Method for determination of related substances of phosphatidylethanolamine by high performance liquid chromatography

Abstract

The present invention provides a method for detecting related substances of functional excipient DSPE-MPEG by high performance liquid chromatography, wherein the chromatographic conditions are as follows: ZORBAX300SB-C18, specification: 4.6mm×150mm×5μm as chromatographic column; 20mmol / L ammonium formate solution as mobile phase A, a mixed solvent of methanol and tetrahydrofuran with a volume ratio of (95-90):(5-10) as mobile phase B, and mobile phases A and B are mixed in a certain proportion for gradient elution. The detection method provided by the present invention can quickly and accurately detect the content of related substances contained in DSPE-MPEG, thereby accurately evaluating the quality of excipients, providing assistance for the screening of liposome pharmaceutical excipients, and having practical promotion and application value.

Description

Technical Field

[0001] The invention belongs to the field of medical detection, and in particular relates to a method for detecting related substances in functional auxiliary material cultured phosphatidylethanolamine. Background Art

[0002] Pharmaceutical excipients are an important component of pharmaceutical preparations. They are the material basis for the production and use of pharmaceutical preparations and determine the performance, safety, effectiveness and stability of pharmaceutical preparations. As a functional pharmaceutical excipient for the preparation of liposome preparations, Peihua phosphatidylethanolamine is a phosphatidylethanolamine compound (DSPE) modified by polyethylene glycol (PEG). Because the methoxyl end capping of the PEG end makes the PEG part inactive and unable to continue to react, Peihua phosphatidylethanolamine is also referred to as (MPEG-DSPE). MPEG-DSPE is an amphiphilic molecule with glycerol as the skeleton. It has the dual characteristics of the amphiphilicity of phospholipids and the hydrophilic polymer of PEG. Phospholipid molecules are arranged in a directional manner in aqueous solution to form a stable bilayer structure. PEGylated phospholipids can more effectively protect liposomes, shield the recognition and uptake of liposomes by the reticuloendothelial system, prolong the circulation time of liposomes in the body, and improve the physical, chemical and biological stability of liposomes. Peihua phosphatidylethanolamine pharmaceutical excipients have been listed in many countries, including China, the United States, Japan, Canada, Germany, and Switzerland.

[0003] Based on the importance and wide application of MPEG-DSPE, it is extremely important to evaluate the quality of MPEG-DSPE. At present, the method that can evaluate the quality of MPEG-DSPE is the "Draft of the Standard for Phosphatidylethanolamine Pharmaceutical Excipients" issued by the Chinese Pharmacopoeia Committee. It uses semi-quantitative thin-layer chromatography to check the related substances of polyethylene glycol monomethyl ether, lysophosphatidylethanolamine, free fatty acids and phosphatidylethanolamine, and at the same time, the limits of the four related substances must be ≤0.5%; but this method can only qualitatively evaluate the quality of MPEG-DSPE, and cannot accurately judge the difference between product quality. Furthermore, the inventor tried to use the thin-layer chromatography method in the public draft to perform a limit check on DSPE and free fatty acids in the MPEG-DSPE test sample. The results showed that its specificity was poor and it was impossible to accurately quantify impurities, and it was impossible to accurately evaluate the quality of the product.

[0004] Therefore, there is an urgent need for a detection method that can quickly and accurately detect related substances in DSPE-MPEG. Summary of the invention

[0005] The present invention aims to provide a method for rapidly and accurately detecting the contents of methoxy polyethylene glycol, lysophosphatidylethanolamine, stearic acid and phosphatidylethanolamine-related substances in the pharmaceutical excipient cultured phosphatidylethanolamine.

[0006] To achieve the above object, the present invention provides a method for detecting related substances in phosphatidylethanolamine, wherein the high performance liquid chromatography method uses a chromatographic column with octadecylsilane bonded silica gel as a filler, and gradient elution is performed with solution A and organic solvent B in a certain proportion;

[0007] Furthermore, the chromatographic column of the present invention is ZORBAX 300SB-C18, with specifications of 4.6 mm×150 mm×5 μm or a chromatographic column with equivalent performance;

[0008] Furthermore, the solution A described in the present invention is a 20mmol / L ammonium formate solution;

[0009] Furthermore, the organic solvent B described in the present invention is a mixed solvent of methanol and tetrahydrofuran in a volume ratio of (95-90):(5-10);

[0010] Preferably, the organic solvent B is a mixed solvent of methanol and tetrahydrofuran in a volume ratio of 92:8;

[0011] Furthermore, the present invention uses solution A and organic solvent B mixed in a certain ratio as a mobile phase for gradient elution, and the gradient ratio is:

[0012] 0min, the volume ratio of solution A to organic solvent B was 50:50;

[0013] 10 min, the volume ratio of solution A to organic solvent B was 28:72;

[0014] 50 min, the volume ratio of solution A to organic solvent B was 0:100;

[0015] 50.1min, the volume ratio of solution A to organic solvent B was 50:50;

[0016] 60 min, the volume ratio of solution A to organic solvent B was 50:50;

[0017] Further, the chromatographic condition flow rate of the present invention is 0.8 to 1.2 mL / min;

[0018] Preferably, the flow rate is 1 mL / min;

[0019] Furthermore, the chromatographic conditions of the present invention have an injection volume of 10 to 20 μl;

[0020] Preferably, the injection volume is 20 μl;

[0021] Furthermore, the chromatographic condition column temperature of the present invention is 20 to 80°C;

[0022] Preferably, the column temperature is 50°C;

[0023] Further, the chromatographic condition detector of the present invention is CAD;

[0024] Further, the chromatographic conditions of the present invention have a nebulizer temperature of 35°C;

[0025] Furthermore, the detection method of the present invention comprises solution preparation: dissolving the cultured phosphatidylethanolamine sample and the related substance reference substance in different solvents respectively, injecting into the chromatograph and recording the chromatogram;

[0026] Furthermore, the phosphatidylethanolamine solution of the present invention is prepared by dissolving in methanol;

[0027] Furthermore, the reference solution of the phosphatidylethanolamine-related substances of the present invention is prepared by dissolving in a mixed solvent of chloroform and methanol in a volume ratio of 7:3;

[0028] Furthermore, the related substances of the phosphatidylethanolamine of the present invention are selected from one or more of methoxy polyethylene glycol, lysophosphatidylethanolamine, stearic acid and phosphatidylethanolamine;

[0029] Furthermore, the detection described in the present invention is qualitative and / or quantitative detection;

[0030] Furthermore, the quantitative detection is to calculate the content of impurities in the cultured phosphatidylethanolamine test solution by a standard curve method.

[0031] According to some specific embodiments of the present invention, the detection method comprises the following steps:

[0032] (1) Test solution: Take an appropriate amount of phosphatidylethanolamine, dissolve it in methanol and dilute it to make a solution containing about 10 mg of phosphatidylethanolamine per 1 ml;

[0033] (2) Reference substance stock solution: Take appropriate amounts of methoxypolyethylene glycol, lysophosphatidylethanolamine, stearic acid and phosphatidylethanolamine reference substances, respectively, add appropriate amounts of chloroform-methanol (volume ratio 7:3) to dissolve and dilute to make a solution containing approximately 0.5 mg of each of methoxypolyethylene glycol, lysophosphatidylethanolamine, stearic acid and phosphatidylethanolamine per 1 ml;

[0034] (3) Reference substance solution: Take 0.2 ml, 0.5 ml, 0.8 ml, 1.0 ml, 1.5 ml and 2.0 ml of the reference substance stock solution and place them in 6 10 ml volumetric flasks respectively, dilute to the mark with methanol and shake well;

[0035] (4) Setting chromatographic conditions: The chromatographic column used was ZORBAX 300SB-C18, with specifications of 4.6 mm×150 mm×5 μm; 20 mmol / L ammonium formate solution was used as mobile phase A, and a mixed solvent of methanol and tetrahydrofuran in a volume ratio of (95-90):(5-10) was used as mobile phase B. Mobile phases A and B were mixed in a certain proportion for gradient elution, and the gradient ratio was: 0-10 min, 50%→28% A; 10-50 min, 28%→0% A; 50-50.1 min, 0%→50% A; 50.1-60 min, 50% A; flow rate was 1 mL / min; injection volume was 20 μl; column temperature was 50° C.; detector was CAD; nebulizer temperature was 35° C.;

[0036] (5) Take 20 μl of each solution from step (1) and (3), inject them into a liquid chromatograph, and record the chromatograms; use the concentration and peak area of ​​the six different concentrations of the reference solution to make a linear regression equation, and calculate the impurity content in the test solution by the standard curve method.

[0037] The present invention has the following beneficial effects:

[0038] The present invention provides a method for detecting related substances in MPEG-DSPE, which can achieve qualitative and quantitative detection of methoxypolyethylene glycol, lysophosphatidylethanolamine, stearic acid and phosphatidylethanolamine related substances contained in MPEG-DSPE, and has the advantages of high accuracy, high sensitivity and good specificity. The detection method provided by the present invention can quickly and accurately detect the content of related substances contained in DSPE-MPEG, so as to accurately evaluate the quality of excipients, provide assistance for the screening of liposome pharmaceutical excipients, and has practical promotion and application value.

[0039] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0040] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The chromatographic column in Example 1 of the present invention is ChromCore C1, and the chromatogram is obtained after the system suitability solution is injected into HPLC;

[0042] Figure 2The chromatographic column in Example 1 of the present invention is ACE5 C4-300, and the chromatogram is obtained after injecting the system suitability solution into HPLC;

[0043] Figure 3 In Example 1 of the present invention, the chromatographic column is Poroshell 120EC-C18, and the chromatogram is obtained after the system suitability solution is injected into HPLC;

[0044] Figure 4 In Example 1 of the present invention, the chromatographic column is ZORBAX SB-C18, and the chromatogram is obtained after the system suitability solution is injected into HPLC;

[0045] Figure 5 In Example 1 of the present invention, the chromatographic column is ZORBAX 300SB-C18, and the chromatogram is obtained after the system suitability solution is injected into HPLC;

[0046] Figure 6 The chromatographic condition in Example 2 of the present invention is that the mobile phase B is acetonitrile, and the chromatogram is obtained after the system suitability solution is injected into HPLC;

[0047] Figure 7 The chromatographic condition in Example 2 of the present invention is that the mobile phase B is methanol, and the chromatogram is obtained after the system suitability solution is injected into HPLC;

[0048] Figure 8 The chromatographic condition mobile phase B in Example 2 of the present invention is a mixed solvent of methanol and tetrahydrofuran in a volume ratio of 95:5, and the system suitability solution is injected into HPLC to obtain a chromatogram;

[0049] Fig. 9 The chromatographic condition mobile phase B in Example 2 of the present invention is a mixed solvent of methanol and tetrahydrofuran in a volume ratio of 90:10, and the system suitability solution is injected into HPLC to obtain a chromatogram;

[0050] Fig.10 The chromatographic condition gradient elution program in Example 3 of the present invention is gradient elution 1, and the chromatogram is obtained after injecting the system suitability solution into HPLC;

[0051] Fig.11 The chromatographic condition gradient elution program in Example 3 of the present invention is gradient elution 2, and the chromatogram is obtained after injecting the system suitability solution into HPLC;

[0052] Fig.12 The chromatographic condition gradient elution program in Example 3 of the present invention is gradient elution 3, and the chromatogram is obtained after injecting the system suitability solution into HPLC;

[0053] Fig.13 is the HPLC detection chromatogram of the blank solution in Example 4 of the present invention;

[0054] Fig.14 is the HPLC detection chromatogram of the test solution in Example 4 of the present invention;

[0055] Fig.15 is a thin layer chromatogram of phosphatidylethanolamine in Comparative Example 1 of the present invention;

[0056] Fig.16 It is the stearic acid thin layer chromatogram in Comparative Example 2 of the present invention. DETAILED DESCRIPTION

[0057] The present invention is completed based on the following findings of the inventors: In order to accurately evaluate the quality of the auxiliary material DSPE-MPEG product, it is necessary to control the content of the functional auxiliary material DSPE-MPEG related substances (≤0.5%), so there is a need for effective separation and detection of DSPE-MPEG and its related substances. However, it is found in the analysis and detection process that not only the related substances and the main component DSPE-MPEG need to be well separated, but also the related substances need to be effectively separated from the adjacent unknown impurity peaks, and at the same time, the peak shape of the related substances must be ensured to achieve accurate quantification. To this end, the inventors fully investigated the detection conditions. First, the chromatographic column was screened, and it was found that the octadecylsilane bonded silica gel column with a pore size of 300A and a particle size of 5μm was the best chromatographic column. Secondly, the types of mobile phase B solvents were screened, and it was found that a mixed solvent of methanol and tetrahydrofuran with a volume ratio of 92:8 was the best mobile phase B. Finally, the mobile phase gradient elution program was screened, and the best gradient ratio was obtained: 0-10min, 50%→28% A; 10-50min, 28%→0% A; 50-50.1min, 0%→50% A; 50.1-60min, 50% A, where A is 20mmol / L ammonium formate solution. Combining the above optimal conditions can not only effectively separate the main component DSPE-MPEG and its related substances, but also the related substances and other adjacent impurities can be well separated and the peak shape of the related substances can be guaranteed, thereby realizing the accurate determination of the content of related substances in DSPE-MPEG.

[0058] The raw materials and equipment used in the specific embodiments of the present invention are all known products and are obtained through commercial purchase.

[0059] In the present invention, the abbreviations involved have the following meanings:

[0060] Phosphatidylethanolamine: MPEG-DSPE or DSPE-MPEG

[0061] Polyethylene glycol monomethyl ether or methoxypolyethylene glycol: mPEG

[0062] Lysophosphatidylethanolamine: LPC

[0063] Free fatty acid or stearic acid: FFA S

[0064] Phosphatidylethanolamine: DSPE

[0065] CAD: Charged Aerosol Detector

[0066] LPC contains a chiral carbon. When the HPLC column is filled with octadecylsilane bonded silica gel, LPC will have two peaks, and its total peak area is the sum of the two LPC peak areas. Therefore, the LPC linear equation is calculated by linear regression of the sum of the two LPC peak areas on the injection concentration.

[0067] Ratio of migration: In thin layer chromatography, timed development is generally used, that is, the distance that the component and the developing agent migrate in the same development time. The ratio of the component's migration distance (L) to the developing agent's migration distance (L0) is called the ratio of migration, referred to as R. f . It is calculated as follows:

[0068] R f =L / L0=distance from the origin to the center of the spot / distance from the origin to the solvent front.

[0069] Example 1

[0070] 1.1 Chromatographic conditions

[0071] Mobile phase: 20 mmol / L ammonium formate as mobile phase A, methanol-tetrahydrofuran (92:8) as mobile phase B;

[0072] Elution method: Gradient elution, see the table below:

[0073] Time (min) 0 10 50 50.1 60 A％ 50 28 0 50 50 B％ 50 72 100 50 50

[0074] Column temperature: 50°C;

[0075] Injection volume: 20 μl;

[0076] Flow rate: 1ml / min;

[0077] Detector: CAD;

[0078] Atomization chamber temperature: 35°C;

[0079] Running time: 60min;

[0080] Reference substance stock solution: Take appropriate amount of methoxypolyethylene glycol, lysophosphatidylethanolamine, stearic acid and phosphatidylethanolamine reference substances respectively, add appropriate amount of chloroform-methanol (volume ratio 7:3) to dissolve and dilute to make a solution containing about 0.5 mg of methoxypolyethylene glycol, lysophosphatidylethanolamine, stearic acid and phosphatidylethanolamine per 1 ml.

[0081] System suitability solution: Take about 100 mg of the cultured phosphatidylethanolamine reference substance, accurately weigh it, place it in a 10 ml volumetric flask, add 1 ml of the reference substance stock solution, add methanol to dissolve and dilute to the scale, and shake well.

[0082] Experimental method: The liquid chromatography columns used were ChromCore C1, specification: 4.6mm×250mm×5μm; ACE5C4-300, specification: 4.6×250mm×5μm; Poroshell 120EC-C18 (pore size ), specifications: 4.6mm×150mm×4μm; ZORBAX SB-C18 (aperture ), specifications: 4.6mm×150mm×3.5μm; ZORBAX 300SB-C18 (aperture ), specification: 4.6mm×150mm×5μm; or its equivalent chromatographic column; test system suitability solution, inject and test according to the above test conditions, record the chromatogram, the results are shown in Table 1 below:

[0083] Table 1 Screening of different chromatographic columns

[0084]

[0085] Example 2

[0086] Chromatographic column: A ZORBAX 300SB-C18 chromatographic column was selected, with specifications of 4.6 mm×150 mm×5 μm. The types of mobile phase B solvents were screened, and other detection conditions were the same as those in Example 1.

[0087] Test process: Use 20mmol / L ammonium formate as mobile phase A, and use acetonitrile, methanol, a mixed solvent of methanol and tetrahydrofuran with a volume ratio of 95:5, a mixed solvent of methanol and tetrahydrofuran with a volume ratio of 92:8, and a mixed solvent of methanol and tetrahydrofuran with a volume ratio of 90:10 as mobile phase B. Inject the test system suitability solution according to the above-mentioned test conditions and record the chromatogram.

[0088] The results are shown in Table 2 below:

[0089]

[0090]

[0091] Example 3

[0092] Chromatographic column: A ZORBAX 300SB-C18 chromatographic column was selected, with specifications of 4.6 mm×150 mm×5 μm; a gradient elution program was selected, and other detection conditions were the same as those in Example 1.

[0093] Test process: The system suitability solution was tested according to the gradient elution 1 to 4 elution programs respectively, and the sample was injected and tested according to the above-mentioned test conditions, and the chromatogram was recorded.

[0094] Gradient elution 1: 0-5 min, 20% A; 5-25 min, 20%→5% A; 25-25.1 min, 5%→20% A; 25.1-35 min, 20% A.

[0095] Gradient elution 2: 0-10 min, 60%→28% A; 10-50 min, 28%→0% A; 50-50.1 min, 0%→60% A; 50.1-60 min, 60% A.

[0096] Gradient elution 3: 0-10 min, 55%→28% A; 10-50 min, 28%→0% A; 50-50.1 min, 0%→55% A; 50.1-60 min, 55% A.

[0097] Gradient elution 4: 0-10 min, 50%→28% A; 10-50 min, 28%→0% A; 50-50.1 min, 0%→50% A; 50.1-60 min, 50% A.

[0098] The results are shown in Table 3 below:

[0099]

[0100] Example 4

[0101] Chromatographic column: a chromatographic column using octadecylsilane bonded silica gel as a filler. In the embodiment of the present invention, a ZORBAX 300SB-C18 chromatographic column is specifically selected, with specifications of 4.6 mm×150 mm×5 μm;

[0102] Mobile phase: 20mmol / L ammonium formate is used as mobile phase A, methanol-tetrahydrofuran (92:8) is used as mobile phase B; mobile phases A and B are gradient eluted in a certain ratio, the specific ratio is as follows:

[0103] 0~10min, 50%→28% A; 10~50min, 28%→0% A; 50~50.1min, 0%→50%A; 50.1~60min, 50%A.

[0104] Column temperature: 50°C;

[0105] Sample chamber temperature: 5°C;

[0106] Injection volume: 20 μl;

[0107] Flow rate: 1ml / min;

[0108] Detector: CAD;

[0109] Atomization chamber temperature: 35°C;

[0110] Running time: 60min;

[0111] Blank solution: Take an appropriate amount of methanol.

[0112] Test solution: Take an appropriate amount of phosphatidylethanolamine, accurately weigh it, dissolve it in methanol and dilute it to make a solution containing about 10 mg per 1 ml.

[0113] Stock solution of reference substances: Take appropriate amount of methoxypolyethylene glycol, lysophosphatidylethanolamine, stearic acid and phosphatidylethanolamine reference substances respectively, add appropriate amount of chloroform-methanol (7:3) to dissolve and dilute to make a solution containing about 0.5 mg of methoxypolyethylene glycol, lysophosphatidylethanolamine, stearic acid and phosphatidylethanolamine per 1 ml.

[0114] Reference substance solution: Take 0.2ml, 0.5ml, 0.8ml, 1.0ml, 1.5ml and 2.0ml of the reference substance stock solution and place them in 6 10ml volumetric flasks respectively, add methanol to dilute to the scale and shake well.

[0115] System suitability solution: Take 100 mg of the cultured phosphatidylethanolamine reference substance, place it in a 10 ml volumetric flask, add 1 ml of the reference substance stock solution, add methanol to dissolve and dilute to the scale, and shake well.

[0116] 4.1 Exclusivity

[0117] Test method: Inject blank solution, test solution, reference solution and system suitability solution into liquid chromatograph respectively and record the chromatogram. Make a linear regression equation based on the concentration and peak area of ​​6 reference solutions with different concentrations and calculate the impurity content in the test solution by the standard curve method.

[0118] Test results: Blank solution ( Fig.13 ) does not interfere with the detection of impurities. System suitability solution ( Figure 5 ) has a resolution of 45.65 between mPEG and adjacent peaks; LPC has two peaks, the first LPC peak has a resolution of 45.65 / 2.92 between adjacent peaks, and the second LPC peak has a resolution of 3.71 / 5.21 between adjacent peaks; FFA SThe separation degree between the adjacent peaks is 3.02 / 25.50; the separation degree between DSPE and the adjacent peaks is 4.46 / 3.89, indicating that the detection method of the present invention has good specificity. The method of the present invention can effectively detect mPEG, LPC, and FFA in cultured phosphatidylethanolamine. S Quantitative detection of DSPE-related substances. Fig.14 ) has a peak area of ​​5.1359. The content of mPEG in the test solution was calculated by the standard curve method to be 2.0%, and the other three related substances were not detected.

[0119] 4.2 Limit of quantification

[0120] Test method:

[0121] Accurately measure 2 ml of the reference stock solution, place it in a 10 ml volumetric flask, dilute to the mark with methanol, and shake well. Dilute step by step to make mPEG, LPC, and FFA S The quantification limit was reached when the peak heights of DSPE and DSPE were both 10 to 50 times of the baseline noise, and this was used as the quantification limit solution.

[0122] Accurately measure 20 μl of the quantitative limit solution, perform the test according to the above test method and chromatographic conditions, and record the chromatogram. The results are shown in the following table:

[0123] Table 4 Results of quantification limits of various related substances

[0124]

[0125]

[0126] Test results: From Table 4, we can see that mPEG, LPC, FFA S The quantitative limits of DSPE and DSPE were 0.1%, 0.1%, 0.01% and 0.01% of DSPE-mPEG, respectively, indicating that the sensitivity of the detection method of the present invention meets the detection requirements.

[0127] 4.3 Linearity

[0128] Test method: Accurately measure an appropriate amount of the reference stock solution and dilute it with methanol into a series of concentration solutions as linear solutions of each component; accurately measure 20 μl of each of the above series of gradient concentration solutions, perform detection according to the above detection method and chromatographic conditions, record the chromatogram, perform linear regression on the peak area Y with concentration X (mg / ml), and calculate the regression equation and correlation coefficient.

[0129] Table 5 Linearity results of various related substances

[0130]

[0131]

[0132] Test results: The results in Table 5 show that mPEG is 0.0103mg / ml~0.1028mg / ml, LPC is 0.0102mg / ml~0.1024mg / ml, and FFA is S In the range of 0.0010mg / ml~0.1021mg / ml and DSPE in the range of 0.0011mg / ml~0.1109mg / ml, the peak area showed a good linear relationship with the measured concentration.

[0133] 4.4 Accuracy

[0134] Test method: Take about 100 mg of the reference substance of phosphatidylethanolamine, weigh it accurately, add 0.5 ml, 1 ml, and 1.5 ml of the reference substance stock solution respectively, place them in 10 ml volumetric bottles, dilute to the scale with methanol, and shake well. Prepare 3 portions in parallel, for a total of 9 portions. Accurately measure 20 μl of each sample and record the chromatogram. The results are shown in the table below:

[0135] Table 6 mPEG accuracy results

[0136]

[0137]

[0138] Table 7 LPC accuracy results

[0139]

[0140] Table 8 FFA S Accuracy results

[0141]

[0142] Table 9 DSPE accuracy results

[0143]

[0144]

[0145] Test results: From Tables 6 to 9, it can be seen that mPEG, LPC, and FFA in phosphatidylethanolamine S The average values ​​of the impurity recoveries of DSPE and DSPE were both within the range of 85% to 110%, and the maximum RSD of the average values ​​of the impurity recoveries was 11.7% (RSD < 15%), indicating that the detection methods of the present invention had good accuracy.

[0146] Comparative Example 1

[0147] Test method: Referring to the thin layer chromatography method of the prior art "Draft Standard Formula of Auxiliary Materials for Phosphatidylethanolamine", the limit test of DSPE and free fatty acids in the MPEG-DSPE test sample was carried out.

[0148] (1) DSPE

[0149] Solvent: chloroform-methanol-water (65:30:4).

[0150] Developing solvent: chloroform-methanol-water (90:18:2).

[0151] Color developer: Take 8 ml of phosphoric acid and place it in a beaker containing about 60 ml of water. Shake well, add 24.4239 g of copper sulfate pentahydrate to dissolve it, and add water to 100 ml to obtain a copper sulfate phosphoric acid solution.

[0152] DSPE reference solution: Weigh 21.10 mg of reference DSPE, place in a 10 ml volumetric flask, add solvent to dissolve and dilute to the scale, shake well, and obtain.

[0153] System suitability solution: Weigh 200.40 mg of the test sample, cultured phosphatidylethanolamine, into a 5 ml volumetric flask, add 0.5 ml of DSPE reference solution, dilute to the mark with solvent, shake well, and the solution is ready.

[0154] Pipette 10 μl of system suitability solution and DSPE reference solution respectively and place them on the same silica gel G 60 The samples were spotted from left to right on the plate, pre-saturated in the developer for 30 min, developed, air-dried, sprayed with copper sulfate phosphoric acid solution, heated at 170°C for 10 min, and taken out for observation.

[0155] Test results: From Fig.15 It can be seen that the R of DSPE in the system suitability solution f The value is d2(3.6cm) / d1(16.5cm)=0.21, and the R f The value is d3(4.7cm) / d1(16.5cm)=0.28, and the R f The values ​​are quite different, indicating that the specificity of the thin layer chromatography method is poor and the spots of DSPE cannot be accurately judged.

[0156] (2) Free fatty acids

[0157] Diluent: chloroform-methanol-water (65:30:4);

[0158] Developing solvent: n-hexane-ether-glacial acetic acid (70:30:1);

[0159] Color developer: the same as the color developer preparation method of "Comparative Example 1 DSPE";

[0160] Free fatty acid stock solution: weigh 49.69 mg of free fatty acid, put it into a 50 ml volumetric flask, add diluent to dissolve and dilute to the scale, shake well, and obtain;

[0161] Free fatty acid solution 1: Accurately measure 2.0 ml of "free fatty acid stock solution" and place it in a 10 ml volumetric bottle. Add diluent to dilute to the mark and shake well to obtain (about 0.2 mg / ml).

[0162] Free fatty acid solution 2: Accurately measure 1.0 ml of "free fatty acid stock solution" and place it in a 10 ml volumetric flask. Add diluent to dilute to the mark and shake well to obtain (about 0.1 mg / ml).

[0163] Free fatty acid solution 3: Accurately measure 0.4 ml of "free fatty acid stock solution" and place it in a 10 ml volumetric flask, add diluent to dilute to the scale, shake well, and obtain (about 0.04 mg / ml);

[0164] Take 10 μl of each of the free fatty acid solutions 1 to 3 and spot from left to right, develop, dry, spray with copper sulfate phosphoric acid solution, and heat at 170°C for 10 minutes.

[0165] Test results: From Fig.16 It can be seen that the size and color depth of the spots of the three free fatty acid solutions 1 to 3 with different concentrations are highly subjective and cannot accurately judge the amount of free fatty acids.

Claims

1. A method for determining related substances of phosphatidylethanolamine by high performance liquid chromatography, characterized in that: The high performance liquid chromatography method uses a chromatographic column with octadecylsilane bonded silica gel as a filler, and performs gradient elution with solution A and organic solvent B in a certain proportion; The solution A is a 20 mmol / L ammonium formate solution, and the organic solvent B is a mixed solvent of methanol and tetrahydrofuran in a volume ratio of (95-90):(5-10); The gradient elution ratio is: 0-10 min, 50%→28% A; 10-50 min, 28%→0% A; 50-50.1 min, 0%→50% A; 50.1-60 min, 50% A; The related substances of the phosphatidylethanolamine include methoxy polyethylene glycol, lysophosphatidylethanolamine, stearic acid and phosphatidylethanolamine.

2. The detection method according to claim 1, characterized in that: The chromatographic column is ZORBAX 300SB-C18, with specifications of 4.6 mm×150 mm×5 μm.

3. The detection method according to claim 1, characterized in that: The organic solvent B is a mixed solvent of methanol and tetrahydrofuran in a volume ratio of 92:

8.

4. The detection method according to claim 1, characterized in that: The chromatographic conditions used meet at least one of the following: flow rate of 0.8-1.2 mL / min; injection volume of 10-20 μl; column temperature of 20-80° C.; detector is CAD.

5. The detection method according to claim 4, characterized in that: The flow rate was 1 mL / min; the injection volume was 20 μl; and the column temperature was 50°C.

6. The detection method according to any one of claims 1 to 5, characterized in that: The cultured phosphatidylethanolamine and its related substances were dissolved in solvents and then tested.

7. The detection method according to claim 6, characterized in that: The solvent for dissolving the purified phosphatidylethanolamine is methanol.

8. The detection method according to claim 6, characterized in that: The solvent for dissolving the phosphatidylethanolamine-related substances is a mixed solvent of chloroform and methanol in a volume ratio of 7:

3.

9. The detection method according to claim 8, characterized in that: The detection is qualitative and / or quantitative detection; the quantitative detection is to calculate the content of related substances in the cultured phosphatidylethanolamine test solution by standard curve method.

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