In-vitro release test method and content analysis method of liposome drug

Abstract

The invention discloses an in-vitro release test method and a content analysis method of a liposome drug, and the method comprises the following steps: mixing a buffer agent and a surfactant in a dissolution cup to obtain a mixed solution, and adding a regulator into the mixed solution to obtain a dissolution medium; adding liposome powder into water for dissolving, and then adding the dissolution medium for dilution to obtain a sample solution; putting the dissolution cup into a dissolution instrument, heating at a preset temperature, and connecting a dialysis tube to the dissolution instrument through an adapter; adding the sample solution into the dialysis tube, mixing the sample solution with the dissolution medium, collecting a drug sample at a specific time point, and analyzing the drug sample to obtain the release rate of the liposome drug. According to the method, the release process of the liposome drug in a living body can be more accurately simulated, and the release rate of the in-vitro release method of the liposome drug is greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of liposome drug testing, in particular to an in vitro release testing method and content analysis method of a liposome drug. Background Art

[0002] In the process of drug development and formulation, liposomes have excellent biocompatibility and targeting, and can improve the solubility, stability and bioavailability of drugs. The in vitro release of liposomal drugs mostly uses dialysis. If the drug is water-soluble, it can pass through the dialysis tube (bag) after being released from the liposomes. Liposomes cannot pass through the dialysis tube (bag) due to their large size, thereby achieving the purpose of separating free drug molecules, thereby analyzing the amount of drug released at a specific time. If the drug has poor solubility in aqueous solution, a solubilizer needs to be added to the dissolution medium to dissolve the drug molecules released from the liposomes, and the dissolved drug molecules can be released from the dialysis tube (bag).

[0003] However, the release rate of the existing in vitro release method of liposome drugs is poor, and the release rate is mainly improved by changing the dissolution device. For example, the public text CN201510600365.2 discloses a test method for in vitro release of liposome drugs, which is to load the liposome drug solution and air into a container together, and use the container's vibration or rotation and other movements to simulate the dynamic effect of blood circulation in the body on the drug, so that the air in the system is dispersed during the movement to form bubbles, and physically collide with the liposomes, thereby destroying the lipid membrane and achieving effective release of the drug. It can be seen from the above-mentioned prior art that although the release rate of liposome drugs can be improved to a certain extent, the vibration or rotation method cannot simulate the complex blood circulation environment in the body, and the degree of damage to the liposomes is difficult to control, which may cause the drug to be released too quickly or unevenly, thereby affecting the release behavior of the drug, resulting in the data obtained in the in vitro release test being difficult to reflect the release of the drug in actual clinical applications. Summary of the invention

[0004] The main purpose of the present invention is to provide a method for testing the in vitro release of liposome drugs, aiming to overcome the technical problem of poor release rate of existing in vitro release methods of liposome drugs.

[0005] In order to achieve the above-mentioned inventive problem, the present invention provides an in vitro release test method of a liposome drug, comprising: Mixing a buffer and a surfactant in a dissolution cup to obtain a mixed solution, and adding a regulator to the mixed solution to obtain a dissolution medium; After the liposome powder is added into water to dissolve, the dissolution medium is added for dilution to obtain a sample solution; The dissolution cup is placed in a dissolution apparatus and heated at a preset temperature, and the dialysis tube is connected to the dissolution apparatus via an adapter; The sample solution is added into the dialysis tube, the sample solution and the dissolution medium are mixed, and drug samples are taken at specific time points to measure the unreleased drug concentration or the released drug concentration in the liposome drug and calculate the release rate of the liposome drug.

[0006] Furthermore, the preset temperature is 36° C. to 38° C., and the volume of the drug sample is 0.5 ml to 1 ml.

[0007] Furthermore, the buffer is 9 mM to 10 mM hydroxyethylpiperazineethanesulfonic acid, the surfactant is 0.7% to 0.8% sodium deoxycholate, and the regulator is sodium hydroxide solution to adjust the pH value of the dissolution medium to 7.4 to 7.6.

[0008] Furthermore, the step of adding the dissolution medium to dilute the liposome powder after dissolving it in water to obtain a sample solution comprises: Add 150 g to 160 g of liposome powder to 3 mL to 5 mL of deionized water and shake vigorously until completely dissolved; The dissolution medium was added to the filtered liposome solution and the mixture was manually shaken to mix until the mixed solution of the liposome solution and the dissolution medium reached 20 mL.

[0009] Furthermore, before the step of connecting the dialysis tube to the dissolution instrument via the rotating shaft, the method further comprises: The dialysis tube is immersed in a 15% to 25% ethanol aqueous solution, the temperature of the ethanol aqueous solution is 20° C. to 30° C., and the immersion time is 50 minutes to 60 minutes; The immersed dialysis tube is cleaned with water, and the cleaned dialysis tube is placed in the dissolution medium for soaking, and the soaking time is 115 minutes to 125 minutes.

[0010] Furthermore, the step of adding the sample solution into the dialysis tube to mix the sample solution and the dissolution medium comprises: The bottom of the dialysis tube and the bottom of the dissolution cup are arranged at a preset distance, wherein the preset distance is 2.5 cm to 3.5 cm; The shaft speed in the dissolution instrument is set to 90 rpm to 110 rpm, and the dissolution instrument is started to rotate the dialysis tube in the dissolution cup, so that the dissolution medium in the dissolution cup diffuses into the dialysis tube through the dialysis membrane in the dialysis tube to mix with the sample solution.

[0011] Furthermore, the step of sampling the drug at a specific time point comprises: A certain volume of the mixture of the dissolution medium and the sample solution is taken out from the dissolution cup as a drug sample, and the specific time point includes but is not limited to: 1 hour, 2 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, and 44 hours.

[0012] Furthermore, the liposome drug is liposome amphotericin B.

[0013] The present application also discloses a method for analyzing the content of liposome drugs, which is used to analyze any of the drug samples described above, comprising: Put 0.9 mL to 1.1 mL of the sample solution into 90 mL to 110 mL of the dissolution medium and mix them to obtain a test solution; The drug sample is injected into a high performance liquid chromatograph for chromatographic analysis to obtain a chromatogram of the drug sample, wherein the chromatographic conditions of the high performance liquid chromatograph include but are not limited to: the mobile phase is composed of a mobile phase A and a mobile phase B, the flow rate of the mobile phase is 1.2 mL / min~1.3 mL / min, the mobile phase A is a mixture of a citric acid buffer (4.2 g / L, adjusted to pH 4.7 with concentrated ammonia water) and methanol, the volume ratio is 9:1, and the mobile phase B is a mixture of acetonitrile and methanol, the volume ratio is 9:1; the chromatographic column is a C18 column, and the column temperature is set to 30°C; the detector is an ultraviolet detector, and the detection wavelength is 383 nm; the injection volume is 10µL~20µL, and the analysis time is 10 minutes~20 minutes; The area of ​​the chromatogram is compared with the peak area of ​​the assay solution to obtain the concentration of the liposome drug in the drug sample.

[0014] Furthermore, the step of comparing the area of ​​the chromatogram with the peak area of ​​the assay solution to obtain the concentration of the liposome drug in the drug sample comprises: Injecting the assay solution into a high performance liquid chromatograph for chromatographic analysis to obtain a reference chromatogram of the assay solution; Calculating the peak area of ​​the reference chromatogram and the peak area of ​​the chromatogram of the drug sample to obtain a proportional relationship between the reference chromatogram and the chromatogram; The actual concentration of the liposome drug in the drug sample is obtained by calculating according to the proportional relationship through the standard curve method.

[0015] Beneficial effects: The in vitro release test method of liposome drugs proposed in this application improves the release efficiency of liposome drugs by adding a specific surfactant to the dissolution medium. The surfactant can reduce the surface tension of the liposome membrane, thereby promoting the release of drug molecules from the liposomes. By adjusting the pH value of the dissolution medium, the dissolution medium is made closer to the actual in vivo situation. The liposome drug powder is mixed and dissolved with water to form a uniform liposome solution, and the solution is added to the dissolution medium containing a buffer and a surfactant for dilution to simulate the release conditions of the drug in the in vivo environment, so as to more accurately reflect the release behavior of the liposome drug in the organism. Further, the dialysis tube is placed in the dissolution instrument and heated at a set temperature to simulate the normal body temperature of the human body. The sample solution is contained in the dialysis tube, and the dissolution medium is filled on the outside of the dissolution instrument. Through the semipermeable membrane of the dialysis tube, the liposome drug in the sample solution can be gradually released into the external dissolution medium. At a specific time point, the dissolution medium outside the dialysis tube is sampled and analyzed to evaluate the release characteristics of the liposome drug. The present application can more accurately simulate the release process of liposome drugs in vivo, and greatly improve the release rate of liposome drugs in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a schematic diagram of the overall steps of an in vitro release test method for a liposome drug in one embodiment of the present invention; Figure 2 1 is a schematic diagram of a release curve of a repeatability study of an in vitro release method in one embodiment of the present invention; Figure 3 Schematic diagram of a study on the permeation rate of amphotericin B in a dialysis tube in one embodiment of the present invention; Figure 4 1 is a schematic diagram of the overall steps of a method for analyzing the content of a liposome drug in one embodiment of the present invention; Figure 5 Schematic diagram of the chromatogram of amphotericin B in one embodiment of the present invention.

[0017] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0019] It will be understood by those skilled in the art that, unless expressly stated, the singular forms "one", "above", and "the" used herein may also include plural forms. It should be further understood that the term "comprising" used in the specification of the present invention refers to the presence of features, integers, steps, operations, elements, modules, and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, modules, components, and / or groups thereof. It should be understood that when an element is said to be "connected" or "coupled" to another element, it may be directly connected or coupled to the other element, or there may be an intermediate element. In addition, the "connection" or "coupling" used herein may include wireless connection or wireless coupling. The term "and / or" used herein includes all or any module and all combinations of one or more associated listed items.

[0020] Those skilled in the art will understand that, unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as those generally understood by those skilled in the art in the field to which the present invention belongs. It should also be understood that terms such as those defined in common dictionaries should be understood to have meanings consistent with the meanings in the context of the prior art, and will not be interpreted with idealized or overly formal meanings unless specifically defined as here.

[0021] Reference Figures 1 to 3 The present invention provides a method for testing the in vitro release of liposome drugs, comprising the following steps: S1: mixing a buffer and a surfactant in a dissolution cup to obtain a mixed solution, and adding a regulator to the mixed solution to obtain a dissolution medium; In step S1, the buffer and surfactant are mixed in the dissolution cup to ensure that they are fully dissolved and evenly distributed. Then, a regulator is added to the mixed solution. The role of the regulator is to adjust the pH value of the dissolution medium to simulate the physiological environment in the human body, thereby ensuring the accuracy and relevance of the test results. Optionally, 10 mM HEPES (4-hydroxyethylpiperazineethanesulfonic acid) and 0.8% sodium deoxycholate can be used as buffers. HEPES can provide a stable pH environment. The concentration of 10 mM is sufficient to provide effective buffering capacity in biological experiments, which can effectively resist pH changes without negatively affecting cells or experimental systems. It can effectively maintain a stable environment within the physiological pH range (about 7.2~7.4) to ensure the consistency of experimental conditions. Sodium deoxycholate helps to simulate the dissolution and release process of liposome drugs in the human body. The concentration of 0.8% is sufficient to simulate the effect of bile salts in the body, which can effectively help the dissolution of drugs in the dissolution medium and promote drug release. At the same time, it can better reflect the level of bile salts in the body, help simulate the behavior of drugs in the digestive tract, and ensure the relevance and accuracy of the experimental results. The amount of regulator added should be precisely controlled according to the required pH value to ensure that the pH value of the dissolution medium matches the human body environment. For example, if you want to simulate the gastric environment, the pH value may need to be adjusted to about 2; if you want to simulate the small intestine environment, the pH value may need to be adjusted to between 6.5 and 7.5.

[0022] S2: adding water to dissolve the liposome powder, and then adding the dissolution medium to dilute it to obtain a sample solution; In step S2, the liposome powder is first added to water for dissolution. This step is to ensure that the liposome drug can be fully dispersed in water to form a uniform sample solution. Alternatively, about 160 mg of liposome powder is weighed, dissolved in 4 mL of water, and then diluted to 20 mL with a dissolution medium. 4 mL of water can effectively dissolve 160 mg of liposome powder, providing enough space for the liposome to be fully dispersed without being overly diluted.

[0023] S3: placing the dissolution cup into a dissolution apparatus for heating at a preset temperature, and connecting the dialysis tube to the dissolution apparatus via an adapter; In step S3, the dissolution apparatus can be selected as an apparatus that complies with the USP1 or USP2 standards, which complies with the specifications of the United States Pharmacopeia (USP) for drug dissolution test equipment. USP1 is the Rotating Basket Apparatus, and USP2 is the Paddle Apparatus. The dialysis tube can be selected as a 300 kDa Float~A~Lyzer® dialysis tube (300 kDa floating dialysis tube), which can effectively perform dialysis within its specific molecular weight cutoff range. The adapter is an adapter designed specifically for the Float~A~Lyzer dialysis tube. The dissolution cup is placed in the water bath of the dissolution apparatus and the temperature is set to 37°C to simulate the normal body temperature of the human body. Heating is to ensure that the temperature of the dissolution medium reaches the temperature of the human body environment, so as to more accurately simulate the release process of the drug in the body. The dialysis tube is connected to the dissolution apparatus through the adapter to ensure that the drug components in the dissolution medium can pass through the membrane of the dialysis tube, while the macromolecular substances are blocked outside, so as to simulate the release process of the drug through the cell membrane in the body.

[0024] S4: adding the sample solution into the dialysis tube to mix the sample solution and the dissolution medium, taking a drug sample at a specific time point, and analyzing the drug sample to obtain the release rate of the liposome drug.

[0025] In step S4, the sample solution is added to the dialysis tube so that the sample solution is fully in contact with the dissolution medium. Under the action of the dissolution medium, the drug components of the sample solution in the dialysis tube will gradually be released and pass through the membrane of the dialysis tube. In order to accurately determine the release rate of the drug, a portion of the dissolution medium is taken out at a specific time point to analyze the drug content. Analysis techniques such as high performance liquid chromatography (HPLC) can be used to accurately determine the drug concentration. During the analysis process, multiple time points can be set to obtain a dynamic curve of drug release, and the sampling amount at each time point is kept consistent, and the sample should be analyzed after sampling to avoid changes in the drug components during sample storage. In addition, the pH value and temperature of the dissolution medium should be kept constant during the experiment to simulate the physiological environment in the human body. In this way, the release curve of liposome drugs in vitro can be obtained, and then its release behavior and efficacy performance in vivo can be evaluated.

[0026] In a specific embodiment, the instrument parameters are pre-set. Add 150 mL of dissolution medium to each of the 6 200 mL dissolution cups, transfer the dissolution cups to the water bath of the dissolution instrument, and set the water bath temperature to 37 ° C. Take out the pretreated dialysis tube, gently shake off the water in the tube, accurately add 1.5 mL of the prepared sample solution to the tube, and cover the lid tightly. Connect the dialysis tube to the lower end of the upper shaft of the dissolution instrument through the adapter, lower the shaft so that the distance between the bottom of the dialysis tube and the bottom of the dissolution cup is about 3 cm, and start the test.

[0027] In one embodiment, after the above method was used to test liposome amphotericin B in the liposome drug, the experimental data shown in Table 1 were obtained: Table 1 It can be seen from this experimental data that, through the test method of the present application, as time goes on, the in vitro release rate of the liposome drug gradually increases. The release rate is low at 2 hours, but at 44 hours, the release rate is close to or exceeds 90%, so that the liposome drug can continuously release the drug components in an in vitro environment, and the release process is relatively stable. In order to further verify the reliability of the experimental results, the present application also performs statistical analysis on the experimental data. The average value (mean) and relative standard deviation (RSD) of the release rate are calculated. The mean reflects the average release rate of the liposome drug during the in vitro release process, while the RSD reflects the consistency of the release rate between different samples. As can be seen from Table 1, as the release time increases, the RSD value gradually decreases, indicating that as time goes on, the difference in release rate between different samples gradually decreases, and the release process tends to be consistent. In another embodiment, the release behavior of the liposome drug is intuitively displayed by drawing a release curve, with time as the horizontal axis and the release rate as the vertical axis, as shown in the attached figure. Figure 2 By comparing the release curves of different samples, we can intuitively see the differences between the samples and the changing trend of the release rate, which shows a gentle upward trend, indicating that the drug ingredients can be released continuously and stably.

[0028] Further, see Attachment Figure 3 In the present application, about 15 mg of amphotericin B is weighed, ultrasonically dissolved with 1 mL of dimethyl sulfoxide, and then diluted to 50 mL with a dissolution medium to obtain a free amphotericin B sample solution, that is, amphotericin B in a free state that is not wrapped by a carrier or other substances and can be directly dissolved in a dissolution medium. The free amphotericin B sample solution is subjected to the above-mentioned step experiment, and the attached Figure 3 The comparison table of free amphotericin B and amphotericin B liposomes (a drug preparation form in which amphotericin B is encapsulated in liposomes) shows that amphotericin B in a free state can be quickly released and pass through the dialysis tube. The experimental results show that about 90% can pass through the dialysis tube within 4 hours and can pass through completely within 8 hours, indicating that the dissolution rate of free amphotericin B is faster. The release rate of amphotericin B liposomes is significantly lower than that of the free form, with less than 20% released within 4 hours and about 30% released within 8 hours, indicating that liposomes can effectively delay the release of drugs, thereby possibly improving the bioavailability and therapeutic effect of drugs, that is, after amphotericin is released from liposomes, it can be well dissolved by the dissolution medium and pass through the dialysis tube to achieve the expected therapeutic effect.

[0029] In summary, the in vitro release test method can effectively improve the release rate of liposome drugs, thereby improving their bioavailability and therapeutic effect.

[0030] In one embodiment, the step of adding water to dissolve the liposome powder and then adding the dissolution medium to dilute the sample solution to obtain the sample solution comprises: Add 150 g to 160 g of liposome powder to 3 mL to 5 mL of deionized water and shake vigorously until completely dissolved; The dissolution medium was added to the filtered liposome solution and the mixture was manually shaken to mix until the mixed solution of the liposome solution and the dissolution medium reached 20 mL.

[0031] In this example, 15 mg of liposome powder was first added to 3 mL of deionized water and shaken vigorously until completely dissolved. The dissolution medium was added to the filtered liposome solution and manually shaken to mix until the mixed solution of the liposome solution and the dissolution medium reached 20 mL.

[0032] In one embodiment, before the step of connecting the dialysis tube to the dissolution apparatus via a rotating shaft, the method further comprises: The dialysis tube is immersed in a 15% to 25% ethanol aqueous solution, the temperature of the ethanol aqueous solution is 20° C. to 30° C., and the immersion time is 50 minutes to 60 minutes; The immersed dialysis tube is cleaned with water, and the cleaned dialysis tube is placed in the dissolution medium for soaking, and the soaking time is 115 minutes to 125 minutes.

[0033] In the above embodiment, the dialysis tube is immersed in a 20% ethanol aqueous solution, the temperature of the ethanol aqueous solution is set to 25°C, and the immersion time is 60 minutes. Afterwards, the immersed dialysis tube is thoroughly washed with water to remove residual ethanol. The washed dialysis tube is then placed in a dissolution medium for a second immersion, and the immersion time is set to 120 minutes to ensure that the inner wall of the dialysis tube is fully moistened to prepare for subsequent in vitro release tests. After completing the pretreatment of the dialysis tube, the dialysis tube is connected to the dissolution instrument via a rotating shaft. The temperature of the dissolution instrument is set to 37°C to simulate the human body temperature conditions to ensure that the test results are closer to the actual situation in the body. The volume of the dissolution medium is 150mL to ensure that the dialysis tube is completely immersed in the dissolution medium. During the experiment, samples are taken and analyzed at regular intervals to monitor the release of liposomal drugs.

[0034] In one embodiment, the step of adding the sample solution into the dialysis tube to mix the sample solution and the dissolution medium comprises: The bottom of the dialysis tube and the bottom of the dissolution cup are arranged at a preset distance, wherein the preset distance is 2.5 cm to 3.5 cm; The rotating shaft speed in the dissolution instrument is set to 90 rpm to 110 rpm, the dissolution instrument is started, the dialysis tube is rotated in the dissolution cup, and the dissolution medium in the dissolution cup diffuses into the dialysis tube through the dialysis membrane in the dialysis tube to mix with the sample solution. In this way, the sample solution can be fully contacted with the dissolution medium, thereby simulating the in vivo environment and more accurately evaluating the release characteristics of the liposome drug.

[0035] In one embodiment, after completing the above steps, timed sampling is started. The sampling time points include but are not limited to: 1 hour, 2 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, and 44 hours. During each sampling, a certain volume of dissolution medium is taken out from the dissolution cup using a micropipette, and an equal volume of fresh dissolution medium is immediately added to keep the volume of the dissolution medium in the dissolution cup constant.

[0036] refer to Figure 4 The present application also includes a method for analyzing the content of liposome drugs, which is used to analyze any of the drug samples described above, comprising: S11: 0.9 mL to 1.1 mL of the sample solution is placed into 90 mL to 110 mL of the dissolution medium and mixed to obtain a test solution; S12: injecting the drug sample into a high performance liquid chromatograph for chromatographic analysis to obtain a chromatogram of the drug sample, wherein the chromatographic conditions of the high performance liquid chromatograph include but are not limited to: the mobile phase is composed of a mobile phase A and a mobile phase B, the flow rate of the mobile phase is 1.2 mL / min~1.3 mL / min, the mobile phase A is a mixture of citric acid buffer (4.2 g / L, adjusted to pH 4.7 with concentrated ammonia water) and methanol, the volume ratio is 9:1, the mobile phase B is a mixture of acetonitrile and methanol, the volume ratio is 9:1; the chromatographic column is a C18 column, the column temperature is set to 30°C; the detector is an ultraviolet detector, the detection wavelength is 383 nm; the injection volume is 10µL~20µL, and the analysis time is 10 minutes~20 minutes; S13: Compare the area of ​​the chromatogram with the peak area of ​​the assay solution to obtain the concentration of the liposome drug in the drug sample.

[0037] In this embodiment, first, 0.9 mL of the sample solution is placed in 100 mL of the dissolution medium for mixing to obtain a test solution. Then, the test solution is injected into a high performance liquid chromatograph for chromatographic analysis. In the chromatographic analysis, the mobile phase consists of mobile phase A and mobile phase B, wherein mobile phase A is a mixture of citric acid buffer (4.2 g / L, adjusted to pH 4.7 with concentrated ammonia) and methanol, with a volume ratio of 9:1, and mobile phase B is a mixture of acetonitrile and methanol, with a volume ratio of 9:1. The chromatographic column is a C18 column, the column temperature is set to 30 ° C, the detector is an ultraviolet detector, and the detection wavelength is 210 nm. The injection volume is 15 µL and the analysis time is 15 minutes. Further, the words include elution gradient parameters, which refers to the technique of changing the composition or strength of the mobile phase during the separation process to optimize the analytical separation. Specifically, referring to Table 2, under this elution gradient, the liposome drug in the drug sample can be effectively separated and detected.

[0038] Table 2 In one embodiment, reference Figure 5 The step of comparing the area of ​​the chromatogram with the peak area of ​​the assay solution to obtain the concentration of the liposome drug in the drug sample comprises: Injecting the assay solution into a high performance liquid chromatograph for chromatographic analysis to obtain a reference chromatogram of the assay solution; Calculating the peak area of ​​the reference chromatogram and the peak area of ​​the chromatogram of the drug sample to obtain a proportional relationship between the reference chromatogram and the chromatogram; The actual concentration of the liposome drug in the drug sample is obtained by calculating according to the proportional relationship through the standard curve method.

[0039] In the above embodiment, the test solution is injected into a high performance liquid chromatograph for chromatographic analysis to obtain a reference chromatogram of the test solution. After the reference chromatogram is obtained, the peak area of ​​the reference chromatogram is compared with the peak area of ​​the chromatogram of the drug sample (e.g. Figure 5 By comparing the reference chromatogram and the drug sample chromatogram, the ratio between the reference chromatogram and the drug sample chromatogram can be determined. A standard curve is drawn based on the standard chromatogram and the peak area. The curve reflects the relationship between the peak area and the drug concentration. By comparing the peak area of ​​the reference chromatogram with the standard curve, the concentration of the liposome drug in the reference solution can be calculated. Finally, the actual concentration of the liposome drug in the drug sample can be calculated based on the concentration of the reference solution and the volume ratio of the reference solution to the drug sample.

[0040] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for testing the in vitro release of liposome drugs, characterized in that: include: Mixing a buffer and a surfactant in a dissolution cup to obtain a mixed solution, and adding a regulator to the mixed solution to obtain a dissolution medium; After the liposome powder is added into water to dissolve, the dissolution medium is added for dilution to obtain a sample solution; The dissolution cup is placed in a dissolution apparatus and heated at a preset temperature, and the dialysis tube is connected to the dissolution apparatus via an adapter; The sample solution is added into the dialysis tube to mix the sample solution and the dissolution medium, and a drug sample is taken at a specific time point, and the drug sample is analyzed to obtain the release rate of the liposome drug.

2. The in vitro release test method of liposome drugs according to claim 1, characterized in that: The preset temperature is 36°C to 38°C, and the volume of the drug sample is 0.5ml to 1ml.

3. The in vitro release test method of liposome drugs according to claim 1, characterized in that: The buffer is 9mM to 10mM hydroxyethylpiperazineethanesulfonic acid, the surfactant is 0.7% to 0.8% sodium deoxycholate, and the regulator is sodium hydroxide solution to adjust the pH value of the dissolution medium to 7.4 to 7.

6.

4. The in vitro release test method of liposome drugs according to claim 1, characterized in that: The step of adding water to dissolve the liposome powder and then adding the dissolution medium to dilute the liposome powder to obtain a sample solution comprises: Add 150 g to 160 g of liposome powder to 3 mL to 5 mL of deionized water and shake vigorously until completely dissolved; The dissolution medium was added to the liposome solution, and the mixture was manually shaken to mix until the mixed solution of the liposome solution and the dissolution medium reached 20 mL.

5. The in vitro release test method of liposome drugs according to claim 1, characterized in that: Before the step of connecting the dialysis tube to the dissolution apparatus via the rotating shaft, the method further comprises: The dialysis tube is immersed in a 15% to 25% ethanol aqueous solution, the temperature of the ethanol aqueous solution is 20° C. to 30° C., and the immersion time is 50 minutes to 60 minutes; The immersed dialysis tube is cleaned with water, and the cleaned dialysis tube is placed in the dissolution medium for soaking, and the soaking time is 115 minutes to 125 minutes.

6. The in vitro release test method of liposome drugs according to claim 1, characterized in that: The step of adding the sample solution into the dialysis tube to mix the sample solution and the dissolution medium comprises: The bottom of the dialysis tube and the bottom of the dissolution cup are arranged at a preset distance, wherein the preset distance is 2.5 cm to 3.5 cm; The shaft speed in the dissolution instrument is set to 90 rpm to 110 rpm, and the dissolution instrument is started to rotate the dialysis tube in the dissolution cup, so that the dissolution medium in the dissolution cup diffuses into the dialysis tube through the dialysis membrane in the dialysis tube to mix with the sample solution.

7. The in vitro release test method of liposome drugs according to claim 1, characterized in that: The step of sampling the drug at a specific time point comprises: A certain volume of the mixture of the dissolution medium and the sample solution is taken out from the dissolution cup as a drug sample, and the specific time point includes but is not limited to: 1 hour, 2 hours, 8 hours, 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, and 44 hours.

8. The in vitro release test method of liposome drugs according to claim 1, characterized in that: The liposome drug is liposome amphotericin B.

9. A method for analyzing the content of liposome drugs, used for analyzing the drug sample according to any one of claims 1 to 8, characterized in that: include: Put 0.9 mL to 1.1 mL of the sample solution into 90 mL to 110 mL of the dissolution medium and mix them to obtain a test solution; The drug sample is injected into a high performance liquid chromatograph for chromatographic analysis to obtain a chromatogram of the drug sample, wherein the chromatographic conditions of the high performance liquid chromatograph include but are not limited to: the mobile phase is composed of a mobile phase A and a mobile phase B, the flow rate of the mobile phase is 1.2 mL / min~1.3 mL / min, the mobile phase A is a mixture of citric acid buffer and methanol, the volume ratio is 9:1, and the mobile phase B is a mixture of acetonitrile and methanol, the volume ratio is 9:1; the chromatographic column is a C18 column, and the column temperature is set to 30°C; the detector is an ultraviolet detector, and the detection wavelength is 383 nm; the injection volume is 10µL~20µL, and the analysis time is 10 minutes~20 minutes; The area of ​​the chromatogram is compared with the peak area of ​​the assay solution to obtain the concentration of the liposome drug in the drug sample.

10. The method for analyzing the content of liposome drugs according to claim 9, characterized in that: The step of comparing the area of ​​the chromatogram with the peak area of ​​the assay solution to obtain the concentration of the liposome drug in the drug sample comprises: Injecting the assay solution into a high performance liquid chromatograph for chromatographic analysis to obtain a reference chromatogram of the assay solution; Calculating the peak area of ​​the reference chromatogram and the peak area of ​​the chromatogram of the drug sample to obtain a proportional relationship between the reference chromatogram and the chromatogram; The actual concentration of the liposome drug in the drug sample is obtained by calculating according to the proportional relationship through the standard curve method.

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