Separation and detection method for impurities in lipid microsphere injection

Abstract

The invention provides a method for separating and detecting impurities in a lipid microsphere injection. The separation and detection method comprises the step of detecting by adopting a high performance liquid chromatography, and the conditions of the high performance liquid chromatography are as follows: the aperture of a chromatographic column filler is # imgabs0 #, the elution mode is gradient elution, a mobile phase A is an aqueous solution of a buffer salt, a mobile phase B is a methanol solution or a methanol aqueous solution of the buffer salt, and the buffer salt is ammonium acetate or ammonium formate. According to the separation and detection method for the impurities of the lipid microsphere injection, eight phospholipid impurities can be effectively separated.

Description

Technical Field

[0001] The present application relates to the technical field of drug analysis, and in particular to a method for separating and detecting impurities in lipid microsphere injection. Background Art

[0002] Ultrasound contrast imaging is a technique that uses contrast agents to enhance backscattered echoes, significantly improving the resolution, sensitivity and specificity of ultrasound diagnosis. With the improvement of instrument performance and the emergence of new ultrasound contrast agents, ultrasound contrast imaging can effectively enhance the two-dimensional ultrasound images and blood flow Doppler signals of solid organs such as the myocardium, liver, kidneys, and brain, and reflect and observe the blood perfusion of normal and diseased tissues, thus becoming a very important and promising development direction for ultrasound diagnosis.

[0003] Octafluoropropane lipid microsphere injection is an ultrasound contrast agent suitable for those whose conventional echocardiography is not clear enough to enhance the identification of the left ventricular endocardial border. It has been marketed in many countries and regions such as the United States, the European Union, and China. It has been recognized clinically and in the market because it can provide patients with more accurate diagnostic results. Phospholipids are key excipients for octafluoropropane lipid microsphere injection, including dipalmitoylphosphatidic acid, dipalmitoylphosphatidylcholine, and phosphatidylethanolamine. These three phospholipids constitute the carrier of microbubbles, which play the role of encapsulating and supporting octafluoropropane gas. The impurities in octafluoropropane lipid microsphere injection are mainly produced by the degradation of the three phospholipid excipients and the production process of octafluoropropane lipid microsphere injection. These impurities have a large molecular weight distribution range and cannot be effectively separated. They can also cause adverse reactions such as hemolysis. Therefore, it is very necessary to study the hemolytic impurities in lipid microsphere injection and establish a detection method for its impurities. Summary of the invention

[0004] Based on this, the present application provides a method that can effectively separate and detect impurities in lipid microsphere injection, which is used for qualitative and quantitative analysis of substances related to lipid microsphere injection and reflects the quality changes of lipid microsphere injection.

[0005] The present application provides a method for separating and detecting impurities in lipid microsphere injection, wherein the impurities include one or more of impurities A to H.

[0006] Among them, impurity A is lysophosphatidylcholine, impurity B is 2-acyl-lysophosphatidylcholine, impurity C is 1-stearoyl-lysophosphatidylcholine, impurity D is dipalmitoylphosphatidylethanolamine, impurity E is 1,3-dipalmitoylglycerol, impurity F is 1,2-dipalmitoylglycerol, impurity G is methoxy polyethylene glycol 5000-lysophosphatidylethanolamine, and impurity H is methoxy polyethylene glycol 5000.

[0007] The separation and detection method includes detection by high performance liquid chromatography, and the conditions of the high performance liquid chromatography include:

[0008] (1) The pore size of the chromatographic column packing is

[0009] (2) The elution method is gradient elution, the mobile phase A is an aqueous solution of a buffer salt, the mobile phase B is a methanol solution of a buffer salt or a methanol aqueous solution, and the buffer salt is ammonium acetate or ammonium formate.

[0010] In some embodiments, the detection method comprises:

[0011] Take any one or more of the reference substances of impurities A to H, dissolve them in a solvent, and prepare a reference substance;

[0012] Take the lipid microsphere injection test object, dissolve or extract it with a solvent to prepare the test sample;

[0013] The high performance liquid chromatography method is used to detect the reference substance and the test substance.

[0014] In some embodiments, the chromatographic column is a C18 chromatographic column, which can be an octadecylsilane bonded silica gel column.

[0015] In some embodiments, the concentration of the buffer salt in the mobile phase A is 5 mM to 20 mM; and / or,

[0016] The concentration of the buffer salt in mobile phase B is 5 mM to 20 mM; and / or,

[0017] The volume percentage of water in the methanol aqueous solution is 0-10%.

[0018] In some embodiments, the conditions of the high performance liquid chromatography method further include: a column temperature of 20°C to 40°C.

[0019] In some embodiments, the gradient elution method is as follows:

[0020] From 0 to 15 min, the volume percentage of mobile phase A was maintained at 53%, and the volume percentage of mobile phase B was maintained at 47%;

[0021] From 15 to 20 min, the volume percentage of mobile phase A decreased from 53% to 15%, and the volume percentage of mobile phase B increased from 47% to 85%;

[0022] From 20 to 35 min, the volume percentage of mobile phase A decreased from 15% to 0%, and the volume percentage of mobile phase B increased from 85% to 100%;

[0023] 35-45 min, keep the volume percentage of mobile phase A at 0% and the volume percentage of mobile phase B at 100%;

[0024] From 45 to 47 min, the volume percentage of mobile phase A increased from 0% to 53%, and the volume percentage of mobile phase B decreased from 100% to 47%;

[0025] From 47 to 52 min, the volume percentage of mobile phase A was maintained at 53%, and the volume percentage of mobile phase B was maintained at 47%.

[0026] In some embodiments, the conditions of the high performance liquid chromatography also include: a flow rate of 1.0 mL / min to 1.3 mL / min.

[0027] In some embodiments, the eluate of the gradient elution is detected using evaporative light scattering detection.

[0028] In some of the embodiments, in the step of detecting the eluent, the evaporative light scattering detection method adopts a closed split mode, the carrier gas flow rate of the evaporative light scattering detection method is 2.0 L / min to 3.5 L / min, and the drift tube temperature of the evaporative light scattering detection method is 90°C to 110°C.

[0029] In some embodiments, the conditions of the high performance liquid chromatography include:

[0030] (1.1) The chromatographic column is an octadecylsilane bonded silica gel column, and the pore size of the filler is

[0031] (2.1) The elution method is gradient elution, the mobile phase A is a 5mM to 20mM buffer salt aqueous solution, the mobile phase B is a 5mM to 20mM buffer salt methanol solution or a methanol aqueous solution, the volume percentage of water in the methanol aqueous solution is 0 to 10%, the buffer salt is ammonium acetate or ammonium formate, and the gradient elution method is as follows:

[0032] From 0 to 15 min, the volume percentage of mobile phase A was maintained at 53%, and the volume percentage of mobile phase B was maintained at 47%;

[0033] From 15 to 20 min, the volume percentage of mobile phase A decreased from 53% to 15%, and the volume percentage of mobile phase B increased from 47% to 85%;

[0034] From 20 to 35 min, the volume percentage of mobile phase A decreased from 15% to 0%, and the volume percentage of mobile phase B increased from 85% to 100%;

[0035] 35-45 min, keep the volume percentage of mobile phase A at 0% and the volume percentage of mobile phase B at 100%;

[0036] From 45 to 47 min, the volume percentage of mobile phase A increased from 0% to 53%, and the volume percentage of mobile phase B decreased from 100% to 47%;

[0037] 47-52 min, maintain the volume percentage of mobile phase A at 53% and the volume percentage of mobile phase B at 47%;

[0038] (3.1) Column temperature is 23°C;

[0039] (4.1) Flow rate: 1.2 mL / min;

[0040] (5.1) The gradient elution eluate is detected by evaporative light scattering detection method, and the drift tube temperature of the evaporative light scattering detection method is 95°C.

[0041] Compared with the prior art, this application has at least the following beneficial effects:

[0042] This application uses a chromatographic column with a suitable pore size and a high performance liquid chromatography method with a suitable mobile phase setting to achieve effective separation of impurities in lipid microsphere injection. The method provided in this application has good separation effect, good specificity, high sensitivity, and a wide range of durability. It fully complies with the guiding principles of methodological validation of the Chinese Pharmacopoeia in terms of system applicability and accuracy, and can effectively and accurately reflect the quality changes of lipid microsphere injection and achieve drug safety control. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the specific implementation methods of the present application or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0044] Figure 1 This is the solution chromatogram of the system suitability of Example 1;

[0045] Figure 2 This is the solution chromatogram of the system suitability of Comparative Example 1. DETAILED DESCRIPTION

[0046] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present application belongs. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0048] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0049] In the present application, "one or more" refers to any one, any two, or any two or more of the listed items.

[0050] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0051] The percentage contents involved in this application, unless otherwise specified, refer to mass percentage for solid-liquid mixing and solid-solid mixing, and refer to volume percentage for liquid-liquid mixing.

[0052] The percentage concentrations mentioned in this application, unless otherwise specified, refer to the final concentration, which refers to the percentage of the added component in the system after the addition of the component.

[0053] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range of instrument control.

[0054] In the present application, the method steps that do not emphasize the temperature usually refer to the method steps performed at normal temperature or room temperature. In this article, normal temperature and room temperature are equivalent and interchangeable, and the specific temperature refers to 22°C to 25°C.

[0055] Unless otherwise specified, all embodiments and optional embodiments of the present application can be combined with each other to form a new technical solution.

[0056] Unless otherwise specified, all technical features and optional technical features of this application can be combined with each other to form a new technical solution.

[0057] Unless otherwise specified, all steps of the present application may be performed sequentially or randomly. For example, the method includes steps (a) and (b), which means that the method may include steps (a) and (b) performed sequentially, or may include steps (b) and (a) performed sequentially. For example, the method may also include step (c), which means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b) and (c), or may include steps (a), (c) and (b), or may include steps (c), (a) and (b), etc.

[0058] If there is no special explanation, the "include" and "comprising" mentioned in this application are open-ended or closed-ended. For example, the "include" and "comprising" may mean that other components not listed may also be included or only the listed components may be included or only the listed components may be included.

[0059] If not specifically stated, in this application, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, any of the following conditions satisfies the condition "A or B": A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0060] "ELSD" herein refers to evaporative light scattering detector and its detection technology. "CAD" herein refers to electrospray detector and its detection technology.

[0061] The phospholipid substances recorded in the relevant reports are all naturally synthesized phospholipids such as soybean lecithin and egg yolk lecithin, and the number of impurities detected is generally no more than 6. However, the phospholipid substances used for analysis in this application are all artificially synthesized, and the number of related impurities generated by the degradation of three phospholipid excipients in octafluoropropane lipid microsphere injection is as high as 8, including 3 pairs of isomers. The existing technology has not yet disclosed a separation and detection method for the relevant impurities.

[0062] Therefore, the present application provides a method for separating and detecting impurities in a lipid microsphere injection solution containing one or more impurities A to H.

[0063] Among them, impurity A: 1-palmitoyl-lysophosphatidylcholine, abbreviated as lysophosphatidylcholine, also known as 1-acyl-lysophosphatidylcholine, English abbreviation LPC, also known as sn-1-LPC.

[0064] Impurity B: 2-palmitoyl-lysophosphatidylcholine, referred to as 2-acyl-lysophosphatidylcholine, also known as 2-acyl-LPC, abbreviated as sn-2-LPC.

[0065] Impurity C: 1-stearoyl-lysophosphatidylcholine, also known as 1-stearoyl-phosphatidylcholine, abbreviated as LSPC.

[0066] Impurity D: Dipalmitoylphosphatidylethanolamine, also known as dipalmitoylphosphatidylethanolamine 16:0PE, abbreviated as DPPE.

[0067] Impurity E: 1,3-dipalmitin.

[0068] Impurity F: 1,2-dipalmitin.

[0069] Impurity G: Methoxypolyethylene glycol 5000-lysophosphatidylethanolamine, also known as PEGylated lysophosphatidylethanolamine, abbreviated as MPEG5000-LPE.

[0070] Impurity H has a chemical name: methoxy polyethylene glycol 5000, abbreviated as MPEG5000.

[0071] Impurities A to H are degradation impurities generated during the production or storage of lipid microsphere injections from dipalmitoylphosphatidic acid, dipalmitoylphosphatidylcholine and phosphatidylethanolamine, excipients for preparing lipid microsphere injections. Further, impurities A to H are suitable as reference standards or control substances for relevant quantitative and qualitative analysis, and then for quality control of lipid microsphere injections.

[0072] Among impurities A to H, impurity A (lysophosphatidylcholine) and impurity B (2-acyl-lysophosphatidylcholine) are isomers of each other. Phospholipids can be hydrolyzed in aqueous solutions, and the ester bonds at the sn1 and sn2 positions can be broken to form free fatty acids and 2-acyl and 1-acyl-lysophospholipids. Impurity E (1,3-dipalmitoylglycerol) and impurity F (1,2-dipalmitoylglycerol) are isomers of each other. When dipalmitoylphosphatidylethanolamine-methoxypolyethylene glycol 5000 is hydrolyzed, the ester bonds at two sites can be broken, and free palmitic acid and impurity G (MPEG5000-LPE) are hydrolyzed. Impurity G contains two isomers.

[0073] In a particularly preferred embodiment, impurities A to H according to the present application are monomeric impurities, most preferably in pure form, preferably with a purity greater than about 95%, preferably with a purity greater than about 98%, and most preferably with a purity greater than about 99%, preferably measured by HPLC.

[0074] The impurities separation and detection method of lipid microsphere injection is carried out by high performance liquid chromatography, and the conditions of the high performance liquid chromatography include:

[0075] (1) The pore size of the chromatographic column packing is

[0076] (2) The elution method is gradient elution, the mobile phase A is an aqueous solution of a buffer salt, the mobile phase B is a methanol solution of a buffer salt or a methanol aqueous solution, and the buffer salt is ammonium acetate or ammonium formate.

[0077] It is understandable that the pore size of the above-mentioned chromatographic column packing can be and any values ​​in between.

[0078] More specifically, the method for separating and detecting impurities in lipid microsphere injection comprises the following steps:

[0079] Take any one or more of the reference substances of impurities A to H, dissolve them in a solvent, and prepare a reference substance;

[0080] Take the lipid microsphere injection test object, dissolve or extract it with a solvent to prepare the test sample;

[0081] The reference substance and the test substance were detected by the above-mentioned high performance liquid chromatography method.

[0082] It is understandable that the analyte of the lipid microsphere injection may be a related substance that may contain any one or more of the impurities A to H.

[0083] In some embodiments, the chromatographic column used in the high performance liquid chromatography method is a C18 chromatographic column, which can be an octadecylsilane bonded silica gel column, that is, the filler of the chromatographic column is octadecylsilane bonded silica gel.

[0084] In some embodiments, the concentration of the buffer salt in the mobile phase A is 5 mM to 20 mM, preferably 5 mM to 10 mM.

[0085] In some embodiments, the concentration of the buffer salt in the mobile phase B is 5 mM to 20 mM, preferably 5 mM to 10 mM.

[0086] In some embodiments, the volume percentage of water in the methanol aqueous solution is 0, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 5%, 10% and any value therebetween.

[0087] In some embodiments, the conditions of the high performance liquid chromatography also include: a column temperature of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C and any value therebetween.

[0088] In some embodiments, the gradient elution method is as follows: from 0 to 5 min, the volume percentage of mobile phase A is maintained at 53%, and the volume percentage of mobile phase B is maintained at 47%; from 5 to 10 min, the volume percentage of mobile phase A decreases from 53% to 15%, and the volume percentage of mobile phase B increases from 47% to 85%; from 10 to 25 min, the volume percentage of mobile phase A decreases from 15% to 0%, and the volume percentage of mobile phase B increases from 85% to 100%; from 25 to 35 min, the volume percentage of mobile phase B is maintained at 100%; from 35 to 37 min, the volume percentage of mobile phase A increases from 0% to 53%, and the volume percentage of mobile phase B decreases from 100% to 47%; from 37 to 42 min, the volume percentage of mobile phase A is maintained at 53%, and the volume percentage of mobile phase B is maintained at 47%.

[0089] In some embodiments, the gradient elution method is as follows: from 0 to 20 min, the volume percentage of mobile phase A is maintained at 53%, and the volume percentage of mobile phase B is maintained at 47%; from 20 to 25 min, the volume percentage of mobile phase A decreases from 53% to 15%, and the volume percentage of mobile phase B increases from 47% to 85%; from 25 to 40 min, the volume percentage of mobile phase A decreases from 15% to 0%, and the volume percentage of mobile phase B increases from 85% to 100%; from 40 to 50 min, the volume percentage of mobile phase B is maintained at 100%; from 50 to 52 min, the volume percentage of mobile phase A increases from 0% to 53%, and the volume percentage of mobile phase B decreases from 100% to 47%; from 52 to 66 min, the volume percentage of mobile phase A is maintained at 53%, and the volume percentage of mobile phase B is maintained at 47%.

[0090] In some embodiments, the gradient elution method is as follows: from 0 to 15 minutes, the volume percentage of mobile phase A is maintained at 53%, and the volume percentage of mobile phase B is maintained at 47%; from 15 to 20 minutes, the volume percentage of mobile phase A decreases from 53% to 15%, and the volume percentage of mobile phase B increases from 47% to 85%; from 20 to 35 minutes, the volume percentage of mobile phase A decreases from 15% to 0%, and the volume percentage of mobile phase B increases from 85% to 100%; from 35 to 45 minutes, the volume percentage of mobile phase B is maintained at 100%; from 45 to 47 minutes, the volume percentage of mobile phase A increases from 0% to 53%, and the volume percentage of mobile phase B decreases from 100% to 47%; from 47 to 52 minutes, the volume percentage of mobile phase A is maintained at 53%, and the volume percentage of mobile phase B is maintained at 47%.

[0091] In some preferred embodiments, the method of gradient elution is as follows:

[0092] From 0 to 15 minutes, the volume percentage of mobile phase A is maintained at 53%, and the volume percentage of mobile phase B is maintained at 47%; from 15 to 20 minutes, the volume percentage of mobile phase A decreases from 53% to 15%, and the volume percentage of mobile phase B increases from 47% to 85%; from 20 to 35 minutes, the volume percentage of mobile phase A decreases from 15% to 0%, and the volume percentage of mobile phase B increases from 85% to 100%; from 35 to 45 minutes, the volume percentage of mobile phase B is maintained at 100%; from 45 to 47 minutes, the volume percentage of mobile phase A increases from 0% to 53%, and the volume percentage of mobile phase B decreases from 100% to 47%; from 47 to 52 minutes, the volume percentage of mobile phase A is maintained at 53%, and the volume percentage of mobile phase B is maintained at 47%. Under this gradient elution mode, the peak height of the impurity chromatographic peak is higher, which is more conducive to improving the detection accuracy.

[0093] In some embodiments, the conditions of the high performance liquid chromatography method further include: a flow rate of 1.0 mL / min, 1.1 mL / min, 1.2 mL / min, 1.3 mL / min, and any value therebetween.

[0094] In some embodiments, the eluent of the gradient elution is detected by evaporative light scattering detection. Optionally, in the step of detecting the eluent, the evaporative light scattering detection method adopts a closed split mode, and optionally, the carrier gas flow rate of the evaporative light scattering detection method is 2.0L / min, 2.2L / min, 2.4L / min, 2.6L / min, 2.8L / min, 3.0L / min, 3.2L / min, 3.4L / min, 3.5L / min and any value therebetween, and further optionally, the drift tube temperature of the evaporative light scattering detection method is 70°C, 72°C, 75°C, 80°C, 85°C, 90°C, 95°C, 100°C, 110°C and any value therebetween, more preferably 90°C to 110°C. When the drift tube temperature is within this range, the appearance of interference peaks can be avoided, thereby improving the accuracy of detection.

[0095] In some embodiments, the conditions for high performance liquid chromatography include:

[0096] (1.1) The chromatographic column is an octadecylsilane bonded silica gel column, and the pore size of the filler is

[0097] (2.1) The elution method is gradient elution, the mobile phase A is a 5mM to 20mM buffer salt aqueous solution, the mobile phase B is a 5mM to 20mM buffer salt methanol solution or a methanol aqueous solution, the volume percentage of water in the methanol aqueous solution is 0 to 10%, the buffer salt is ammonium acetate or ammonium formate, and the gradient elution method is as follows:

[0098] From 0 to 15 min, the volume percentage of mobile phase A was maintained at 53%, and the volume percentage of mobile phase B was maintained at 47%;

[0099] From 15 to 20 min, the volume percentage of mobile phase A decreased from 53% to 15%, and the volume percentage of mobile phase B increased from 47% to 85%;

[0100] From 20 to 35 min, the volume percentage of mobile phase A decreased from 15% to 0%, and the volume percentage of mobile phase B increased from 85% to 100%;

[0101] 35-45 min, keep the volume percentage of mobile phase A at 0% and the volume percentage of mobile phase B at 100%;

[0102] From 45 to 47 min, the volume percentage of mobile phase A increased from 0% to 53%, and the volume percentage of mobile phase B decreased from 100% to 47%;

[0103] 47-52 min, maintain the volume percentage of mobile phase A at 53% and the volume percentage of mobile phase B at 47%;

[0104] (3.1) Column temperature is 23°C;

[0105] (4.1) Flow rate: 1.2 mL / min;

[0106] (5.1) The gradient elution eluate is detected by evaporative light scattering detection method, and the drift tube temperature of the evaporative light scattering detection method is 95°C.

[0107] The following are specific embodiments. The purpose is to further explain the present application in detail to help the technical personnel and researchers in the field to further understand the present application. The relevant technical conditions do not constitute any limitation to the present application. Any form of modification made within the scope of the claims of the present application is within the scope of protection of the claims of the present application.

[0108] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods. Experimental methods without specific conditions specified in the examples are carried out according to conventional conditions, such as those described in literature or books or methods recommended by manufacturers.

[0109] Example 1

[0110] Organic impurities in actual samples were separated and detected by high performance liquid chromatography assisted by evaporative light detection.

[0111] (1) Sample

[0112] Test solution: phospholipid stock solution in octafluoropropane lipid microsphere injection.

[0113] Impurity A~H individual reference substance stock solutions: Take impurities A~H separately, add solvent to dissolve and prepare individual impurity reference substance stock solutions containing approximately 50ug / ml per 1mL.

[0114] Impurity A~H mixed reference stock solution: Accurately pipette an appropriate amount of impurity A~H individual reference stock solution into the same volumetric flask and dilute to the scale with solvent. Each reference stock solution contains approximately 50ug / ml of a single impurity per 1mL.

[0115] System suitability solution (sample): Accurately pipette the impurity A~H mixed reference substance stock solution into a 0.5 ml volumetric flask, add 1.5 ml of the test solution, and dilute to the scale with solvent.

[0116] (2) Liquid chromatography conditions

[0117] Chromatographic column: Octadecylsilane bonded silica gel as filler (C18 4.6mm×250mm, 5μm )

[0118] Mobile phase A: 5 mM ammonium acetate in water

[0119] Mobile phase B: 5 mM ammonium acetate in methanol

[0120] Column temperature: 25°C; injection volume: 100 μl, flow rate: 1.0 mL / min.

[0121] Evaporative light detector parameters: drift tube temperature 90°C, carrier gas flow rate 3.0L / min

[0122] Gradient Program:

[0123]

[0124] (3) Results analysis

[0125] Figure 1 The chromatogram of the system suitability solution of this embodiment is shown, and the retention time and separation degree of each substance in the system suitability solution are shown in the following table:

[0126] Impurity Name Retention time RT / min Separation Impurity H 23.3 \ Impurity G 26.1 9.38 Impurity B 27.3 5.32 Impurity A 27.7 1.69 Impurity C 30.1 10.83 DPPE-PEG5000 34.1 13.11 DPPA 34.5 1.27 Impurity D 36.4 6.65 Impurity E 37.3 5.60 Impurity F 37.8 2.30 DPPC 40.6 8.47

[0127] Note: “\” means the first peak has no separation. Separation refers to the separation degree between the following peaks and the previous peak.

[0128] As can be seen from the above table, Example 1 can effectively separate impurities A to H, and can further effectively separate impurities A to H from octafluoropropane lipid microsphere injection. Figure 1 It can be seen that the peaks of each substance are sharp and the sensitivity is good.

[0129] Determination method

[0130] Accurately measure 100 μL of system suitability solution, impurity A~H mixed reference linear standard solution, and test solution, respectively, inject and measure, and record the chromatogram.

[0131] Example 2

[0132] It is basically the same as Example 1, except that a CAD detector is used instead of the evaporative light detector in Example 1, and the CAD detector parameters are: atomizer temperature 35°C.

[0133] Results analysis of this example

[0134] The retention time comparison of the peaks in the sample and the reference substance is shown in the following table:

[0135] Impurity Name Retention time RT / min Separation Impurity H 22.2 \ Impurity G 26.2 6.89 Impurity B 27.3 1.97 Impurity A 27.6 2.38 Impurity C 30.1 1.78

[0136] Note: “\” means the first peak has no separation. Separation refers to the separation degree between the following peaks and the previous peak.

[0137] The comparison shows that Example 2 effectively separates impurities A to C, G, and H from the octafluoropropane lipid microsphere injection, and the DPPE-PEG5000 and DPPA peaks are severely broadened, encapsulating 1,3-dipalmitin and 1,2-dipalmitin.

[0138] Embodiments 3 to 6

[0139] The method is basically the same as Example 1, except that the temperature of the drift tube of the evaporative light detector in the chromatographic conditions is 75° C., 85° C., 100° C., and 110° C., respectively.

[0140] By comparing the system suitability solution chromatograms of Example 1 and Examples 3 to 6, and combining the cutoff time of the glycerol propylene glycol peak, it can be found that as the drift tube temperature increases, the glycerol chromatographic peak broadens and the cutoff time decreases. When the drift tube temperature is above 90°C, the propylene glycol and glycerol peaks no longer interfere with other impurities. Propylene glycol and glycerol are excipients for octafluoropropane lipid microsphere injection, and the two usually peak together.

[0141] The glycerol propylene glycol peak cutoff time of the system applicability solution chromatographic analysis of Example 1 and Examples 3 to 6 is listed in the following table:

[0142] serial number Example 1 Example 3 Example 4 Example 5 Example 6 Drift tube temperature (℃) 90 75 85 100 110 Glycerol propylene glycol peak cut-off time (min) 12 36 13 4 3

[0143] Example 7

[0144] It is basically the same as Example 1, except that the gradient program is as follows:

[0145]

[0146] Example 8

[0147] It is basically the same as Example 1, except that the gradient program is as follows:

[0148]

[0149] Example 9

[0150] It is basically the same as Example 1, except that the gradient program is as follows:

[0151]

[0152] The peak heights of the chromatographic peaks of the relevant substances in the system applicability solution chromatograms of Example 1 and Examples 7 to 9 are listed in the following table:

[0153]

[0154] Combined with the above table, it can be seen that under each gradient elution condition, the specificity of each impurity detection meets the requirements. It can be seen from the table that in the system applicability solution chromatogram of Example 1, the peak height of each chromatographic peak is slightly larger. Combined with the gradient running time, the gradient elution condition of Example 1 is better.

[0155] Example 10

[0156] The method is substantially the same as Example 1, except that the flow rate in the chromatographic conditions is 1.2 ml / min, the column temperature is 23° C., and the drift tube temperature of the ELSD detector is 95° C. The separation between adjacent chromatographic peaks in each impurity peak in the solution chromatogram of the system suitability is not less than 1.82.

[0157] Embodiment 11

[0158] The method is substantially the same as Example 1, except that the flow rate in the chromatographic conditions is 1.2 ml / min, the column temperature is 27° C., and the drift tube temperature of the ELSD detector is 105° C. The separation between adjacent chromatographic peaks in each impurity peak in the solution chromatogram of the system suitability is not less than 1.68.

[0159] Example 12

[0160] The method is substantially the same as Example 1, except that the flow rate in the chromatographic conditions is 1.3 ml / min, the drift tube temperature of the ELSD detector is 100° C., and the carrier gas flow rate is 2.7 L / min. The separation between adjacent chromatographic peaks in each impurity peak in the solution chromatogram of the system suitability is not less than 1.64.

[0161] Embodiment 13

[0162] The method is substantially the same as Example 1, except that the flow rate in the chromatographic conditions is 1.2 ml / min, the drift tube temperature of the ELSD detector is 100° C., and the carrier gas flow rate is 3.3 L / min. The separation between adjacent chromatographic peaks in each impurity peak in the system suitability solution chromatogram is not less than 1.48.

[0163] Embodiment 14

[0164] The method is substantially the same as Example 1, except that the flow rate in the chromatographic conditions is 1.2 ml / min. The separation degree between each impurity peak and the adjacent chromatogram in the solution chromatogram of the system suitability is not less than 1.70.

[0165] Embodiment 15

[0166] The same as Example 14, except that the chromatographic column is C18 4.6mm250mm, 5μm

[0167] Although the peak order of palmitoyl glyceride in the system suitability solution chromatogram changed, baseline separation was achieved between the substances, and the separation degree between adjacent chromatographic peaks in each impurity peak was not less than 1.50.

[0168] Example 16

[0169] Basically the same as Example 14, except that the drift tube temperature is 100°C.

[0170] Although the peak order of palmitoyl glyceride in the system suitability solution chromatogram changed, baseline separation was achieved between the substances, and the separation degree between adjacent chromatographic peaks in each impurity peak was not less than 1.50.

[0171] Comparative Example 1

[0172] The same as Example 14, except that the chromatographic column has a pore size of

[0173] System suitability solution chromatogram results are shown in Figure 2 ,Depend on Figure 2 It can be seen that the conventional aperture is The C18 column showed that the retention of DPPE-PEG4000, DPPA and DPPC as well as impurities E and F was enhanced, but effective separation was not achieved, and the baseline rose after 38 min. This indicates that under this chromatographic condition, The same separation effect cannot be achieved with other chromatographic columns.

[0174] Impurity Name Retention time RT / min Separation Impurity H 22.6 \ Impurity G 25.8 1.55 Impurity B 26.2 / Impurity A 26.5 / Impurity C 31.6 14.13 DPPE-PEG5000 35.5 / DPPA 39.0 / DPPC 39.5 / Impurity E 40.3 / Impurity F 40.2 /

[0175] Note: “\” means the first peak has no separation degree. Separation degree refers to the separation degree between the following peak and the previous peak. “ / ” means the adjacent peaks are very close and cannot be effectively separated.

[0176] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0177] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several modifications and improvements can be made, which all belong to the scope of protection of the present application. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent of this application shall be based on the contents of the attached claims, and the description and drawings can be used to explain the contents of the claims.

Claims

1. A method for separating and detecting impurities in lipid microsphere injection, characterized in that: The impurities include one or more of impurities A to H, Among them, impurity A is lysophosphatidylcholine, impurity B is 2-acyl-lysophosphatidylcholine, impurity C is 1-stearoyl-lysophosphatidylcholine, impurity D is dipalmitoylphosphatidylethanolamine, impurity E is 1,3-dipalmitoylglycerol, impurity F is 1,2-dipalmitoylglycerol, impurity G is methoxy polyethylene glycol 5000-lysophosphatidylethanolamine, and impurity H is methoxy polyethylene glycol 5000. The separation and detection method includes detection by high performance liquid chromatography, and the conditions of the high performance liquid chromatography include: (1) The pore size of the chromatographic column packing is (2) The elution method is gradient elution, the mobile phase A is an aqueous solution of a buffer salt, the mobile phase B is a methanol solution of a buffer salt or a methanol aqueous solution, and the buffer salt is ammonium acetate or ammonium formate.

2. The method for separating and detecting impurities in lipid microsphere injection according to claim 1, characterized in that: The detection method comprises: Take any one or more of the reference substances of impurities A to H, dissolve them in a solvent, and prepare a reference substance; Take the lipid microsphere injection test object, dissolve or extract it with a solvent to prepare the test sample; The high performance liquid chromatography method is used to detect the reference substance and the test substance.

3. The method for separating and detecting impurities of lipid microsphere injection according to claim 1, characterized in that: The chromatographic column is a C18 chromatographic column, which can be an octadecylsilane bonded silica gel column.

4. The method for separating and detecting impurities in lipid microsphere injection according to claim 1, characterized in that: The concentration of the buffer salt in mobile phase A is 5 mM to 20 mM; and / or, The concentration of the buffer salt in mobile phase B is 5 mM to 20 mM; and / or, The volume percentage of water in the methanol aqueous solution is 0-10%.

5. The method for separating and detecting impurities in lipid microsphere injection according to claim 1, characterized in that: The conditions of the high performance liquid chromatography also include: the column temperature is 20°C to 40°C.

6. The method for separating and detecting impurities in lipid microsphere injection according to claim 1, characterized in that: The method of the gradient elution is as follows: From 0 to 15 min, the volume percentage of mobile phase A was maintained at 53%, and the volume percentage of mobile phase B was maintained at 47%; From 15 to 20 min, the volume percentage of mobile phase A decreased from 53% to 15%, and the volume percentage of mobile phase B increased from 47% to 85%; From 20 to 35 min, the volume percentage of mobile phase A decreased from 15% to 0%, and the volume percentage of mobile phase B increased from 85% to 100%; 35-45 min, keep the volume percentage of mobile phase A at 0% and the volume percentage of mobile phase B at 100%; From 45 to 47 min, the volume percentage of mobile phase A increased from 0% to 53%, and the volume percentage of mobile phase B decreased from 100% to 47%; From 47 to 52 min, the volume percentage of mobile phase A was maintained at 53%, and the volume percentage of mobile phase B was maintained at 47%.

7. The method for separating and detecting impurities in lipid microsphere injection according to claim 1, characterized in that: The conditions of the high performance liquid chromatography also include: a flow rate of 1.0 mL / min to 1.3 mL / min.

8. The method for separating and detecting impurities in lipid microsphere injection according to any one of claims 1 to 7, characterized in that: The eluate of the gradient elution was detected by evaporative light scattering detection.

9. The method for separating and detecting impurities in lipid microsphere injection according to any one of claims 1 to 7, characterized in that: In the step of detecting the eluent, the evaporative light scattering detection method adopts a closed split mode, the carrier gas flow rate of the evaporative light scattering detection method is 2.0L / min to 3.5L / min, and the drift tube temperature of the evaporative light scattering detection method is 90°C to 110°C.

10. The method for separating and detecting impurities in lipid microsphere injection according to claim 1, characterized in that: The conditions of the high performance liquid chromatography include: (1.1) The chromatographic column is an octadecylsilane bonded silica gel column, and the pore size of the filler is (2.1) The elution method is gradient elution, the mobile phase A is a 5mM to 20mM buffer salt aqueous solution, the mobile phase B is a 5mM to 20mM buffer salt methanol solution or a methanol aqueous solution, the volume percentage of water in the methanol aqueous solution is 0 to 10%, the buffer salt is ammonium acetate or ammonium formate, and the gradient elution method is as follows: From 0 to 15 min, the volume percentage of mobile phase A was maintained at 53%, and the volume percentage of mobile phase B was maintained at 47%; From 15 to 20 min, the volume percentage of mobile phase A decreased from 53% to 15%, and the volume percentage of mobile phase B increased from 47% to 85%; From 20 to 35 min, the volume percentage of mobile phase A decreased from 15% to 0%, and the volume percentage of mobile phase B increased from 85% to 100%; 35-45 min, keep the volume percentage of mobile phase A at 0% and the volume percentage of mobile phase B at 100%; From 45 to 47 min, the volume percentage of mobile phase A increased from 0% to 53%, and the volume percentage of mobile phase B decreased from 100% to 47%; 47-52 min, maintain the volume percentage of mobile phase A at 53% and the volume percentage of mobile phase B at 47%; (3.1) Column temperature is 23°C; (4.1) Flow rate: 1.2 mL / min; (5.1) The gradient elution eluate is detected by evaporative light scattering detection method, and the drift tube temperature of the evaporative light scattering detection method is 95°C.

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