Method for analyzing and detecting lipid auxiliary material for lipidosome

Abstract

The invention relates to an analysis and detection method of lipid auxiliary materials for lipidosome, which adopts reversed-phase high performance liquid chromatography, and can detect one or more of phosphatidyl ethanolamine, hydrogenated soybean phosphatidylcholine, cholesterol and sodium distearoyl phosphatidyl glycerol for lipidosome under the same chromatographic condition. The analysis method disclosed by the invention is simple and rapid to operate, relatively low in cost, high in sensitivity, strong in specificity, good in repeatability and suitable for analysis and detection of other products with similar lipid auxiliary materials.

Description

Technical Field

[0001] The present invention relates to the field of chemical analysis technology, and in particular to a high performance liquid phase analysis method for simultaneously detecting DSPE-MPEG2000, HSPC, DSPG-Na and cholesterol. Background Art

[0002] Liposomes are drug vesicles with a closed central water cavity formed by lipid self-assembly, and more than ten injectable liposome products have been approved for marketing worldwide. An exemplary liposome drug preparation is doxorubicin hydrochloride liposomes developed and launched by Sequus in 1995, which is mainly used to treat ovarian cancer, HIV-related Kaposi's sarcoma and multiple myeloma. In the production process, lipid excipients such as phosphatidylethanolamine (also known as polyoxyethylene / polyethylene glycol distearoylphosphatidylethanolamine, DSPE-MPEG2000), hydrogenated soybean phosphatidylcholine (HSPC) and cholesterol are often used; another exemplary liposome drug preparation is amphotericin B liposomes for injection, which has been approved for marketing in China, the United States, Europe and other countries / regions, and its lipid excipients include hydrogenated soybean phosphatidyl (HSPC), sodium distearoylphosphatidylglycerol (DSPG-Na) and cholesterol.

[0003] In the prior art, thin layer chromatography can be used to detect individual liposome excipients. Although the main components can be separated to a certain extent, the operation is cumbersome, and the reproducibility and quantitative accuracy are poor, and the content of each component cannot be accurately indicated. The Chinese Pharmacopoeia records the detection of cholesterol by HPLC-ELSD method, and the HPLC-ELSD method is also widely used in the detection of phospholipids. However, the detection process needs to go through the steps of enrichment extraction, and the method is too cumbersome, and different detection methods are used to detect a variety of lipid excipients for liposomes, which will greatly increase the time cost and elution solvent, and even cause the loss of chromatographic column. Therefore, a universal analytical method is urgently needed for detecting lipid excipients for liposomes, providing a basis for its quality control. Summary of the invention

[0004] To address the deficiencies in the prior art, the present application provides a method for analyzing and detecting lipid excipients for liposomes. The method is simple and universal and is suitable for simultaneously detecting hydrogenated phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, cholesterol and sodium distearoylphosphatidylglycerol.

[0005] Compared with the prior art, the first aspect of the present application provides a method for simultaneously detecting multiple liposome excipients by using reversed-phase high performance liquid chromatography.

[0006] In an exemplary embodiment, the excipient is a lipid excipient, including one or more of hydrogenated phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, cholesterol and sodium distearoylphosphatidylglycerol, and optionally, all four of the above.

[0007] In an exemplary embodiment, the method comprises the following steps:

[0008] S1. Prepare solution;

[0009] Prepare reference solution;

[0010] Prepare test sample solution;

[0011] S2. Detection:

[0012] Separately pipette the reference solution and the test solution into the high performance liquid chromatograph for detection and record the chromatogram.

[0013] In an exemplary embodiment, in the high performance liquid chromatography detection in step S2, the chromatographic conditions are:

[0014] Mobile phase: a combination of mobile phase A and mobile phase B, wherein mobile phase A is a volatile salt solution, preferably a methanol solution of ammonium formate or ammonium acetate, and mobile phase B is water or ultrapure water.

[0015] In an exemplary embodiment, the ratio of the mobile phase A to the mobile phase B is (95:5)-(99:1) (v / v%), preferably, 95:5, 96:4, 97:3, 98:2 or 99:1, and more preferably, 97:3.

[0016] In an exemplary embodiment, the concentration of ammonium formate in the mobile phase A is 10 mM.

[0017] In an exemplary embodiment, in the high performance liquid chromatography detection, other chromatographic conditions are:

[0018] Chromatographic column: C18 chromatographic column, i.e. octadecylsilane bonded silica gel column;

[0019] Chromatographic column model: YMC-Triart C18 S-3μm, 4.6mm×150mm;

[0020] Injection volume: 10 μL-50 pL; preferably, 20 μL;

[0021] Elution mode: isocratic elution;

[0022] Flow rate: 0.5mL / min-1.5mL / min; preferably, 0.5mL / min-1.0mL / min or 1.0mL / min-1.5mL / min; more preferably, 0.5mL / min, 0.6mL / min, 0.7mL / min, 0.8mL / min, 0.9mL / min, 1.0mL / min, 1.1mL / min, 1.2mL / min, 1.3mL / min, 1.4mL / min or 1.5mL / min;

[0023] Chromatographic column temperature: 45°C-60°C; preferably, 45°C, 50°C, 55°C or 60°C.

[0024] In an exemplary embodiment, in the HPLC detection, the detector is a charged aerosol detector (CAD) detector.

[0025] In an exemplary embodiment, the detection condition of the detector is:

[0026] Detector temperature: 35℃ or 55℃;

[0027] Filter constant: 0.1s-10.0s;

[0028] Data acquisition frequency: 2Hz-100Hz.

[0029] In an exemplary embodiment, preferably, the detection conditions of the detector are:

[0030] Detector temperature: 35°C;

[0031] Filter constant: 10.0s;

[0032] Data acquisition frequency: 10Hz; or

[0033] Detector temperature: 55°C;

[0034] Filter constant: 10.0s;

[0035] Data acquisition frequency: 10Hz.

[0036] In an exemplary embodiment, most preferably, in the high performance liquid chromatography detection, the chromatographic conditions are:

[0037] The volume ratio of mobile phase A and mobile phase B is 97:3.

[0038] Flow rate: 1.2mL / min,

[0039] Column temperature: 55°C,

[0040] CAD detector filter constant: 10.0s,

[0041] Detector temperature: 35°C,

[0042] Data acquisition frequency: 10 Hz;

[0043] Injection volume: 20 μL.

[0044] In an exemplary embodiment, in step S1, the reference solution is prepared by:

[0045] Add the standard substances of phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, cholesterol and sodium distearoylphosphatidylglycerol into the same volumetric flask, add a solvent to dissolve and prepare a reference substance stock solution; dilute the reference substance stock solution to obtain a reference substance solution.

[0046] In an exemplary embodiment, in step S1, the method for preparing the test solution is:

[0047] Add the test samples of hydrogenated phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, cholesterol and sodium distearoylphosphatidylglycerol into the same volumetric flask, and add solvent to dissolve them to prepare the test sample solution.

[0048] In an exemplary embodiment, the solvent is an organic solvent, which may be methanol and / or chloroform; optionally, the solvent is a combination of methanol and chloroform.

[0049] In an exemplary embodiment, the volume ratio of methanol to chloroform is (90-98):(10-2), preferably, 95:5.

[0050] In an exemplary embodiment, in the reference solution and the test solution, the concentration of hydrogenated phosphatidylethanolamine may be 2.94 μg / mL-0.5 mg / mL; the concentration of hydrogenated soybean phosphatidylcholine may be 2.08 μg / mL-0.5 mg / mL; the concentration of sodium distearoylphosphatidylglycerol may be 0.69 μg / mL-0.5 mg / mL; and the concentration of cholesterol may be 0.56 μg / mL-0.5 mg / mL;

[0051] Preferably, the concentrations of hydrogenated phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, cholesterol and sodium distearoylphosphatidylglycerol are all 0.25 mg / mL.

[0052] In an exemplary embodiment, the method further includes:

[0053] S3. Analysis:

[0054] According to the chromatograms of the reference solution and the test solution, calculate the contents of hydrogenated phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, cholesterol and sodium distearoylphosphatidylglycerol in the test sample.

[0055] In an exemplary embodiment, in step S3, the contents of hydrogenated phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, cholesterol and sodium distearoylphosphatidylglycerol in the test sample are calculated using the following formula:

[0056]

[0057] Wherein, Au is the main peak area of ​​the test sample; Ws is the sample weight of the reference sample in mg; Wu is the sample weight of the test sample in mg; p is the labeled content of the reference sample; As is the main peak area of ​​the reference sample; Vs is the dilution multiple of the reference sample, and Vu is the dilution multiple of the test sample.

[0058] The second aspect of the present application provides an analytical detection method for excipients in a liposome product, wherein the excipient is a lipid excipient, comprising using the above method to detect the content of the lipid excipient in the liposome product, wherein the lipid excipient is one or more of hydrogenated phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, cholesterol or sodium distearoylphosphatidylglycerol.

[0059] The analytical method described in the present application has the advantages of simple operation, rapid elution, low cost, strong universality, strong specificity, good system adaptability, good linear relationship, and wide linear range.

[0060] Other features and advantages of the present application will be described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0062] Figure 1 It is the chromatogram of the reference substance solution in Example 1;

[0063] Figure 2 The chromatogram of the test solution in Example 1;

[0064] Figure 3 The chromatogram is the specificity test result of Example 2;

[0065] Figure 4-Figure 9 This is the chromatogram of the repeatability test results of Example 2;

[0066] Figure 10-Figure 17 This is a chromatogram of the durability test results of Example 2;

[0067] Figure 18-Figure 22They are chromatograms of mobile phases 1-5 in Example 3 respectively. DETAILED DESCRIPTION

[0068] The embodiments described below are used to understand the technical solutions of the present invention. The patent protection scope of the present invention is not limited thereto. Non-creative adjustments and improvements made based on the present invention all fall within the protection scope of the present invention.

[0069] The raw materials and equipment used in the specific implementation manner of the present invention are all known products and can be obtained by purchasing commercially available products.

[0070] Example 1. Determination of lipid excipient content in the test sample

[0071] 1.1 Reagents used: Except for ultrapure water prepared in the laboratory, the following reagents are commercially available products.

[0072] Test products: DSPE-MPEG2000 (purchased from Lipoid), HSPC (purchased from Lipoid), cholesterol (purchased from Nippon Seika Co., Ltd.), and sodium distearoylphosphatidylglycerol DSPG-Na (purchased from Lipoid);

[0073] Reference substances: reference-grade DSPE-MPEG2000 (purchased from Yuanye Biotechnology Co., Ltd.), HSPC (purchased from Lipoid Co., Ltd.), DSPG-Na (purchased from Lipoid Co., Ltd.) and cholesterol (purchased from China National Institute for Food and Drug Control);

[0074] Methanol (purchased from Fisher), anhydrous methanol (purchased from Xilong Scientific), chloroform (purchased from Komiou Company), and ammonium formate (purchased from Sigma Company).

[0075] 1.2 Solution preparation:

[0076] Preparation of solvent: Anhydrous methanol and chloroform were mixed in a volume ratio of 95:5 (v / v%);

[0077] Preparation of mobile phase: ammonium formate is added to methanol to prepare a 10 mM ammonium formate methanol solution as mobile phase A; take the mobile phase A, add ultrapure water as mobile phase B, and prepare a mobile phase with a volume ratio of ammonium formate methanol solution: water = 97:3 (v / v%);

[0078] Preparation of test solution: Add DSPE-MPEG2000, HSPC, DSPG-Na and cholesterol test samples into the same volumetric flask, add solvent to dissolve, and quantitatively dilute to a test solution containing approximately 0.25 mg of each substance per 1 mL of mixed solution;

[0079] Preparation of reference solution: Add reference-grade DSPE-MPEG2000, HSPC, DSPG-Na and cholesterol standards to the same volumetric flask, dissolve with solvent, and quantitatively dilute to a reference stock solution with a concentration of 2.5 mg / mL for each substance. Quantitatively dilute the reference stock solution to prepare a reference solution containing approximately 0.25 mg of each substance per 1 mL of solution.

[0080] 1.3 Chromatographic detection conditions:

[0081] Take 20 μL of the reference solution and the test solution respectively and inject them into the high performance liquid chromatograph for detection and record the chromatogram. The detection conditions are as follows:

[0082] Chromatographic column: YMC-Triart C18 S-3μm, 4.6mm×150mm;

[0083] Detector: CAD;

[0084] Mobile phase: ammonium formate methanol solution: water = 97:3 (v / V%);

[0085] Column temperature: 55 °C;

[0086] Elution mode: isocratic elution;

[0087] Flow rate: 1.2 mL / min;

[0088] Acquisition frequency: 10Hz;

[0089] Filter constant: 10.0s;

[0090] Detector temperature: 35°C.

[0091] 1.4 Determination of lipid excipient content in the test sample

[0092] The following formula was used to calculate the content of each lipid excipient in the test sample:

[0093]

[0094] Wherein, Au is the main peak area of ​​each lipid excipient in the test sample; Ws is the sample weight of each lipid excipient reference substance in mg; Wu is the sample weight of each lipid excipient test sample in mg; p is the labeled content of each lipid excipient reference substance; As is the main peak area of ​​each lipid excipient reference substance; Vs is the dilution multiple of each lipid excipient reference substance, and Vu is the dilution multiple of each lipid excipient test sample.

[0095] The chromatographic test results of the reference solution and the test solution are as follows Figure 1 , Figure 2 shown.

[0096] according to Figure 1 , Figure 2 It can be seen that under the chromatographic detection conditions, the four excipients can be detected normally.

[0097] Example 2. Methodological Investigation

[0098] 2.1 Exclusivity

[0099] Take the reference solution of Example 1 and inject one injection under the same chromatographic conditions as Example 1, and take the solvent as a blank solution and inject one injection, analyze in the same way and record the chromatogram.

[0100] Test results: The chromatograms recorded for the reference solution and blank solution are as follows: Figure 3 shown.

[0101] according to Figure 3 It can be seen that the blank solution has no interference at the sample peak, and the mixed solution has a good peak, indicating that the method of this application has good specificity.

[0102] 2.2 System Applicability

[0103] The reference solution in Example 1 was injected 5 times under the same chromatographic conditions as in Example 1, and the chromatograms were recorded. The relative standard deviation of the peak area of ​​each chromatogram was calculated based on the peak area to evaluate the system suitability. The results are shown in Table 1.

[0104] Table 1 System suitability results

[0105] serial number DSPE-MPEG2000 DSPG-Na HSPC 1 HSPC 2 cholesterol 1 30.7753 19.7900 7.0974 19.7184 21.9237 2 30.6537 19.8192 7.1457 19.7270 21.9091 3 30.7470 19.8436 7.1376 19.7324 22.0258 4 30.6500 19.7244 7.0946 19.7695 21.9614 5 30.6050 19.7921 7.1033 19.5754 21.8059 RSD 0.23％ 0.23％ 0.34％ 0.38％ 0.37％

[0106] According to Table 1, after 5 injections, the relative standard deviation RSD of the peak area of ​​each substance is ≤0.40%, and the results show that the system applicability of this method is good.

[0107] 2.3 Repeatability

[0108] Six test solutions and six reference solutions were prepared in parallel, wherein the preparation methods of the test solutions and the reference solutions were the same as those in Example 1.

[0109] Take the above 6 portions of the test solution and the reference solution, inject one injection each according to the same chromatographic conditions as in Example 1, analyze and detect in the same way, and record the chromatogram. Detect the four lipid excipients and calculate the content of each excipient in the test sample. The test results are shown in Table 2 and Figure 4-9 shown.

[0110] Table 2 Repeatability test results

[0111] serial number DSPE-MPEG2000 DSPG-Na HSPC cholesterol 1 102.4％ 94.7％ 108.2％ 104.6％ 2 101.5％ 95.5％ 111.3％ 102.4％ 3 100.8％ 94.5％ 109.0％ 102.7％ 4 99.4％ 95.2％ 109.2％ 102.8％ 5 100.0％ 94.8％ 108.7％ 103.2％ 6 99.0％ 92.7％ 107.8％ 102.9％ RSD (n=6) 1.29％ 1.05％ 1.12％ 0.76％

[0112] The results showed that in the repeatability test, the RSDs of the contents of the four lipid excipients were all less than 1.5% (n=6), and the repeatability of the method of the present application was good.

[0113] 2.4 Linear Relationship

[0114] Preparation of stock solutions: Take appropriate amounts of DSPE-MPEG2000 reference substance, HSPC reference substance, DSPG-Na reference substance and cholesterol reference substance, respectively, and place them in 50 ml volumetric bottles, add the solvent in Example 1, and adjust the volume to obtain 4 kinds of reference substance stock solutions; wherein, the concentration of each substance in the reference substance stock solutions is: DSPE-MPEG2000 2.35mg / ml, HSPCs 2.50mg / ml, DSPG-Na is 1.36 mg / ml, Cholesterol is 0.45 mg / ml.

[0115] Preparation of linear solutions: Dilute the stock solutions of each auxiliary material reference substance step by step to 25%, 50%, 100%, 150% and 200% limit concentrations, respectively, as linear solutions of each auxiliary material component.

[0116] The linear solutions of the above-mentioned limiting concentrations were taken and tested under the same chromatographic conditions as in Example 1. The chromatographic peak area-concentration graph was plotted to obtain the standard curves of the lipid excipients. The results are shown in Table 3.

[0117] Table 3 Linear relationship investigation results

[0118] Components Linear equations <![CDATA[R 2 ]]> Phosphatidylethanolamine y=119.090x+2.454 0.997 Hydrogenated Soy Phosphatidylcholine y=91.831x+5.190 0.993 Sodium Distearoylphosphatidylglycerol y=90.567x+2.494 0.998 cholesterol y=77.129x+4.133 0.995

[0119] Conclusion: As shown in Table 3, according to the results of the linear relationship investigation, the concentration of each lipid excipient component has a good linear relationship in the range of 0.11 mg / ml to 5.00 mg / ml.

[0120] 2.5 Limit of quantitation

[0121] Take the linear solution of each component in 2.4, for example, with a concentration of 25%, dilute it appropriately, and use the signal-to-noise ratio method to dilute it to an S / N of about 10. The corresponding sample concentration is the quantitative limit concentration, and the resulting solution is the quantitative limit solution.

[0122] The quantitative limit solutions of the above components were injected once each, and the detection was carried out under the same chromatographic conditions as in Example 1 to obtain the quantitative limit concentrations of the lipid excipients. The results are shown in Table 4.

[0123] Table 4 Quantitation limit results

[0124] Components Limit of Quantitation Phosphatidylethanolamine 2.94 μg / mL Hydrogenated Soy Phosphatidylcholine 2.08 μg / mL Sodium Distearoylphosphatidylglycerol 0.69 μg / mL cholesterol 0.56 μg / mL

[0125] According to Table 4, the quantitative limits of DSPE-MPEG2000, HSPC, DSPG-Na and cholesterol are 2.94, 2.08, 0.69 and 0.56 μg / mL, respectively.

[0126] 2.6 Durability

[0127] The chromatographic detection conditions were adjusted to detect the test solution in Example 1 to examine the durability of the detection method of this application. The detection conditions and results are shown in Tables 5 and Figure 10-17 As shown:

[0128] Table 5 Durability consideration factors

[0129]

[0130] From Table 5 and Figure 10-17 It can be seen that this method has excellent durability under the above-mentioned consideration factors.

[0131] Example 3 Investigation of mobile phase

[0132] The reference solution in Example 1 was tested using the following mobile phases 1-5.

[0133] Wherein, the composition of mobile phase 1-5 is as follows:

[0134] Mobile phase 1: 100 mM ammonium formate methanol solution-water, volume ratio 97:3;

[0135] Mobile phase 2: 50 mM ammonium formate methanol solution-water, volume ratio 97:3;

[0136] Mobile phase 3: 25 mM ammonium formate methanol solution-water, volume ratio 97:3;

[0137] Mobile phase 4: 20 mM ammonium formate methanol solution-water, volume ratio 97:3;

[0138] Mobile phase 5: 0 mM ammonium formate methanol solution-water, volume ratio of 97:3.

[0139] The detection conditions are the same as those in Example 1.

[0140] The test results of each component in the reference solution are as follows Figure 18-22 As shown:

[0141] When using mobile phase 1-2 for detection, the lipid excipients cannot be separated normally.

[0142] When mobile phases 3-5 were used for detection, the separation effect of various lipid excipients was poor.

[0143] Therefore, when the ratio of the mobile phase A to the mobile phase B is (95:5)-(99:1) (v / v%), preferably, 95:5, 96:4, 97:3, 98:2 or 99:1, more preferably, 97:3, and the concentration of ammonium formate is 10 mM, the separation and detection effect of each lipid excipient is optimal.

[0144] The present application describes multiple embodiments, but the description is exemplary rather than restrictive, and it is obvious to a person skilled in the art that there may be more embodiments and implementations within the scope of the embodiments described in the present application.

Claims

1. A method for simultaneously detecting multiple liposome excipients, wherein the excipients are lipid excipients, including one or more of hydrogenated phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, cholesterol or sodium distearoylphosphatidylglycerol, and detecting by reverse phase high performance liquid chromatography, the method comprising the following steps: S1. Prepare solution; Prepare reference solution; Prepare test sample solution; S2. Detection: Separately pipette the reference solution and the test solution into a high performance liquid chromatograph for detection and record the chromatogram; Among them, in the high performance liquid chromatography detection, the chromatographic conditions are: Mobile phase: a combination of mobile phase A and mobile phase B, wherein mobile phase A is a volatile salt solution and mobile phase B is water or ultrapure water; The volume ratio of the mobile phase A to the mobile phase B is (95:5)-(99:1).

2. The method according to claim 1, wherein the mobile phase A is a methanol solution of ammonium formate or ammonium acetate, and the concentration of ammonium formate in the mobile phase A is 10 mM.

3. The method according to claim 1, wherein the volume ratio of mobile phase A to mobile phase B is 95:5, 96:4, 97:3, 98:2 or 99:1; more preferably, 97:

3.

4. The method according to claim 1, wherein the other chromatographic conditions are: Chromatographic column: octadecylsilane bonded silica gel column; Chromatographic column model: YMC-TriartC18 S-3μm, 4.6mm×150mm; Injection volume: 10 μL-50 μL; preferably, 20 μL; Detector: Charged aerosol detector; Elution mode: isocratic elution; Flow rate: 0.5mL / min-1.5mL / min; preferably, 0.5mL / min-1.0mL / min or 1.0mL / min-1.5mL / min; more preferably, 0.5mL / min, 0.6mL / min, 0.7mL / min, 0.8mL / min, 0.9mL / min, 1.0mL / min, 1.1mL / min, 1.2mL / min, 1.3mL / min, 1.4mL / min or 1.5mL / min; Chromatographic column temperature: 45°C-60°C; preferably, 45°C, 50°C, 55°C or 60°C.

5. The method according to claim 4, wherein the condition of the detector is Detector temperature: 35℃ or 55℃; Filter constant: 0.1s-10.0s; Data acquisition frequency: 2Hz-100Hz; Preferably, the detector temperature: 35°C; Filter constant: 10.0s; Data acquisition frequency: 10Hz; or Detector temperature: 55°C; Filter constant: 10.0S; Data acquisition frequency: 10Hz.

6. The method according to any one of claims 1 to 5, wherein the chromatographic conditions when the high performance liquid chromatograph is used for detection are: The volume ratio of mobile phase A and mobile phase B is 97:

3. Flow rate: 1.2mL / min, Column temperature: 55°C, Detector filter constant: 10.0s, Detector temperature: 35°C, Data acquisition frequency: 10Hz, Injection volume: 20 μL.

7. The method according to claim 1, in step S1, the reference solution is prepared by: Adding the standard substances of phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, cholesterol and sodium distearoylphosphatidylglycerol into the same volumetric flask, adding a solvent to dissolve and prepare a reference substance stock solution; diluting the reference substance stock solution to obtain a reference substance solution; The test solution preparation method is: Add the test samples of phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, cholesterol and sodium distearoylphosphatidylglycerol into the same volumetric flask, and add solvent to dissolve them to prepare the test sample solution; In the reference solution and the test solution, the concentration of hydrogenated phosphatidylethanolamine may be 2.94 μg / mL-0.5 mg / mL; the concentration of hydrogenated soybean phosphatidylcholine may be 2.08 μg / mL-0.5 mg / mL; the concentration of sodium distearoylphosphatidylglycerol may be 0.69 μg / mL-0.5 mg / mL; the concentration of cholesterol may be 0.56 μg / mL-0.5 mg / mL; preferably, the concentrations of hydrogenated phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, cholesterol and sodium distearoylphosphatidylglycerol may all be 0.25 mg / mL.

8. The method according to claim 7, wherein the solvent is methanol and / or chloroform; optionally, the solvent is a combination of methanol and chloroform, wherein: The volume ratio of methanol to chloroform is 95:

5.

9. The method according to claim 1, further comprising: S3. Analysis: According to the chromatograms of the reference solution and the test solution, calculate the contents of hydrogenated phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, cholesterol and sodium distearoylphosphatidylglycerol in the test sample.

10. The method according to claim 9, in step S3, the content of hydrogenated phosphatidylethanolamine, hydrogenated soybean phosphatidylcholine, cholesterol and sodium distearoylphosphatidylglycerol in the test sample is calculated by the following formula: in, Au is the main peak area of ​​the test sample; Ws is the sample weight of the reference sample in mg; Wu is the sample weight of the test sample in mg; p is the labeled content of the reference sample; As is the main peak area of ​​the reference sample; Vs is the dilution multiple of the reference sample, and Vu is the dilution multiple of the test sample.

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