Method for detecting content of polysorbate 80 in liposome preparation
Through high-performance liquid chromatography and external standard method combined with specific mobile phases and detection conditions, the accuracy of polysorbate 80 detection in liposome preparations was solved, and efficient and accurate quantitative detection effect was achieved.
Patent Information
- Application Number
- CN202510484036.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Priority Date
- 2025-04-11
- Filing Date
- 2025-04-17
- Publication Date
- 2025-07-11
AI Technical Summary
The prior art is difficult to accurately and quantitatively detect polysorbate 80 in liposome preparations, especially in the presence of liposome components and different esterification degrees and polyoxyethylene polymerization degrees, the detection results are seriously disturbed, resulting in inaccurate detection results.
Using high-performance liquid chromatography combined with external standard method, quantitative detection of polysorbate 80 was performed by preparing a standard curve solution, a calibration standard solution and a sample solution to be tested, using an octadecyl silane-bonded silica gel chromatography column, gradient elution and CAD detector, combined with specific mobile phases and detection conditions.
The accurate quantity detection of polysorbate 80 in liposome preparations is achieved, with low operation difficulty, high accuracy and good repeatability, and meets drug safety requirements.
Smart Images
Figure CN120294235A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of detection, and in particular to a method for detecting the content of polysorbate 80 in liposomal preparations. Background Art
[0002] Polysorbate 80 (Tween 80) is a non-ionic surfactant with the chemical name of polyoxyethylene 20 sorbitan monooleate and the molecular formula of C 64 H 124 O 26 , and its hydrophilic-lipophilic balance value HLB is 15.0. Polysorbate 80 is a light yellow to orange-yellow viscous liquid with a slight special odor, a slightly bitter and astringent taste, and a warm feeling; it is easily soluble in organic solvents such as water, ethanol, methanol, and ethyl acetate, but is very slightly soluble in mineral oil. It is stable to electrolytes, weak acids, and weak bases, but will gradually saponify when exposed to strong acids and strong bases; the oleate component is easily oxidized and needs to be stored sealed in a dark, cool and dry place.
[0003] Due to its excellent emulsifying and solubilizing properties, polysorbate 80 is widely used in the fields of medicine, food, etc., and can improve the clarity and stability of drugs. However, it has hemolytic, sensitizing, and potential hepatotoxic effects, and the dosage needs to be strictly controlled and impurities need to be removed when used in injections.
[0004] According to the General Principles for Biological Products 3203 in the 2020 Edition of the Chinese Pharmacopoeia, the determination of the residual amount of polysorbate 80 uses the ammonium cobalt thiocyanate colorimetric method: polyethoxy reacts with ammonium cobalt thiocyanate to form a blue complex, which is dissolved in dichloromethane and then quantified by colorimetry. However, this method has defects such as insufficient sensitivity, low accuracy, and poor safety. The conventional alternative method is size exclusion chromatography-evaporative light scattering detection (SEC-ELSD), which can quantify polysorbate 80 after sample treatment.
[0005] With the development of the pharmaceutical industry, liposomal preparations are widely used in vaccines and drugs. Since polysorbate 80 contains not only monooleate, but also a large amount of dioleate, trioleate, etc. with different degrees of esterification, and there are also different degrees of polyoxyethylene polymerization, this makes polysorbate 80 form a large number of relatively wide peak shapes on the reverse phase chromatography, making it difficult to perform quantitative calculations. And the components of liposomes overlap with these components and are difficult to separate, which will seriously interfere with the detection results. The current methods are difficult to accurately quantify the total content of polysorbate 80. In order to ensure the safety of drugs, it is urgent to develop a detection method that can accurately quantitatively detect polysorbate 80 in liposomal preparations. Summary of the Invention
[0006] To solve the deficiencies of the prior art, the present invention provides a method for detecting the content of polysorbate 80 in liposomal preparations, which has low operation difficulty, high accuracy, and good repeatability.
[0007] To achieve the object of the present invention, the following scheme is proposed: A method for detecting the content of polysorbate 80 in a liposome preparation, comprising the following steps: (1) Prepare a polysorbate 80 standard curve solution, a calibration standard solution, and a blank control solution; perform sample treatment on the standard curve solution, the calibration standard solution, the blank control solution, and the sample to be tested respectively; Among them, the polysorbate 80 reference substance is prepared into a reference stock solution with ultrapure water, and the concentration of the reference stock solution is 1 mg / mL; the reference stock solution is diluted successively to obtain 5 standard curve solutions (STD1 - STD5) with concentrations of 0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.7 mg / mL, and 0.9 mg / mL respectively; Among them, the polysorbate 80 reference substance is prepared into a calibration standard solution with ultrapure water, and the concentration of the calibration standard solution is 0.5 mg / mL; Among them, the blank control solution is ultrapure water; Among them, the sample to be tested solution is the test solution; preferably, when processing the test sample, two portions are taken and processed simultaneously as parallel samples.
[0008] The specific steps of sample treatment are as follows: Take 200 μl of each sample, place it in a 1.5 ml centrifuge tube, add 100 μl of 2M NaOH solution, centrifuge instantaneously, then water bath at 90 °C for 45 min, then water bath at room temperature until the temperature reaches room temperature, centrifuge instantaneously, add 900 μl of 0.25M formic acid + 80% ethanol solution, mix well, centrifuge at 10000×g at 8 °C for 5 min, and take the supernatant to obtain.
[0009] The preparation steps of the 2M NaOH solution are as follows: Weigh 40 g of NaOH and dissolve it with 500 ml of ultrapure water to obtain. The shelf life is 6 months.
[0010] The preparation steps of the 0.25M formic acid + 80% ethanol solution are as follows: Take 0.235 ml of formic acid, add 4.765 ml of ultrapure water and 20 ml of absolute ethanol, mix well to obtain. It is prepared and used immediately, and the volume ratio of formic acid, water, and absolute ethanol is 9.4:190.6:800.
[0011] (2) Use high performance liquid chromatography (CAD detector) to detect the standard curve solution, the calibration standard solution, the blank control solution, and the sample to be tested solution: Chromatographic column: Octadecylsilyl silica gel chromatographic column, size 4.6 mm × 50 mm, particle size 2.7 μm; Mobile phase A: 0.01% formic acid solution; The preparation process of 0.01% formic acid solution is as follows: Measure 1000 ml of ultrapure water, add 0.1 ml of formic acid, and mix well to obtain it. The shelf life is 7 days.
[0012] Mobile phase B: Acetonitrile solution; Column temperature: 40 °C; Flow rate: 1.5 ml / min; Auto-sampler temperature: 15 °C; Detection conditions of CAD detector: Nebulizer temperature 35 °C, acquisition frequency 5 Hz, filtering 3.6 s; The elution method is gradient elution, and the elution conditions are as follows: .
[0013] The injection volume is 15 - 30 μl, and the preferred injection volume in the present invention is 20 μl.
[0014] Detection injection method: Use the mobile phase to fully equilibrate the high performance liquid chromatography system; Inject at least one blank control solution; Inject 5 needles of calibration standard solution; Inject 1 needle each of standard curve solutions (STD1 - STD5); Inject 1 needle each of parallel samples of the test sample solution; For every 6 injections of the test sample solution, inject 1 needle of calibration standard solution as the system suitability solution.
[0015] Requirements during the detection process are as follows: The relative standard deviation (RSD) of the peak area of polyethoxylated sorbitol in 5 consecutive needles of calibration standard solution should be ≤ 3.0%; The relative error between the average value of the results of the content of polysorbate 80 in 5 consecutive needles of calibration standard solution and the theoretical value should be ≤ 3.0%, relative error = measured value / theoretical value - 1; The relative standard deviation (RSD) of the peak area of polyethoxylated sorbitol between the system suitability solution and the calibration standard should be ≤ 3.0%.
[0016] (3) According to the external standard method, using the logarithm of the peak area of polyethoxylated sorbitol in the standard curve solution as the ordinate y, and the logarithm of the concentration of polysorbate 80 in the standard curve solution as the abscissa x, calculate the linear regression equation; Substitute the peak area of polyethoxylated sorbitol in the test solution into the regression equation to calculate the content of polysorbate 80 in the test solution.
[0017] When calculating using the external standard method, it includes the following content: Due to the coincidence of the solvent interference peak generated during the gradient process of the chromatographic conditions with the peak of polyethoxylated sorbitol, it is necessary to use the chromatogram of ultrapure water as the background, and subtract the chromatogram of ultrapure water from all chromatograms before performing the integration process.
[0018] Calculate the regression equation with the logarithm of the concentration of the standard curve solution (STD1 - STD5) and the logarithm of the corresponding peak area of polyethoxylated sorbitol, and the linear correlation coefficient R 2It should be ≥ 0.98.
[0019] Use the average of the concentration results of two parallel samples as the content result of polysorbate 80 in the test sample. The absolute value of the relative deviation of the concentration results of the two parallel samples should be ≤ 2.0%, and the relative deviation = (Parallel 1 - Parallel 2) / (Parallel 1 + Parallel 2).
[0020] The beneficial effects of the present invention are as follows: This method can accurately quantitatively detect polysorbate 80 in liposomal preparations, with low operation difficulty, high accuracy, and good repeatability. Description of the Drawings
[0021] Figure 1 It is the high-performance liquid chromatography (HPLC) chromatogram of the standard curve solution in Example 1; Figure 2 It is the HPLC chromatogram of the calibration standard solution in Example 1; Figure 3 It is the HPLC chromatogram of the blank control solution in Example 1; Figure 4 It is the HPLC chromatogram of the test sample solution in Example 1; Figure 5 It is the HPLC chromatogram of the negative control in Experiment 1; Figure 6 It is the HPLC chromatogram of the standard curve solution in Experiment 2; Figure 7 It is the standard curve graph in Experiment 2; Figure 8 It is the HPLC chromatogram of the repeatability test in Experiment 3; Figure 9 It is the HPLC chromatogram of the 40% spiked solution in Experiment 5; Figure 10 It is the HPLC chromatogram of the 100% spiked solution in Experiment 5; Figure 11 It is the HPLC chromatogram of the 150% spiked solution in Experiment 5; Figure 12 It is the HPLC chromatogram of Mobile Phase Selection Condition 1 in Experiment 6; Figure 13 It is the HPLC chromatogram of Mobile Phase Selection Condition 2 in Experiment 6; Figure 14 It is the HPLC chromatogram of Mobile Phase Selection Condition 3 in Experiment 6; Figure 15 It is the HPLC chromatogram of Mobile Phase Selection Condition 4 and the present invention in Experiment 6; Figure 16 It is the HPLC chromatogram of selecting different gradient elution conditions in Experiment 7; Figure 17High performance liquid chromatography (HPLC) chromatograms for Experiment 8 under the conditions of 1 M NaOH alkali concentration, water bath heating at 90 °C for 10 min, 20 min, and 30 min; Figure 18 High performance liquid chromatography (HPLC) chromatograms for Experiment 8 under the conditions of 1 M NaOH alkali concentration, water bath heating at 95 °C for 10 min, 20 min, and 30 min; Figure 19 High performance liquid chromatography (HPLC) chromatograms for Experiment 8 under the conditions of 1 M NaOH alkali concentration, water bath heating at 100 °C for 10 min, 20 min, and 30 min; Figure 20 High performance liquid chromatography (HPLC) chromatograms for Experiment 8 under the conditions of 2 M NaOH alkali concentration, water bath heating at 90 °C for 10 min, 20 min, 30 min, and 60 min. Detailed implementation method
[0022] Example 1 As Figure 1 shown, this example provides a method for detecting the content of polysorbate 80 in liposome preparations, including the following steps: The instrument equipment involved in the method of this example is shown in Table 1: Table 1 Instrument statistics table The reagents involved are as follows: Formic acid: Sinopharm Chemical Reagent Co., Ltd., mass spectrometry grade; Acetonitrile: Fisher Chemical, HPLC grade; Absolute ethanol: Sinopharm Chemical Reagent Co., Ltd., HPLC grade; Sodium hydroxide: Sinopharm Chemical Reagent Co., Ltd., pharmaceutical grade.
[0023] (1) Sample treatment Preparation of reference stock solution: Weigh 100 mg of polysorbate 80 standard substance, dissolve it with ultrapure water and make up the volume to 100 ml, and mix well to obtain.
[0024] Preparation of standard curve solution: Take the reference stock solution and dilute it according to Table 2; Table 2 Concentration table of standard curve solution Preparation of calibration standard solution: Weigh 50 mg of polysorbate 80 standard substance, dissolve it with ultrapure water and make up the volume to 100 ml, and mix well to obtain.
[0025] Blank control solution: Ultrapure water.
[0026] Test sample solution: Recombinant herpes zoster vaccine product, in which the stock solution is gE protein, and the adjuvant is the self-made MRJ103 adjuvant of our company, mainly containing cationic liposomes (composed of DOTAP, DOPC, and cholesterol), QS-21, and CpG 1018. Other components include polysorbate 80, sucrose, histidine, etc.
[0027] Take 200 μl of each of the above standard curve solutions (STD1~STD5), calibration standard solution, blank control solution, and test sample solution into 1.5 ml centrifuge tubes, add 100 μl of 2M NaOH solution, centrifuge instantaneously, then heat in a water bath at 90 °C for 45 min, cool to room temperature in a normal temperature water bath, centrifuge instantaneously, add 900 μl of 0.25M formic acid + 80% ethanol solution, mix well, centrifuge at 10000×g at 8 °C for 5 min, and take the supernatant to obtain the solution. Among them, ultrapure water is used as the blank control, and two test sample solutions are prepared as parallel 1 and parallel 2.
[0028] (2)High performance liquid chromatography detection Turn on the high performance liquid chromatograph, connect the chromatographic column, use 0.01% formic acid solution as mobile phase A and acetonitrile as mobile phase B, and set the detection parameters as follows: column temperature 40 °C; flow rate 1.5 ml / min; autosampler temperature 15 °C; select CAD detector, nebulizer temperature 35 °C, acquisition frequency 5 Hz, filter 3.6 s; injection volume 20 μl; gradient elution.
[0029] The gradient elution conditions are: According to the gradient elution method, use the mobile phase to equilibrate the chromatographic column until there are no interfering peaks at the elution peak position of polyethoxylated sorbitol (about 5.1 min). Inject a blank control solution again, then inject 5 needles of the calibration standard solution; inject 1 needle of each of the standard curve solutions (STD1~STD5); inject 1 needle of each parallel sample of the sample to be measured. For every 6 injections of the sample to be measured, inject 1 needle of the calibration standard solution as the system suitability solution.
[0030] (3)Calculation of test results Figures 1 - 4 They are the chromatograms of the standard curve solution, calibration standard solution, blank control solution, and test sample solution respectively.
[0031] Since the solvent interference peak generated during the gradient process of the chromatographic conditions coincides with the polyethoxylated sorbitol peak, it is necessary to use the ultrapure water chromatogram as the background, and subtract the ultrapure water chromatogram from all chromatograms before performing the integration process.
[0032] Calculate the regression equation with the logarithm of the concentration of the standard curve solution (STD1~STD5) and the logarithm of the corresponding polyethoxylated sorbitol peak area, and the linear correlation coefficient R2 It should be ≥ 0.98. Calculate the content of polysorbate 80 in the test sample by the regression equation.
[0033] Use the average value of the concentration results of two parallel samples as the content result of polysorbate 80 in the test sample. The absolute value of the relative deviation of the concentration results of the two parallel samples should be ≤ 2.0%, and the relative deviation = (parallel 1 - parallel 2) / (parallel 1 + parallel 2).
[0034] Experiment 1 Conduct a specificity test on the detection method provided in Example 1.
[0035] Inject the negative control sample according to the experimental procedure of Example 1, such as Figure 5 As shown, there is no interference at the target peak for the negative control, and the specificity meets the requirements.
[0036] Experiment 2 Conduct a linearity and range test on the detection method provided in Example 1.
[0037] Reference stock solution: Weigh 102.03 mg of polysorbate 80 reference substance, dissolve it with ultrapure water and make up the volume to 100 ml, and mix well.
[0038] Prepare the standard curve solutions according to Table 3: Table 3 Concentration dilution table of standard curve solutions Conduct the detection according to the detection method of Example 1. The detection results are as shown in Figure 6 As shown, the original data is shown in Table 4: Table 4 Peak areas corresponding to the standard curve solutions Perform linear fitting on the logarithm of the concentration of the standard curves STD1 - STD5 and the logarithm of the corresponding peak areas, and obtain the regression equation Log (peak area) = 0.7673 × Log (concentration μg / ml) - 1.4963, R² = 0.9995. The standard curve is shown in Figure 7 .
[0039] In the range of 102.03 μg / ml to 918.27 μg / ml, there is a good linear relationship between the logarithm of the concentration and the logarithm of its peak area. The correlation coefficient R 2 = 0.9995, which is greater than 0.98. The linearity of Example 1 is good and the detection range is wide.
[0040] Experiment 3 Conduct a repeatability (precision) test on the detection method provided in Example 1.
[0041] Take the test sample (i.e., the sample to be tested) in the same batch of Example 1, and prepare six samples in parallel. Then, conduct the detection and result calculation according to the experimental procedure in Example 1, and record the peak areas of the samples.
[0042] The detection results are as Figure 8 shown, and the original data is shown in Table 5: Table 5 Peak areas corresponding to the test sample solution The relative standard deviation of the concentrations of the 6 test sample solutions is 1.5%, which is less than 4.0%, indicating that the method provided in Example 1 has good repeatability.
[0043] Experiment 4 Conduct an intermediate precision test on the detection method provided in Example 1.
[0044] Experimenters A and B prepared standard curve solutions, calibration standard solutions, blank control solutions, and 6 parallel test sample solutions for testing according to the operation method in Example 1, and the results are shown in Table 6: Table 6 Relative standard deviation values of the concentrations of the test sample solutions The relative standard deviation of the determination results of polysorbate 80 in 12 test sample solutions among different experimenters is 1.3%, which meets the requirement that the relative standard deviation is less than 6%, indicating that the intermediate precision of the method provided in Example 1 is good, and the method is stable and reliable.
[0045] Experiment 5 Conduct an accuracy test on the method provided in Example 1.
[0046] The theoretical content of polysorbate 80 in the test sample solution in Example 1 is 500 μg / ml. Prepare a spiked solution of polysorbate 80 accurately, and add it to the test sample to prepare three spiked samples with high, medium, and low levels of 40%, 100%, and 150% respectively.
[0047] Conduct the detection and calculation according to the method in Example 1. The detection results of the three spiked samples are respectively Figure 9 , Figure 10 , Figure 11 , and the calculation results are shown in Table 7: Table 7 Recovery values corresponding to different spiked solutions The spiked recoveries are between 100% and 112%, and the detection results are stable and have good repeatability, indicating that the method provided in Example 1 has high accuracy.
[0048] Experiment 6 Regarding the screening experiment of the mobile phase, the details are as follows: Table 8 Mobile phase condition table Inject the solution after pretreatment with polysorbate 80 and interfering excipients, and the results are as follows: Figures 12 - 15 They are the chromatograms of mobile phase selection conditions 1, 2, 3, 4 and the present invention respectively. Among them, the peak of polysorbate 80 at 7.5 - 10 min is the target peak of polyethoxylated sorbitol, and the arrow indicates the interfering peak; Regarding the selection of mobile phase A, by adjusting the pH of the mobile phase to acidic, the peak shape of the excipient interfering peak can be made more single, which is beneficial to the separation from polyethoxylated sorbitol. In addition to formic acid, other available volatile acids include acetic acid, trifluoroacetic acid, etc. Adjust the dosage of the acid to the minimum dosage that meets the usage requirements to ensure that the baseline interference of the CAD detector is small enough.
[0049] Regarding the selection of mobile phase B, choosing acetonitrile as the mobile phase can avoid the interference of degradation impurities. Using acetonitrile as mobile phase B can reduce the chromatographic retention difference of polyethoxylated sorbitol with different degrees of polymerization, resulting in a higher peak response and a narrower peak width.
[0050] Experiment 7 Regarding the screening experiment of the elution gradient, it is as follows: Table 9 Conditions table of gradient 1 Table 10 Conditions table of gradient 2 Table 11 Conditions table of gradient 3 Table 12 Conditions table of gradient elution of the present invention The results are as Figure 16 shown. The sample treatment process of the gradient elution conditions of the present invention is different from those of gradient 1 to gradient 3, resulting in an increase in the resolution of the interfering peak after the peak. Gradient 2, gradient 3 and the present invention adopt higher flow rates and gradient changes, making the peaks of polyethoxylated sorbitol more concentrated and convenient for integration.
[0051] Experiment 8 Regarding the screening experiment of sample treatment conditions, it is as follows: The degradation peak 2 of the excipient generated by simple base degradation may interfere with the integration of the polyethoxylated sorbitol peak. Adopt stronger conditions to further degrade the excipient and screen the mildest conditions that meet the exclusion of excipient interference.
[0052] Take 200 μl of the sample and place it in a 2-ml brown crimp vial. Add 100 μl of NaOH solution with different concentrations, seal it with an 11-mm aluminum crimp cap, and place it in a water bath at different temperatures for different times. After cooling to room temperature, add 900 μl of 1 M formic acid + 80% ethanol solution, mix well, centrifuge at 10,000×g for 5 min at 8°C, and take the supernatant to obtain the sample.
[0053] The first group of experiments: Using a 1 M NaOH alkali concentration, heat in a water bath at 90°C for 10 min, 20 min, and 30 min. The chromatograms are as Figure 17 shown; The second group of experiments: Using a 1 M NaOH alkali concentration, heat in a water bath at 95°C for 10 min, 20 min, and 30 min. The chromatograms are as Figure 18 shown; The third group of experiments: Using a 1 M NaOH alkali concentration, heat in a water bath at 100°C for 10 min, 20 min, and 30 min. The chromatograms are as Figure 19 shown; The fourth group of experiments: Using a 2 M NaOH alkali concentration, heat in a water bath at 90°C for 10 min, 20 min, 30 min, and 60 min. The chromatograms are as Figure 20 shown.
[0054] Conclusion: When using 1 M NaOH for degradation, the degradation peak 2 of the excipient was not completely converted to the degradation peak 3 of the excipient within 30 min. In order to reduce the analysis time and improve the analysis efficiency, no longer degradation time was carried out.
[0055] When using 2 M NaOH for degradation, the degradation peak 2 of the excipient can be completely converted to the degradation peak 3 of the excipient by degrading for 30 min and 60 min at 90°C. The degradation time between 30 min and 60 min can be selected.
[0056] The 1 M formic acid + 80% ethanol solution is a usable concentration. The actual formic acid concentration can be other concentrations that make the final test solution acidic, such as 0.25 M formic acid + 80% ethanol solution. Ethanol plays a role in assisting dissolution. Other solvents that can dissolve oleate and are miscible with water in any proportion can also be used, such as isopropanol, acetone, etc.
[0057] The above embodiments are only used to illustrate the technical ideas and features of the present invention, and do not represent the only or limit the present invention. Those skilled in the art should understand that without departing from the scope of the present invention, various changes or equivalent substitutions made to the present invention all fall within the scope of protection of the present invention.
Claims
1. A method for detecting the content of polysorbate 80 in a liposomal preparation, characterized in that: After sample treatment, high performance liquid chromatography is used for detection; An octadecylsilyl bonded silica gel chromatographic column is used as the chromatographic column; Mobile phase A is a 0.01% formic acid solution; Mobile phase B is an acetonitrile solution; The detector is a CAD detector; The gradient elution conditions are: 。 2. The method for detecting the content of polysorbate 80 in the liposomal preparation according to claim 1, characterized in that, The liposomal preparation contains DOTAP and / or DOPC.
3. The method for detecting the content of polysorbate 80 in the liposomal preparation according to claim 1, characterized in that, The sample treatment steps are as follows: Take 200 μl of each sample, place it in a 1.5 ml centrifuge tube, add 100 μl of 2M NaOH, centrifuge instantaneously, then heat in a water bath at 90 °C for 45 min, then cool to room temperature in a water bath at normal temperature, centrifuge instantaneously, add 900 μl of a mixed solution of formic acid and ethanol, mix well, centrifuge at 10000×g at 2 - 8 °C for 5 min, and take the supernatant.
4. The method for detecting the content of polysorbate 80 in the liposome preparation according to claim 3, characterized in that, The concentration of formic acid is 0.25M, ethanol is 80%, and the volume ratio of formic acid, water, and absolute ethanol is 9.4:190.6:
800.
5. The method for detecting the content of polysorbate 80 in the liposomal preparation according to claim 1, characterized in that, When using high performance liquid chromatography for detection, the substance to be detected is polyethoxysorbitol.
6. The method for detecting the content of polysorbate 80 in the liposomal preparation according to claim 1, characterized in that, The specifications of the octadecylsilyl bonded silica gel chromatographic column are 4.6 mm × 50 mm, and the particle size is 2.7 μm.
7. The method for detecting the content of polysorbate 80 in the liposome preparation according to claim 1, characterized in that, The detection conditions of the CAD detector are: the atomization chamber temperature is 35 °C, the acquisition frequency is 5 Hz, and the filtering is 3.6 s.
8. The method for detecting the content of polysorbate 80 in the liposomal preparation according to claim 1, characterized in that, During the gradient elution process, the flow rate of the mobile phase is 0.5 - 2 ml / min, the column temperature is 30 - 50 °C, and the temperature of the autosampler is 10 - 30 °C.
9. The method for detecting the content of polysorbate 80 in the liposomal preparation according to claim 1, characterized in that, The flow rate of the mobile phase is 1.5 ml / min, the column temperature is 40 °C, and the temperature of the autosampler is 15 °C.