CN112129871B - Method for detecting contents of DOPE and M5 phospholipids in composite phospholipid liposome - Google Patents
Method for detecting contents of DOPE and M5 phospholipids in composite phospholipid liposome Download PDFInfo
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention establishes an HPLC-ELSD method for simultaneously measuring the composite phospholipid lipidIn particular, to methods for two phospholipids (DOPE, M5) in the COVID19LPP vaccine. Performing gradient elution with 0.1% trifluoroacetic acid in redistilled water as mobile phase A and methanol as mobile phase B, and performing linear correlation coefficient square (R) of chromatographic peak areas and mass concentrations of two phospholipids, DOPE and M52) More than 0.993, the average recovery rate of the two phospholipids is between 100 and 102 percent, and the RSD is less than 2.1 percent. The method does not need solvent extraction, can directly analyze the composite phospholipid liposome, realizes good separation of DOPE and M5 from other components, has the characteristics of good reproducibility, high stability, simple and quick pretreatment and easy operation, can simultaneously determine the content of DOPE and M5 in the composite phospholipid liposome, meets the related requirements of quantitative analysis, and is suitable for screening and verification of the preparation process of the composite phospholipid liposome, stability test and product quality inspection.
Description
Technical Field
The invention relates to a quality control method of an mRNA liposome vaccine, in particular to a detection method for simultaneously detecting the contents of two phospholipids, namely DOPE and M5 in a composite phospholipid liposome.
Background
The liposome refers to a micro-vesicular body formed by encapsulating a medicament in a lipid bilayer, is commonly used as an anti-tumor medicament carrier, and is developed into a mature novel targeted preparation. The liposome is limited in development due to poor stability, mainly because the phospholipid is easily oxidized and decomposed during storage; phospholipids are not single compounds, but are mixtures of substances with the same backbone structure but different fatty acid chains, thereby limiting the detection of phospholipid materials. Because the components of the liposome are complex, the conventional liposome generally contains main functional auxiliary materials such as cholesterol, phospholipid and the like, and degradation products of the phospholipid and impurities of the degradation products are more and complex, the detection of various phospholipids in the liposome is difficult to a certain extent.
The Chinese pharmacopoeia 2010 clearly indicates that the raw and auxiliary materials used in the injection should be strictly controlled according to the production links such as sources, processes and the like and should meet the quality requirements for injection. Meanwhile, according to the industrial standard of medicine quality, as a functional pharmaceutic adjuvant, the content of phospholipid in a pharmaceutic preparation needs to be accurately quantified, but the existing literature method is very complex, and certain difficulty is brought to the research of the pharmaceutic preparation.
The research means of phospholipid are various, and the current methods for measuring the content of phospholipid mainly comprise a high performance liquid chromatography, a molybdenum blue colorimetric method, an ultraviolet spectrophotometry method, a fluorescence method, a thiocyanatonium iron-ammonium color development method, a thin layer chromatography, a nuclear magnetic resonance method and the like. In practice, detection of a particular phospholipid usually requires a particular detection method. CN101692038 provides a method for determining phospholipid in liposome by a colorimetric method; researchers Zhao Bao Qin provided a method for measuring the content of phospholipid in a phospholipid soft capsule by a molybdenum blue colorimetric method (measuring the content of phospholipid in a linolenic acid phospholipid soft capsule, ginseng research, 2015(03: 36-37)); gongjin inflammation provides a color development method of ammonium ferrocyanide for measuring the content of phospholipid in lecithin (the color development method of ammonium ferrocyanide is used for measuring the content of total phospholipid in egg yolk lecithin, 2018,039(008):319-322), and the reagents used in the methods relate to various instruments, have complex operation steps and cannot effectively separate various phospholipid components. Hatziantoniou S uses thin layer chromatography in combination with a flame ion detector to separate and analyze liposome phospholipid composition, but the mobile phase is complex and the chromatographic peak shape is poorly effective (Method of heterogeneous analysis of lipid compositions using HPTLC/FID [ J ]. Methods in molecular biology, (Clifton, N.J.; 2010,606(606):363 368)).
HPLC is the most common detection method at present, high performance liquid chromatography has advantages in the separation and detection of phospholipid, and the separation of phospholipid by high performance liquid chromatography can avoid the destruction of phospholipid molecular structure and obtain more accurate molecular structure information. The detector for detecting phospholipid includes ultraviolet, evaporative light scattering, differential refraction and mass spectrum detectors. Ultraviolet detectors (UV) and evaporative light detectors (ELSD) are commonly used detectors. Most of the reported phospholipid HPLC detection methods are ultraviolet detector detection, and the phospholipid lacks strong chromophore, so that the ultraviolet absorption performance is poor, and the sensitivity is low. Phospholipid molecules can also be derivatized to increase the ultraviolet absorption of the phospholipid. The phospholipid is changed into a compound with a chromophoric group through a chemical reaction, or the phospholipid molecule is directly connected with the chromophoric group or a fluorescent group, and then an ultraviolet or fluorescence detector is used for detecting. However, the derivatization reaction process is complex, the pretreatment step is complicated, the influence on the accuracy of the quantitative result is large, and the operation is complicated. Studies of Yang and Wen show that the UV detector is greatly influenced by phospholipid source and is difficult to realize accurate quantification (HPLC-RI method for rapidly and accurately determining the content of soybean phosphatidylcholine, analysis and test report 2004(05): 118-. The differential refraction detector is less affected by the source, but has lower sensitivity, can not be used for gradient elution, has high requirements on the environment and is more affected by the temperature (HPLC-RID method for measuring lysolecithin and fatty acid in PEG modified liposome, China journal of medical industry, 2015,046(007): 750-. The detection method reported by Diaz-Lopez et al uses electrospray (CAD) detector, acetonitrile-methanol-ammonia-glacial acetic acid as mobile phase A, and methanol-ammonia-glacial acetic acid as mobile phase B for gradient elution, which has high requirements for detector, and very complicated sample pretreatment and elution conditions, which are unfavorable for operation (quantitative of functionalized nanoparticles purifying polymeric capsules of fluorescent dye by phase HPLC with a charged particle adsorbed detector. pharmaceutical and biological Analysis, 2008, 48:702 707). The detection of phospholipids using mass spectrometry detectors is carried out by the methods of the prior art, such as the fluorescence (HPLC analysis of phospholipids [ J ]. Fujian analysis test, 2007,016(001):71-74) and the Rampler E (Rampler E, Schoeny H, Mitic B M, et al, Simultaneous non-polar and polar lipid analysis by on-line combination of HILIC, RP and high resolution MS [ J ]. analysis, 2018:10.1039.C7AN01984J), but the mass spectrometry detectors are costly and not easy to popularize.
The evaporative scattered light detector is a non-selective detector, and the application is not limited by the type of a sample. The evaporative light detector has relatively higher sensitivity and is suitable for gradient elution, so that the evaporative light detector is more widely applied to phospholipid detection. However, in the conventional process of measuring phospholipid content by HPLC-ELSD, after extracting total fat from the material, further enrichment of phospholipid by column chromatography, solid phase microextraction and other methods is often required, and the steps are complicated, which not only prolongs the sample processing time, but also causes partial loss of phospholipid and affects the accuracy of measurement, such as HPLC-ELSD analysis of 6 kinds of phospholipid content in the gonad of stichopus japonicus (Zhuyao, Chen Hui, Luhang, etc., Dai university of Ogao, 2015, 30(04): 422) and chromatography analysis in portable oil consumption liquid chromatography-mass spectrometry (Herchi W, Sakouhi F, Khaled S, et al. Chemistry,2011,129(2): p.437-442).
The composite phospholipid liposome is a new emerging technology in recent years, is a spherical drug carrier which is composed of different phospholipids arranged at intervals and cholesterol and has similar biomembrane structure and phospholipid bilayers with different phase regions, and is characterized in that two phospholipids with different phase transition temperatures are adopted as membrane materials (one is higher than normal temperature and the other is lower than normal temperature) in the preparation process, the temperature is controlled to be 60 ℃ in the preparation process, in addition, the phospholipid material with high phase transition temperature is added to influence the rigidity of the liposome, and the rigidity improvement can weaken the adsorption of a conditioning mechanism on the surface of the liposome so as to slow down the clearance of the liposome from the circulation. Thus, varying the proportions of phospholipid material within a certain range affects the rigidity of the complex phospholipid liposomes. In addition, because different phase regions are formed in the composite phospholipid liposome, the prepared liposome can greatly increase the drug-loading rate of some difficultly soluble drugs. Compared with the traditional liposome, the liposome has better stability and bioavailability, and overcomes the problems of low drug loading rate, low inclusion rate and the like of the traditional liposome.
DOPE is a commonly used helper lipid, and M5 is one of the currently highly efficient cationic lipids. In recent years, with the wider application of cationic lipid-mediated small-molecule drugs and biomolecules such as proteins and RNAs in vivo and in vitro, the research on the composite phospholipid liposome is more and more intensive, DOPE and M5 are more and more concerned as functional auxiliary materials with good effects, the corresponding detection methods are few, and the detection of DOPE and M5 in the total substance of the composite phospholipid liposome is not reported.
Disclosure of Invention
In order to solve the problems, the invention develops a novel method for simultaneously measuring the contents of DOPE and M5 with good selectivity, convenience, rapidness and accuracy by combining the self characteristics of a self-developed COVID19LPP vaccine preparation (mRNA composite phospholipid liposome vaccine) on the basis of the traditional phospholipid detection method, and realizes the simultaneous separation and detection of the contents of DOPE and M5 in the composite phospholipid liposome for the first time.
The DOPE is named as dioleoyl phosphatidylethanolamine in Chinese and has a molecular formula of C41H78NO8P, molecular weight 744.0337, CAS: 4004-05-1, the structural formula is shown in formula I.
M5 is neutral phospholipid, can be used as lipid component in vaccine reagent, and has a structural formula shown in formula II.
The composite phospholipid liposome usually contains two phospholipid components, such as COVID19LPP vaccine preparation, and DOPE and M5 phospholipid, and the phospholipid detection method in the prior art is complex and tedious in operation process, but DOPE and M5 cannot be detected simultaneously, so that the quality control of the composite phospholipid liposome vaccine is difficult greatly. Therefore, how to rapidly and effectively detect DOPE and M5 from the composite phospholipid liposome simultaneously is the basis for improving the quality control level of the composite phospholipid liposome vaccine.
However, due to the similarity of the phospholipid backbone structure and the complex components of the phospholipid complex liposome, and the degradation products of phospholipid and impurities thereof, the separation of DOPE and M5 has certain difficulty. The COVID19LPP vaccine is an mRNA composite phospholipid liposome vaccine independently developed by the applicant, besides phospholipids, the liposome vaccine also contains mRNA, protamine and other components, and further increases the difficulty for separating DOPE and M5. The HPLC-ELSD method provided by the invention can be used for simultaneously detecting the contents of the compound phospholipid liposome, particularly DOPE and M5 in the COVID19LPP vaccine, and is simple and convenient to operate and suitable for popularization.
The invention aims to establish a method for simply, efficiently, accurately and simultaneously detecting DOPE and M5 in the composite phospholipid liposome, so as to determine the proportion of the content of each component in the composite phospholipid liposome and provide a certain experimental basis for the quality control of the liposome.
The invention provides a method for simultaneously detecting the contents of two phospholipids, namely DOPE and M5 by high performance liquid chromatography, which adopts the high performance liquid chromatography-evaporative light scattering detector to detect, wherein a mobile phase A is 0.1% trifluoroacetic acid solution, a mobile phase B is methanol, and gradient elution is carried out.
The preparation method of the mobile phase A comprises the following steps: add 1.0mL trifluoroacetic acid into 1.0L redistilled water and mix well. The mobile phase B adopts methanol as chromatographic grade methanol.
The retention of cationic lipids can be enhanced by adding 0.1% (v/v) trifluoroacetic acid (TFA) to the mobile phase, achieving good separation of DOPE, M5 from other components in the liposomes.
Further, the gradient elution procedure is as follows:
further, the high performance liquid chromatography conditions are as follows: the chromatographic column is a C18 chromatographic column, the flow rate of a mobile phase is 0.4mL/min, and the column temperature is 35 ℃. The C18 chromatographic column is Agilent Poroshell 120EC-C18 pore size 12nm, 3.0 × 100mm, 2.7 μm.
Further, the detection conditions of the evaporative light scattering detector are as follows: evaporator temperature 70 ℃, atomizer temperature 70 ℃, gas flow Rate 1.6L/min, Date Rate: 40 Hz.
Further, the method also comprises standard preparation, wherein the standard preparation comprises the preparation of an M5 standard and the preparation of a DOPE standard, and the main steps are as follows: m5 or DOPE was dissolved in ethanol to give stock solutions of 32mg/mL, and then diluted with methanol to give working solutions of 2.0, 1.0, 0.50, 0.25 and 0.125mg/mL, respectively, for use in calibration curve samples.
Preparation of M5 stock solution: precisely weighing 32mg of M5, dissolving the M5 into 32mg/mL solution by using ethanol, and mixing the solution by vortex. The stock solution was stored in a refrigerator at 4 ℃ as a stock solution of M5.
Preparing M5 working solution: stock M5 was taken and diluted with chromatographic methanol to the following concentrations: 2.0, 1.0, 0.50, 0.25 and 0.125 mg/mL. The diluted series was used as M5 working solution for the standard curve sample.
Preparing DOPE stock solution: precisely weighing 32mg of DOPE, dissolving the DOPE into 32mg/mL solution by using ethanol, and mixing the solution by vortex. As DOPE stock solution, the stock solution was stored in a refrigerator at 4 ℃.
Preparing DOPE working solution: DOPE stock was taken and diluted with chromatographic methanol to the following concentrations: 2.0, 1.0, 0.5, 0.25 and 0.125 mg/mL. The diluted series of solutions were used as DOPE working solutions for the standard curve samples.
Further, the method also comprises sample treatment, and the main steps of the sample treatment are as follows: a200. mu.L sample was taken into a centrifuge tube, 200. mu.L of redistilled water and 400. mu.L of 2% Triton 100 were added to the centrifuge tube, and mixed well, and reacted at room temperature for 1 hour.
The COVID19LPP vaccine is an mRNA composite phospholipid liposome vaccine independently developed by the applicant, and besides phospholipids, the liposome vaccine also contains mRNA, protamine and other components, so that Triton (Triton) needs to be added to open the liposome to dissolve the phospholipids during analysis, and interference of other components and impurities needs to be effectively avoided.
Further, the preparation method of the 2% Triton 100 comprises the following steps: add 20. mu.L Triton 100 to 980. mu.L redistilled water, dissolve in 37 ℃ water bath, mix well.
Further, when the method is used for detection, the sample injection amount is 30 mu L.
Further, the method further comprises data analysis.
The method for detecting the contents of two phospholipids, namely DOPE and M5 by high performance liquid chromatography comprises the following steps: standard preparation, sample treatment, sample detection and data analysis.
Sequentially measuring the chromatographic curves of M5(0.125-2.0mg/mL), DOPE (0.125-2.0mg/mL) and the chromatogram of the test sample with different concentration gradients.
The data analysis steps are as follows: respectively taking logarithms to perform linear fitting according to the concentrations of M5 and DOPE and peak areas on chromatographic curves to obtain a linear equation and a square R of a linear correlation coefficient2. And calculating the contents of M5 and DOPE in the test solution according to respective fitted straight lines through different retention times and peak areas on chromatographic curves of M5 and DOPE in the test solution. Through experimental calculation, the linear correlation coefficient square (R) of the chromatographic peak areas and the mass concentrations of the two phospholipids of DOPE and M52) More than 0.993, the average recovery rate of the two phospholipids is between 100 and 102 percent, and the RSD is less than 2.1 percent.
In another aspect, the invention provides the use of the high performance liquid chromatography detection method for simultaneously detecting the contents of two phospholipids, namely DOPE and M5 in the composite phospholipid liposome.
Due to the similarity of the phospholipid backbone structure, the complex components of the liposome, and the degradation products of the phospholipid and the impurities thereof, the separation of DOPE and M5 has certain difficulty. The high performance liquid chromatography detection method provided by the invention can be used for simultaneously detecting the contents of two phospholipids, namely DOPE and M5 in the composite phospholipid liposome.
Further, the composite phospholipid liposome is a COVID19LPP vaccine.
The invention establishes a simple and rapid method for simultaneously measuring the composite phospholipid liposome by a reversed-phase high performance liquid chromatography-evaporation photodetector (HPLC-ELSD), in particular to a method for measuring two phospholipids (DOPE, M5) in a COVID19LPP vaccine. To be provided withGradient elution was carried out using a chromatographic column (Agilent Poroshell 120EC-C18 pore size 12nm, 3.0 × 100mm, 2.7 μ M) as the stationary phase, 0.1% trifluoroacetic acid in redistilled water as mobile phase A, chromatographic methanol as mobile phase B, and the square of the linear correlation coefficient (R) between the chromatographic peak area and the mass concentration of two phospholipids, DOPE and M52) More than 0.993, the average recovery rate of the two phospholipids is between 100 and 102 percent, and the RSD is less than 2.1 percent. The method does not need solvent extraction, can directly analyze the composite phospholipid liposome, has the characteristics of good reproducibility, high stability and simple pretreatment, can simultaneously determine the content of DOPE and M5 in the composite phospholipid liposome, meets the related requirements of quantitative analysis, realizes the separation and quantification of the two lipids in the LPP liposome for the first time, and is suitable for screening and verification of the preparation process of the composite phospholipid liposome, stability test and product quality inspection.
The invention has the following beneficial effects:
(1) the pretreatment process is simplified, the mobile phase preparation and the sample pretreatment process are included, solvent extraction is not needed, and the composite phospholipid liposome can be directly analyzed.
(2) And 0.1% (v/v) trifluoroacetic acid (TFA) is added into the mobile phase to enhance the retention of cationic lipid, so that the good separation of DOPE and M5 from other components in the liposome is realized, the time is saved, and errors brought by additional operation to experiments are avoided.
(3) The method is faster, simple and easy to operate, and simultaneously separates and detects the content of DOPE and M5 in the composite phospholipid liposome for the first time.
(4) Good reproducibility and high stability.
Drawings
FIG. 1, the M5 control map obtained by the detection in example 1, the retention time is 14.179min
FIG. 2, DOPE control map obtained by detection in example 1, retention time is 20.239min
FIG. 3, example 2, a graph for measuring the content of DOPE and M5 in a test sample solution
Detailed Description
The invention will be described in further detail below with reference to the drawings and examples, which are intended to facilitate the understanding of the invention without limiting it in any way.
Example 1 Standard Curve plotting
Preparation of first, stock solution and working solution
Preparation of M5 stock solution: precisely weighing 32mg of M5, dissolving the M5 into 32mg/mL solution by using ethanol, and mixing the solution by vortex. The stock solution was stored in a refrigerator at 4 ℃ as a stock solution of M5.
Preparing M5 working solution: stock M5 was taken and diluted with chromatographic methanol to the following concentrations: 2.0, 1.0, 0.50, 0.25 and 0.125 mg/mL. The diluted series was used as M5 working solution for the standard curve sample.
Preparing DOPE stock solution: precisely weighing 32mg of DOPE, dissolving the DOPE into 32mg/mL solution by using ethanol, and mixing the solution by vortex. As DOPE stock solution, the stock solution was stored in a refrigerator at 4 ℃.
Preparing DOPE working solution: DOPE stock was taken and diluted with chromatographic methanol to the following concentrations: 2.0, 1.0, 0.5, 0.25 and 0.125 mg/mL. The diluted series of solutions were used as DOPE working solutions for the standard curve samples.
Second, HPLC-ELSD detection
Taking five standard curve samples with the concentration of 0.125, 0.25, 0.5, 1.0 and 2.0mg/ml of M5 and DOPE, wherein the sample injection amount is 30 mu L, and carrying out HPLC-ELSD detection under the specific detection conditions:
the high performance liquid chromatography column is Agilent Poroshell 120EC-C18 pore size 12nm, 3.0 x 100mm, 2.7 μm, the flow rate of mobile phase is 0.4mL/min, the column temperature is 35 ℃; evaporative light scattering detector evaporator temperature 70 ℃, atomizer temperature 70 ℃, gas flow Rate 1.6L/min, Date Rate: 40 Hz; the mobile phase A is 0.1 percent trifluoroacetic acid solution, the mobile phase B is methanol, and gradient elution is carried out, wherein the gradient elution procedure is as follows:
| time (min) | Mobile phase A (%) | Mobile phase B (%) |
| 0 | 50 | 50 |
| 10 | 0 | 100 |
| 18 | 0 | 100 |
| 19 | 50 | 50 |
| 24 | 50 | 50 |
The preparation method of the mobile phase A comprises the following steps: add 1.0mL trifluoroacetic acid into 1.0L redistilled water and mix well. The mobile phase B adopts methanol as chromatographic grade methanol.
Wherein, the chromatograms of the M5(2.0mg/mL) and DOPE (0.125mg/mL) reference solutions are respectively shown in FIG. 1 and FIG. 2, and it can be seen that M5 shows a peak at 14.179min and DOPE shows a peak at 20.239 min.
According to the detection result, linear regression is carried out by taking the logarithm of the peak area of y and the logarithm of the injection concentration of x, and the regression equations are respectively 1.2133x +5.3755, R2＝0.9939；y＝1.211x+5.3855，R20.9996. The result shows that the linear relation between M5 and DOPE is good in the range of 0.125-2.0 mg/mL.
EXAMPLE 2 sample testing
A batch of vaccine finished products (batch number COVID19 LPP-200414) is adopted, 200 mu L of the vaccine finished products are taken to be a centrifuge tube, 200 mu L of secondary distilled water and 400 mu L of 2% Triton 100 (the preparation of the 2.0% Triton 100: 20 mu L of Triton 100 is measured to be added to 980 mu L of secondary distilled water, dissolved in water bath at 37 ℃ and mixed uniformly) are added to the centrifuge tube, mixed uniformly, reacted for 1 hour at room temperature, the HPLC-ELSD method is adopted to detect the phospholipid content in the vaccine finished products according to the chromatographic conditions of the example 1, and the chromatogram is recorded, and the result is shown in figure 3.
As can be seen from FIG. 3, the method can separate two phospholipids, DOPE and M5, from the sample, and the DOPE and M5 in the liposome can be detected simultaneously with good separation effect, and are not interfered by impurity peaks.
Through detection, calculation and analysis, the content of DOPE and M5 in the sample with the obtained batch number COVID19 LPP-200414 is shown in Table 1, and the content of DOPE and M5 in the liposome can be determined to be reliable by comparing with the theoretical input amount (4 mg/mL).
TABLE 1 content of DOPE and M5 in the test articles
| Batches of | COVID19 LPP-200414 |
| M5 mg/ml | 3.99 |
| DOPE mg/ml | 3.84 |
Example 3 repeatability test
The same batch of vaccine finished product (batch number COVID19 LPP-200416) was taken, 200. mu.L was put into a centrifuge tube, 200. mu.L of redistilled water and 400. mu.L of 2% Triton 100 (preparation of 2.0% Triton 100: 20. mu.L Triton 100 was weighed and added to 980. mu.L redistilled water, dissolved in a water bath at 37 ℃ and mixed) were added into the centrifuge tube, mixed and reacted at room temperature for 1 hour to prepare a sample, 6 portions were paralleled and measured under the chromatographic conditions of example 1, and the results are shown in Table 2 in terms of peak area.
TABLE 2 results of the repeatability tests
As can be seen from Table 2, the peak areas of M5 and DOPE of the 6 samples are shown to be very close, and the variances RSD are 0.69% and 0.85%, respectively, and are both less than 1%, indicating that the detection method is very good in repeatability.
Example 4 recovery test
After 100. mu.L of the finished vaccine (lot number COVID19 LPP-200415) was put into a 1.5mL centrifuge tube, 100. mu.L of DOPE or M5 control solution (concentration 2.4, 4.0, 5.0mg/mL, respectively) was precisely added, and then test samples were prepared according to the preparation method of example 2, to obtain test sample solutions of 3 concentrations, each of which was 3 parts in parallel. The measurement was carried out under the chromatographic conditions of example 1, and the chromatogram was recorded. The results are shown in Table 3.
TABLE 3 recovery rate test results
As can be seen from Table 3, the recovery rate of M5 in the composite phospholipid liposome measured by the detection method is 98.3-103.5%, the average recovery rate is 101.08%, and the average relative standard deviation is 2.07%; the recovery rate of DOPE is between 99.1% and 103.9%, the average recovery rate is 100.91%, and the average relative standard deviation is 2.04%, which shows that the recovery rate results of DOPE and M5 sample application in the composite phospholipid liposome measured by the method are very good.
Example 5 comparison of the present Process with the mobile phase composition of the prior art
This example compares the mobile phase composition of the method with that of other methods in the prior art, wherein, other method 1 is HPLC-RID method of Liangmin et al for measuring lysolecithin in liposome (HPLC-RID method for measuring lysolecithin and fatty acid in PEG modified liposome, J.Med.J.M. 2015,046(007): 750-; other method 2 is that the HPLC-ELSD method adopted by Lissajou et al measures DSPE-PEG2000 in cisplatin liposome (the HPLC-ELSD method measures DSPE-PEG2000 content in cisplatin liposome [ J ]. Nature's college newspaper of Hubei university: Nature's edition, 2016(04): 285) -287); other methods 3 are HPLC-ELSD method adopted by Zhao Qi Yan et al to determine the phospholipid content in peanut (direct sample HPLC-ELSD method to determine phospholipid content [ J ] in peanut, Chinese oil 2018,43(03): 131-; other method 4 is the Analysis of human blood for phospholipid species by normal phase liquid Chromatography by Uran S et al (Analysis of phospholipid species in human blood coating using normal-phase liquid Chromatography [ J ]. Journal of Chromatography B,2001,758(2): 265) -275), the results are shown in Table 4.
TABLE 4 mobile phase composition comparison
As can be seen from Table 4, the mobile phase adopted by the invention has simpler composition, only consists of two components, is more convenient to prepare and simple and convenient to operate, and DOPE and M5 can be completely separated by elution; while the mobile phase adopted by other methods respectively needs three or more components, the preparation process is more complicated, but the method cannot be used for simultaneously detecting DOPE and M5. The invention adopts a simpler mobile phase composition, can simultaneously obtain an ideal detection result aiming at the contents of two phospholipids, namely DOPE and M5 in the composite phospholipid liposome, and further proves the simplicity, the rapidness and the high efficiency of the method.
Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (9)
1. A method for simultaneously detecting the contents of two phospholipids, namely DOPE and M5 by high performance liquid chromatography is characterized in that the method adopts the high performance liquid chromatography-evaporative light scattering detector for detection, the mobile phase A is 0.1% trifluoroacetic acid solution, the mobile phase B is methanol, and gradient elution is carried out, wherein the gradient elution procedure is as follows:
The structural formula of M5 is shown as the following formula:
the chromatographic column adopted by the high performance liquid chromatography is a C18 chromatographic column.
2. The method of claim 1, wherein the high performance liquid chromatography conditions are: the flow rate of the mobile phase was 0.4mL/min and the column temperature was 35 ℃.
3. The method of claim 1 or 2, wherein the evaporative light scattering detector detects conditions as: evaporator temperature 70 ℃, atomizer temperature 70 ℃, gas flow Rate 1.6L/min, Data Rate: 40 Hz.
4. The method of claim 1, further comprising a standard preparation, the standard preparation comprising the preparation of a M5 standard and the preparation of a DOPE standard, the main steps being: m5 or DOPE was dissolved in ethanol to give stock solutions of 32mg/mL, and then diluted with methanol to give working solutions of 2.0, 1.0, 0.50, 0.25 and 0.125mg/mL, respectively, for use in calibration curve samples.
5. The method of claim 1, further comprising sample processing, the main steps of the sample processing being: a200. mu.L sample was taken into a centrifuge tube, 200. mu.L of redistilled water and 400. mu.L of 2% Triton 100 were added to the centrifuge tube, and mixed well, and reacted at room temperature for 1 hour.
6. The method of claim 5, wherein said 2% Triton 100 is prepared by: add 20. mu.L Triton 100 to 980. mu.L redistilled water, dissolve in 37 ℃ water bath, mix well.
7. The method of claim 1, wherein the amount of sample is 30 μ L when the method is performed.
8. Use of the method of any one of claims 1-7 for simultaneous detection of the content of both DOPE and M5 in a complex phospholipid liposome.
9. The use of claim 8, wherein the complex phospholipid liposome is a COVID19LPP vaccine.
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