Method for detecting impurities in lecithin

Abstract

The application discloses a method for detecting impurities in cultured phosphatidylethanolamine, which comprises detecting DSPE, 1-StePE-MPEG, unknown impurities, MPEG2000 and SA, and comprises the following steps: preparing a test sample solution; preparing a control solution; preparing a reference solution; and performing thin layer chromatography determination. The method for detecting impurities in cultured phosphatidylethanolamine can effectively detect various impurities in the cultured phosphatidylethanolamine by using the thin layer chromatography, and can also effectively detect impurities with low concentration, has good detection effect, good stability, high accuracy of results and strong practicability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of purity inspection of organic compounds, and particularly to a method for inspecting impurities in cultured phosphatidylethanolamine. BACKGROUND

[0002] Cultured phosphatidylethanolamine (DSPE-MPEG2000) is a pharmaceutical excipient, which is a polyethylene glycol (PEG) derivative of 1,2-distearoylphosphatidylethanolamine (DSPE). The PEG part is inactivated and cannot continue to react because the end of the polyethylene glycol is capped with a methoxy group, so it is called MPEG. When the molecular weight of PEG is 2000, DSPE-MPEG is also called DSPE-MPEG2000.

[0003] The synthesis raw material of DSPE-MPEG2000 is DSPE and MPEG2000. The raw material may be introduced into DSPE-MPEG2000 due to incomplete reaction. At the same time, DSPE-MPEG2000 is prone to degradation during synthesis and storage, generating impurities such as DSPE, 1-stePE-MPEG2000 (molecular formula as follows), MPEG2000 and SA (stearic acid), affecting its purity and quality, and further affecting the use effect.

[0004] The structural formula of 1-stePE-MPEG2000 is as follows:

[0005]

[0006] The ultraviolet absorption wavelength of DSPE-MPEG2000 is low (205 nm), at the end of the ultraviolet absorption wavelength, and is easily disturbed by other solvents, so ultraviolet is not generally used as a detector. The evaporative light scattering detector (ELSD) as a detector is not affected by gradient elution, and is a commonly used detector in the prior art.

[0007] However, the present inventors found at least the following technical problems in the process of implementing the technical scheme of the embodiments of the present application:

[0008] When using the evaporative light scattering detector (ELSD) as a detector, the response signal and the sample amount of the component may have a non-linear relationship in a larger sample injection range. The response signal of the RID is large, and can be used as a detector for content determination, but when detecting related substances, the amount of related substances is small, and the signal-to-noise ratio does not meet the requirements, so it is difficult to achieve effective detection of impurities, the measurement accuracy is low, and it does not have practical reference value. SUMMARY

[0009] The application solves the problems of great difficulty and low accuracy in detecting impurities in the cultured phosphatidylethanolamine by providing a method for detecting impurities in the cultured phosphatidylethanolamine.

[0010] To solve the above technical problems, the application provides a method for detecting impurities in the cultured phosphatidylethanolamine, which comprises detecting DSPE, 1-StePE-MPEG and unknown impurities, and the steps are as follows:

[0011] S1, preparing a test sample solution: a proper amount of DSPE-MPEG2000 is weighed and dissolved in a mixed solvent of chloroform-methanol, and then the test sample solution is prepared by constant volume;

[0012] S2, preparing a first control solution: a proper amount of DSPE, 1-StePE-MPEG and DSPE-MPEG2000 control samples are weighed and dissolved in a mixed solvent of chloroform-methanol-water, and then the first control solution is prepared by constant volume;

[0013] S3, preparing a first reference solution: a proper amount of DSPE-MPEG2000 is weighed and dissolved in the first control solution prepared in step S2, and then the first reference solution is prepared by constant volume;

[0014] S4, thin layer chromatography: 10 μl of the test sample solution, the first control solution and the first reference solution are taken respectively and spotted on a thin layer plate, and then the thin layer plate is developed in a first developing agent, and the thin layer plate is taken out, preheated and treated, and then cooled to room temperature, sprayed with a first color developing agent, heated and treated, and visually inspected, and the color depth of the spots on the test sample solution thin layer plate is compared with that of the spots on the first control solution thin layer plate.

[0015] In a preferred embodiment of the application, the detection of the impurity MPEG2000 is further included, and the steps are as follows:

[0016] S2-1, preparing a second control solution: a proper amount of MPEG2000 control sample is weighed and dissolved in a mixed solvent of chloroform-methanol-water, and then the second control solution is prepared by constant volume;

[0017] S3-1, preparing a second reference solution: a proper amount of DSPE-MPEG2000 is weighed and dissolved in the second control solution prepared in step S2-1, and then the second reference solution is prepared by constant volume;

[0018] S4-1, thin layer chromatography determination: take 10 μl of the test solution prepared in claim 1, the second control solution and the second reference solution respectively, spot on the thin layer plate, place the thin layer plate in the first developing agent, take out the thin layer plate, preheat, cool to room temperature after treatment, spray the second color developing agent, visual inspection, compare the color depth of the spots on the test solution thin layer plate with the spots on the second control solution thin layer plate.

[0019] In a preferred embodiment of the present application, the detection of impurity SA is also included, and the steps are as follows:

[0020] S2-2, preparation of the third control solution: weigh an appropriate amount of SA control product, dissolve in the mixed solvent of chloroform-methanol-water, and prepare the third control solution by constant volume;

[0021] S3-2, preparation of the third reference solution: weigh an appropriate amount of DSPE-MPEG2000, dissolve in the third control solution prepared in step S2-2, and prepare the third reference solution by constant volume;

[0022] S4-2, thin layer chromatography determination: take 10 μl of the test solution prepared in claim 1, the third control solution and the third reference solution respectively, spot on the thin layer plate, spot, place the thin layer plate in the second developing agent, take out the thin layer plate, dry, spray the first color developing agent, then heat treatment, visual inspection, compare the color depth of the spots on the test solution thin layer plate with the spots on the third control solution thin layer plate.

[0023] In a preferred embodiment of the present application, in S1, the concentration of DSPE-MPEG2000 in the test solution is 40 mg / ml.

[0024] In a preferred embodiment of the present application, in S2, in the first control solution, the concentrations of DSPE, 1-StePE-MPEG and DSPE-MPEG2000 are 0.2 mg / ml, 0.48 mg / ml and 0.41 mg / ml respectively.

[0025] In a preferred embodiment of the present application, the first developing agent is a mixture of chloroform-methanol-water-glacial acetic acid with a volume ratio of 94:14:1.6:0.4.

[0026] In a preferred embodiment of the present application, the first color developing agent is a phosphoric acid solution of copper sulfate.

[0027] In a preferred embodiment of the present application, the second color developing agent is bismuth potassium iodide reagent.

[0028] In a preferred embodiment of the present application, the preheating process has a temperature of 100 DEG C or above and a time of 2-3 minutes.

[0029] In a preferred embodiment of the present application, the heating process has a temperature of 160-170 DEG C and a time of 10-15 minutes.

[0030] The present application has the advantages that the method for detecting impurities in cultured phosphatidylethanolamine has the characteristics of fast detection speed and low cost, can effectively detect various impurities, and can effectively detect impurities with low concentration, has good detection effect, good stability, high accuracy of results, and strong practicability. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is an effect diagram of 1-StePE-MPEG2000, DSPE and DSPE-MPEG2000 control solution, SA control solution, MPEG2000 control solution using the thin layer chromatography condition for detecting DSPE, 1-StePE-MPEG2000 and unknown impurities for thin layer chromatography determination;

[0032] Figure 2 is an effect diagram of the test solution after oxidative damage, alkali damage, acid damage, light damage and heat damage using the thin layer chromatography condition for detecting DSPE, 1-StePE-MPEG2000 and unknown impurities for thin layer chromatography determination;

[0033] Figure 3 is an effect diagram of 1-StePE-MPEG2000, DSPE and DSPE-MPEG2000 control solution, SA control solution, MPEG2000 control solution using the thin layer chromatography condition for detecting MPEG2000 for thin layer chromatography determination;

[0034] Figure 4 is an effect diagram of the test solution after oxidative damage, alkali damage, acid damage, light damage and heat damage using the thin layer chromatography condition for detecting MPEG2000 for thin layer chromatography determination;

[0035] Figure 5 is an effect diagram of 1-StePE-MPEG2000, DSPE and DSPE-MPEG2000 control solution, SA control solution, MPEG2000 control solution using the thin layer chromatography condition for detecting SA for thin layer chromatography determination. DETAILED DESCRIPTION

[0036] The advantages and features of the present application will be more readily understood by those skilled in the art from the detailed description of the preferred embodiments of the present application taken with reference to the accompanying drawings, so as to make the scope of protection of the present application more clear and explicit.

[0037] Referring to Figures 1-5 The embodiments of the present application include:

[0038] Embodiment 1

[0039] A detection method of DSPE, 1-StePE-MPEG2000, MPEG2000, SA and unknown impurities in phosphatidylethanolamine

[0040] The specific detection method is thin layer chromatography, and the specification of the thin layer plate used is: TLC Silica gel 60 F254, Merck.

[0041] The developing agent and color developing agent used include:

[0042] The first developing agent: a mixed solution of chloroform-methanol-water-glacial acetic acid with a volume ratio of 94:14:1.6:0.4.

[0043] The second developing agent: a mixed solution of n-hexane-diethyl ether-glacial acetic acid with a volume ratio of 70:30:1.

[0044] The first color developing agent: a phosphoric acid solution of copper sulfate, which is prepared by weighing 10 g of anhydrous copper sulfate, dissolving it in 8 ml of phosphoric acid and 100 ml of water.

[0045] The second color developing agent: bismuth potassium iodide (Dragendorff's) reagent, which is prepared immediately before use, and the specific preparation method is: weighing 0.85 g of bismuth subnitrate, dissolving it in 10 ml of acetic acid and 40 ml of water, and shaking vigorously, as solution A; weighing 8 g of potassium iodide, dissolving it in 20 ml of water, as solution B; taking 4 ml of the mixed solution of solution A and solution B in equal proportions, and adding 20 ml of dilute acetic acid solution with a mass concentration of 20%.

[0046] The specific detection steps are as follows:

[0047] First step, preparation of test solution:

[0048] An appropriate amount of DSPE-MPEG2000 is dissolved in a mixed solvent of chloroform-methanol (volume ratio 1:1), and the volume is adjusted to prepare a test solution with a DSPE-MPEG2000 concentration of 40 mg / ml.

[0049] Second step, preparation of control solution:

[0050] Preparation of the first control solution: a certain amount of DSPE control, 1-StePE-MPEG control and DSPE-MPEG2000 control were dissolved in chloroform-methanol-water (volume ratio of 65:25:4) mixed solvent, and the first control solution was prepared by constant volume. In the first control solution, the concentrations of DSPE, 1-StePE-MPEG and DSPE-MPEG2000 were 0.2 mg / ml, 0.48 mg / ml and 0.41 mg / ml respectively, which were equivalent to 0.5%, 1.2% and 1.0% of the corresponding substance concentrations in the test solution.

[0051] Preparation of the second control solution: a certain amount of MPEG2000 control was dissolved in chloroform-methanol-water (volume ratio of 65:25:4) mixed solvent, and the second control solution with a concentration of 0.52 mg / ml of MPEG2000 was prepared by constant volume. In the second control solution, the concentration of MPEG2000 was equivalent to 1.3% of the corresponding substance content in the test solution.

[0052] Preparation of the third control solution: a certain amount of SA control was dissolved in chloroform-methanol-water (volume ratio of 65:25:4) mixed solvent, and the third control solution with a concentration of 0.20 mg / ml of SA was prepared by constant volume. In the third control solution, the concentration of SA was equivalent to 0.5% of the corresponding substance content in the test solution.

[0053] Step 3, preparation of reference solution:

[0054] Preparation of the first reference solution: a certain amount of DSPE-MPEG2000 was dissolved in the first control solution, and the first reference solution was prepared by constant volume. In the first reference solution, the concentration of DSPE-MPEG2000 was 40 mg / ml.

[0055] Preparation of the second reference solution: a certain amount of DSPE-MPEG2000 was dissolved in the second control solution, and the second reference solution was prepared by constant volume. In the second reference solution, the concentration of DSPE-MPEG2000 was 40 mg / ml.

[0056] Preparation of the third reference solution: a certain amount of DSPE-MPEG2000 was dissolved in the third control solution, and the third reference solution was prepared by constant volume. In the third reference solution, the concentration of DSPE-MPEG2000 was 40 mg / ml.

[0057] Step 4, detection:

[0058] A, detection of DSPE, 1-StePE-MPEG2000 and unknown impurities

[0059] Take 10 μl of the above test sample solution, the first control solution and the first reference solution respectively, and spot them on the thin layer plate. Then, place the thin layer plate in the above first developing agent for development. Take out the thin layer plate, heat it above 100°C for 2-3 minutes, cool it to room temperature, spray the above first color developing agent, and then heat it at 160-170°C for 10-15 minutes until all the color developing spots appear. Visual inspection shows that if the color depth of the impurity spots is as deep or shallower than that of the corresponding spots of the control solution, then the content of DSPE in the sample DSPE-MPEG2000 to be detected is not more than 0.5%, the content of 1-StePE-MPEG is not more than 1.2%, and the content of unknown impurities is not more than 1.0%.

[0060] B. Detection of MPEG2000 impurities

[0061] Take 10 μl of the above test sample solution, the second control solution and the second reference solution respectively, and spot them on the thin layer plate. Place the thin layer plate in the above first developing agent for development. Take out the thin layer plate, heat it above 100°C for 2-3 minutes, cool it to room temperature, spray the second color developing agent, and then heat it at 160-170°C for 10-15 minutes until all the color developing spots appear. Visual inspection shows that if the color depth of the impurity spots is as deep or shallower than that of the corresponding spots of the control solution, then the content of MPEG2000 impurities in the sample DSPE-MPEG2000 to be detected is not more than 1.3%.

[0062] C. Detection of SA impurities

[0063] Take 10 μl of the above test sample solution, the third control solution and the third reference solution respectively, and spot them on the thin layer plate. After spotting, place the thin layer plate in the second developing agent for development. Take out the thin layer plate, dry it, spray the above first color developing agent, and then heat it at 160-170°C for 10-15 minutes until all the color developing spots appear. Visual inspection shows that if the color depth of the impurity spots is as deep or shallower than that of the corresponding spots of the control solution, then the content of SA impurities in the sample DSPE-MPEG2000 to be detected is not more than 0.5%.

[0064] The result is determined as follows. If the color of the above impurity spots is not deeper than that of the corresponding control spots, then the purity of the sample DSPE-MPEG2000 to be detected meets the requirements. Otherwise, the purity does not meet the requirements.

[0065] The performance of the detection method is analyzed as follows.

[0066] (1) First, the solutions are prepared as follows.

[0067] DSPE control solution: take DSPE, accurately weighed, dissolved in chloroform-methanol-water (volume ratio of 65:25:4) as the solvent, and diluted to make a solution containing 0.2 mg of DSPE per 1 ml.

[0068] 1-StePE-MPEG2000 control solution: take 1-StePE-MPEG2000, accurately weighed, dissolved in chloroform-methanol-water (volume ratio of 65:25:4) as the solvent, and diluted to make a solution containing 0.48 mg of 1-StePE-MPEG2000 per 1 ml.

[0069] DSPE-MPEG2000 control solution: take DSPE-MPEG2000, accurately weighed, dissolved in chloroform-methanol-water (volume ratio of 65:25:4) as the solvent, and diluted to make a solution containing 0.4 mg of DSPE-MPEG2000 per 1 ml.

[0070] MPEG2000 control solution: take MPEG2000, accurately weighed, dissolved in chloroform-methanol (volume ratio of 1:1) as the solvent, and diluted to make a solution containing 0.52 mg of MPEG2000 per 1 ml.

[0071] SA control solution: take SA, accurately weighed, dissolved in chloroform-methanol (volume ratio of 1:1) as the solvent, and diluted to make a solution containing 0.2 mg of SA per 1 ml.

[0072] Test solution: take DSPE-MPEG2000 sample, accurately weighed, dissolved in chloroform-methanol (volume ratio of 1:1) as the solvent to make a solution containing 40 mg of DSPE-MPEG2000 per 1 ml.

[0073] Three impurities (1-StePE-MPEG2000, DSPE and DSPE-MPEG2000) control solution: accurately take 1-StePE-MPEG2000, DSPE and DSPE-MPEG2000, dissolved in chloroform-methanol-water (volume ratio of 65:25:4) as the solvent to make a solution containing 0.48 mg of 1-StePE-MPEG2000, 0.2 mg of DSPE, 0.4 mg of DSPE-MPEG2000 per 1 ml, as the three impurities control solution.

[0074] Three impurities control reference solution: take DSPE-MPEG2000 sample, accurately weighed, dissolved in three impurities control solution as the solvent to make a solution containing 40 mg of DSPE-MPEG2000 sample per 1 ml, as the three impurities control reference solution.

[0075] MPEG2000 reference solution: take DSPE-MPEG2000 sample, accurately weighed, with MPEG2000 control solution as solvent, prepared into a solution containing 40 mg DSPE-MPEG2000 sample per 1 ml, as MPEG2000 reference solution.

[0076] SA reference solution: take DSPE-MPEG2000 sample, accurately weighed, with SA control solution as solvent, prepared into a solution containing 40 mg DSPE-MPEG2000 sample per 1 ml, as SA reference solution.

[0077] (II) Forced degradation method of test solution

[0078] Oxidative destruction: take 0.1 ml of test solution, add 1 μl of 30% H2O2, mix well, react at room temperature for 1 hour, and reserve.

[0079] Alkaline destruction: take 0.1 ml of test solution, add 1 μl of 0.1 mol / L NaOH solution, mix well, react at room temperature for 1 hour, and reserve.

[0080] Acid destruction: take 0.1 ml of test solution, add 1 μl of 1 mol / L HCL, mix well, react at 60℃ for 10 minutes, and then react at room temperature for 50 minutes, and reserve.

[0081] Light destruction: take 0.2 ml of test solution, place it under 15000 lx light for 24 hours, and reserve.

[0082] Heat destruction: take an appropriate amount of DSPE-MPEG2000 sample and place it at 80℃ for 1 hour, accurately weigh the DSPE-MPEG2000 sample after heat destruction, accurately weigh, with chloroform-methanol (1:1) as solvent, prepared into a solution containing 40 mg per 1 ml, and reserve.

[0083] (III) Specificity verification

[0084] Take 10 μl of the above prepared 1-StePE-MPEG2000, DSPE and DSPE-MPEG2000 control solution, SA control solution, MPEG2000 control solution and point them on the same point of the thin layer plate, and determine them by the thin layer chromatography conditions for detecting DSPE, 1-StePE-MPEG2000 and unknown impurities, and the specificity test results of impurity inspection system are shown in Figure 1 .

[0085] Results and analysis: from Figure 1It can be seen that in the three impurity (1-StePE-MPEG2000, DSPE and DSPE-MPEG2000) inspection systems, the Rf values of DSPE and 1-StePE-MPEG2000 are different from the Rf value of DSPE-MPEG2000, and do not overlap with the Rf value of DSPE-MPEG2000, wherein the resolution of 1-StePE-MPEG2000 and DSPE, and the resolution of DSPE-MPEG2000 and 1-StePE-MPEG2000 are all greater than 1. SA is above the main spot, and MPEG2000 does not develop color under this inspection system, so SA and MPEG2000 do not interfere with the inspection of DSPE and 1-StePE-MPEG2000 and unknown impurities.

[0086] The test sample solutions subjected to oxidative destruction, alkaline destruction, acid destruction, light destruction and heat destruction are tested by spotting using the thin layer chromatography conditions for detecting DSPE, 1-StePE-MPEG2000 and unknown impurities, and the results are shown in Table 2. Figure 2 Figure 2 It can be seen that acid destruction and alkaline destruction can destroy a small amount of 1-StePE-MPEG2000 and unknown impurities, and the destroyed unknown impurities are well separated from the main spot. Therefore, the inspection method for DSPE, 1-StePE-MPEG2000 and unknown impurities is good in specificity.

[0087] 10 μl of each of the above-prepared 1-StePE-MPEG2000, DSPE and DSPE-MPEG2000 control solutions, SA control solution and MPEG2000 control solution is spotted on the same point of a thin layer plate, and the spotting test is performed using the thin layer chromatography conditions for detecting MPEG2000, and the results are shown in Table 3. Figure 3 Figure 3 It can be seen that in the MPEG2000 inspection system, the Rf value of MPEG2000 does not overlap with the Rf value of the main spot, and the resolution with the main spot is greater than 1. SA and DSPE do not develop color under this inspection condition, and the developed color spots of 1-StePE-MPEG2000 and MPEG2000 are not adjacent, so SA, DSPE and 1-StePE-MPEG2000 do not interfere with the inspection of the impurity MPEG2000.

[0088] The test sample solutions subjected to oxidative destruction, alkaline destruction, acid destruction, light destruction and heat destruction are tested by spotting using the thin layer chromatography conditions for detecting MPEG2000, and the results are shown in Table 4. Figure 4 Figure 4 It can be seen that acid destruction can destroy 1-StePE-MPEG2000, and alkaline destruction can destroy MPEG2000. Therefore, the inspection method for MPEG2000 is good in specificity. ​​​

[0089] The 1-StePE-MPEG2000, DSPE and DSPE-MPEG2000 control solutions, SA control solution and MPEG2000 control solution prepared above were each spotted 10 μl on a thin layer plate, and the spotting test was performed under the thin layer chromatography conditions for detecting SA, and the results are shown in Table 1. Figure 5 Figure 5 It can be seen that under the SA checking system, the Rf value of SA does not overlap with the Rf value of the main spot, and the separation degree of the main spot is large. MPEG2000 does not develop color under the checking condition, and the Rf value of 1-StePE-MPEG2000 and DSPE is 0, so MPEG2000, DSPE and 1-StePE-MPEG2000 do not interfere with the checking of the impurity SA.

[0090] The test sample solutions after oxidative destruction, alkali destruction, acid destruction, light destruction and heat destruction were subjected to spotting test under the thin layer chromatography conditions for detecting SA, and unknown impurities were destroyed in all the test sample solutions. The alkali destruction can destroy obvious unknown impurities and SA, and the unknown impurities do not interfere with the checking of SA. Therefore, the checking method of SA is good in specificity.

[0091] (IV) Detection limit of each impurity

[0092] The 1-StePE-MPEG2000, DSPE and DSPE-MPEG2000 control solutions, MPEG2000 control solution and SA control solution were diluted step by step, and the diluted solutions were subjected to thin layer chromatography test until the spots of each component were almost not obvious. The specific dilution concentration changes and detection limit concentrations of each impurity are shown in the following table.

[0093]

[0094] (V) Durability

[0095] A. The influence of the organic phase ratio of the mobile phase on the checking of impurities was investigated, and the detection method was that the developing agent was chloroform-methanol-water-glacial acetic acid (volume ratio 92:16:1.2:0.4).

[0096] The test results showed that each impurity was well separated from the main spot and was not affected by the developing agent.

[0097] B. The influence of the thin layer plate on the detection of impurities was investigated, and the detection method was that the Merck thin layer plate was replaced by a domestic thin layer plate (G254, Qingdao Bangkai).

[0098] The test results showed that each impurity was well separated from the main spot and was not affected by the thin layer plate.

[0099] ​From the above verification, the application can be used for detecting each related substance of the cultured phosphatidylethanolamine, and the test result is stable and reliable, and each index meets the relevant requirements.

[0100] The above is only an embodiment of the application, and does not limit the patent scope of the application, and any equivalent structure or equivalent process transformation using the content of the application specification and drawings, or direct or indirect application in other related technical fields, are also included in the patent protection scope of the application.

Claims

1. A method for detecting impurities in lecithin, characterized in that, The detection of DSPE, 1-StePE-MPEG and unknown impurities includes the following steps: S1, preparing a test solution: a certain amount of DSPE-MPEG2000 is weighed and dissolved in a mixed solvent of chloroform-methanol-methanol, and then diluted to prepare a test solution; S2, preparing a first control solution: a certain amount of DSPE, 1-StePE-MPEG and DSPE-MPEG2000 control samples are weighed and dissolved in a mixed solvent of chloroform-methanol-water, and then diluted to prepare a first control solution; S3, preparing a first reference solution: a certain amount of DSPE-MPEG2000 is weighed and dissolved in the first control solution prepared in step S2, and then diluted to prepare a first reference solution; S4, thin layer chromatography determination: 10 μL of the test solution, the first control solution and the first reference solution are taken respectively and spotted on a thin layer plate, then the thin layer plate is placed in a first developing agent for development, the thin layer plate is taken out, preheated and treated, after the treatment is completed, the thin layer plate is cooled to room temperature, then a first color developing agent is sprayed, and then heated and treated, and the color depth of the spots on the test solution thin layer plate is compared with that of the first control solution thin layer plate; The first developing agent is a mixture of chloroform-methanol-water-glacial acetic acid with a volume ratio of 94:14:1.6:0.4; The first color developing agent is a copper sulfate phosphoric acid solution.

2. The method for detecting impurities in cultured phosphatidylethanolamine according to claim 1, characterized in that, The detection of impurity MPEG2000 also includes the following steps: S2-1, preparing a second control solution: a certain amount of MPEG2000 control sample is weighed and dissolved in a mixed solvent of chloroform-methanol-water, and then diluted to prepare a second control solution; S3-1, preparing a second reference solution: a certain amount of DSPE-MPEG2000 is weighed and dissolved in the second control solution prepared in step S2-1, and then diluted to prepare a second reference solution; S4-1, thin layer chromatography determination: 10 μL of the test solution, the second control solution and the second reference solution prepared in claim 1 are taken respectively and spotted on a thin layer plate, then the thin layer plate is placed in the first developing agent for development, the thin layer plate is taken out, preheated and treated, after the treatment is completed, the thin layer plate is cooled to room temperature, a second color developing agent is sprayed, and then heated and treated, and the color depth of the spots on the test solution thin layer plate is compared with that of the second control solution thin layer plate; The second color developing agent is a bismuth potassium iodide reagent.

3. The method for detecting impurities in cultured phosphatidylethanolamine according to claim 1, characterized in that, The detection of impurity SA also includes the following steps: S2-2, preparing a third control solution: a certain amount of SA control sample is weighed and dissolved in a mixed solvent of chloroform-methanol-water, and then diluted to prepare a third control solution; S3-2, preparing a third reference solution: a certain amount of DSPE-MPEG2000 is weighed and dissolved in the third control solution prepared in step S2-2, and then diluted to prepare a third reference solution; S4-2, thin layer chromatography determination: take 10 μl of the test solution prepared in claim 1, the third control solution and the third reference solution respectively, and spot on the thin layer plate, after spotting, place the thin layer plate in the second developing agent, take out the thin layer plate, dry, spray the first color developing agent, then heat treatment, visual inspection, compare the color depth of the spots on the test solution thin layer plate with the spots on the third control solution thin layer plate; The second developing agent is a mixture of n-hexane-ethyl ether-glacial acetic acid mixed in a volume ratio of 70:30:

1.

4. The method for detecting impurities in cultured phosphatidylethanolamine according to claim 1, characterized in that, In S1, the concentration of DSPE-MPEG2000 in the test solution is 40 mg / ml.

5. The method for detecting impurities in cultured phosphatidylethanolamine according to claim 1, characterized in that, In S2, in the first control solution, the concentrations of DSPE, 1-StePE-MPEG and DSPE-MPEG2000 are 0.2 mg / ml, 0.48 mg / ml and 0.41 mg / ml respectively.

6. The method for detecting impurities in cultured phosphatidylethanolamine according to claim 1 or 2, characterized in that, The process conditions of the preheating treatment are: temperature above 100℃, time 2-3 minutes.

7. The method for detecting impurities in cultured phosphatidylethanolamine according to claim 1 or 3, characterized in that, The process conditions of the heating treatment are: temperature 160-170℃, time 10-15 minutes.

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