A method for detecting lipid components in lipid nanospheres

Abstract

The present invention provides a method for detecting lipid components in lipid nanospheres, which adopts a high performance liquid chromatography-electrospray ionization detector method for detection, comprising: (1) detecting a standard solution of the lipid components, performing linear regression calculation on the peak area in the chromatogram using the concentration of the standard solution, and constructing a standard curve; (2) detecting the lipid nanosphere solution, identifying the lipid components in the lipid nanospheres according to the retention time in the chromatogram, and calculating the concentration of the lipid components according to the peak area of ​​the lipid components in the chromatogram and the above-mentioned standard curve.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug analysis and relates to a quality control method for lipid nanospheres encapsulating mRNA. Background Art

[0002] In recent years, mRNA therapeutics have garnered increasing attention. Currently under development, mRNA therapeutics are used to treat diseases such as bacterial and viral infections and tumors. Compared to traditional vaccines, mRNA offers significant safety advantages. mRNA does not insert into gene sequences and cause mutations, can be degraded by normal cells, and its half-life can be altered through sequence modification and delivery vectors. More importantly, by regulating RNA sequences, mRNA can trigger the body's own production of targeted antibody proteins, making it particularly suitable for treating emerging diseases such as the novel coronavirus.

[0003] However, due to the large molecular weight and dense negative charge of mRNA, naked mRNA molecules cannot directly enter human cells to achieve protein expression. After in vivo administration, the transfection process for mRNA to cross the cell membrane barrier requires the assistance of a delivery vector. At the same time, an excellent delivery vector should also be able to protect the mRNA through physical packaging, enhance mRNA stability, and increase the overall expression efficiency of the mRNA by assisting in various steps of transfection.

[0004] After years of practical validation, lipid nanoparticles (LNPs) are currently recognized as the most mature and efficient mRNA delivery vectors. These nanoparticles are solid spherical nanoparticles. Typically, a negatively charged mRNA molecule is surrounded by a positively charged phospholipid (Cationic lipid) and compressed at the center of the LNP. The outer layer contains PEGylated lipids, which form a stable colloidal formulation in aqueous solution. This structure not only effectively prevents rapid mRNA degradation in vivo through physical encapsulation but also facilitates endocytosis by fusing with cell membranes. Subsequently, the cationic lipids in the LNPs can disrupt endosomes through various pathways, allowing the antigen-encoding mRNA to escape into the cytoplasm.

[0005] Due to the novelty of lipid nanosphere technology, current pharmacopoeias and published literature do not yet include methods for qualitative and quantitative detection of lipid nanospheres. The difficulty in detecting lipid nanospheres lies in the fact that the various lipids that comprise them lack UV absorption and their similar polarity makes separation difficult. Currently, for substances that do not absorb UV light, general-purpose detectors are used, including differential refractive index (RI), mass spectrometry (MS), electrospray ionization (CAD), and evaporative light scattering (ELSD). RI has low sensitivity for most substances (approximately 10–5 g / ml) and is generally not used for trace analysis. RI is also significantly affected by fluctuations in ambient temperature and mobile phase composition, making gradient elution impossible and generally reserved for carbohydrate analysis. Mass spectrometry (MS) is expensive, cumbersome, and primarily used for qualitative analysis, with poor quantitative accuracy. Electrospray ionization (CAD) and evaporative light scattering (ELSD) detectors are more suitable for lipid analysis.

[0006] The working principle of an electrospray ionization detector (CAD) is to convert analytes into solute particles. The particle size increases with the analyte content. The solute particles collide with positively charged nitrogen particles, transferring charge to the particles. The larger the solute particles, the more charged they are. The charge of the solute particles is measured by a highly sensitive electrostatic detector, and the resulting signal current is proportional to the solute content. The principle of an evaporative light scattering detector (ELSD) is to generate sample microparticles or droplets through atomization and evaporation. The solute particles enter a light detection cell and pass through a laser beam. The light scattered by the solute particles is collected and detected by an electron multiplier tube. But CAD has advantages over ELSD (Kayori Takahashi, et al., Quantitative comparison of a corona-charged aerosol detector and an evaporative light-scattering detector for the analysis of a synthetic polymer by supercriticalfluid chromatography. Journal of Chromatography A, 1193 (2008) 151–155; ZhenLong, et al., A non-derivative method for the quantitative analysis ofisosteroidal alkaloids from Fritillaria by high performance liquidchromatography combined with charged aerosol detection. Talanta 151 (2016) 239–244), mainly manifested in: 1. High sensitivity. For the same compound, the detection limit of the electrospray ionization detector is on average one order of magnitude lower than that of the evaporative light scattering detector; 2. Linearity and precision are better than those of ELSD; 3. The response of different compounds is consistent. Since the evaporative light scattering detector ultimately measures light by the scattering of particles, and the degree of scattering of different substances is different, and since the particles precipitated in the solution at high temperature are irregular, the degree of scattering of light by their different surfaces is also different, so the response between compounds is very inconsistent; 4. Better durability, because each machine has an optimal condition fixed before leaving the factory and cannot be changed, and is less affected by the environment. Summary of the Invention

[0007] In view of the lack of existing methods for analyzing the lipid components of lipid nanospheres, the present invention provides a method for qualitative and quantitative detection of the lipid components in lipid nanospheres by high performance liquid chromatography electrospray ionization detector.

[0008] The present invention provides a method for detecting lipid components in lipid nanospheres, which adopts high performance liquid chromatography-electrospray ionization detector method for detection, comprising:

[0009] (1) Detecting a standard solution of lipid components, performing linear regression calculation on the peak area in the chromatogram according to the concentration of the standard solution, and constructing a standard curve;

[0010] (2) Detecting the lipid nanosphere solution, identifying the lipid components in the lipid nanospheres according to the retention time in the chromatogram, and calculating the concentration of the lipid components according to the peak area of ​​the lipid components in the chromatogram and the above-mentioned standard curve.

[0011] Preferably, the dryer temperature of the electrospray detector is 50°C, the acquisition frequency is 1-20 Hz, and the filtration constant is 1-3 s; the chromatographic column filler is octadecyl silica gel, the column temperature is 25-60°C, the flow rate is 0.6-1.5 mL / min, the injection volume is 5-50 μL, the mobile phase A is a 0.01M-0.5M triethylamine acetic acid aqueous solution, the mobile phase B is a 0.01M-0.5M triethylamine acetic acid methanol solution, and the gradient elution is as follows:

[0012] .

[0013] Preferably, the chromatographic column is an Xbridge Peptide BEH C18 chromatographic column.

[0014] Preferably, the electrospray detector has a dryer temperature of 50° C., a collection frequency of 10 Hz, and a filtration constant of 1 s.

[0015] Preferably, the column temperature is 55°C.

[0016] Preferably, the flow rate is 1.0 mL / min.

[0017] Preferably, the injection volume is 10 μL.

[0018] Preferably, the mobile phase A is a 0.01 M triethylamine acetic acid aqueous solution, and the mobile phase B is a 0.01 M triethylamine acetic acid methanol solution.

[0019] Preferably, the lipid standard solution comprises at least six groups of linear solutions with decreasing concentrations.

[0020] Preferably, the solvents of the lipid component standard solution and the lipid nanosphere solution are one or more of methanol, ethanol, isopropanol, and dimethyl sulfoxide.

[0021] This invention provides, for the first time, a method for qualitative and quantitative detection of lipid components in lipid nanospheres. This method utilizes conventional high-performance liquid chromatography (HPLC), is simple to operate, and offers excellent specificity, sensitivity, linearity, precision, and accuracy, with a short single analysis time. This method can be used for rapid in-process analysis and quality control of lipid nanospheres. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Standard curve of lipid 1;

[0023] Figure 2 : Standard curve of lipid 2;

[0024] Figure 3 : Standard curve of lipid 3;

[0025] Figure 4 : Standard curve of lipid 4;

[0026] Figure 5 : Chromatogram of lipid nanosphere test solution. DETAILED DESCRIPTION

[0027] The present invention is described in detail below with reference to specific embodiments. However, the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

[0028] As used herein, "lipid" refers to a class of organic compounds that includes, but is not limited to, esters of fatty acids. Lipids are insoluble in water but are soluble in many organic solvents. They are generally divided into at least three categories: (1) "simple lipids," which include fats and oils as well as waxes; (2) "compound lipids," which include phospholipids and glycolipids; and (3) "derivative lipids," such as steroids. Lipids can also be classified as "cationic lipids," "anionic lipids," and "neutral lipids," based on the charge they carry at a selected pH.

[0029] "Cationic lipids" refers to lipids that carry a net positive charge at a selected pH, such as physiological pH. Common cationic lipids include, but are not limited to, 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenoyloxy-N,N-dimethylaminopropane (DLenDMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearoyl-N,N-dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 1,2-Dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2K); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol) and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), etc.

[0030] "Neutral lipid" refers to any of a number of lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, cholesterol, phospholipids such as diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, distearoylphosphatidylcholine, cerebrosides, and diacylglycerols.

[0031] "Anionic lipids" refer to lipids that carry a negative charge at a selected pH. Common anionic lipids include, but are not limited to, dioleoylphosphatidylglycerol (DOPG), distearoylphosphatidylethanolamine (DPSE), diacylphosphatidylserine, diacylphosphates, diacylphosphatidylinositols, diacylglycerol hemisuccinate, diacylglycerol hemiglutarate, cholesterol hemisuccinate, cholesterol hemiglutarate, and the like.

[0032] In addition, the lipids of the present invention also include "polymer-conjugated lipids," i.e., compounds obtained by covalently linking a polymer to a lipid. Polymer-conjugated lipids can include phospholipid polyethylene glycols (e.g., DPPE-PEG, dipalmitoylphosphatidylethanolamine-polyethylene glycol, DMPE-PEG, myristoylphosphatidylethanolamine-polyethylene glycol), ceramide polyethylene glycols (e.g., C16-Ceramide-PEG, palmitoylceramide-polyethylene glycol), glyceride polyethylene glycols (e.g., DMG-PEG, myristoylglycerol polyethylene glycol), and the like.

[0033] "Nucleic acid" refers to a polymer containing at least two deoxyribonucleotides (DNA) or ribonucleotides (RNA) in single- or double-stranded form. Oligonucleotides are a general term for a class of short nucleotide chains with fewer than 20 bases (including nucleotides within deoxyribonucleic acid DNA or ribonucleic acid RNA). RNA includes messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), etc. Unless specifically limited, the term encompasses both natural and synthetic nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly indicated sequence.

[0034] Naturally produced mRNA is a type of single-stranded RNA that carries genetic information and guides protein synthesis, transcribed from a single strand of DNA. Using a gene as a template, mRNA is transcribed according to the principle of complementary base pairing. The mRNA contains base sequences corresponding to specific functional segments of the DNA molecule, serving as a direct template for protein biosynthesis and determining the amino acid sequence of the peptide chain of the gene's protein product. Although mRNA only accounts for 2% to 5% of total cellular RNA, it is the most abundant and metabolically active type of RNA, with the shortest half-life, being degraded within minutes to hours of synthesis. Synthetic mRNA can be used in vaccine development. Two types of mRNA are used for vaccine development: non-replicating mRNA and self-amplifying mRNA. Non-replicating mRNA is 2 to 3 kb in length and consists of a m7Gp3N cap, a 5' untranslated region (UTR), an open reading frame encoding an antigen, a 3' untranslated region (UTR), and a poly A tail, with a relatively simple structure. Self-amplifying mRNA is a complex structure, measuring 9 to 10 kb in size. In addition to its non-amplified structure, it also contains nonstructural protein gene sequences and a subgenomic promoter. Self-amplifying mRNA not only encodes antigens but also contains sequences required for viral replication, giving it the ability to replicate, thereby significantly increasing protein expression.

[0035] In the present invention, "lipid nanospheres" refer to nanoparticles formed by fully or partially encapsulating nucleic acids within liposomes formed from lipids, wherein the nanoparticles are smaller than 100 nanometers in at least one dimension. Lipid nanospheres can be used to deliver fully or partially encapsulated nucleic acids to cells. Those skilled in the art can select appropriate lipids based on the properties of the various lipids, the nucleic acids to be encapsulated, or the intended use of the lipid nanospheres.

[0036] The method of the present invention is described below using lipid nanospheres containing four lipid components, lipid 1 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000, DMG-PEG2K, manufacturer: Nof America Corporation), lipid 2 (cholesterol, manufacturer: Nippon Fine Chemical), lipid 3 (distearoyl phosphatidylcholine, DSPC, manufacturer: Nippon Fine Chemical), and lipid 4, as an example. The target concentrations of the four lipid components in the prepared nanosphere solution are 248 μg / mL, 982 μg / mL, 521 μg / mL, and 2757 μg / mL, respectively. The structure of lipid 4 is shown in formula (I). For details, please see invention patent application No. 202010275644.4.

[0037] Formula (I).

[0038] The lipid nanospheres contain an mRNA sequence with a total length of 1000 nt. In addition to the antigen-encoding sequence, they also include a 5' end cap structure, 5' and 3' UTRs, and a long polyadenylic acid tail (polyA tail) encoded by the plasmid, which can stably and efficiently express the target protein in cells. Lipid nanospheres are prepared using the above-mentioned lipids 1-4 and mRNA using known methods, such as the method disclosed in WO2017180917A2. The target mRNA concentration in the resulting lipid nanosphere solution is 200 μg / mL. The above-mentioned lipid nanospheres can be used as a new coronavirus vaccine. Those skilled in the art should understand that the method of the present invention is not limited to this lipid nanosphere. Lipid nanospheres containing other lipid components, other mRNAs, and other concentrations can also be detected using the method of the present invention.

[0039] Preparation of lipid component standard solutions

[0040] Accurately weigh the four lipid component standards and dissolve them in methanol to obtain a standard stock solution containing the four lipid components. The concentration of each lipid component in the standard stock solution is approximately 1 / 3 to 1 / 2 of the target concentration of the four lipid components in the lipid nanospheres. In the following examples, the concentration of lipid 1 in the standard stock solution is 0.1 mg / mL, lipid 2 is 0.4 mg / mL, lipid 3 is 0.2 mg / mL, and lipid 4 is 1.2 mg / mL.

[0041] The standard stock solutions containing each lipid component were diluted with methanol to obtain a series of linear standard solutions containing different concentrations of each lipid component. To minimize error, the number of diluted standard solutions should be at least six.

[0042] Dilute the standard stock solution according to Table 1 to obtain linear solutions 1-6:

[0043] Table 1: Linear solution preparation method

[0044] .

[0045] Preparation of lipid nanosphere test solution

[0046] The lipid nanosphere solution was diluted so that the solubility of each lipid component was within the concentration range of the linear solution. In the following examples, 50 μL of the lipid nanosphere solution was added to 950 μL of methanol and mixed to obtain a lipid nanosphere test solution.

[0047] Parameter setting of high performance liquid chromatography-electrospray ionization detector

[0048] The electrospray ionization detector (manufacturer: Thermo Fisher Scientific Inc.; model: Corona Veo) had a high dryer temperature (50°C), an acquisition frequency of 1-20 Hz, and a filtration constant of 1-3 s. The high performance liquid chromatography (HPLC) column (manufacturer: Thermo Fisher Scientific Inc.; model: U3000) was packed with octadecyl silica gel, the column temperature was 25-60°C, the flow rate was 0.6-1.5 mL / min, the injection volume was 5-50 μL, the mobile phase A was a 0.01 M-0.5 M triethylamine acetic acid aqueous solution, and the mobile phase B was a 0.01 M-0.5 M triethylamine acetic acid methanol solution. Gradient elution was performed. The elution gradient is shown in Table 2:

[0049] Table 2: Elution gradient

[0050] .

[0051] Constructing a standard curve

[0052] The standard linear solutions of the above-mentioned lipid components were detected separately using the above-mentioned high performance liquid chromatography-electrospray ionization detector. The data processing software Chromeleon (manufacturer: Thermo Fisher Scientific Inc.) was used to perform linear regression calculation based on the peak area in the chromatogram with the lipid component concentration to construct the standard curve of each component.

[0053] Qualitative and quantitative analysis of lipid components in lipid nanospheres

[0054] The lipid nanosphere solution was detected using the high performance liquid chromatography-electrospray ionization detector, and each lipid component was identified according to the retention time in the chromatogram. The concentration of each lipid component was calculated based on the peak area of ​​each lipid component using the standard curve of each component.

[0055] Example 1

[0056] The parameters of the high performance liquid chromatography-electrospray ionization detector were as follows: the dryer temperature of the electrospray ionization detector was high (50°C), the acquisition frequency was 10 Hz, and the filter constant was 1 s; the chromatographic column was an Xbridge Peptide BEH C18 column (manufacturer: Waters Corporation), the column temperature was 55°C, the flow rate was 1.0 mL / min, the injection volume was 10 μL, the mobile phase A was a 0.01 M triethylamine acetic acid solution in water, the mobile phase B was a 0.01 M triethylamine acetic acid solution in methanol, and the gradient elution was as shown in Table 3:

[0057] Table 3: Elution gradient

[0058]

[0059] Respectively for " Preparation of lipid component standard solutions The linear solutions of the standard lipid components obtained in the "Section 3" were tested. The retention times of lipids 1-4 obtained are shown in Table 4:

[0060] Table 4: Retention time and resolution of lipid components

[0061] .

[0062] The resolution in Table 4 is the quotient of the retention time difference between two adjacent peaks in the chromatogram and the average base width of the two peaks. In Table 4, the resolution data to the right of lipid 2 represents the resolution between lipid 1 and lipid 2, the resolution data to the right of lipid 3 represents the resolution between lipid 2 and lipid 3, and the resolution data to the right of lipid 4 represents the resolution between lipid 3 and lipid 4. As can be seen in Table 4, the resolution of each lipid component is greater than 1.5, indicating that the method has good specificity and is suitable for lipid quantitative analysis.

[0063] according to" Constructing a standard curve The standard curves of lipids 1-4 were obtained by the method described in the " Figure 1-4 The linear regression equation for lipid 1 is y = -0.0012 x 2 + 0.1444 x + 0.0125 (R² = 0.9991), the linear regression equation for lipid 2 is y = -0.0003 x 2 + 0.1159 x - 0.3046 (R² = 0.9995), the linear regression equation for lipid 3 is y = -0.0004 x 2 + 0.1339 x - 0.0617 (R² = 0.9990), the linear regression equation for lipid 4 is y = -7E-05 x2 + 0.0896 x + 0.6655 (R² = 0.9996). Within the detection concentration range of each lipid component, the coefficient of determination (R²) of the linear regression equation for each component was above 0.999, indicating that the linearity curve obtained by this method is good.

[0064] right" Preparation of lipid nanosphere test solution The lipid nanosphere test solution obtained from the "part was tested, and the chromatogram obtained was as follows Figure 5 As shown. Figure 5 From the retention time of each peak, it can be seen that reference numeral 1 represents lipid 1, reference numeral 2 represents lipid 2, reference numeral 3 represents lipid 3, and reference numeral 4 represents lipid 4. Figure 5 Substituting the peak areas of each lipid component into the standard curves for each lipid component, the measured concentrations of each lipid component in the lipid nanosphere test solution were calculated as follows: lipid 1: 12.8303 μg / mL, lipid 2: 47.9453 μg / mL, lipid 3: 25.2547 μg / mL, and lipid 4: 143.5796 μg / mL. The dilution factor between the lipid nanosphere test solution and the lipid nanosphere solution was 20-fold. Therefore, the calculated concentrations of each lipid component in the lipid nanosphere test solution were multiplied by the dilution factor of 20 to obtain the measured concentrations of each lipid component in the lipid nanosphere solution: lipid 1: 256.606 μg / mL, lipid 2: 958.906 μg / mL, lipid 3: 505.094 μg / mL, and lipid 4: 2871.592 μg / mL. The measured solubility of each lipid component in the lipid nanosphere solution was divided by the theoretical concentration of each lipid component, and the content of each lipid component was obtained as follows: lipid 1: 103%, lipid 2: 98%, lipid 3: 97%, lipid 4: 104%.

[0065] Example 2

[0066] In this example, the repeatability and precision of the method of the present invention were tested.

[0067] Tester A repeated the test six times using the method of Example 1. The results are shown in Table 5:

[0068]

[0069] Tester B repeated the test six times using the method of Example 1. If as shown in Table 6:

[0070]

[0071] The results of testers A and B are summarized in Table 7:

[0072]

[0073] The lipid content in Tables 5-7 is expressed as the ratio of the measured concentration to the theoretical concentration of each lipid component. The above test results show that the relative standard deviation (RSD) of the test results for the same sample by the same tester is less than 5%, and for some lipid components, the RSD is less than 1%, demonstrating the good reproducibility of the method of the present invention. Even for different testers, the RSD of the test results for the same sample is less than 10%, and for some lipid components, the RSD is only 1%, demonstrating the excellent precision of the method of the present invention.

[0074] Example 3

[0075] This example tests the stability of lipid component standard solutions and lipid nanosphere test solutions over time.

[0076] Taking the above-mentioned linear solution 4 as an example, the stability of the standard solution of the lipid component over time was tested using the method of Example 1. The results are shown in Table 8:

[0077]

[0078] The stability of the lipid nanosphere test solution over time was tested using the method of Example 1. The results are shown in Table 9:

[0079]

[0080] The above results show that after the standard solutions of each lipid component and the lipid nanosphere test solution were placed at room temperature for 24 hours, the relative standard deviation of the concentration of each lipid component was less than 5%, indicating that the standard solutions and lipid nanosphere test solutions used in the present invention have excellent stability.

[0081] Example 4

[0082] This example tests the accuracy of the method of the present invention.

[0083] Take 50 μL of 0.2 mg / mL mRNA solution and dilute it with 950 μL of linear solution 1. The concentration of each lipid component in the obtained accuracy test sample 1 is equivalent to about 50% of the concentration of each lipid component in the above lipid nanosphere test solution, and the final mRNA concentration is consistent with the mRNA concentration in the lipid nanosphere test solution. Prepare three parallel test samples 1, 2, and 3.

[0084] Using the same method, linearization solution 3 and linearization solution 5 were used in place of linearization solution 1 to obtain accuracy test samples 4, 5, 6, 7, 8, and 9. In accuracy test samples 4-6, the concentration of each lipid component was approximately equal to the concentration of each lipid component in the lipid nanosphere test solution, and the final mRNA concentration was consistent with the mRNA concentration in the lipid nanosphere test solution. In accuracy test samples 7-9, the concentration of each lipid component was equivalent to approximately 150% of the concentration of each lipid component in the lipid nanosphere test solution, and the final mRNA concentration was consistent with the mRNA concentration in the lipid nanosphere test solution.

[0085] The accuracy test samples were detected using the high performance liquid chromatography-electrospray detector in Example 1, and the measured concentrations of the lipid components in the accuracy test samples were calculated based on the standard curve obtained in Example 1.

[0086] The recovery rate was obtained by dividing the measured concentration of each lipid component by the theoretical concentration. The recovery rates of the sample solutions for each accuracy test are shown in Table 10:

[0087]

[0088] The results show that the recovery rates of the lipid components detected by the method of the present invention are in the range of 94.2% to 110.2%, and the relative standard deviations are all less than 4%, indicating that the accuracy of the method of the present invention is excellent.

Claims

1. A method for detecting lipid components in lipid nanospheres (LNPs), characterized in that: Detection was performed using high performance liquid chromatography-electrospray ionization detection, including: (1) Detecting a standard solution of lipid components, performing a linear regression calculation on the peak area in the chromatogram using the concentration of the standard solution to construct a standard curve; (2) Detecting the lipid nanosphere solution, identifying the lipid components in the lipid nanospheres according to the retention time in the chromatogram, and calculating the concentration of the lipid components according to the peak area of ​​the lipid components in the chromatogram and the above-mentioned standard curve, wherein the mobile phase A in the high performance liquid chromatography is a 0.01M~0.5M aqueous solution of triethylamine acetic acid, and the mobile phase B is a 0.01M~0.5M methanol solution of triethylamine acetic acid, and gradient elution is performed, and the elution gradient is as follows: ， The chromatographic column was packed with octadecyl silica gel, the column temperature was 25–60°C, the flow rate was 0.6–1.5 mL / min, and the injection volume was 5–50 μL; The lipid nanospheres contain four lipid components, which are 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000, cholesterol, distearoyl phosphatidylcholine and lipids represented by formula (I): 。 2. The method according to claim 1, wherein: The dryer temperature of the electrospray detector was 50 °C, the acquisition frequency was 1–20 Hz, and the filtration constant was 1–3 s.

3. The method according to claim 2, wherein The chromatographic column is an Xbridge Peptide BEH C18 chromatographic column.

4. The method according to claim 2, characterized in that The electrospray detector had a dryer temperature of 50° C., a collection frequency of 10 Hz, and a filtration constant of 1 s.

5. The method according to claim 2, characterized in that The column temperature was 55°C.

6. The method according to claim 2, wherein The flow rate was 1.0 mL / min.

7. The method according to claim 2, characterized in that The injection volume was 10 μL.

8. The method according to claim 2, wherein The mobile phase A is a 0.01M triethylamine acetic acid aqueous solution, and the mobile phase B is a 0.01M triethylamine acetic acid methanol solution.

9. The method according to any one of claims 1 to 8, characterized in that The standard solution of the lipid component comprises at least six groups of linear solutions with decreasing concentrations.

10. The method according to any one of claims 1 to 8, characterized in that The solvents of the lipid component standard solution and the lipid nanosphere solution are one or more of methanol, ethanol, isopropanol, and dimethyl sulfoxide.

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