Method for analyzing lipid-related substances

Abstract

The invention belongs to the field of medicine analysis, and provides a method for analyzing lipid-related substances, which adopts a high performance liquid reversed phase chromatography, can effectively detect the lipid-related substances in an octafluoropropane lipid microsphere injection and degraded impurities in the lipid-related substances, and is high in separation degree and good in durability.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of pharmaceutical analysis, and particularly relates to an analysis method for lipid related substances in octafluoropropane lipid microsphere injection. BACKGROUND

[0002] The octafluoropropane lipid microsphere injection is an ultrasonic contrast agent, which is used for enhancing the recognition of left ventricular endocardial boundary for those whose conventional echocardiography is not clear enough. The auxiliary materials of the octafluoropropane lipid microsphere injection include sodium chloride, propylene glycol, glycerol, a lipid mixture, sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate heptahydrate and water for injection.

[0003] The lipid mixture comprises dipalmitoyl phosphatidylcholine (chemical name: 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, abbreviated as DPPC), dipalmitoyl phosphatidic acid sodium (chemical name: 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid sodium, abbreviated as DPPA-NA) and phosphatidyl ethanolamine (chemical name: N-(methoxy polyethylene glycol amine 5000 carbamoyl)-1,2-dipalmitoyl-sn-glycero-3-phosphatidyl ethanolamine monosodium salt, abbreviated as DPPE-PEG5000).

[0004] The lipid mixture in the product will be degraded to produce impurities during the preparation process and storage process, which will be introduced into the drug, and finally affect the quality of the drug, and further affect the safety of drug use, so it is necessary to fully study the lipid related substances in the product to ensure the controllable quality.

[0005] WO2016109400A2 discloses an HPLC detection method of a lipid blend containing DPPC, DPPA and PEG5000-DPPE, which uses a C18 column and a mobile phase containing water, methanol, ammonium acetate and triethylamine, and does not disclose other chromatographic conditions, nor further determines and studies the lipid related degradation impurities. At present, there is an urgent need for an analysis method for lipid and related substances with strong specificity, high accuracy, excellent separation effect and good durability, which can provide effective support for the production process and quality control. SUMMARY

[0006] The technical problem to be solved by the present application is to provide an analysis method for lipid related substances.

[0007] The analysis method of the present application adopts high performance liquid reverse phase chromatography, in which the mobile phase A is an ammonium acetate solution, and the mobile phase B is a mixed solution of methanol and isopropyl alcohol.

[0008] In some embodiments, the concentration of the ammonium acetate solution in the mobile phase A is 0.01-0.04 mol / L, preferably, the concentration of the ammonium acetate solution is 0.02-0.04 mol / L; preferably, the concentration of the ammonium acetate solution is 0.04 mol / L.

[0009] In some embodiments, an acid is added to the mobile phase A to adjust the pH value; preferably, the kind of the added acid is volatile acid; preferably, the kind of the added acid is formic acid or acetic acid; preferably, the kind of the added acid is formic acid.

[0010] In some embodiments, an acid is added to the mobile phase A to adjust the pH value; preferably, the pH value is adjusted to 2.0-2.3; preferably, the pH value is 2.05-2.25; preferably, the pH value is 2.15.

[0011] In some embodiments, the volume ratio of methanol to isopropanol in the mobile phase B is 30-35:70-65; preferably, the volume ratio of methanol to isopropanol is 35:65.

[0012] In some embodiments, the chromatographic column is selected from a reversed-phase chromatographic column with octadecylsilane bonded silica gel as the filler; in some typical embodiments, the chromatographic column is Welch Xtimate C18 with a specification of 4.6 mm x 250 mm, 5 μm or Waters Atlantis T3 with a specification of 4.6 mm x 250 mm, 5 μm.

[0013] In some typical embodiments, the gradient elution conditions are as follows:

[0014]

[0015] In the elution process, the sum of the proportion of the mobile phase A and the proportion of the mobile phase B is 100%; wherein the proportion of the mobile phase A refers to the percentage of the mobile phase A in the total volume of the eluent, and the proportion of the mobile phase B refers to the percentage of the mobile phase B in the total volume of the eluent.

[0016] In some embodiments, the column temperature is 50-65°C; in some embodiments, the column temperature is 55-65°C; in some typical embodiments, the column temperature is 60-65°C; in some typical embodiments, the column temperature is 60°C.

[0017] In some embodiments, the flow rate is 0.5-1.5 mL / min; in some typical embodiments, the flow rate is 0.5-1.1 mL / min; in some typical embodiments, the flow rate is 0.6-1.1 mL / min; in some typical embodiments, the flow rate is 0.6-1.0 mL / min; in some typical embodiments, the flow rate is 0.6-0.8 mL / min; in some typical embodiments, the flow rate is 0.7 mL / min.

[0018] In some embodiments, the analysis method is performed on a high performance liquid chromatograph, using a universal detector; in some typical embodiments, the analysis method is performed on a high performance liquid chromatograph, using an evaporative light scattering detector or an electro-spray detector or a mass spectrometer detector; in some typical embodiments, the analysis method is performed on a high performance liquid chromatograph, using an evaporative light scattering detector or an electro-spray detector; in some typical embodiments, the analysis method is performed on a high performance liquid chromatograph, using an evaporative light scattering detector, and the temperature of the drift tube thereof is 60-70℃, and the flow rate of the carrier gas thereof is 1.6 L per minute; preferably, the temperature of the drift tube is 65℃.

[0019] In another aspect, the present application provides an analysis method of lipid-related substances, characterized in that:

[0020] The analysis method is performed on a high performance liquid chromatograph; a reverse phase chromatographic column is used, and the reverse phase chromatographic column uses octadecylsilane-bonded silica gel filler as the filler;

[0021] The analysis method uses an evaporative light scattering detector, and the temperature of the drift tube thereof is 60-70℃, and the flow rate of the carrier gas thereof is 1.6 L per minute;

[0022] The column temperature of the analysis method is 50-65℃;

[0023] The mobile phase A of the analysis method is an ammonium acetate solution, the concentration of the ammonium acetate solution is 0.04 mol / L, the mobile phase B is a mixed solution of methanol and isopropyl alcohol, and the volume ratio of methanol to isopropyl alcohol is 35:65;

[0024] The flow rate of the analysis method is 0.5-1.5 mL / min;

[0025] Gradient elution is performed according to the following table:

[0026]

[0027] And during the elution process, the sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%; wherein the proportion of mobile phase A refers to the percentage of the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of the total volume of the eluent.

[0028] In some experimental schemes, the present application provides a method for analyzing lipid-related substances, characterized in that:

[0029] The analysis method is performed on a high-performance liquid chromatograph; a reversed-phase chromatographic column is used, which uses octadecylsilane-bonded silica gel filler as the filler, and the reversed-phase chromatographic column is Welch Xtimate C18 with a specification of 4.6 mm x 250 mm, 5 μm or Waters Atlantis T3 with a specification of 4.6 mm x 250 mm, 5 μm;

[0030] The analysis method uses an evaporative light scattering detector, and the drift tube temperature is 60-70°C, and the carrier gas flow rate is 1.6 L per minute;

[0031] The analysis method has a column temperature of 50-65°C;

[0032] The analysis method uses ammonium acetate solution as the mobile phase A, the concentration of the ammonium acetate solution is 0.04 mol / L, and formic acid is used to adjust the pH value to 2.15; and a mixed solution of methanol and isopropanol is used as the mobile phase B, and the volume ratio of methanol to isopropanol is 35:65;

[0033] The analysis method has a flow rate of 0.6-1.1 mL / min;

[0034] Gradient elution is performed according to the following table:

[0035]

[0036]

[0037] And during the elution process, the sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%; wherein the proportion of mobile phase A refers to the percentage of mobile phase A in the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of the eluent.

[0038] In one specific experimental scheme, the present application provides a method for analyzing lipid-related substances, characterized in that:

[0039] The analysis method is performed on a high-performance liquid chromatograph; a reversed-phase chromatographic column is used, which uses octadecylsilane-bonded silica gel filler as the filler, and the reversed-phase chromatographic column is Welch Xtimate C18 with a specification of 4.6 mm x 250 mm, 5 μm;

[0040] The analysis method uses an evaporative light scattering detector, and the drift tube temperature is 65°C, and the carrier gas flow rate is 1.6 L per minute;

[0041] The analysis method has a column temperature of 60°C;

[0042] The mobile phase A of the analysis method is an ammonium acetate solution, the concentration of the ammonium acetate solution is 0.04 mol / L, and the pH value is adjusted to 2.15 by formic acid; the mobile phase B is a mixed solution of methanol and isopropyl alcohol, and the volume ratio of methanol to isopropyl alcohol is 35:65;

[0043] The flow rate of the analysis method is 0.7 mL / min;

[0044] Gradient elution is performed according to the following table:

[0045]

[0046] And during the elution process, the sum of the proportion of mobile phase A and the proportion of mobile phase B is 100%; wherein the proportion of mobile phase A refers to the percentage of mobile phase A in the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of the eluent.

[0047] In some embodiments, the related substances in the lipid include LPPE-PEG5000, the structural formula of which is as follows, wherein n = 113:

[0048]

[0049] In some embodiments, the related substances in the lipid include one or more of DPPA, DPPC, DPPE-PEG5000, the structural formulas of which are as follows, and in the structural formula of DPPE-PEG5000, n = 113:

[0050]

[0051] In some embodiments, the related substances in the lipid include one or more of PA, LPPA, LPPC, mPEG5000, the structural formulas of which are as follows, and in the structural formula of mPEG5000, n = 113:

[0052]

[0053] In some embodiments, the related substances in the lipid are selected from one or more of DPPA, DPPC, DPPE-PEG5000, PA, LPPA, LPPC or mPEG5000, the structures of which are as described above.

[0054] It is particularly pointed out that the lipid mixture can be degraded during the preparation process and storage, and dipalmitoyl phosphatidylcholine (DPPC) can be degraded to produce lysophosphatidylcholine (LPPC) and palmitic acid (PA), dipalmitoyl phosphatidic acid (DPPA) can be degraded to produce lysophosphatidic acid (LPPA) and palmitic acid, and dipalmitoyl phosphatidyl ethanolamine (DPPE-PEG5000) can be degraded to produce dipalmitoyl lysophosphatidyl ethanolamine (LPPE-PEG5000) and polyethylene glycol 5000 monomethyl ether (mPEG5000). According to the Guiding Principles for Microparticle Preparations in the Fourth Part of the Chinese Pharmacopoeia 2020, the degradation products, especially lysophospholipids, need to be controlled.

[0055] The beneficial effects of the present application: the method of the present application can effectively detect the lipids DPPC, DPPA, DPPE-PEG5000 and their degradation impurities PA, LPPA, LPPC, LPPE-PEG5000 and mPEG5000 in octafluoropropane lipid microsphere injection, and the separation degree between the compound chromatographic peak and the adjacent peak is greater than 1.2. The analysis method of the present application has strong specificity, good precision, high accuracy, excellent separation effect and good durability. The method of the present application can quickly and accurately perform quantitative analysis of lipids and degradation impurities in octafluoropropane lipid microsphere injection, ensure the quality control of octafluoropropane lipid microsphere injection, provide effective support for its production process and quality control, and also provide important reference for the quality control of other medical products. BRIEF DESCRIPTION OF DRAWINGS

[0056] Figure 1 The chromatogram of the system suitability solution of Example 2.

[0057] Figure 2 The chromatogram of the system suitability solution of Example 2.

[0058] Figure 3 The chromatogram of the system suitability solution of Example 2.

[0059] Figure 4 The chromatogram of the system suitability solution of Example 12.

[0060] Figure 5 The chromatogram of the system suitability solution of Example 13. DETAILED DESCRIPTION

[0061] The present application is described in more detail below, but the following examples do not constitute any limitation on the scope of the present application. Methanol (TEDIA, USA, chromatographically pure); isopropanol (ROE, USA, chromatographically pure); ammonium acetate (ROE, USA, chromatographically pure); formic acid (ROE, USA, chromatographically pure); water for experiment is ultra-pure water made by Merck Millipore ultra-pure water instrument; lysophosphatidylcholine control and lysophosphatidic acid control are purchased from Avanti; palmitic acid control and polyethylene glycol 5000 monomethyl ether control are purchased from Macklin; dipalmitoyl phosphatidylcholine control and dipalmitoyl phosphatidic acid control are purchased from Lipoid.

[0062] The lipid names, abbreviations and structural formulas in the present application are as follows.

[0063]

[0064]

[0065] The method of the present application is not limited to the above-mentioned three lipids and their five degradation impurities, any combination of any combination of one to five impurities selected from the above-mentioned impurities in the octafluoropropane lipid microsphere injection separated and determined by the method of the present application falls within the scope of the present application, and any combination of one to five impurities containing the above-mentioned impurities in the octafluoropropane lipid microsphere injection separated and determined by the method of the present application falls within the scope of the present application.

[0066] Example 1 Preparation method of impurity lysophosphatidylethanolamine (LPPE-PEG5000)

[0067] Synthesis of polyethylene glycol monomethyl ether 5000 and p-nitrophenyl chloroformate: take 460 mg of polyethylene glycol monomethyl ether 5000, 2.5 mg of toluene, 62 mg of sodium bicarbonate, stir and heat to 60-90°C, then add 65 mg of p-nitrophenyl chloroformate, synthesize polyethylene glycol 5000 monomethyl ether (4-nitrophenyl) carbonate. This step is achieved by esterification.

[0068] Introduction of L-lysophosphatidylethanolamine: the obtained polyethylene glycol 5000 monomethyl ether (4-nitrophenyl) carbonate is reacted with 50 mg of L-lysophosphatidylethanolamine and 247 mg of sodium bicarbonate at 60-90°C, the filtrate is rotary evaporated to remove the organic solvent to obtain a yellow solid, the yellow solid is dissolved in dichloromethane, and column chromatography is carried out with dichloromethane:methanol (20:1) as the developing agent, thereby synthesizing lysophosphatidylethanolamine (LPPE-PEG5000), this step is completed by condensation reaction.

[0069] 1H NMR (CDCI3): δ 0.879-0.891 (t, 3H), 1.256-1.261 (m, 24H), 1.597 (m, 2H), 2.304-2.316 (m, 2H), 3.379-3.393 (m, 3H), 3.529-3.546 (m, 2H), 3.554-3.764 (m, 354H), 3.967 (m, 2H), 4.069 (m, 1H), 4.116-4.218 (m, 4H), 6.734 (m, 1H).

[0070] System suitability test of Example 2

[0071] Instrument: Agilent high performance liquid chromatograph (Agilent, USA);

[0072] Column: Welch Xtimate C18 (4.6 mm x 250 mm, 5 μm) or a column of equivalent performance

[0073] Column temperature: 60 °C

[0074] Flow rate: 0.7 ml / min

[0075] Injection volume: 50 μL

[0076] Detector: Evaporative light scattering detector (ELSD)

[0077] Drift tube temperature: 65 °C

[0078] Carrier gas flow rate: 1.6 L / min

[0079] Mobile phase A: acetate buffer (take 3.08 g of ammonium acetate, dissolve and dilute to 1000 ml with water, and adjust the pH to 2.15 with formic acid)

[0080] Mobile phase B: methanol-isopropanol (35:65)

[0081] Elution gradient:

[0082]

[0083]

[0084] The solution was prepared as follows:

[0085] Blank excipient solution: namely a buffer salt solution, take about 103.5 g of propylene glycol, about 126.2 g of glycerol, about 2.34 g of sodium dihydrogen phosphate monohydrate, about 2.16 g of disodium hydrogen phosphate dodecahydrate and about 4.87 g of sodium chloride, dissolve in water and dilute to 1000 ml.

[0086] Blank solvent: isopropanol-trichloromethane-blank adjuvant solution (80:10:110).

[0087] Polyethylene glycol 5000 monomethyl ether positioning solution: take polyethylene glycol 5000 monomethyl ether control product, precision, add solvent to dissolve and dilute to make about 15 μg in 1 ml solution, shake, ready.

[0088] Cultured lysophosphatidyl ethanolamine positioning solution: take cultured lysophosphatidyl ethanolamine control product, precision, add solvent to dissolve and dilute to make about 60 μg in 1 ml solution, shake, ready.

[0089] Lysophosphatidyl choline positioning solution: take lysophosphatidyl choline control product, precision, add solvent to dissolve and dilute to make about 30 μg in 1 ml solution, shake, ready.

[0090] Lysophosphatidic acid positioning solution: take lysophosphatidic acid control product, precision, add solvent to dissolve and dilute to make about 60 μg in 1 ml solution, shake, ready.

[0091] Palmitic acid positioning solution: take palmitic acid control product, precision, add solvent to dissolve and dilute to make about 30 μg in 1 ml solution, shake, ready.

[0092] Dipalmitoyl phosphatidyl choline positioning solution: take dipalmitoyl phosphatidyl choline control product, precision, add solvent to dissolve and dilute to make about 800 μg in 1 ml solution, shake, ready.

[0093] Dipalmitoyl phosphatidic acid positioning solution: take dipalmitoyl phosphatidic acid control product, precision, add solvent to dissolve and dilute to make about 90 μg in 1 ml solution, shake, ready.

[0094] Cultured phosphatidyl ethanolamine positioning solution: take cultured phosphatidyl ethanolamine control product, precision, add solvent to dissolve and dilute to make about 600 μg in 1 ml solution, shake, ready.

[0095] Control stock solution 1: take polyethylene glycol 5000 monomethyl ether control product, cultured lysophosphatidyl ethanolamine control product, lysophosphatidyl choline control product, lysophosphatidic acid control product, palmitic acid control product, precision, add solvent to dissolve and dilute to make about 15 μg in 1 ml solution, shake, ready.

[0096] Control solution 2: about 18 mg of dipalmitoyl phosphatidic acid (DPPA) control, about 160 mg of dipalmitoyl phosphatidylcholine (DPPC) control and about 120 mg of cultured phosphatidylethanolamine (DPPE-PEG5000) control were precisely weighed into a 200 ml flask, dissolved and diluted with solvent to the mark, and shaken to give control solution 2.

[0097] Control solution: appropriate amounts of control solution 1 and control solution 2 were taken into different flasks, dissolved and diluted with solvent to the appropriate concentration, and shaken to give the control solution. (The concentrations of polyethylene glycol 5000 monomethyl ether were 2.4 μg / ml, 4.8 μg / ml, 12 μg / ml, 16 μg / ml, and 24 μg / ml, respectively; the concentrations of cultured lysophosphatidyl ethanolamine were 11.2 μg / ml, 22.4 μg / ml, 56 μg / ml, 74.7 μg / ml, and 112 μg / ml, respectively; the concentrations of lysophosphatidylcholine were 7.5 μg / ml, 15 μg / ml, 37.5 μg / ml, 56.25 μg / ml, and 75 μg / ml, respectively; the concentrations of lysophosphatidic acid were 1.2 μg / ml, 2.4 μg / ml, 6 μg / ml, 9 μg / ml, and 12 μg / ml, respectively; the concentrations of palmitic acid were 6 μg / ml, 12 μg / ml, 30 μg / ml, 45 μg / ml, and 60 μg / ml, respectively; the concentrations of dipalmitoyl phosphatidic acid were 22.5 μg / ml, 30 μg / ml, 45 μg / ml, 60 μg / ml, and 90 μg / ml, respectively; the concentrations of dipalmitoyl phosphatidylcholine were 200 μg / ml, 266.7 μg / ml, 400 μg / ml, 533.3 μg / ml, and 800 μg / ml, respectively; and the concentrations of cultured phosphatidylethanolamine were 150 μg / ml, 200 μg / ml, 300 μg / ml, 400 μg / ml, and 600 μg / ml, respectively.)

[0098] System suitability solution: 1 ml of control solution 1 and 10 ml of control solution 2 were taken into the same 20 ml flask, diluted with solvent to the mark, and shaken to give the system suitability solution.

[0099] Test solution: an appropriate amount of octafluoropropane lipid microsphere injection solution was taken to give the test solution.

[0100] 50 μl of the above solution was precisely measured and injected into the chromatograph, and the chromatogram was recorded.

[0101] Results:

[0102] The chromatogram of the system suitability solution is shown in Figure 1. Figure 1, the peak sequence is mPEG5000 (retention time 10.653 min), LPPE-PEG5000 (retention time 13.142 min), LPPC (retention time 19.200 min), LPPA (retention time 19.887 min), DPPE-PEG5000 (retention time 21.866 min), PA (retention time 25.059 min), DPPC (retention time 41.386 min), DPPA (retention time 44.007 min), and the separation between each adjacent chromatographic peak is greater than 1.2, and the baseline is smooth.

[0103] The chromatogram of the blank solvent is shown in Figure 2 .

[0104] The chromatogram of the test sample solution is shown in Figure 3 , the peak time of mPEG5000 is 10.550 min, the peak time of LPPE-PEG5000 is 13.054 min, the peak time of LPPC is 20.724 min, the peak time of LPPA is 21.675 min, the peak time of DPPE-PEG5000 is 23.964 min, the peak time of PA is 28.510 min, the peak time of DPPC is 47.831 min, and the peak time of DPPA is 51.467 min.

[0105] Forced degradation test of Example 3

[0106] An appropriate amount of octafluoropropane lipid microsphere injection was taken and subjected to acid, alkali, oxidation, high temperature, and light destruction, and a blank destruction solution was prepared in the same manner. An evaporative light detector (ELSD) was used for detection. The destruction test method is shown in Table 1.

[0107] Table 1 Forced degradation test method

[0108]

[0109]

[0110] Precisely take 50 μl of each of the above solutions, and inject them into a liquid chromatograph according to the chromatographic conditions of Example 2. The test results are shown in Tables 2 and 3.

[0111] Table 2 Alkali destruction and acid destruction degradation test results

[0112]

[0113] Table 3 Oxidation destruction, high temperature destruction, and light destruction degradation test results

[0114]

[0115] Note: Material balance (%) = [A total (destruction) / C destruction] / [A total (non-destruction) / C non-destruction]

[0116] The results show that octafluoropropane lipid microspheres injection is relatively stable under the conditions of oxidative destruction and light destruction; it is degraded under the conditions of acid destruction, alkali destruction and high temperature destruction, and the main degradation products include polyethylene glycol 5000 monomethyl ether, LPPE-PEG5000, lysophosphatidylcholine, lysophosphatidic acid and palmitic acid, and no unknown impurities exceeding the limit of 0.5% are found under each condition. The separation degree of the main peak and the front and rear impurities is good. The material balance rate of octafluoropropane lipid microspheres injection under each forced degradation condition is between 90.0% and 110.0%. The method has good specificity.

[0117] Example 4 Linearity and Range

[0118] An appropriate amount of cultured phosphatidylethanolamine reference substance, dipalmitoyl phosphatidylcholine reference substance and dipalmitoyl phosphatidic acid reference substance and each impurity reference substance was precisely weighed and prepared into a series of linear solutions. 10 μl of each linear solution of cultured phosphatidylethanolamine, dipalmitoyl phosphatidylcholine and dipalmitoyl phosphatidic acid and 50 μl of each linear solution of impurities were precisely taken and injected into the liquid chromatograph, and the chromatogram was recorded. The logarithmic value of the peak area was taken as the vertical coordinate (y), and the logarithmic value of the concentration (μg / ml) was taken as the horizontal coordinate (x), and the standard curve was drawn. The test results are shown in Tables 4-11.

[0119] Table 4 Linear test results of cultured phosphatidylethanolamine

[0120]

[0121]

[0122] Table 5 Linear test results of dipalmitoyl phosphatidylcholine

[0123]

[0124] Table 6 Linear test results of dipalmitoyl phosphatidic acid

[0125]

[0126] Table 7 Linear test results of polyethylene glycol 5000 monomethyl ether

[0127]

[0128] Table 8 Linear test results of LPPE-PEG5000

[0129]

[0130]

[0131] Table 9 Linear test results of lysophosphatidylcholine

[0132]

[0133] Table 10 Linear test results of lysophosphatidic acid

[0134]

[0135] Table 11 Linear test results of palmitic acid

[0136]

[0137]

[0138] The results show that the concentration of phosphatidyl ethanolamine in the range of 150.1 μg / ml to 600.3 μg / ml, the peak area and the concentration show a good linear relationship, the correlation coefficient r≥0.99, the y-axis intercept deviation≤2.0%, meet the verification requirements.

[0139] The concentration of dipalmitoyl phosphatidyl choline in the range of 200.0 μg / ml to 800.0 μg / ml, the peak area and the concentration show a good linear relationship, the correlation coefficient r≥0.99, the y-axis intercept deviation≤2.0%, meet the verification requirements.

[0140] The concentration of dipalmitoyl phosphatidic acid in the range of 24.3 μg / ml to 97.3 μg / ml, the peak area and the concentration show a good linear relationship, the correlation coefficient r≥0.99, the y-axis intercept deviation≤2.0%, meet the verification requirements.

[0141] The concentration of polyethylene glycol 5000 monomethyl ether in the range of 0.545 μg / ml to 24.551 μg / ml, the LOG peak area and the LOG concentration show a good linear relationship, the correlation coefficient r≥0.99, meet the verification requirements.

[0142] The concentration of LPPE-PEG5000 in the range of 2.317 μg / ml to 115.169 μg / ml, the LOG peak area and the LOG concentration show a good linear relationship, the correlation coefficient r≥0.99, the y-axis intercept deviation≤2.0%, meet the verification requirements.

[0143] The concentration of lysophosphatidyl choline in the range of 0.732 μg / ml to 73.920 μg / ml, the LOG peak area and the LOG concentration show a good linear relationship, the correlation coefficient r≥0.99, meet the verification requirements.

[0144] The lyso-phosphatidic acid concentration was in the range of 0.969 μg / ml to 12.452 μg / ml, the LOG peak area and the LOG concentration had a good linear relationship, the correlation coefficient r was greater than or equal to 0.99, and the verification requirement was met.

[0145] The palmitic acid concentration was in the range of 0.919 μg / ml to 61.000 μg / ml, the LOG peak area and the LOG concentration had a good linear relationship, the correlation coefficient r was greater than or equal to 0.99, and the verification requirement was met.

[0146] Example 5 Accuracy test

[0147] Blank excipient: the blank excipient solution of Example 2.

[0148] Blank solvent: the blank solvent of Example 2.

[0149] Reference solution: the reference solution of Example 2.

[0150] A. Phosphatidyl ethanolamine, dipalmitoyl phosphatidyl choline and dipalmitoyl phosphatidic acid accuracy test

[0151] 80% recovery rate solution: 0.8 ml of 2 volumes of the reference stock solution was precisely taken, placed in a 2 ml volumetric flask, dissolved and diluted with the solvent to the calibration mark, and shaken uniformly; it was obtained, and 3 portions were prepared in parallel.

[0152] 100% recovery rate solution: 1.0 ml of 2 volumes of the reference stock solution was precisely taken, placed in a 2 ml volumetric flask, dissolved and diluted with the solvent to the calibration mark, and shaken uniformly; it was obtained, and 6 portions were prepared in parallel.

[0153] 120% recovery rate solution: 1.2 ml of 2 volumes of the reference stock solution was precisely taken, placed in a 2 ml volumetric flask, dissolved and diluted with the solvent to the calibration mark, and shaken uniformly; it was obtained, and 3 portions were prepared in parallel.

[0154] 10 μl of the reference solution and the recovery rate solution were taken and injected into the liquid chromatograph, and the chromatogram was recorded, and the recovery rate was calculated according to the linear equation of each impurity, and the results are shown in Tables 12-14.

[0155] Table 12 Accuracy test results of cultured phosphatidyl ethanolamine

[0156]

[0157] Table 13 Accuracy test results of dipalmitoyl phosphatidyl choline

[0158]

[0159] Table 14 Accuracy test results of dipalmitoyl phosphatidic acid

[0160]

[0161]

[0162] B. Polyoxyl 5000, LPPE-PEG 5000, lysophosphatidylcholine, lysophosphatidic acid, palmitic acid accuracy test

[0163] 50% recovery solution: precisely pipette 250 μl of the control stock solution 1 into a 10 ml volumetric flask, add sample lipid solution, dissolve and dilute to the mark, shake well; obtained, prepared in parallel 3 parts.

[0164] 100% recovery solution: precisely pipette 500 μl of the control stock solution 1 into a 10 ml volumetric flask, add sample lipid solution, dissolve and dilute to the mark, shake well; obtained, prepared in parallel 6 parts.

[0165] 150% recovery solution: precisely pipette 750 μl of the control stock solution 1 into a 10 ml volumetric flask, add sample lipid solution, dissolve and dilute to the mark, shake well; obtained, prepared in parallel 3 parts.

[0166] Take 50 μl of the control solution and the recovery solution, inject into the liquid chromatograph, record the chromatogram, calculate the recovery according to the linear equation of each impurity, the test results are shown in Table 15-Table 19.

[0167] Table 15 Polyoxyl 5000 accuracy test results

[0168]

[0169] Table 16 LPPE-PEG 5000 accuracy test results

[0170]

[0171]

[0172] Table 17 lysophosphatidylcholine accuracy test results

[0173]

[0174] Table 18 lysophosphatidic acid accuracy test results

[0175]

[0176] Table 19 palmitic acid accuracy test results

[0177]

[0178]

[0179] The results show that the average recovery of polyethylene glycol 5000 monomethyl ether is 97.9%, RSD is 3.4% (n=9); the average recovery of LPPE-PEG5000 is 93.0%, RSD is 3.0% (n=9); the average recovery of lysophosphatidylcholine is 99.5%, RSD is 1.7% (n=9); the average recovery of lysophosphatidic acid is 99.8%, RSD is 3.7% (n=9); the average recovery of palmitic acid is 106.0%, RSD is 8.1% (n=9), and the method has good accuracy.

[0180] Example 6 Durability test

[0181] To investigate the interference ability of the method itself to the variable test factors, we investigated the durability of the method, and the factors investigated mainly include: column temperature, flow rate, drift tube temperature, pH value, different batches of chromatographic column. The durability chromatographic conditions are shown in Table 20.

[0182] Table 20 Durability chromatographic conditions

[0183] Serial number Flow rate (ml / min) Column temperature (°C) Drift tube temperature (°C) pH value Different brand of chromatographic column Chromatographic condition 1 0.7 60 65 2.15 Chromatographic column 1 Chromatographic condition 2 0.6 60 65 2.15 Chromatographic column 1 Chromatographic condition 3 0.8 60 65 2.15 Chromatographic column 1 Chromatographic condition 4 0.7 55 65 2.15 Chromatographic column 1 Chromatographic condition 5 0.7 65 65 2.15 Chromatographic column 1 Chromatographic condition 6 0.7 60 60 2.15 Chromatographic column 1 Chromatographic condition 7 0.7 60 70 2.15 Chromatographic column 1 Chromatographic condition 8 0.7 60 65 2.05 Chromatographic column 1 Chromatographic condition 9 0.7 60 65 2.25 Chromatographic column 1 Chromatographic condition 10 0.7 60 65 2.15 Chromatographic column 2

[0184] Note: 1, chromatographic condition 1 is normal condition;

[0185] 2, Column 1: Welch Xtimate C18 (4.6mm x 250mm, 5μm); Column 2: Welch Xtimate C18 (4.6mm x 250mm, 5μm).

[0186] A. Method durability for incubated phosphatidylethanolamine, dipalmitoylphosphatidylcholine and dipalmitoylphosphatidic acid

[0187] Under each durability condition, 10 μL of the incubated phosphatidylethanolamine, dipalmitoylphosphatidylcholine and dipalmitoylphosphatidic acid control solution and test solution in Example 2 were injected into the liquid chromatograph, and the chromatogram was recorded. The content of octafluoropropane lipid microspheres injection under each variable condition was investigated, and the test results are shown in Tables 21-22.

[0188] Table 21 Durability test-control solution results

[0189]

[0190] Table 22 Durability test-test solution results

[0191]

[0192] The results show that the ratio of the measured content of each lipid of the test sample to the content under standard conditions is between 90.0% and 110.0% after adjusting the chromatographic condition parameters within a certain range, and the theoretical plate number of each lipid peak in the reference solution is greater than 1500, and the method has good robustness.

[0193] B. Robustness of the method for polyethylene glycol 5000 monomethyl ether, LPPE-PEG5000, lysophosphatidylcholine, lysophosphatidic acid, and palmitic acid

[0194] Under each robustness condition, 50 μL of the polyethylene glycol 5000 monomethyl ether, LPPE-PEG5000, lysophosphatidylcholine, lysophosphatidic acid, and palmitic acid reference solution and 50 μL of the test sample solution in Example 2 were injected into the liquid chromatograph, and the chromatogram was recorded to investigate the content of the octafluoropropane lipid microsphere injection under each variable condition. The test results are shown in Tables 23-24.

[0195] Table 23 Robustness test - system suitability results

[0196]

[0197]

[0198] Table 24 Robustness test - impurity detection results of the test sample solution

[0199]

[0200] The results show that the difference between the content of each impurity under different conditions and the content under standard conditions is less than 0.5%, and the difference between the total impurity content and the content under standard conditions is less than 1.0%, and the method has good robustness.

[0201] The inventors have explored the mobile phase, gradient elution conditions, etc., as shown in the following Examples 7-14.

[0202] Example 7

[0203] Instrument: Agilent high performance liquid chromatograph (Agilent, USA);

[0204] Chromatographic column: Welch Xtimate C18 (specification: 4.6 mm x 250 mm, 5 μm)

[0205] Column temperature: 60°C

[0206] Flow rate: 0.5 ml / min

[0207] Injection volume: 20 μL

[0208] Detector: Evaporative light scattering detector (ELSD)

[0209] Drift tube temperature: 65 °C

[0210] Carrier gas flow rate: 1.6 L / min

[0211] Mobile phase A: 0.01 mol / L acetate buffer (take 0.77 g of ammonium acetate, dissolve and dilute to 1000 ml with water, and adjust the pH value to 4 with formic acid)

[0212] Mobile phase B: mobile phase A-isopropanol (volume ratio 5:95)

[0213] Elution gradient:

[0214]

[0215] Sample preparation: same as example 2.

[0216] Measurement: take 20 μL of blank solvent and system suitability solution, inject into liquid chromatograph, and record chromatogram.

[0217] Results: lysophosphatidic acid (LPPA) peak, lysophosphatidylcholine (LPPC) peak, cultured lysophosphatidylethanolamine (PEG5000-LPPE) peak and cultured phosphatidylethanolamine (PEG5000-DPPE) peak are all around 25 min, and dipalmitoyl phosphatidic acid (DPPA) peak and dipalmitoyl phosphatidylcholine (DPPC) peak are all around 38 min, which cannot be separated and analyzed.

[0218] Example 8

[0219] Instrument: Agilent high performance liquid chromatograph (Agilent, USA);

[0220] Chromatographic column: Welch Xtimate C18 (specification 4.6 mm x 250 mm, 5 μm)

[0221] Column temperature: 60 °C

[0222] Flow rate: 0.7 ml / min

[0223] Injection volume: 50 μL

[0224] Detector: evaporative light scattering detector (ELSD)

[0225] Drift tube temperature: 65 °C

[0226] Carrier gas flow rate: 1.6 L / min

[0227] Mobile phase A: 0.01 mol / L acetate buffer (take 0.77 g of ammonium acetate, dissolve and dilute to 1000 ml with water, and adjust the pH value to 6 with formic acid)

[0228] Mobile phase B: Mobile phase A - isopropanol (5:95 by volume)

[0229] Elution gradient:

[0230]

[0231] Sample preparation: same as Example 2.

[0232] Measurement: 50 μL of the blank solvent and the system suitability solution were injected into the liquid chromatograph, and the chromatogram was recorded.

[0233] Results: mPEG5000, PEG5000-LPPE and palmitic acid could be effectively separated; lysophosphatidic acid, lysophosphatidylcholine and PEG5000-DPPE were still inseparable; the separation degree of DPPA and DPPC was less than 1.2.

[0234] Example 9

[0235] Instrument: Agilent high performance liquid chromatograph (Agilent, USA);

[0236] Chromatographic column: Welch Xtimate C18 (4.6 mm x 250 mm, 5 μm)

[0237] Column temperature: 60°C

[0238] Flow rate: 0.7 ml / min

[0239] Injection volume: 50 μL

[0240] Detector: evaporative light scattering detector (ELSD)

[0241] Drift tube temperature: 65°C

[0242] Carrier gas flow rate: 1.6 L / min

[0243] Mobile phase A: 0.04 mol / L acetate buffer (take 3.08 g of ammonium acetate, dissolve and dilute to 1000 ml with water, and adjust the pH value to 2.15 with formic acid)

[0244] Mobile phase B: Mobile phase A - isopropanol (5:95 by volume)

[0245] Elution gradient:

[0246]

[0247] Sample preparation: same as Example 2.

[0248] Measurement: 50 μL of the blank solvent and the system suitability solution were injected into the liquid chromatograph, and the chromatogram was recorded.

[0249] Result: The separation degree of impurities LPPC, LPPA, and phosphatidylethanolamine prepared by culture is less than 1.2, and DPPA and DPPC are not separated, which is not applicable.

[0250] Example 10

[0251] Instrument: Agilent high performance liquid chromatograph (Agilent, USA);

[0252] Chromatographic column: Welch Xtimate C18 (4.6 mm x 250 mm, 5 μm)

[0253] Column temperature: 60℃

[0254] Flow rate: 0.5 ml / min

[0255] Injection volume: 50 μL

[0256] Detector: Evaporative light scattering detector (ELSD)

[0257] Drift tube temperature: 65℃

[0258] Carrier gas flow rate: 1.6 L / min

[0259] Mobile phase A: 0.01 mol / L acetate buffer (take 3.08 g of ammonium acetate, dissolve and dilute to 4000 ml with water, and adjust the pH value to 2.31 with formic acid)

[0260] Mobile phase B: Mobile phase A-isopropanol (5:95 by volume)

[0261] Elution gradient:

[0262]

[0263] Sample preparation: same as Example 2.

[0264] Measurement: Take 50 μL of blank solvent, system suitability solution, and test sample solution, inject into the liquid chromatograph, and record the chromatogram.

[0265] Result: The peaks of DPPC and DPPA in the test sample solution are coincided and cannot be separated, which is not applicable.

[0266] Example 11

[0267] Instrument: Agilent high performance liquid chromatograph (Agilent, USA);

[0268] Chromatographic column: Welch Xtimate C18 (4.6 mm x 250 mm, 5 μm)

[0269] Column temperature: 60℃

[0270] Flow rate: 0.5 ml / min

[0271] Injection volume: 50 μL

[0272] Detector: Evaporative light scattering detector (ELSD)

[0273] Drift tube temperature: 65 °C

[0274] Carrier gas flow rate: 1.6 L / min

[0275] Mobile phase A: 0.04 mol / L acetate buffer (take 3.08 g of ammonium acetate, dissolve and dilute to 1000 ml with water, and adjust the pH value to 2.15 with formic acid)

[0276] Mobile phase B: isopropanol-methanol (70:30)

[0277] Elution gradient:

[0278]

[0279] Sample preparation: the same as Example 2.

[0280] Measurement: 50 μL of the blank solvent, system suitability solution, and test sample solution were injected into the liquid chromatograph, and the chromatogram was recorded.

[0281] Results: the LPPA chromatographic peak was bifurcated, the LPPC chromatographic peak overlapped with the LPPA chromatographic peak, and the chromatographic peak was bifurcated, which was not applicable.

[0282] Example 12

[0283] Instrument: Agilent high performance liquid chromatograph (Agilent, USA);

[0284] Chromatographic column: Agient Zorbax SB-C8 (specification 3.0 mm x 150 mm, 3.5 μm) or a chromatographic column with equivalent performance

[0285] Flow rate: 1.0 ml / min

[0286] Injection volume: 20 μL

[0287] Detector: Evaporative light scattering detector (ELSD)

[0288] Drift tube temperature: 65 °C

[0289] Carrier gas flow rate: 1.6 L / min

[0290] Mobile phase A: 0.01 mol / L acetate buffer (take 0.77 g of ammonium acetate, dissolve and dilute to 1000 ml with water, and adjust the pH value to 8.5 with phosphoric acid)

[0291] Mobile phase B: Acetonitrile - Isopropanol (80:20)

[0292] Elution gradient:

[0293]

[0294] Sample preparation:

[0295] Blank solvent: Isopropanol: Chloroform: Water (50:5:45)

[0296] System suitability solution, test solution as in Example 2.

[0297] Measurement: 20 μL of the blank solvent, system suitability solution, test solution were injected into the liquid chromatograph and the chromatograms were recorded.

[0298] Results: DPPC did not elute, LPPA and LPPE-PEG5000 resolution < 1.2. Chromatogram of system suitability solution is shown in Figure 2. Figure 4 .

[0299] mPEG5000 eluted at 6.583 min, LPPA at 8.712 min, LPPE-PEG5000 at 8.907 min, LPPC at 12.002 min, PA at 14.091 min, DPPE-PEG5000 at 15.071 min, and DPPA at 19.911 min.

[0300] Example 13

[0301] Instrument: Agilent high performance liquid chromatograph (Agilent, USA)

[0302] Column: Waters Atlantis T3 (4.6 mm x 250 mm, 5 μm) or equivalent

[0303] Column temperature: 60 °C

[0304] Flow rate: 0.7 ml / min

[0305] Injection volume: 50 μL

[0306] Detector: Evaporative light scattering detector (ELSD)

[0307] Drift tube temperature: 65 °C

[0308] Carrier gas flow rate: 1.6 L / min

[0309] Mobile phase A: 0.04 mol / L acetate buffer (take 3.08 g of ammonium acetate, dissolve and dilute to 1000 ml with water, and adjust the pH value to 2.15 with formic acid)

[0310] Mobile phase B: methanol-isopropanol (35:65)

[0311] Elution gradient:

[0312]

[0313] Sample preparation: the same as Example 2.

[0314] Measurement: take 50 μL of the blank solvent, system suitability solution, and test sample solution, inject into the liquid chromatograph, and record the chromatogram.

[0315] Results: each impurity is well separated, with the smallest separation degree of 1.9, meeting the requirements, and the test sample solution chromatogram is shown in Figure 5 .

[0316] Example 14

[0317] Instrument: Agilent high performance liquid chromatograph (Agilent, USA)

[0318] Chromatographic column: Waters Atlantis T3 (4.6 mm x 250 mm, 5 μm) or a chromatographic column with equivalent performance

[0319] Column temperature: 50°C

[0320] Flow rate: 1.1 ml / min

[0321] Injection volume: 50 μL

[0322] Detector: evaporative light scattering detector (ELSD)

[0323] Drift tube temperature: 65°C

[0324] Carrier gas flow rate: 1.6 L / min

[0325] Mobile phase A: 0.04 mol / L acetate buffer (take 3.08 g of ammonium acetate, dissolve and dilute to 1000 ml with water, and adjust the pH value to 2.15 with formic acid)

[0326] Mobile phase B: methanol-isopropanol (35:65) as mobile phase B

[0327] Elution gradient:

[0328]

[0329]

[0330] Sample preparation: same as Example 2.

[0331] Measurement: 50 μL of the blank solvent, system suitability solution, and test solution were injected into the liquid chromatograph, and the chromatogram was recorded.

[0332] Result: each impurity was well separated, with the minimum separation being >1.2, which met the requirements.

Claims

1. A method for analyzing lipid-related substances, characterized in that: High performance liquid reverse phase chromatography was used, where mobile phase A was ammonium acetate solution and mobile phase B was a mixed solution of methanol and isopropanol.

2. The method according to claim 1, characterized in that, The concentration of ammonium acetate solution in mobile phase A is 0.01–0.04 mol / L; preferably, the concentration of ammonium acetate solution is 0.02–0.04 mol / L; preferably, the concentration of ammonium acetate solution is 0.04 mol / L.

3. The method according to claim 1, characterized in that, The pH value is adjusted by adding acid to the mobile phase A; preferably, the type of acid added is a volatile acid; preferably, the type of acid added is formic acid or acetic acid; preferably, the type of acid added is formic acid.

4. The method according to claim 1, characterized in that, Acid is added to mobile phase A to adjust the pH value; preferably, the pH value is adjusted to 2.0 to 2.3; preferably, the pH value is 2.05 to 2.25; preferably, the pH value is 2.

15.

5. The method according to claim 1, characterized in that, The volume ratio of methanol to isopropanol in the mobile phase B is 30-35:70-65; preferably, the volume ratio of methanol to isopropanol is 35:

65.

6. The method according to claim 1, characterized in that, The gradient elution conditions used in the high-performance liquid reverse reverse chromatography method are as follows: ； During the elution process, the sum of the proportions of mobile phase A and mobile phase B is 100%; where the proportion of mobile phase A refers to the percentage of mobile phase A in the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of the eluent.

7. The method according to claim 1, characterized in that, The chromatographic column used in the high-performance liquid reversed-phase chromatography (HPLC-RP-CP) is an octadecylsilane-bonded silica gel column; preferably, the column is a Welch Xtimate C18 with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm, or a Waters Atlantis T3 with dimensions of 4.6 mm × 250 mm and a diameter of 5 μm; the column temperature is 50–65 °C; preferably, 55–65 °C; preferably, 60–65 °C; preferably, 60 °C; the flow rate is 0.5–1.5 mL / min; preferably, 0.5–1.1 mL / min; preferably, 0.6–1.1 mL / min; preferably, 0.6–1.0 mL / min; preferably, 0.6–0.8 mL / min; preferably, 0.7 mL / min.

8. The method according to claim 1, characterized in that, The analytical method is performed on a high-performance liquid chromatograph (HPLC) using a general-purpose detector; preferably, the analytical method is performed on an HPLC using an evaporative light scattering (EVL) detector, an electro-fogging detector, or a mass spectrometer detector; preferably, the analytical method is performed on an HPLC using an evaporative light scattering (EVL) detector or an electro-fogging detector; preferably, the analytical method is performed on an HPLC using an evaporative light scattering (EVL) with a drift tube temperature of 60–70°C and a carrier gas flow rate of 1.6 L / min; preferably, the drift tube temperature is 65°C.

9. The method according to claim 1, characterized in that, The chromatographic column used in the high performance liquid reverse phase chromatography method is packed with octadecylsilane-bonded silica gel. An evaporative light scatterer is used, with a drift tube temperature of 60–70°C and a carrier gas flow rate of 1.6 L / min. The column temperature is 50–65℃; Mobile phase A is an ammonium acetate solution with a concentration of 0.04 mol / L, and the pH is adjusted to 2.15 with formic acid; mobile phase B is a mixed solution of methanol and isopropanol, with a volume ratio of methanol to isopropanol of 35:

65. The flow rate is 0.5–1.5 mL / min; The elution gradient conditions are: ； During the elution process, the sum of the proportions of mobile phase A and mobile phase B is 100%; where the proportion of mobile phase A refers to the percentage of mobile phase A in the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of the eluent.

10. The method according to claim 1, characterized in that, The chromatographic column used in the high performance liquid reversed phase chromatography method is either a Welch Xtimate C18 (4.6 mm × 250 mm, 5 μm) or a Waters Atlantis T3 (4.6 mm × 250 mm, 5 μm). An evaporative light scatterer is used, with a drift tube temperature of 60–70°C and a carrier gas flow rate of 1.6 L / min. The column temperature is 50–65℃; Mobile phase A is an ammonium acetate solution with a concentration of 0.04 mol / L, and the pH is adjusted to 2.15 with formic acid; mobile phase B is a mixed solution of methanol and isopropanol, with a volume ratio of methanol to isopropanol of 35:

65. The flow rate is 0.6–1.1 mL / min; The elution gradient conditions are: ； During the elution process, the sum of the proportions of mobile phase A and mobile phase B is 100%; where the proportion of mobile phase A refers to the percentage of mobile phase A in the total volume of the eluent, and the proportion of mobile phase B refers to the percentage of mobile phase B in the total volume of the eluent.

Images