A method for detecting MPL in a sample and its related applications

Abstract

The present invention discloses a method for detecting MPL in a sample and its related applications, relating to the field of biological detection. The adsorbent includes aluminum phosphate or a product obtained by mixing aluminum hydroxide and a phosphate solution, which can effectively adsorb free MPL in the sample, thereby achieving effective separation of free MPL and MPL bound to liposomes in the sample. By detecting the difference in MPL content in the sample before and after adsorption, the proportion of free MPL and MPL bound to liposomes in the sample can be determined. This method has the advantages of being easy to implement, low cost, and high efficiency. It does not require expensive reagents and instruments, and can quickly and accurately calculate the encapsulation rate of MPL in liposomes, providing a new approach for quality control of adjuvants or vaccines containing MPL.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a method for detecting MPL in a sample and related applications thereof. Background Art

[0002] MPLA, also known as MPL, is a phospholipid A in Chinese. It is primarily extracted from the innermost lipid A of lipopolysaccharide (LPS) in the cell wall of Gram-negative bacteria. It is an amphiphilic structure that is key to the toxicity and immunogenicity of Gram-negative bacteria, capable of eliciting an immune response.

[0003] MPL targets Toll-like receptor 4 (TLR4) and possesses advantages such as strong immunogenicity, a well-defined mechanism, and low toxicity. It activates macrophages, particularly dendritic cells (DCs), promoting the differentiation of antigen-specific CD4+ T cells and the production of IFN-γ, thereby inducing a Th1 immune response. MPL is widely used as an adjuvant in vaccines, allergy medications, and immunotherapy to enhance immune responses. It has been incorporated into several vaccine products, including the cervical cancer vaccine Cervarix® and the hepatitis B vaccine Fendrix®.

[0004] Because MPL alone has significant toxic side effects, it is often combined with other ingredients to form adjuvant systems, such as AS01 and AS02. In adjuvant systems, MPL is often tightly bound to lipids, thereby reducing its toxic side effects while retaining its adjuvant effect.

[0005] Currently, MPL detection methods can only detect the total amount of MPL in a sample and cannot distinguish between free MPL and MPL bound to liposomes. To ensure batch-to-batch consistency of MPL bound to liposomes and improve vaccine safety, a new detection method that can distinguish between free and liposome-bound MPL is urgently needed.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The object of the present invention is to provide a method for detecting MPL in a sample and related applications thereof.

[0008] The present invention is achieved in that:

[0009] In a first aspect, an embodiment of the present invention provides a method for detecting MPL in a sample, comprising the following steps: mixing the sample with an adsorbent, taking the portion not bound to the adsorbent, and detecting the MPL content as the MPL content bound to liposomes; wherein the adsorbent comprises adsorbent A and / or adsorbent B; the adsorbent A comprises aluminum phosphate; and the preparation method of the adsorbent B comprises: mixing aluminum hydroxide and a phosphate solution.

[0010] In a second aspect, embodiments of the present invention provide the use of the methods described in the preceding embodiments or the reagents used to implement the methods described in the preceding embodiments in distinguishing free MPL from liposome-bound MPL or in quality control of products containing free MPL and / or liposome-bound MPL.

[0011] The present invention has the following beneficial effects:

[0012] The present invention uses aluminum phosphate, or a product obtained by mixing aluminum hydroxide and a phosphate solution, as a new adsorbent to effectively adsorb free MPL in a sample, thereby achieving efficient separation of free MPL from liposome-bound MPL. By measuring the difference in MPL content in the sample before and after adsorption, the proportion of free MPL to liposome-bound MPL in the sample can be determined. This method is easy to implement, low-cost, and highly efficient. It does not require expensive reagents and instrumentation, and can quickly and accurately calculate the MPL encapsulation efficiency in liposomes, providing a new approach for quality control of MPL-containing products (such as adjuvants or vaccines). DETAILED DESCRIPTION

[0013] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0014] In one aspect, an embodiment of the present invention provides a method for detecting MPL in a sample, comprising the following steps:

[0015] The sample is mixed with an adsorbent, and the portion not bound to the adsorbent is taken for testing the MPL content, which is used as the MPL content bound to the liposome;

[0016] Wherein, the adsorbent includes adsorbent A and / or adsorbent B; the adsorbent A includes aluminum phosphate; and the preparation method of the adsorbent B includes: mixing aluminum hydroxide and phosphate solution.

[0017] In some embodiments, the phosphate solution contains phosphate ions, and the phosphate ions include (dihydrogen phosphate ion), (hydrogen phosphate ion) and (phosphate ion) any one or more.

[0018] In some embodiments, in the adsorbent B, the molar ratio of the aluminum ions to the phosphate ions is 37:22-50.

[0019] The inventors discovered that when using an adsorbent for adsorption treatment, if the phosphate ion concentration is too low, both liposome-bound MPL and free MPL will be adsorbed. If the phosphate ion concentration is too high, neither liposome-bound MPL nor free MPL will be significantly adsorbed. By adjusting the molar ratio of aluminum ions to phosphate ions, the adsorbent's adsorption of free MPL can be significantly enhanced, while adsorbing liposome-bound MPL.

[0020] The 37:22-50 ratio may specifically be any one of 37:22, 37:24, 37:26, 37:28, 37:30, 37:32, 37:34, 37:36, 37:38, 37:40, 37:42, 37:44, 37:46, 37:48 and 37:50, or a range between any two of them.

[0021] In some embodiments, in the adsorbent B, the concentration of the aluminum ions is in the range of any one or any two of 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 and 10 mg / mL.

[0022] In some embodiments, the concentration of phosphate ions in the adsorbent B is 2.2, 5, 10, 15, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 14 450, 460, 470, 480, 490, and 500 mM.

[0023] In some embodiments, the phosphate solution includes any one or more of PB, PBS, PBST, KPBS, and TPBS.

[0024] Specifically, the main components of PBS (Phosphate-Buffered Saline) are: The main components of PB (Phosphate Buffer): PBST (PBS + Tween-20), PBS with Tween-20 (non-ionic surfactant). KPBS (Potassium PBS, potassium salt PBS), Alternative (such as KCl, ) TPBS (Tris-PBS, Tris-buffered PBS), Tris-HCl is added to PBS to enhance the buffering capacity.

[0025] In some embodiments, the pH of the phosphate solution is 5-8, specifically any one of 5, 5.2, 5.4, 5.6, 5.8, 6, 6.2, 6.4, 6.6, 6.8, 7, 7.2, 7.4, 7.6, 7.8 and 8, or a range between any two of them.

[0026] In some embodiments, the mixed system of aluminum hydroxide and phosphate solution contains sodium chloride, and the mass volume fraction of the sodium chloride in the mixed system is 0.8% to 5% (w / v). The presence of sodium chloride in the mixed system of aluminum hydroxide and phosphate solution includes two situations: the phosphate solution used is a phosphate solution containing sodium chloride, such as PBS; or the phosphate solution used is a phosphate solution that does not contain sodium chloride. In this case, sodium chloride is added to the phosphate solution or system that does not contain sodium chloride (such as PB + NaCl) to bring the effective concentration of sodium chloride in the system to the above-mentioned set range. The effective concentration of sodium chloride in the system can specifically be any one of 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4%, 4.2%, 4.4%, 4.6%, 4.8%, and 5%, or a range between any two of them.

[0027] In some embodiments, the volume ratio of the sample to the adsorbent is 1-3:1-3, specifically any one of 1:1, 1:2, 1:3, 2:1, 2:3, 3:1 and 3:2, or a range between any two of them.

[0028] In some embodiments, the mass volume fraction of sodium chloride in the mixed system of the adsorbent and the sample is 0.4% to 2.5% (w / v), specifically any one of 0.4%, 0.6%, 0.8%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4% and 2.5%, or a range between any two of them.

[0029] In some embodiments, the method further comprises: detecting the MPL content of the sample not treated with the adsorbent as the total amount of MPL in the sample; the proportion of free MPL in the sample = (total amount of MPL - content of MPL bound to liposomes) / total amount of MPL × 100%.

[0030] In some embodiments, the method for detecting the MPL content includes any one or more of the following: high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS / MS), and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS).

[0031] In some embodiments, the sample comprises a solution containing MPL; the solution containing MPL comprises an adjuvant or a vaccine.

[0032] In some embodiments, the MPL includes any one or more of the native form of MPL, 2D-MPL (di-deacylated MPL), 3D-MPL (3-O-deacylated MPL), and 6D-MPL (hexa-deacylated MPL).

[0033] The native form of MPL is derived from the natural hydrolysis product of lipopolysaccharide (LPS) from Gram-negative bacteria (e.g., Salmonella), retaining a single phosphate group and intact lipid chains. 2D-MPL: A derivative of native MPL that has been partially deacylated to remove two acyl chains (typically branched). 3D-MPL: A derivative of native MPL that has been deacylated at the 3-position (selectively removing the acyl chain at the 3-O position). 6D-MPL: A derivative of native MPL that has been fully deacylated to remove all six acyl chains.

[0034] In some embodiments, the liposomes include any one or more of neutral liposomes and cationic liposomes.

[0035] Specifically, neutral liposomes are typically composed of neutral (uncharged) lipids (e.g., phosphatidylcholine (PC), cholesterol) and have a net surface charge of zero, while positive liposomes are typically composed of cationic lipids (e.g., DOTAP, DDAB) and / or cholesterol and have a positive surface charge.

[0036] In addition, embodiments of the present invention provide the use of the methods described in the preceding embodiments or the reagents used to implement the methods described in the preceding embodiments in distinguishing free MPL from liposome-bound MPL or in quality control of products containing free MPL and / or liposome-bound MPL.

[0037] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0038] Example 1

[0039] This example provides a method for detecting the content of MPL (applicable to different forms of MPL, such as native form and 3D-MPL) as follows:

[0040] (1) Experimental instruments and reagents

[0041] HPLC: Agilent 1260 Infinity II, equipment number: ME-D-070 (J);

[0042] Chromatographic column: Shim-pack GIS 5μm C18 4.6×150mm (HSS) No.: C18-A-23-018;

[0043] Mobile phase A: 95% methanol-triethylamine-glacial acetic acid (volume ratio 1000:1:1);

[0044] Mobile phase B: isopropanol-triethylamine-glacial acetic acid (volume ratio 1000:1:1);

[0045] MPL (purity: 93.17%);

[0046] Solvent: isopropanol-tetrahydrofuran (volume ratio 1:2).

[0047] (2) Chromatographic conditions

[0048] Mobile phase A: 95% methanol-triethylamine-glacial acetic acid (volume ratio 1000:1:1);

[0049] Mobile phase B: isopropanol-triethylamine-glacial acetic acid (volume ratio 1000:1:1);

[0050] Flow rate: 1 ml / min; detector: ELSD (drift tube temperature: 45°C, carrier gas: 1.5 L / min); column temperature: 45°C; injection volume: 100 μl; gradient elution; time: 35 min;

[0051] Use mobile phase A and mobile phase B to perform gradient elution according to the elution gradient shown in Table 1.

[0052] Table 1 Elution gradient

[0053]

[0054] (3) Preparation of reference solution

[0055] Reference substance mother solution: Weigh 5.367 mg of MPL solid powder (purity: 93.17%) and dissolve it in a 25 ml volumetric flask with solvent, and dilute to the mark. The concentration of the reference substance mother solution is 200 μg / ml.

[0056] Standard curve: Take 50μl, 100μl, 150μl, 200μl, and 250μl of the mother solution respectively into 5 1.5ml EP tubes, and add solvent to make the volume to 1ml. In this way, standard curves of 10μg / ml, 20μg / ml, 30μg / ml, 40μg / ml, and 50μg / ml were prepared.

[0057] Table 2 Standard curve

[0058]

[0059] Table 3 Injection sequence

[0060]

[0061] (4) Preparation of test solution

[0062] Take 200μl of the sample solution at room temperature and add 800μl of solvent (use a pipette to take the solvent), dilute the sample 5-fold, and inject it as the test solution. The sample must be diluted at least 4-fold to be completely dissolved.

[0063] Example 2

[0064] This embodiment provides a method for detecting MPL in a sample, which includes the following steps.

[0065] 1. Before adsorption

[0066] Before adsorption with an adsorbent, 2.5 mL of the sample not treated with an adsorbent was taken and added to 2.5 mL of ultrapure water to test the MPL content (see Example 1 for the detection method). The obtained result was used as the total amount of MPL in the sample to be tested.

[0067] 2. Adsorption

[0068] 2.5 mL of the sample to be tested was mixed evenly with 2.5 mL of the adsorbent, and adsorbed at room temperature for 15 minutes. After centrifugation at 6000 rpm for 5 minutes, the supernatant was collected for MPL content detection (see Example 1 for the detection method). The obtained result was used as the MPL content bound to the liposomes.

[0069] The adsorbent is prepared by taking an aluminum hydroxide solution, adding PB (monobasic sodium phosphate and disodium hydrogen phosphate, pH 6.0) to the aluminum hydroxide solution at a molar ratio of aluminum ions to phosphate ions of 1.48:1, and then adding a NaCl solution. Mixing for more than 30 minutes, the mixture is then diluted with water to a final concentration of 2 mg / ml of Al ions, 50 mM of PB, and 1.8% (w / v) of NaCl. It should be noted that the final concentration of Al ions in the adsorbent-sample mixture is 1 mg / ml, the concentration of PB in the mixture is 25 mM, and the concentration of NaCl in the mixture is 0.9% (w / v).

[0070] 3. Calculation

[0071] The proportion of free MPL in the sample was calculated based on the following formula:

[0072] The proportion of free MPL in the sample = (total MPL amount - content of MPL bound to liposomes) / total MPL amount × 100%.

[0073] Example 3

[0074] The method of Example 2 was used to detect native MDL (samples 1-2) and 3D-MDL (samples 3-4), and the results are as follows.

[0075] Table 4 Results of detection of MPL content and encapsulation efficiency

[0076]

[0077] Note: Encapsulation efficiency = MPL concentration after adsorption by adsorbent / MPL concentration after mixing with ultrapure water * 100%.

[0078] Example 4

[0079] The adsorption effects of adsorbents containing different molar ratios of aluminum ions and phosphate ions on free MPL and MPL bound to liposomes were verified.

[0080] This example sets up multiple experimental groups and control groups. The MPL concentration detection method refers to Example 1, which is as follows:

[0081] Control 1: The MPL concentration of 0.1 mg / ml free MPL solution was directly detected; Samples 1-1 to 1-11: Based on the method of Example 2, adsorbents with different molar ratios of aluminum ions and phosphate ions were set, and 0.1 mg / ml free MPL solutions were detected respectively; Sample 1-12: The adsorbent in Example 2 was replaced by aluminum phosphate, and MPL detection was performed on 0.1 mg / ml free MPL solution; Control 2: The MPL concentration of 0.1 mg / ml solution of MPL combined with neutral liposomes (composed of cholesterol and DOPC) (neutral-MPL) was directly detected; Samples 2-1 to 2-11: Based on the method of Example 2, adsorbents with different molar ratios of aluminum ions and phosphate ions were set, and 0.1 mg / ml neutral-MPL solution was detected respectively; Sample 2-12: The adsorbent in Example 2 was replaced by aluminum phosphate, and MPL detection was performed on 0.1 mg / ml neutral-MPL solution.

[0082] The results are as follows.

[0083] Table 5 Comparison of adsorbents with different molar ratios of aluminum ions and phosphate ions

[0084]

[0085] Note: The concentrations of Al, Al in aluminum phosphate, PB, and NaCl recorded in the "Preparation" column are their effective concentrations in the mixture of sample and adsorbent; the concentrations of MPL and neutral-MPL recorded in the table are the concentrations of MPL and neutral-MPL in the sample; pH is the pH of the mixture of sample and adsorbent.

[0086] Samples 1-1 to 1-11 show that when the aluminum ion concentration in aluminum hydroxide is 1 mg / ml and the phosphate ion concentration is 60 mM, the adsorbent cannot completely adsorb free MPL. However, when the phosphate ion concentration is ≤50 mM, the adsorbent can completely adsorb free MPL. Samples 2-1 to 2-11 show that when the aluminum ion concentration in aluminum hydroxide is 1 mg / ml and the phosphate ion concentration is 5-15 mM, the adsorbent will adsorb liposome-bound MPL. However, when the phosphate ion concentration is ≥22 mM, the adsorbent no longer adsorbs liposome-bound MPL. Therefore, when the aluminum ion concentration in aluminum hydroxide is 1 mg / ml and the phosphate ion concentration is 22-50 mM, that is, when the molar ratio of aluminum ion to phosphate ion is 37:22-50, the adsorbent only adsorbs free MPL and does not adsorb liposome-bound MPL, thus enabling the separation of free MPL from liposome-bound MPL. It can be seen from samples 1-12 and 2-12 that aluminum phosphate can completely adsorb free MPL but does not adsorb MPL bound to liposomes.

[0087] Example 5

[0088] Verify the effects of different adsorbents on MPL detection.

[0089] Adsorbents containing aluminum ions and phosphate ions in different molar ratios (based on the adsorbent of Example 2, with a molar ratio of aluminum:phosphate ions of 37:22 to 37:50) and aluminum phosphate were used as adsorbents, and MPL was detected based on the method of Example 2. Sample addition and recovery were verified to determine the accuracy and applicability of the method, as follows.

[0090] Control 3: Direct MPL concentration detection of 100 μg / ml neutral-MPL solution;

[0091] Samples 3-1 to 3-7: 100 μg / ml neutral-MPL solution (or additional MPL solutions with final concentrations of 10 μg, 50 μg, and 100 μg) were prepared based on the method of Example 2, wherein the concentration of Al ions (Al in aluminum hydroxide) in the mixture was set to 1 mg / mL, and the concentration of phosphate ions in the mixture was set to 22 mM;

[0092] Samples 3-8 to 3-14: 100 μg / ml neutral-MPL solution (or additional MPL solutions with final concentrations of 10 μg, 50 μg, and 100 μg) were prepared based on the method of Example 2, wherein the concentration of Al ions (Al in aluminum hydroxide) in the mixture was set to 1 mg / mL, and the concentration of phosphate ions in the mixture was set to 50 mM;

[0093] Samples 3-15 to 3-21: Based on the method of Example 2, a solution of 100 μg / ml neutral-MPL (or additional free MPL or neutral / MPL, the additional added concentrations are 10 μg / mL, 50 μg / mL and 100 μg / mL), wherein the adsorbent is replaced with aluminum phosphate, and the effective concentration of aluminum ions of aluminum phosphate in the mixture is 1 mg / mL.

[0094] Table 6 Sample recovery

[0095]

[0096] Note: The concentrations of Al, Al in aluminum phosphate, PB, and NaCl recorded in the "Preparation" column are their respective effective concentrations in the mixture of sample and adsorbent, and the pH is the pH of the mixture of sample and adsorbent.

[0097] The results show that the recoveries of samples with different concentrations of MPL added (samples 3-2 to 3-4, samples 3-9 to 3-11, and samples 3-16 to 3-18) were all close to 0%, indicating that aluminum phosphate or adsorbents obtained by mixing aluminum hydroxide and phosphate solution (aluminum:phosphate ion molar ratio = 37:22 to 37:50) can effectively adsorb free MPL;

[0098] The recoveries of samples spiked with different concentrations of "neutral-MPL" (samples 3-5 to 3-7, samples 3-12 to 3-14, and samples 3-19 to 3-21) were close to 100%, indicating that aluminum phosphate or the adsorbent obtained by mixing aluminum hydroxide and phosphate solution (aluminum:phosphate ion molar ratio = 37:22 to 37:50) does not adsorb "neutral-MPL". This also proves that the quality control measures of this analytical method are effective and the analytical data are reliable.

[0099] Example 6

[0100] Effect of different sodium chloride concentrations on detection

[0101] The adsorption effects of adsorbents containing different sodium chloride concentrations on free MPL and MPL bound to liposomes were verified.

[0102] This example sets up multiple experimental groups and control groups. The MPL concentration detection method refers to Example 1, which is as follows:

[0103] Control 4: The MPL concentration of a 0.15 mg / ml free MPL solution was directly detected; Samples 4-1 to 4-5: Based on the method of Example 2, adsorbents with different sodium chloride concentrations were set, and 0.15 mg / ml free MPL solutions were detected respectively; Control 5: The MPL concentration of a 0.15 mg / ml solution of MPL bound to neutral liposomes (neutral-MPL) was directly detected; Samples 5-1 to 5-5: Based on the method of Example 2, adsorbents with different sodium chloride concentrations were set, and 0.15 mg / ml neutral-MPL solutions were detected respectively.

[0104] Table 7 Test results

[0105]

[0106] Note: The concentrations of Al, PB, and NaCl recorded in the "Preparation" column are their respective functional concentrations in the mixture of sample and adsorbent, and the pH is the pH of the mixture of sample and adsorbent.

[0107] Samples 4-1 to 4-5 show that when the aluminum ion concentration in aluminum hydroxide is maintained at 1 mg / ml and the phosphate ion concentration is maintained at 25 mM, the adsorbent cannot completely adsorb free MPL when the sodium chloride concentration is 0%. However, when the sodium chloride concentration is ≥ 0.4%, the adsorbent can completely adsorb free MPL. Samples 5-1 to 5-5 show that when the aluminum ion concentration in aluminum hydroxide is maintained at 1 mg / ml and the phosphate ion concentration is maintained at 25 mM, the adsorbent does not adsorb liposome-bound MPL when the sodium chloride concentration is between 0% and 2.5%. Therefore, when the sodium chloride concentration is between 0.4% and 2.5%, the adsorbent can effectively adsorb free MPL but does not adsorb liposome-bound MPL.

[0108] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for detecting MPL in a sample, characterized in that: It includes the following steps: The sample is mixed with an adsorbent, and the portion not bound to the adsorbent is taken for testing the MPL content, which is used as the MPL content bound to the liposome; Wherein, the adsorbent includes adsorbent A and / or adsorbent B; the adsorbent A includes aluminum phosphate; the preparation method of the adsorbent B includes: mixing aluminum hydroxide and phosphate solution; The phosphate solution contains phosphate ions, and the phosphate ions include 、 and Any one or more of; in the adsorbent B, the molar ratio of aluminum ions to phosphate ions is 37:22~50; The mixed system of aluminum hydroxide and phosphate solution contains sodium chloride, and the concentration of the sodium chloride is 0.4% to 2.5%, which is the concentration of sodium chloride in the mixture of the sample and the adsorbent; The effective concentration of aluminum ions in adsorbent A in the mixture of sample and adsorbent is 1 mg / mL.

2. The method according to claim 1, characterized in that In the adsorbent B, the concentration of the aluminum ions is 0.1-10 mg / mL, and the concentration of the phosphate ions is 2.2-500 mM.

3. The method according to claim 1, characterized in that The phosphate solution includes: any one or more of PB, PBS, PBST, KPBS and TPBS; The pH of the phosphate solution is 5-8.

4. The method according to any one of claims 1 to 3, characterized in that The method further comprises: detecting the MPL content of the sample not treated with the adsorbent as the total amount of MPL in the sample; The proportion of free MPL in the sample = (total MPL amount - content of MPL bound to liposomes) / total MPL amount × 100%.

5. The method according to any one of claims 1 to 3, characterized in that The method for detecting the MPL content includes any one or more of the following: high performance liquid chromatography detection, liquid chromatography-mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

6. The method according to any one of claims 1 to 3, characterized in that The sample includes a solution containing MPL; the solution containing MPL includes an adjuvant or a vaccine.

7. The method according to any one of claims 1 to 3, characterized in that The MPL includes any one or more of the natural form of MPL, 2D-MPL, 3D-MPL and 6D-MPL; The liposomes include any one or more of neutral liposomes and cationic liposomes.

8. Use of the method according to any one of claims 1 to 7 in distinguishing free MPL from liposome-bound MPL or in quality control of a product containing free MPL and / or liposome-bound MPL.

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