samples were taken as the test objects, their mass was weighed, and phosphate buffer was added for dilution. The mass ratio of liposome sample to phosphate buffer was 1:10. After mixing, the liposome sample to be tested was obtained.

[0071] The liposome samples to be tested were separated by ultrafiltration using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa. The filtrate was collected as the first detection solution, and the remaining part was the preliminary release treatment material.

[0072] The first test solution was detected by high performance liquid chromatography to obtain the peak area. Based on the pre-established correspondence between peak area and related substance concentration, the concentration of each related substance in the first test solution was calculated. After summing, the concentration was multiplied by the volume of the first test solution to obtain the mass of the free related substances.

[0073] A surface release treatment solution was added to the preliminary release treatment material for the first release treatment. The surface release treatment solution consisted of Tween-20 and phosphate buffer, with a mass ratio of Tween-20 to phosphate buffer of 1:2500. The amount added was 5 times the amount of the molecular weight cutoff. After treatment at 4°C and shaking at 100 rpm for 30 min, ultrafiltration was performed using an ultrafiltration membrane with a molecular weight cutoff of 100 kDa. The filtrate was collected as the second detection solution, and the remaining part was the secondary release treatment material.

[0074] The second detection solution was detected by high performance liquid chromatography (HPLC) to obtain the peak area. Based on the pre-established correspondence between peak area and related substance concentration, the concentration of each related substance in the second detection solution was calculated. After summing, the concentration was multiplied by the volume of the second detection solution to obtain the mass of the membrane-bound related substances.

[0075] A second release treatment was performed by adding a vesicle disruption solution to the sample to be released; the vesicle disruption solution consisted of Tween-20, ethanol, and phosphate buffer in a mass ratio of 1:30:50.

[0076] The amount of vesicle destruction treatment solution added was 5 times the amount of the retained substance. After shaking at 1000 rpm for 30 min at 37℃, the solution was centrifuged at 12000 rpm for 10 min. The supernatant was then filtered through a 0.22 μm filter membrane to obtain the third detection solution.

[0077] The third detection solution was detected by high performance liquid chromatography (HPLC) to obtain the peak area. Based on the pre-established correspondence between peak area and related substance concentration, the concentration of each related substance in the third detection solution was calculated. After summing, the concentration was multiplied by the volume of the third detection solution to obtain the mass of related substances loaded in the capsule.

[0078] Based on this, the distribution mass of liposome-related substances at different spatial locations of liposomes is obtained. After summing these masses as a whole and combining them with the mass of liposomes, the mass percentage content of liposome-related substances can be obtained.

[0079] Example 2

[0080] The difference between this embodiment and Example 1 is that the mass ratio of Tween-20 to phosphate buffer is 1:100.

[0081] Example 3

[0082] The difference between this embodiment and Embodiment 1 is that the mass ratio of Tween-20 to phosphate buffer is 1:5000.

[0083] Example 4

[0084] The difference between this embodiment and Embodiment 1 is that the vesicle destruction treatment solution includes Tween-20, ethanol, and phosphate buffer, with a mass ratio of 1:5:200.

[0085] Example 5

[0086] The difference between this embodiment and Embodiment 1 is that the vesicle destruction treatment solution includes Tween-20, ethanol, and phosphate buffer, with a mass ratio of 1:17:125.

[0087] Combining the detection results of Examples 1, 2, and 3, it was found that the deviation in the mass percentage content of liposome-related substances was within the error range (±0.01). Specifically, the mass percentage content of liposome-related substances in Examples 1, 2, and 3 were 0.52%, 0.53%, and 0.51%, respectively. During the process, particle size change analysis was performed on the liposome sample and the secondary release treatment material in Example 1. Specifically, the absolute difference between the average particle size of the secondary release treatment material and the average particle size of the liposome sample was calculated and divided by the liposome sample to obtain the particle size change rate. Similarly, the particle size change rates for Examples 2 and 3 were obtained. It can be seen that the particle size change rates in Examples 1 and 2 were both less than the preset threshold (10%), indicating that the liposome structure had not undergone significant changes. However, the particle size change rate in Example 3 was greater than the preset threshold, indicating that the liposome structure stability had decreased.

[0088] Meanwhile, the chromatographic detection results of the second detection solution in Examples 1, 2 and 3 were obtained. After extracting the peak area and peak number, it was found that the peak area of ​​the same related substance in Examples 1, 2 and 3 decreased sequentially, and no new impurity peaks appeared.

[0089] In summary, it can be determined that when the proportion of Tween-20 is too low, some membrane-bound related substances are not released sufficiently, and the target peak response in the second detection solution is reduced (Example 3); when the proportion of Tween-20 is too high, some liposome structures may be destroyed prematurely, causing premature release of the related substances loaded in the vesicle (Example 2); when the mass ratio of Tween-20 to phosphate buffer is 1:2500, the release of membrane-bound related substances can be achieved well, while maintaining the integrity of the liposome vesicle structure (Example 1).

[0090] Based on the detection results of Examples 1, 4 and 5, it was found that the deviation in the mass percentage content of liposome-related substances was within the error range (±0.01).

[0091] Chromatograms of the third detection solution obtained by high performance liquid chromatography in Examples 1, 4 and 5 were obtained. The number of peaks and peak area of ​​the corresponding chromatograms were extracted respectively. The results showed that new impurity peaks appeared in all cases. The peak areas of the corresponding peaks in Examples 4, 5 and 1 increased in sequence. Based on this, it can be determined that the component ratio of the vesicle destruction treatment solution in Example 1 is the optimal ratio.

[0092] The terms "comprising" and "having," and any variations thereof, in the specification, claims, and accompanying drawings of this application are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such process, method, product, or apparatus. The terms "first," "second," and "third," etc., are used to distinguish different objects, etc., and do not indicate a sequence, nor do they limit "first," "second," and "third" to different types.

[0093] In the description of the embodiments of this application, terms such as "exemplary," "for example," or "for instance" are used to indicate examples, illustrations, or explanations. Any embodiment or design described as "exemplary," "for example," or "for instance" in the embodiments of this application should not be construed as being more preferred or advantageous than other embodiments or designs. Specifically, the use of terms such as "exemplary," "for example," or "for instance" is intended to present the relevant concepts in a concrete manner.

[0094] In the description of the embodiments of this application, unless otherwise stated, " / " means "or". For example, A / B can mean A or B. The "and / or" in the text is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, and B exists alone. In addition, in the description of the embodiments of this application, "multiple" means two or more.

[0095] In some processes described in the embodiments of this application, multiple operations or steps are included in a specific order. However, it should be understood that these operations or steps may not be executed in the order they appear in the embodiments of this application, or they may be executed in parallel. The sequence number of the operation is only used to distinguish different operations, and the sequence number itself does not represent any execution order. In addition, these processes may include more or fewer operations, and these operations or steps may be executed sequentially or in parallel, and these operations or steps may be combined.

[0096] The above are merely specific embodiments of this application, but the protection scope of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the protection scope of this application. Therefore, the protection scope of this application should be determined by the scope of the claims.

Claims

1. A method for detecting and verifying liposome-related substances, characterized in that, The method includes the following steps: S1. After diluting the liposome sample, obtain the liposome sample to be tested; S2. Perform ultrafiltration separation on the liposome sample to be tested, collect the filtrate as the first detection solution, and the remaining part is the preliminary release treatment material; S3. After adding surface release treatment liquid to the preliminary release treatment material for the first release treatment, perform ultrafiltration separation treatment, collect the filtrate as the second detection liquid, and the remaining part is the secondary release treatment material. S4. After adding vesicle destruction treatment fluid to the secondary release treatment material for the second release treatment, separate and obtain the third detection fluid. S5. Use chromatographic detection methods to detect the first detection solution, the second detection solution and the third detection solution respectively, and obtain the mass of free related substances, the mass of membrane-bound related substances and the mass of encapsulated related substances. S6. After summing the masses of the free-state related substances, the membrane-bound related substances, and the encapsulated related substances, and combining this with the mass of the liposome sample, obtain the mass percentage of related substances in the liposome sample.

2. The method for detecting and verifying liposome-related substances according to claim 1, characterized in that, The method for diluting the liposome sample is as follows: the liposome sample is added to an aqueous medium and mixed thoroughly; wherein the mass ratio of the liposome sample to the aqueous medium is 1:5-50.

3. The method for detecting and verifying liposome-related substances according to claim 1, characterized in that, The ultrafiltration separation uses an ultrafiltration membrane of 50kDa-200kDa.

4. The method for detecting and verifying liposome-related substances according to claim 1, characterized in that, The surface release treatment liquid comprises a nonionic surfactant and an aqueous medium, wherein the mass ratio of the nonionic surfactant to the aqueous medium is 1:100-5000.

5. The method for detecting and verifying liposome-related substances according to claim 4, characterized in that, The method of adding a surface release treatment liquid to the initially released material for the first release treatment includes: Add 1-10 times the mass of surface release treatment solution to the initial release treatment material, and shake at 50-200 rpm for 10-60 minutes at 2℃-15℃.

6. The method for detecting and verifying liposome-related substances according to claim 1, characterized in that, The vesicle destruction treatment solution comprises a nonionic surfactant, an organic co-solvent, and an aqueous medium, wherein the mass ratio of the nonionic surfactant, the organic co-solvent, and the aqueous medium is 1:5-30:50-200. The organic co-solvent is one or more of ethanol, isopropanol, and methanol.

7. The method for detecting and verifying liposome-related substances according to claim 1, characterized in that, The method for separating and obtaining the third detection liquid after adding vesicle destruction treatment fluid to the secondary release treatment material for the second release treatment includes: Add 2-10 times the mass of vesicle destruction solution to the secondary release treatment material, and treat it at 500rpm-1500rpm for 10min-60min in an environment of 25℃-45℃. After centrifuging at 8000rpm-15000rpm for 5min-15min, collect the supernatant. The supernatant was filtered through a 0.20μm-0.45μm filter membrane to obtain the third detection solution.

8. A method for detecting and verifying liposome-related substances according to claim 2, 4, or 6, characterized in that, The aqueous medium is one of deionized water, phosphate buffer, or physiological saline.

9. A method for detecting and verifying liposome-related substances according to claim 4 or 6, characterized in that, The nonionic surfactant is one or more of the following: Tween surfactants, poloxamer surfactants, and polyoxyethylene surfactants.

10. The method for detecting and verifying liposome-related substances according to claim 1, characterized in that, The method of obtaining the mass of free related substances, membrane-bound related substances, and encapsulated related substances by detecting the first, second, and third detection solutions respectively using chromatographic detection methods includes: The first, second, and third detection solutions were detected by chromatographic detection methods to obtain the chromatographic response parameters of the corresponding detection solutions. Based on the pre-established correspondence between chromatographic response parameters and related substance concentrations, the concentrations of related substances in the corresponding detection solutions are determined. Calculate the product of the concentration of the relevant substance and the volume of the corresponding detection solution to obtain the mass of the relevant substance in free state, the mass of the relevant substance in membrane-bound state, and the mass of the relevant substance in the capsule-loaded state.

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