A method for determining related substances in liposome adjuvants and its application

Abstract

This invention discloses a method for determining related substances in liposome adjuvants and its applications, relating to the field of pharmaceutical testing. The method is a high-performance liquid chromatography (HPLC) method. Mobile phase A comprises an aqueous solution containing trifluoroacetic acid; mobile phase B comprises a mixed solution of methanol and acetonitrile containing trifluoroacetic acid. Through a specific elution procedure, the simultaneous detection of multiple target substances in liposome adjuvants is achieved, including oleic acid, ketocholesterol, two isomers of Lyso-DOTAP, and two isomers of Lyso-DOPC. This method exhibits good specificity, high sensitivity, and good resolution, providing a new approach for evaluating the efficacy, safety, and stability of liposome adjuvants.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical testing, and more specifically, to a method for determining related substances in liposome adjuvants and its application. Background Technology

[0002] Adjuvants are substances that can enhance specific immune responses. They can be mainly divided into two types: one type is immunogenic, such as Bordetella pertussis, acid-fast bacilli (Mycobacterium tuberculosis), and Gram-negative bacilli; the other type is non-immunogenic, such as aluminum hydroxide adjuvant, calcium phosphate, mineral oil emulsions, and surfactants. The most commonly used adjuvants include Freund's adjuvant, aluminum hydroxide adjuvant, and liposomes. Liposomes are currently one of the most effective carriers for transporting various antigens into cells.

[0003] Liposomes used as vaccine adjuvant systems typically contain phospholipids, cholesterol, and immunostimulants. Liposomes are synthetically produced lipid vesicles with a single or multilayered membrane-like structure, possessing both adjuvant and carrier effects. As vaccine adjuvants, liposomes can simultaneously enhance humoral and cellular immunity. Phospholipids are an important component of liposomes and are crucial materials for forming the bilayer. Phospholipids used in vaccine adjuvant systems mainly include cationic phospholipids and neutral phospholipids. Commonly used cationic phospholipids include DOTAP (2,3-dioleoyl-trimethylammonium propane chloride) and DDA (Dioctadecyl Dimethyl Ammonium Chloride); commonly used neutral phospholipids include DOPC (Dioleoyl Phosphatidylcholine), DMPC (Dimyristoyl Phosphatidylcholine), and DMPG (Dimyristoyl Phosphatidylglycerole). Lysophospholipids (LP) are intermediates in the synthesis of phospholipids and are also their main degradation and metabolic products. They exist in small amounts in cell membranes and tissues; excessive amounts in the body can cause the rupture of red blood cells, leading to hemolysis or cell necrosis. Therefore, accurately determining the lysophospholipid content is one of the key indicators for assessing the quality stability and safety of liposomes. The main degradation and metabolites of cholesterol are ketolites, etc. Therefore, accurately determining the ketolite content is also one of the key indicators for assessing the quality stability and safety of liposomes.

[0004] ELSD detectors, as a versatile detector, have been widely used in the field of pharmaceutical testing. Numerous studies have investigated the application of ELSD in the determination of lysophosphatidylcholine in small-molecule liposomal drugs. Currently, there are few reports in the literature on analytical methods for related substances (lysophosphatidylcholine, oleic acid, ketone cholesterol, etc.) in liposomal adjuvants in biological products such as vaccines; the detection is particularly challenging when the antigen and liposomal adjuvant are in the same solution; and the ability to effectively separate lysophosphatidylcholine isomers is also required. Existing methods cannot simultaneously determine related substances of all components in liposomal adjuvants.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a method for determining related substances in liposome adjuvants and its application.

[0007] This invention is implemented as follows:

[0008] In a first aspect, embodiments of the present invention provide a method for determining a target substance in a liposome adjuvant, comprising: detecting the target substance in a test sample using high-performance liquid chromatography (HPLC); the chromatographic method comprising gradient elution using mobile phase A and mobile phase B, wherein mobile phase A comprises an aqueous solution containing trifluoroacetic acid; mobile phase B comprises a mixed solution of methanol and acetonitrile containing trifluoroacetic acid; the elution conditions of the chromatography are as follows: from 0 to 20 to 30 min, the volume percentage of mobile phase A decreases from 15% to 25% to 0 to 10%, and the volume percentage of mobile phase B increases from 75% to 85% to 90% to 100%; from 20 to 30 min to 30 to 40 min, the volume percentage of mobile phase A is maintained at 0% to 10%, and the volume percentage of mobile phase B is maintained at 90% to 100%; the target substance comprises: components of the liposome adjuvant and / or degradation products and / or metabolites of the components.

[0009] Secondly, embodiments of the present invention provide the application of a reagent combination in the preparation of a product for determining a target substance in a liposome adjuvant, the reagent combination comprising reagents for implementing the method as described in the foregoing embodiments; the target substance comprising: a component of the liposome adjuvant and / or a metabolite of the component.

[0010] Thirdly, embodiments of the present invention provide a product for determining a target substance in a liposome adjuvant, comprising: a reagent for implementing the method as described in the foregoing embodiments.

[0011] Fourthly, embodiments of the present invention provide the application of the method described in the foregoing embodiments in the quality assessment of liposome adjuvants or in the detection of the effectiveness, safety and / or stability of liposome adjuvants.

[0012] The present invention has the following beneficial effects:

[0013] This invention provides a method for simultaneously detecting target substances in liposome adjuvants, the target substances including cationic phospholipids DOTAP, neutral phospholipids DOPC, cholesterol, and their related substances. The related substances mainly include lysophospholipids (two isomers of Lyso-DOTAP and two isomers of Lyso-DOPC), oleic acid, and ketocholesterol. The method has good separation effect and provides a new approach for evaluating the effectiveness, safety, and stability of liposome adjuvants. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 The HPLC chromatogram of the control solution (Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, ketone cholesterol: 20 μg / ml) is shown below.

[0016] Figure 2 The HPLC chromatogram of test solution-1 (containing antigen protein, DOTAP, DOPC, cholesterol, etc.) is shown.

[0017] Figure 3 The HPLC chromatogram of test solution-2 (containing DOPC, cholesterol, etc.);

[0018] Figure 4 The HPLC chromatogram of test solution-3 (containing DOTAP, DOPC, cholesterol, etc.) is shown.

[0019] Figure 5 The HPLC chromatogram is for the degradation of DOTAP acid (containing two isomers of Lyso-DOTAP);

[0020] Figure 6 The HPLC chromatogram is for the acid degradation of DOPC (containing two isomers of Lyso-DOPC);

[0021] Figure 7 The HPLC chromatogram of the control solution (Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, ketone cholesterol, DOTAP, DOPC, cholesterol: 25 μg / ml) is shown below.

[0022] Figure 8The HPLC chromatogram is shown for the spiked solution of the test sample (Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, ketone cholesterol: 25 μg / ml).

[0023] Figure 9 The HPLC chromatogram of the control solution (DOTAP, DOPC, cholesterol: 61, 298, 106 μg / ml);

[0024] Figure 10 The HPLC chromatogram of the control solution (DOTAP, DOPC, cholesterol: 61, 298, 106 μg / ml);

[0025] Figure 11 The HPLC chromatogram is shown for the control solution (DOTAP, DOPC, cholesterol: 61, 298, 106 μg / ml). Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0027] To address the problems of existing technologies, this invention constructs a novel detection process for target substances in liposome adjuvants, enabling the simultaneous detection of multiple target substances such as lysophospholipids (Lyso-DOTAP, Lyso-DOPC), oleic acid, and ketocholesterol. It can effectively separate the two isomers of Lyso-DOTAP and Lyso-DOPC. This method is highly specific, simple to operate, has good separation, and high sensitivity, providing a pathway for the detection of the efficacy, stability, and safety of liposome adjuvants.

[0028] On one hand, embodiments of the present invention provide a method for determining a target substance in a liposome adjuvant, comprising: detecting the target substance in a test sample using high performance liquid chromatography;

[0029] The chromatographic method includes gradient elution using mobile phase A and mobile phase B, wherein mobile phase A comprises an aqueous solution containing trifluoroacetic acid; and mobile phase B comprises a mixed solution of methanol and acetonitrile containing trifluoroacetic acid; the elution conditions for the chromatography are as follows:

[0030] From 0 to 20 to 30 minutes, the volume percentage of mobile phase A decreases from 15% to 25% to 0% to 10%, while the volume percentage of mobile phase B increases from 75% to 85% to 90% to 100%.

[0031] From 20 to 30 minutes to 30 to 40 minutes, the volume percentage of mobile phase A is maintained at 0 to 10%, and the volume percentage of mobile phase B is maintained at 90% to 100%.

[0032] In some embodiments, the elution conditions for the chromatography are as follows:

[0033] From 0 to 24 to 26 minutes, the volume percentage of mobile phase A decreases from 18% to 22% to 0% to 5%, while the volume percentage of mobile phase B increases from 78% to 82% to 95% to 100%.

[0034] From 24 to 26 minutes to 34 to 36 minutes, the volume percentage of mobile phase A is maintained at 0 to 5%, and the volume percentage of mobile phase B is maintained at 95% to 100%.

[0035] In some embodiments, the volume fraction of trifluoroacetic acid in the mobile phase A in the aqueous solution is 0.05% to 0.15%, specifically any one or any two of 0.05%, 0.06%, 0.08%, 0.10%, 0.12%, 0.14%, and 0.15%.

[0036] In some embodiments, the volume fraction of trifluoroacetic acid in the mobile phase B in the mixed solution of methanol and acetonitrile is 0.05% to 0.15%, specifically within any one or any two of the following: 0.05%, 0.06%, 0.08%, 0.10%, 0.12%, 0.14%, and 0.15%; the mixing volume ratio of methanol and acetonitrile is 1 to 5:1 to 5, specifically within any one or any two of the following: 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 2:3, 2:5, 3:1, 3:2, 3:4, 3:5, 4:1, 4:3, 4:5, 5:1, 5:2, 5:3, and 5:4.

[0037] In some embodiments, the gradient elution flow rate is 0.5~1.5 mL / min, specifically any one or any two of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, and 1.5 mL / min.

[0038] In some embodiments, the chromatographic column used in the chromatography includes a reversed-phase column, which includes any one of C8 and C18; the specifications of the column are a column length of 50-250 mm × an inner diameter of 2.1-4.6 mm, and a packing particle size of 1.7-10 μm. Specifically, the column length can be any one or any two of 50, 100, 150, 200, and 250 mm; the inner diameter can be any one or any two of 2.1, 3.0, and 4.6 mm; and the packing particle size can be any one or any two of 1.7, 2.6, 3, 3.5, 5, and 10 μm.

[0039] In some embodiments, the chromatographic conditions of the chromatographic method include: a column temperature of 30-50°C and an injection volume of 10-50 μL. Specifically, the column temperature can be any one or any two of 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50°C; the injection volume can be any one or any two of 10, 15, 20, 25, 30, 35, 40, 45, and 50 μL.

[0040] In some embodiments, prior to the detection, the method further includes: taking a liposome adjuvant sample solution, diluting it, centrifuging it, and taking the supernatant as the sample to be tested.

[0041] In some embodiments, the liposome adjuvant sample may be a sample containing an antigen or a sample not containing an antigen.

[0042] In some embodiments, the amount of liposome adjuvant sample solution taken can be 0.2 to 2 mL, specifically any one or any two of 0.2, 0.5, 1, 1.5, and 2 mL.

[0043] In some embodiments, the diluent used for dilution includes any one or more combinations of methanol, ethanol, and acetonitrile.

[0044] In some embodiments, the dilution factor is 5 to 10 times, specifically any one or any two of 5, 6, 7, 8, 9, and 10 times.

[0045] In some embodiments, the centrifugation conditions include: 3000~15000 rpm, 3~10 min. Specifically, the rotation speed can be any one or any two of the following: 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 1500 rpm; the centrifugation time can be any one or any two of the following: 3, 4, 5, 6, 7, 8, 9, 10 min.

[0046] In some embodiments, the target substance includes: components of the liposome adjuvant and / or degradation products and / or metabolites of the components. The detection method provided in this invention is applicable to the simultaneous detection of components of the liposome adjuvant and their metabolites and / or degradation products.

[0047] In some embodiments, the components include at least one of cationic phospholipid DOTAP, neutral phospholipid DOPC, and cholesterol.

[0048] In some embodiments, the degradation products and / or metabolites of the component include at least one of lysophospholipids, oleic acid, and ketocholesterol. The lysophospholipids include: Lyso-DOTAP-1, Lyso-DOTAP-2 (an isomer of Lyso-DOTAP), Lyso-DOPC-1, and Lyso-DOOC-2 (an isomer of Lyso-DOPC).

[0049] The structural formula of Lyso-DOTAP-1 is:

[0050] ;

[0051] The structural formula of Lyso-DOTAP-2 is:

[0052] ;

[0053] The structural formula of Lyso-DOPC-1 is:

[0054] ;

[0055] The structural formula of Lyso-DOOC-2 is:

[0056] .

[0057] In some embodiments, the method further includes: obtaining a control solution, performing chromatographic detection on the control solution under the same detection conditions as described in any of the foregoing embodiments, calculating a linear regression equation using the logarithm of the concentration of the control solution and the logarithm of the corresponding peak area, and calculating the content of each target substance in the test solution using the linear regression equation.

[0058] In some embodiments, the detector used for detection includes an ELSD detector.

[0059] In some embodiments, the ELSD detector is an AllChrom 6000 or an Agilent G7102A.

[0060] In some embodiments, the AllChrom 6000 operates with a drift tube temperature of 40–80°C, a gas flow rate of 1.0–3 L / min, and the impactor position is open. Optionally, the drift tube temperature can be any one or any two of 40, 45, 50, 55, 60, 65, 70, 75, and 80°C. The gas flow rate can be any one or any two of 1, 1.2, 1.4, 1.5, 1.6, 1.8, 2, 2.5, and 3 L / min.

[0061] On the other hand, embodiments of the present invention provide the application of reagent combinations in the preparation of products for determining target substances in liposome adjuvants, the reagent combinations comprising reagents for implementing the methods described in any of the foregoing embodiments; the target substances comprising: components of the liposome adjuvant and / or metabolites of the components.

[0062] On the other hand, embodiments of the present invention also provide a product for determining a target substance in a liposome adjuvant, comprising: a reagent for implementing the method as described in any of the foregoing embodiments.

[0063] In some embodiments, the types of products include reagents and kits.

[0064] In some embodiments, the reagents for implementing the method as described in any of the foregoing embodiments include: mobile phase A and mobile phase B as described in any of the foregoing embodiments.

[0065] In some embodiments, the reagent for implementing the method as described in any of the foregoing embodiments further includes the diluent described in any of the foregoing embodiments.

[0066] In some embodiments, the reagents for implementing the method as described in any of the foregoing embodiments further include any one or more combinations of standards, quality control products, reagents for preparing standards, and reagents for preparing quality control products.

[0067] Furthermore, embodiments of the present invention also provide the application of the methods described in any of the foregoing embodiments in the quality assessment of liposome adjuvants or in the detection of the effectiveness, safety and / or stability of liposome adjuvants.

[0068] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0069] Example 1

[0070] A method for determining a target substance in a liposome adjuvant, the method being high-performance liquid chromatography, specifically including the following steps.

[0071] 1.1 Chromatographic conditions

[0072] (1) Chromatographic column: Reversed-phase C18 column (Spolar C18), with specifications of 250×4.6mm and packing particle size of 5μm; column temperature of 40℃;

[0073] (2) Mobile phase: Mobile phase A is an aqueous solution containing 0.1 v / v% trifluoroacetic acid, and mobile phase B is a mixed solution of methanol and acetonitrile containing 0.1 v / v% trifluoroacetic acid (the volume ratio of methanol to acetonitrile is 1:1).

[0074] (3) Gradient elution program: The elution time is 35 min; the specific gradient elution program is as follows:

[0075] Table 1 Elution Procedure

[0076]

[0077] (4) Detector: AllChrom 6000 ELSD detector is used, the drift tube temperature is 45℃, the gas flow rate is 1.5L / min, and the impactor position is open;

[0078] (5) The flow rate of the mobile phase is 1.0 mL / min; the injection volume is 30 μl.

[0079] 1.2 Solution Preparation

[0080] Reference stock solution: Take appropriate amounts of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, and ketone cholesterol reference standards, dissolve them in anhydrous ethanol, and dilute to prepare a solution containing 100 μg of each component per 1 ml.

[0081] Control solutions: Take an appropriate amount of the control stock solution and add 80% ethanol to prepare solutions containing 5, 10, 20, 30, and 50 μg per ml, respectively.

[0082] Test solution-1: Accurately measure 0.2 ml of liposome adjuvant (containing appropriate amounts of antigen protein, DOTAP, DOPC, cholesterol, etc.), add 0.8 ml of anhydrous ethanol to dissolve, shake well, centrifuge the solution at 5000 rpm for 5 min, and take the supernatant to obtain test solution-1.

[0083] Test solution-2: Accurately measure 0.2 ml of liposome adjuvant (containing appropriate amounts of DOPC, cholesterol, etc.), add 0.8 ml of anhydrous ethanol to dissolve, shake well, centrifuge the solution at 5000 rpm for 5 min, and take the supernatant to obtain test solution-2.

[0084] Test solution-3: Accurately measure 0.2 ml of liposome adjuvant (containing appropriate amounts of DOTAP, DOPC, cholesterol, etc.), add 0.8 ml of anhydrous ethanol to dissolve, shake well, centrifuge the solution at 5000 rpm for 5 min, and take the supernatant to obtain test solution-3.

[0085] 1.3 Determination Method

[0086] Inject 30 μL of the control solution and test solutions 1-3 respectively. The results are as follows: Figures 1-4 As shown.

[0087] Example 2 Specificity

[0088] The specificity of the method in Example 1 was examined using blank solution, control solution, test solution, and spiked test solution.

[0089] 2.1 The chromatographic conditions are the same as in Example 1.

[0090] 2.2 Solution preparation:

[0091] Blank solution: Take 0.2 ml of purified water, add 0.8 ml of anhydrous ethanol to dissolve, shake well, centrifuge the solution at 5000 rpm for 5 min, and take the supernatant to obtain the blank solution.

[0092] Reference stock solution: Take appropriate amounts of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, ketone cholesterol, DOTAP, DOPC and cholesterol reference standards, dissolve and dilute with anhydrous ethanol to prepare a solution containing 100 μg of each component per 1 ml.

[0093] Control solutions: Take an appropriate amount of the control stock solution and add 80% ethanol to prepare solutions containing 5, 15, 25, 35, and 50 μg per ml, respectively.

[0094] DOTAP acid degradation solution: Take 0.5 ml of 4 mg / ml DOTAP control stock solution, add 0.4 ml of 1N hydrochloric acid solution, then add 1.1 ml of anhydrous ethanol, mix well, and let stand at room temperature for 4 h.

[0095] DOPC acid degradation solution: Take 0.5 ml of 4 mg / ml DOPC control stock solution, add 0.4 ml of 1N hydrochloric acid solution, then add 1.1 ml of anhydrous ethanol, mix well, and let stand at room temperature for 4 h.

[0096] Test solution: Accurately measure 0.2 ml of liposome adjuvant (containing appropriate amounts of antigen protein, DOTAP, DOPC, cholesterol, etc.), add 0.8 ml of anhydrous ethanol to dissolve, shake well, centrifuge the solution at 5000 rpm for 5 min, and take the supernatant to obtain the test solution.

[0097] Spiked solution for test sample: Accurately measure 2.0 ml of liposome adjuvant (containing appropriate amounts of antigen protein, DOTAP, DOPC, cholesterol, etc.) and 2.5 ml of control stock solution, place them in the same 10 ml volumetric flask, dilute to volume with diluent, and shake well; centrifuge the solution at 5000 rpm for 5 min, and collect the supernatant.

[0098] 2.3 The results are shown in Table 2 and Figures 5-8 As shown, the other components do not interfere with the detection of the two isomers of Lyso-DOTAP, the two isomers of Lyso-DOPC, oleic acid, and ketone cholesterol, indicating that this method is highly specific.

[0099] Table 2 Specificity Results Table

[0100]

[0101] The results showed that the remaining components in the blank solution and the test solution did not interfere with the detection of the two isomers of Lyso-DOTAP, the two isomers of Lyso-DOPC, oleic acid, and ketone cholesterol, and could effectively separate the two isomers of Lyso-DOTAP and Lyso-DOPC. Figures 5-6 This indicates strong specificity. The relative retention times (RRTs) of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, ketone cholesterol, and DOPC were 0.28, 0.37, 0.62, 0.74, and 1.0, respectively. Figures 7-8 .

[0102] Example 3 Linear

[0103] The linear response of the method in Example 1 was tested with the concentration range of each target substance being 5 μg / ml to 50 μg / ml. The results showed that the logarithm of the response value (peak area) was proportional to the logarithm of the concentration of the analyte in the sample within the given range, as detailed below.

[0104] 3.1 The chromatographic conditions are the same as in Example 1.

[0105] 3.2 Solution preparation:

[0106] Reference stock solution: Take appropriate amounts of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid and ketone cholesterol reference standards, dissolve and dilute with anhydrous ethanol to prepare a solution containing 100 μg of each component per 1 ml.

[0107] Linear solutions: Take an appropriate amount of the control stock solution and add 80% ethanol to prepare solutions containing 5, 10, 20, 25, and 50 μg per ml, respectively.

[0108] 3.3 The test results are shown in Table 3:

[0109] Table 3 Linear Results

[0110]

[0111] The results showed that Lyso-DOTAP-1 exhibited good linearity in the range of 4.54 μg / ml to 45.45 μg / ml, Lyso-DOPC-1 exhibited good linearity in the range of 4.90 μg / ml to 49.00 μg / ml, oleic acid exhibited good linearity in the range of 5.32 μg / ml to 53.22 μg / ml, and ketone cholesterol exhibited good linearity in the range of 5.22 μg / ml to 52.18 μg / ml, all meeting the acceptance criteria.

[0112] Example 4 Limit of Quantitation (LOQ) 4.1 Chromatographic conditions are the same as in Example 1.

[0113] 4.2 Solution Preparation

[0114] Limit of Quantification Solution: Take a solution containing 5 μg per 1 ml of the linear solution from step 3.2 in Example 3.

[0115] 4.3 The test results are shown in Table 4:

[0116] Table 4 Results of Limit of Quantification

[0117]

[0118] The results showed that the limit of quantitation (LOQ) concentrations of Lyso-DOTAP-1 was 4.54 μg / ml, Lyso-DOPC-1 was 4.90 μg / ml, oleic acid was 5.32 μg / ml, and ketol cholesterol was 5.22 μg / ml. The LOQs of each component met the acceptance criteria of a signal-to-noise ratio ≥10.

[0119] Example 5 Accuracy

[0120] The method provided in Example 1 was used to determine the difference between the measured results and the true values ​​when measuring the target substances Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, and ketone cholesterol, thereby confirming whether the method can obtain accurate test results.

[0121] 5.1 The chromatographic conditions are the same as in Example 1.

[0122] 5.2 Solution preparation:

[0123] Reference stock solution-1: Take appropriate amounts of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, and ketone cholesterol reference standards, dissolve and dilute them with anhydrous ethanol to prepare a solution containing 25 μg of each component per 1 ml.

[0124] Reference stock solution-2: Take appropriate amounts of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, and ketone cholesterol reference standards, dissolve and dilute them with anhydrous ethanol to prepare a solution containing 100 μg of each component per 1 ml.

[0125] Reference stock solution-3: Take appropriate amounts of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, and ketone cholesterol reference standards, dissolve and dilute them with anhydrous ethanol to prepare a solution containing 200 μg of each component per 1 ml.

[0126] Test solution: Accurately measure 0.2 ml of liposome adjuvant (containing appropriate amounts of antigen protein, DOTAP, DOPC, cholesterol, etc.), add 0.8 ml of anhydrous ethanol to dissolve, shake well, centrifuge the solution at 5000 rpm for 5 min, and take the supernatant to obtain the test solution.

[0127] Accuracy Solution 1 (20%, relative to 25 μg / ml): Accurately measure 2.0 ml of liposome adjuvant (containing appropriate amounts of antigen protein, DOTAP, DOPC, cholesterol, etc.) and 2.5 ml of control stock solution-1, place them in the same 10 ml volumetric flask, dilute to volume with anhydrous ethanol, and shake well; centrifuge the solution at 5000 rpm for 5 min, and collect the supernatant; prepare 3 parallel aliquots.

[0128] Accuracy Solution 2 (100%, relative to 25 μg / ml): Accurately measure 2.0 ml of liposome adjuvant (containing appropriate amounts of antigen protein, DOTAP, DOPC, cholesterol, etc.) and 2.5 ml of control stock solution-2, place them in the same 10 ml volumetric flask, dilute to volume with anhydrous ethanol, and shake well; centrifuge the solution at 5000 rpm for 5 min, and collect the supernatant; prepare 3 parallel aliquots.

[0129] Accuracy Solution 3 (200%, relative to 25 μg / ml): Accurately measure 2.0 ml of liposome adjuvant (containing appropriate amounts of antigen protein, DOTAP, DOPC, cholesterol, etc.) and 2.5 ml of control stock solution-3, place them in the same 10 ml volumetric flask, dilute to volume with anhydrous ethanol, and shake well; centrifuge the solution at 5000 rpm for 5 min, and collect the supernatant; prepare 3 parallel aliquots.

[0130] 5.3 The test results are shown in Figure 5.

[0131] Table 5. Accuracy Results for Each Target Substance

[0132]

[0133] The results showed that the accuracy of each target substance at the three levels met the acceptance criteria of a recovery rate between 70.0% and 130.0% and an RSD ≤ 15.0%.

[0134] Example 6: Precision

[0135] Precision refers to the degree of similarity between results obtained from multiple measurements of the same homogeneous sample under specified test conditions. The degree of similarity between results obtained by the same analyst under identical conditions is called repeatability. This involves parallel determination of three spiked solutions of the same concentration of sample, and calculation of the mean recovery rate and RSD.

[0136] 6.1 The chromatographic conditions are the same as in Example 1.

[0137] 6.2 Solution Preparation

[0138] Reference stock solution: Take appropriate amounts of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid, and ketone cholesterol reference standards, dissolve and dilute with anhydrous ethanol to prepare a solution containing 100 μg of each component per 1 ml.

[0139] Spiked solution for test sample: Accurately measure 2.0 ml of liposome adjuvant (containing appropriate amounts of antigen protein, DOTAP, DOPC, cholesterol, etc.) and 2.5 ml of control stock solution, place them in the same 10 ml volumetric flask, dilute to volume with diluent, and shake well; centrifuge the solution at 5000 rpm for 5 min, and collect the supernatant; prepare 3 parallel samples.

[0140] 6.3 The test results are shown in Table 6:

[0141] Table 6 Repeatability Results

[0142]

[0143] The results showed that the RSDs of the recoveries of Lyso-DOTAP-1, Lyso-DOPC-1, oleic acid and ketone cholesterol in the three spiked solutions were 1.8%, 1.1%, 1.3% and 1.4%, respectively, and the repeatability met the acceptance criterion of RSD ≤ 5.0%.

[0144] Example 7 Verification of different mobile phases

[0145] 7.1 Chromatographic conditions

[0146] The chromatographic conditions were largely the same as in Example 1, except that the mobile phase B was a methanol solution containing 0.1 v / v% trifluoroacetic acid.

[0147] 7.2 Solution Preparation

[0148] Control solution: Take appropriate amounts of DOTAP, DOPC and cholesterol control standards, dissolve and dilute with anhydrous ethanol to prepare a solution containing 61 μg of DOTAP, 298 μg of DOPC and 106 μg of cholesterol per 1 mL.

[0149] 7.3 Determination Method

[0150] Inject 30 μL of the reference solution, and the results are as follows: Figure 9 As shown.

[0151] The results showed that when methanol was used as the organic phase, the elution times of DOPC and cholesterol were reversed, and the elution times of cholesterol and DOPC were relatively close, resulting in poor separation.

[0152] Example 8 Different elution gradients 1

[0153] 8.1 Chromatographic conditions

[0154] The chromatographic conditions were largely the same as in Example 1, except for the gradient elution program, as detailed below.

[0155] Table 7 Elution Procedure

[0156]

[0157] 8.2 Solution Preparation

[0158] Control solution: Take appropriate amounts of DOTAP, DOPC, and cholesterol control standards, dissolve and dilute with 80% ethanol to prepare a solution containing 61 μg of DOTAP, 298 μg of DOPC, and 106 μg of cholesterol per 1 ml.

[0159] 8.3 Determination Method

[0160] Inject 30 μL of the reference solution, and the results are as follows: Figure 10 As shown.

[0161] The results showed that after adjusting the elution gradient, DOPC and cholesterol were co-eluted, but effective separation was not achieved.

[0162] Example 9 Different elution gradients 2

[0163] 8.1 Chromatographic conditions

[0164] The chromatographic conditions were largely the same as in Example 1, except for the gradient elution program, as detailed below.

[0165] Table 8 Elution Procedure

[0166]

[0167] 9.2 Solution Preparation

[0168] Control solution: Take appropriate amounts of DOTAP, DOPC, and cholesterol control standards, dissolve and dilute with 80% ethanol to prepare a solution containing 61 μg of DOTAP, 298 μg of DOPC, and 106 μg of cholesterol per 1 ml.

[0169] 9.3 Determination Method

[0170] Inject 30 μL of the reference solution, and the results are as follows: Figure 11 As shown.

[0171] The results showed that after adjusting the elution gradient, the elution times of DOPC and cholesterol were relatively close, and baseline separation was not achieved, resulting in poor separation performance.

[0172] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for determining a target substance in a liposome adjuvant, characterized in that, It includes: The target substance in the test sample was detected by high performance liquid chromatography. The chromatographic method includes gradient elution using mobile phase A and mobile phase B, wherein mobile phase A comprises an aqueous solution containing trifluoroacetic acid; and mobile phase B comprises a mixed solution of methanol and acetonitrile containing trifluoroacetic acid; the gradient elution conditions are as follows: From 0 to 25 minutes, the volume percentage of mobile phase A decreases from 15% to 25% to 0% to 10%, while the volume percentage of mobile phase B increases from 75% to 85% to 90% to 100%. From 25 min to 30-40 min, the volume percentage of mobile phase A is maintained at 0-10%, and the volume percentage of mobile phase B is maintained at 90%-100%. The target substance includes: components of the liposome adjuvant and / or degradation products and / or metabolites of the components; The volume fraction of trifluoroacetic acid in the mobile phase A in the aqueous solution is 0.05%~0.15%; The volume fraction of trifluoroacetic acid in the mobile phase B in the mixed solution of methanol and acetonitrile is 0.05%~0.15%, and the volume ratio of methanol to acetonitrile is (1~5):(1~5). The components include at least one of cationic phospholipid DOTAP, neutral phospholipid DOPC, and cholesterol; The degradation products and / or metabolites of the component include at least one of lysophospholipids, oleic acid and ketocholesterol; The lysophospholipids include: Lyso-DOTAP-1, Lyso-DOTAP-2 (an isomer of Lyso-DOTAP-1), Lyso-DOPC-1, and Lyso-DOOC-2 (an isomer of Lyso-DOPC-1). The structural formula of Lyso-DOTAP-1 is: ； The structural formula of Lyso-DOTAP-2 is: ； The structural formula of Lyso-DOPC-1 is: ； The structural formula of Lyso-DOOC-2 is: 。 2. The method according to claim 1, characterized in that, The gradient elution flow rate is 0.5~1.5 mL / min.

3. The method according to claim 1, characterized in that, The chromatographic column used in the chromatographic method includes a reversed-phase column, which includes either C8 or C18; the specifications of the chromatographic column are a column length of 50~250mm × an inner diameter of 2.1~4.6mm, and a packing particle size of 1.7~10μm; The chromatographic conditions of the method include: column temperature of 30~50℃ and injection volume of 10~50μL.

4. The method according to claim 1, characterized in that, Before the detection, the method further includes: taking a liposome adjuvant sample solution, diluting it, centrifuging it, and taking the supernatant as the sample to be tested; The diluent used for dilution includes any one or more combinations of methanol, ethanol, and acetonitrile; The centrifugation conditions include: 3000~15000 rpm, 3~10 min.

5. The method according to any one of claims 1 to 4, characterized in that, The detectors used for the detection include ELSD detectors.

6. The application of the method according to any one of claims 1 to 5 in the quality assessment of liposome adjuvants or in the detection of the efficacy, safety and / or stability of liposome adjuvants.

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