CN117491546A - Detection method for content of PEGylated lipid - Google Patents
Detection method for content of PEGylated lipid Download PDFInfo
- Publication number
- CN117491546A CN117491546A CN202310795704.1A CN202310795704A CN117491546A CN 117491546 A CN117491546 A CN 117491546A CN 202310795704 A CN202310795704 A CN 202310795704A CN 117491546 A CN117491546 A CN 117491546A
- Authority
- CN
- China
- Prior art keywords
- mobile phase
- content
- pegylated lipid
- detecting
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 150000002632 lipids Chemical class 0.000 title claims abstract description 55
- 238000001514 detection method Methods 0.000 title claims description 7
- 238000000034 method Methods 0.000 claims abstract description 56
- 239000000243 solution Substances 0.000 claims description 77
- 238000012360 testing method Methods 0.000 claims description 56
- 239000000523 sample Substances 0.000 claims description 49
- 238000010828 elution Methods 0.000 claims description 19
- 239000003085 diluting agent Substances 0.000 claims description 18
- 239000013558 reference substance Substances 0.000 claims description 18
- 239000002202 Polyethylene glycol Substances 0.000 claims description 17
- 229920001223 polyethylene glycol Polymers 0.000 claims description 17
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 16
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 13
- 239000005695 Ammonium acetate Substances 0.000 claims description 13
- 229940043376 ammonium acetate Drugs 0.000 claims description 13
- 235000019257 ammonium acetate Nutrition 0.000 claims description 13
- 239000012488 sample solution Substances 0.000 claims description 13
- 239000002245 particle Substances 0.000 claims description 10
- 230000005526 G1 to G0 transition Effects 0.000 claims description 9
- 125000002947 alkylene group Chemical group 0.000 claims description 9
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 8
- 238000006116 polymerization reaction Methods 0.000 claims description 8
- 125000000217 alkyl group Chemical group 0.000 claims description 7
- 239000000126 substance Substances 0.000 claims description 7
- 238000005303 weighing Methods 0.000 claims description 7
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 5
- 125000003827 glycol group Chemical group 0.000 claims description 5
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 claims description 4
- 239000000377 silicon dioxide Substances 0.000 claims description 4
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 4
- 239000012498 ultrapure water Substances 0.000 claims description 4
- OQKFGIANPCRSSK-UHFFFAOYSA-N azanium;methanol;acetate Chemical group [NH4+].OC.CC([O-])=O OQKFGIANPCRSSK-UHFFFAOYSA-N 0.000 claims description 2
- 238000010790 dilution Methods 0.000 claims description 2
- 239000012895 dilution Substances 0.000 claims description 2
- 125000002960 margaryl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 125000001421 myristyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 125000000913 palmityl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- 125000002889 tridecyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims description 2
- 238000012795 verification Methods 0.000 abstract description 9
- 238000003908 quality control method Methods 0.000 abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 238000005259 measurement Methods 0.000 description 13
- 238000011084 recovery Methods 0.000 description 13
- 239000012490 blank solution Substances 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 239000012535 impurity Substances 0.000 description 7
- 238000000926 separation method Methods 0.000 description 5
- 239000012085 test solution Substances 0.000 description 5
- 230000000052 comparative effect Effects 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000010812 external standard method Methods 0.000 description 4
- 238000007689 inspection Methods 0.000 description 4
- 238000011835 investigation Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 239000012491 analyte Substances 0.000 description 3
- 125000004432 carbon atom Chemical group C* 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 125000005647 linker group Chemical group 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 229940054967 vanquish Drugs 0.000 description 3
- 238000013353 HPLC-CAD method Methods 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 125000004429 atom Chemical group 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 235000014113 dietary fatty acids Nutrition 0.000 description 2
- 238000012377 drug delivery Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000194 fatty acid Substances 0.000 description 2
- 229930195729 fatty acid Natural products 0.000 description 2
- 150000004665 fatty acids Chemical class 0.000 description 2
- -1 genetic vaccines Substances 0.000 description 2
- 238000012417 linear regression Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000002105 nanoparticle Substances 0.000 description 2
- 125000006353 oxyethylene group Chemical group 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 1
- 241000764238 Isis Species 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- OGBUMNBNEWYMNJ-UHFFFAOYSA-N batilol Chemical class CCCCCCCCCCCCCCCCCCOCC(O)CO OGBUMNBNEWYMNJ-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000007872 degassing Methods 0.000 description 1
- 229910001873 dinitrogen Inorganic materials 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 125000005842 heteroatom Chemical group 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 108700021021 mRNA Vaccine Proteins 0.000 description 1
- 229940126582 mRNA vaccine Drugs 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000004451 qualitative analysis Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 235000019166 vitamin D Nutrition 0.000 description 1
- 239000011710 vitamin D Substances 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
- 235000019168 vitamin K Nutrition 0.000 description 1
- 239000011712 vitamin K Substances 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
- G01N30/6052—Construction of the column body
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/64—Electrical detectors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/86—Signal analysis
- G01N30/8624—Detection of slopes or peaks; baseline correction
- G01N30/8631—Peaks
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/884—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample organic compounds
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides a novel method for measuring the content of PEGylated lipid based on a C18 chromatographic column. The determination method provided by the invention can be used for rapidly and accurately detecting the content of mPEG-DMG-2K and analogues thereof in a sample, meets verification standards in the aspects of system applicability, specificity, linearity, repeatability, intermediate precision, accuracy, solution stability and the like, has better durability, and can play an important role in quality control of related products.
Description
Technical Field
The invention belongs to the field of chemical analysis, and particularly relates to a content determination method of polyethylene glycol lipid.
Background
Liposomes are widely used to deliver nucleic acid drugs, genetic vaccines, anti-tumor drugs, small molecule drugs, polypeptide drugs, or protein drugs. The liposome has different forms and particle sizes, wherein the lipid nanoparticle (lipid nanoparticle, LNP) is one of the most popular drug delivery technologies at present as a non-traditional liposome. As one of key components of LNP, polyethylene glycol chain segment of polyethylene glycol lipid can improve hydrophilicity of LNP, form hydration film on LNP surface, prevent aggregation of particles in storage process, reduce non-specific cell uptake in drug delivery process, avoid LNP from being rapidly cleared by immune system, improve LNP stability under physiological condition, and make its pharmaceutical preparation fully exert drug effect.
mPEG-DMG-2K has been shown to function as a pegylated lipid in LNP delivery systems for commercial vaccines, such as mRNA vaccines developed by Moderna against covd-19. mPEG-DMG-2K is less polar and is difficult to elute on C18 columns, whereas C18 columns are popular in analytical work, especially the most widely used liquid columns for reverse phase chromatography. Therefore, how to detect the content of mPEG-DMG-2K and pegylated lipids having a similar structure using C18 chromatography column is a challenge in routine analysis. Therefore, the establishment of a widely-used content detection method of mPEG-DMG-2K and analogues thereof based on a C18 chromatographic column has important significance for the research and development of related polyethylene glycol lipid, LNP-pharmaceutical compositions and preparations thereof and the quality control in production.
Summary of The Invention
In order to overcome the defects of the prior art, the invention aims to provide a content detection method of mPEG-DMG-2K and analogues thereof based on a C18 chromatographic column, which can effectively elute the mPEG-DMG-2K and the analogues thereof, and accurately and efficiently determine the content of a sample.
The above object of the present invention is achieved by the following technical solutions,
one embodiment of the invention:
the method for detecting the content of the PEGylated lipid is characterized in that a reference substance solution and a test substance solution are respectively diluted and then detected by adopting a high performance liquid chromatography, wherein a detector of the high performance liquid chromatography is an electrospray detector, and a chromatographic column of the high performance liquid chromatography takes octadecylsilane chemically bonded silica as a stationary phase;
the elution mode of the detection method is gradient elution;
the structural formula of the polyethylene glycol lipid isWherein B is 1 、B 2 Each independently is a bond or C 1-3 An alkylene group; l (L) 1 、L 2 Each independently is a bond, -O-, -C (=o) O-, or-OC (=o) -; r is R 1 、R 2 Each independently is C 4-20 An alkyl group; l (L) d Is- (CH) 2 ) i -、-(CH 2 ) i OC(＝O)(CH 2 ) i -、-(CH 2 ) i OC(＝O)NH(CH 2 ) i -、-(CH 2 ) i NHC(＝O)(CH 2 ) i -、-(CH 2 ) i NHC(＝O)O(CH 2 ) i -any one of the following, wherein i is an integer from 1 to 4; n is the polymerization degree of polyethylene glycol chain and is selected from the integer of 4-250.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a novel method for measuring the content of PEGylated lipid based on a C18 chromatographic column, which solves the problem that mPEG-DMG-2K and analogues thereof are difficult to elute in the traditional reversed phase chromatographic method adopting the C18 chromatographic column and cannot be used for measuring the content. The determination method provided by the invention can be used for rapidly and accurately detecting the content of mPEG-DMG-2K and analogues thereof in a sample, meets verification standards in the aspects of system applicability, specificity, linearity, repeatability, intermediate precision, accuracy, solution stability and the like, has better durability, and can play an important role in quality control of related products.
1. Detailed description of the invention
The present invention has been described in detail with respect to specific embodiments, however, it is to be understood that it is by way of illustration and not limitation, and that various changes and modifications within the scope of the invention will become apparent to those skilled in the art.
The description in the cited documents of the present invention is different from the description of the present invention, and controls the present invention; this principle is directed to all references throughout the specification.
1.1 description of the terms
In the present invention, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The disclosures of all patents and other publications cited herein are incorporated by reference in their entirety. To the extent that any description of a term herein conflicts with any document incorporated by reference, the description and illustration of the following term shall govern. Unless otherwise indicated, each term has the following meaning.
In the present invention, "each independently is/is selected from/preferred" means that different objects may each independently be/is selected from/preferred any of their given options or ranges. For example, "B 1 、B 2 Each independently is a bond or C 1-3 The description of alkylene "indicates that B 1 The specific form of the catalyst is independent of B 2 Can be selected from the group consisting of a bond, C 1 Alkylene, C 2 Alkylene or C 3 Alkylene groups and vice versa.
In the present invention, unless otherwise indicated, the terms "comprising," "including," and "containing," and similar referents in the specification and claims are to be construed to be "including but not limited to" or "comprising" in an open and inclusive sense.
In the present invention, an object "including but not limited to" a certain range means that the object may be selected from, but not limited to, the range, and that there may be exceptions within the range that are expressly excluded by the present invention for the successful implementation of the present invention as a screening criterion.
In the present invention, unless otherwise specified, a numerical range means a group of all integers and non-integers in the range, including both endpoints. The numerical intervals can be represented by short horizontal lines (such as 1-6), wavy lines (such as 1-6), up to/to (such as 1-6, 1-6), and the like. For example, a concentration of 4-6g/L may be an integer (4, 5 or 6) or a non-integer (e.g., 4.4, 5.1, 5.88, etc.) within the interval). As another example, an "integer from 1 to 3" represents a group of 1, 2, 3. When numerical intervals are used to describe numbers, groups of integers are generally defaulted. For example, - (CH) 2 ) 1-4 -default representation-CH 2 -、-(CH 2 ) 2 -、-(CH 2 ) 3 -、-(CH 2 ) 4 -a group of members. As another example of the use of a catalyst,default representationA group of components. However, when numerical intervals are used to describe the average of numbers, it may be a non-integer. For example, a polyethylene glycol segment- (CH) 2 CH 2 O) n When n is an average or number average degree of polymerization selected from 44 to 46, n may be an integer (44, 45 or 46) or a non-integer (e.g., 44.64, 45.81, etc.).
In the present invention, the divalent linking group is not particularly limited, and any one of the two linking terminals may be selected when it is linked to another group. For example, an amide bond is used as a divalent linking group between GroupA and GroupB, and the specific linking may be GroupA-C (=O) NH-GroupB or GroupA-NHC (=O) -GroupB.
In the present invention, structural representation of the group is referred to byMarking the bond, e.g->Represents a group-G or G-, and (2)>Represents a group-CH 3 Or CH (CH) 3 -。
In the present invention, "bond" means a bond only and does not contain any atom.
In the present invention, a "linker" contains at least one atom.
In the present invention, the number of carbon atoms in a group may be represented by a subscript number of C, which does not contain a contribution of a substituent unless otherwise specified. For example, C 1-12 Meaning "having 1 to 12 carbon atoms". As another example, C 1-10 Alkylene means any alkylene group having 1 to 10 carbon atoms, i.e. C 1 、C 2 、C 3 、C 4 、C 5 、C 6 、C 7 、C 8 、C 9 、C 10 Any one of alkylene groups.
In the present invention, if there is a difference between the structure described herein and the name of the structure, the structure itself should have a greater weight.
In the present invention, the molecular weight of a polymer (including but not limited to pegylated lipids) by default refers to the average molecular weight. When not specifically defined, the average molecular weight is generally the number average molecular weight (M n ) Either the molecular weight of the polydisperse blocks or species can be described or the monodisperse blocks or species can be described. Unless otherwise indicated, the molecular weight is measured in daltons (Da). The molecular weight of the polyethylene glycol chain may also be expressed in terms of "degree of polymerization", specifically referring to the number of repeating units (oxyethylene units) therein. Accordingly, the average value or the number average value of the number of repeating units is preferably represented by "average polymerization degree" or "number average polymerization degree".
In the present invention, unless otherwise specified, the molecular weight of the polymer is generally selected from the numerical range of.+ -. 10% of the given value, and some cases may be amplified to.+ -. 15% but not more than.+ -. 20%. For example, a "pegylated lipid having a molecular weight of 2000Da" will generally be described as having a molecular weight selected from the range of 2000 Da.+ -. 10% (i.e., 1800-2200 Da).
In the present invention, "lipids" are also known as "lipids," including but not limited to esters of fatty acids, and are characterized by generally poor solubility in water, but are soluble in many non-polar organics. Although lipids generally have poor solubility in water, certain classes of lipids (e.g., lipids modified with polar groups) have limited water solubility and may be soluble in water under certain conditions. Known types of lipids include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins (e.g., vitamins A, D, E and K), monoglycerides, diglycerides, triglycerides, phospholipids, and the like. In the present invention, the lipid may be synthetic or derived (isolated or modified) from natural sources or compounds.
In the present invention, the "polyethylene glycol segment" comprises n repeating oxyethylene units (-CH) 2 CH 2 O-or-OCH 2 CH 2 (-), n is the degree of polymerization.
In the present invention, "pegylated lipid" refers to a compound in which a lipid and a polyethylene glycol segment are bonded by several covalent chemical bonds, wherein the lipid moiety and the segment adjacent to the polyethylene glycol segment are allowed to contain a heteroatom group.
In the present invention, "mPEG-DMG-2K" means that the structure isIs a compound of (a).
In the invention, the high performance liquid chromatography (high performance liquid chromatography) is called HPLC for short, which is a chromatographic analysis technology and is characterized in that a high pressure infusion pump, a high sensitivity detector and a high performance particle stationary phase are adopted, and the method can be used for component separation, component qualitative and quantitative analysis in a liquid mixture.
In the invention, an electric fog detector (charged aerosol detector) is called CAD for short, the working principle is that an eluent is atomized to form particles, the particles are dried by an evaporating pipe and collide with charged nitrogen gas, so that the surfaces of the analyte particles are positively charged, and finally, the charge quantity of the surfaces of the analyte particles is measured by an electrometer, so that the chromatographic peak area is related to the charge quantity of the surfaces of the analyte particles.
In the present invention, "HPLC-CAD" means an HPLC method using a CAD detector.
In the present invention, the "C18 column" is also called "ODS column" and refers to a chromatographic column using octadecylsilane chemically bonded silica as a stationary phase.
1.2 method for measuring content of PEGylated lipid by HPLC-CAD method
One embodiment of the invention:
the method for detecting the content of the PEGylated lipid is characterized in that a reference substance solution and a test substance solution are respectively diluted and then detected by adopting a high performance liquid chromatography, wherein a detector of the high performance liquid chromatography is an electrospray detector, and a chromatographic column of the high performance liquid chromatography takes octadecylsilane chemically bonded silica as a stationary phase;
the elution mode of the detection method is gradient elution;
the structural formula of the polyethylene glycol lipid isWherein B is 1 、B 2 Each independently is a bond or C 1-3 An alkylene group; l (L) 1 、L 2 Each independently is a bond, -O-, -C (=o) O-, or-OC (=o) -; r is R 1 、R 2 Each independently is C 4-20 An alkyl group; l (L) d Is- (CH) 2 ) i -、-(CH 2 ) i OC(＝O)(CH 2 ) i -、-(CH 2 ) i OC(＝O)NH(CH 2 ) i -、-(CH 2 ) i NHC(＝O)(CH 2 ) i -、-(CH 2 ) i NHC(＝O)O(CH 2 ) i -any one of the following, wherein i is an integer from 1 to 4; n is the polymerization degree of polyethylene glycol chain and is selected from the integer of 4-250.
In one embodiment of the present invention, R 1 、R 2 Each independently is C 10-20 An alkyl group; more preferably each independently is tridecyl, tetradecyl, hexadecyl or heptadecyl; further preferably R 1 、R 2 Are the same alkyl groups.
In a specific embodiment of the invention, the structure of the pegylated lipid is selected from any one of the following:
preferably selected from any one of the following:
more preferably
In a specific embodiment of the invention, the molecular weight of the pegylated lipid is between 200 and 20000Da, preferably between 1000 and 3000Da, more preferably between 1400 and 2600Da, most preferably 2000Da.
In a specific embodiment of the invention, the pore size of the stationary phase is selected fromPreferably +.>More preferably +.>
In a specific embodiment of the invention, the particle size of the stationary phase is selected from 2 to 15. Mu.m, preferably 3 to 10. Mu.m, most preferably 5. Mu.m.
In one embodiment of the present invention, the mobile phase of the high performance liquid chromatography comprises mobile phase a and mobile phase B; wherein the mobile phase A is ultrapure water, and the mobile phase B is ammonium acetate methanol solution; preferably, the ammonium acetate concentration in the mobile phase B is 0.4-0.6g/L, more preferably 0.4g/L, 0.5g/L or 0.6g/L, most preferably 0.6g/L.
In a specific embodiment of the invention, the ammonium acetate concentration in mobile phase B is 0.4g/L.
In a specific embodiment of the invention, the ammonium acetate concentration in mobile phase B is 0.5g/L.
In a specific embodiment of the invention, the ammonium acetate concentration in mobile phase B is 0.6g/L.
In a specific embodiment of the invention, mobile phase B is also used to dilute the control solution and/or the test solution.
In one embodiment of the invention, mobile phase B is formulated using the following method: weighing 0.6g of ammonium acetate, adding 1L of methanol for dissolution, filtering, and carrying out ultrasonic degassing for 20-80min.
In a specific embodiment of the invention, the flow rate of the mobile phase is 0.5 to 1.5mL/min, preferably 0.8 to 1.2mL/min, more preferably 1.0mL/min.
In one embodiment of the invention, the flow rate of the mobile phase is 0.8mL/min.
In one embodiment of the invention, the flow rate of the mobile phase is 1.0mL/min.
In one embodiment of the invention, the flow rate of the mobile phase is 1.2mL/min.
In one embodiment of the invention, the gradient elution procedure is as follows:
0-T 1 min, mobile phase B: gradient of 50-80% to 100%, mobile phase A: the balance;
T 1 -T 2 min, mobile phase B:100%;
T 2 -T 3 min, mobile phase B: changing from 100% gradient to 50-80%, mobile phase A: the balance;
T 4 min, stopping;
wherein T is 1 、T 2 、T 3 、T 4 An integer selected from 1 to 60, and wherein any one of the time intervals is composed of 1 to 2 sub-intervals;
preferably the gradient elution procedure is selected from any one of the following:
procedure (1):
0-20min, mobile phase B:50% gradient to 100%, mobile phase a: the balance;
20-30min, mobile phase B:100%;
30-31min, mobile phase B: from 100% gradient to 50%, mobile phase a: the balance;
stopping for 40 min;
procedure (2):
0-20min, mobile phase B:60% gradient to 100%, mobile phase a: the balance;
20-30min, mobile phase B:100%;
30-31min, mobile phase B: from 100% gradient to 60%, mobile phase a: the balance;
stopping for 40 min;
procedure (3):
0-10min, mobile phase B:50% gradient to 70%, mobile phase a: the balance;
10-20min, mobile phase B:70% gradient to 100%, mobile phase a: the balance;
20-30min, mobile phase B:100%;
30-31min, mobile phase B: from 100% gradient to 50%, mobile phase a: the balance;
stopping for 40 min;
more preferably procedure (3).
In a specific embodiment of the invention, the control solution is prepared by the following method: taking 0.02-0.2 g of polyethylene glycol lipid reference substance, precisely weighing, precisely adding 10-100 mL of diluent, dissolving and quantitatively diluting to prepare a reference substance solution with the concentration of the reference substance of 1 mg/mL.
In one embodiment of the invention, the sample concentration of the control solution after dilution is 200 mug/mL.
In one specific embodiment of the invention, the test solution is formulated by the following method: taking 0.02-0.2 g of the PEGylated lipid sample, precisely weighing, precisely adding 10-100 mL of diluent, dissolving and quantitatively diluting to prepare a sample solution with the concentration of the sample of 1 mg/mL.
In a specific embodiment of the invention, the sample solution is diluted to a sample concentration of 100-300. Mu.g/mL, preferably 140-260. Mu.g/mL, more preferably 200. Mu.g/mL.
In one embodiment of the invention, the sample size for chromatographic testing is 6-10. Mu.L, preferably 8. Mu.L.
In one embodiment of the invention, the sample loading for chromatographic testing is 6. Mu.L.
In one embodiment of the invention, the sample size for chromatographic testing is 8. Mu.L.
In one embodiment of the invention, the sample loading for chromatographic testing is 10. Mu.L.
In a specific embodiment of the invention, the column temperature for chromatographic testing is 30-50 ℃, preferably 35-45 ℃, more preferably 40 ℃.
In one embodiment of the invention, the column temperature for chromatographic testing is 35 ℃.
In one embodiment of the invention, the column temperature for chromatographic testing is 40 ℃.
In one embodiment of the invention, the column temperature for chromatographic testing is 45 ℃.
In a specific embodiment of the invention, the content of said pegylated lipid in the test solution is calculated by an external standard method.
In a specific embodiment of the invention, the temperature of the electrospray detector is 30 ℃, 50 ℃ or 70 ℃, preferably 50 ℃.
In a specific embodiment of the invention, the chromatographic column is selected from any of XBIridge BEH 300C 18, agilent Zorbax300SB C18, agilent Poroshell EC-C18, phenomenex Kinetex C, titank C18, ACE ExcEL C18-AR, ACE ExcEL C18-Amide, cadeza HS-C18, pnulips BP300A-C18, preferably pnulips BP300A-C18.
In a specific embodiment of the invention, the length of the chromatographic column is selected from 100-300mm, preferably 250mm.
In a specific embodiment of the invention, the diameter of the chromatographic column is selected from 4-8mm, preferably 4.6mm.
In one embodiment of the invention, the method for detecting the content of the PEGylated lipid comprises the following steps:
(1) Determining a specificity spectrogram: preparing blank solution, reference substance solution, test substance solution and reference substance-test substance mixed solution, respectively performing chromatographic test to confirm that blank component has no interference, and determining retention time of the component to be tested.
(2) And (3) making a standard curve: preparing a series of reference substance solutions with gradient concentration, performing chromatographic test to obtain parameters such as peak area, calculating a linear regression equation of concentration and peak area, and drawing a linear curve.
(3) And (3) content measurement: and (3) diluting the sample solution (preferably to about 200 mug/mL), performing chromatographic test, recording a chromatogram, obtaining parameters such as peak area and the like, and calculating the content of the component to be detected by an external standard method.
2. Description of the drawings
FIG. 1 is a chromatogram of a blank solution;
FIG. 2 is a chromatogram of a mPEG-DMG-2K control solution;
FIG. 3 is a chromatogram of a mPEG-DMG-2K test solution;
FIG. 4 is a chromatogram of a mPEG-DMG-2K mixed solution;
FIG. 5 is a linear regression plot of peak area of mPEG-DMG-2K component versus its concentration;
FIG. 6 is a chromatogram of a non-gradient elution comparative example;
FIG. 7 is a chromatogram of a gradient elution comparative example.
3. Detailed description of the preferred embodiments
(1) HPLC instrument configuration
Pump unit: vanquish Pump;
a detector: vanquish CAD Detector (detector temperature: 50 ℃);
chromatographic column (analytical column): a C18-group of the components,5μm，4.6×250mm；
protective column: a C18-group of the components,5μm，4.0×10mm
column incubator: vanquish Column Compartment;
work station: chromelon7.
(2) Equipment and method for manufacturing the same
Analytical balance (graduation value 0.1 mg);
pipette (200.0. Mu.L gauge);
pipette (1.0 mL).
(3) Reagent(s)
Methanol (chromatographic grade);
ammonium acetate (chromatographic grade);
mPEG-DMG-2K test;
mPEG-DMG-2K control.
Example 1: solution formulation and sample preparation
Mobile phase a: ultrapure water, and ultrasonically deaerated for at least 10 minutes.
Mobile phase B: to 1L of methanol was added 0.6g of ammonium acetate and the mixture was degassed by ultrasound for at least 20min.
Solution 1: accurately weighing 100.0mg of mPEG-DMG-2K reference substance, dissolving with a proper amount of diluent, and fixing the volume into a 100mL volumetric flask to obtain the product;
solution 2: accurately weighing 100.0mg of mPEG-DMG-2K test sample, dissolving with a proper amount of diluent, and fixing the volume into a 100mL volumetric flask to obtain the product;
in all examples, the diluent (blank solution) was mobile phase B, unless otherwise specified.
Example 2: chromatographic test
HPLC-CAD method is used for detecting the content of mPEG-DMG-2K in the sample solution, and the chromatographic conditions are as follows:
column oven temperature: 40 ℃;
flow rate: 1.0mL/min;
sample injection amount: 8. Mu.L;
mobile phase a: ultrapure water;
mobile phase B:0.6g/L ammonium acetate in methanol;
gradient elution procedure:
0-10min, mobile phase B:50% gradient to 70%, mobile phase a: the balance;
10-20min, mobile phase B:70% gradient to 100%, mobile phase a: the balance;
20-30min, mobile phase B:100%;
30-31min, mobile phase B: from 100% gradient to 50%, mobile phase a: the balance;
and stopping the operation after 40 min.
The content of mPEG-DMG-2K in the sample solution is calculated by an external standard method, and the formula is as follows:
wherein w is the content of the component to be detected in the sample; ar is the average peak area of the components to be detected in the reference substance solution, and As is the peak area of the components to be detected in the sample solution; cr is the concentration (mug/mL) of the component to be detected in the reference substance solution; p is the purity of the component reference substance to be detected, and Cs is the concentration (mg/mL) of the solution of the sample to be detected.
Example 3: methodological verification
Example 3.1: system applicability test
2.0mL of the solution 1 is taken and placed in a 10mL volumetric flask, the volume is fixed by using a diluent solution, and the solution is uniformly mixed and shaken to obtain a system applicability solution (standard solution with the marked quantity of 100%). The sample was continuously fed 6 times, and the result was shown in Table 1, and the RSD of the peak area was 0.27% (. Ltoreq.5.0%), indicating that the method system was well-suited.
Table 1, system applicability results
Example 3.2: specificity test
A proprietary test solution was formulated according to table 2 and the retention time was tested. The test results are shown in FIGS. 1-4, the blank solution has no interference, and the retention time of the mPEG-DMG-2K component is 24.442min.
TABLE 2 specific formulation Table
| Sample of | Diluent solution constant volume (mL) | Solution 1 (mL) | Solution 2 (mL) |
| Blank solution | 10.0 | 0 | 0 |
| mPEG-DMG-2K reference substance | 10.0 | 2.0 | 0 |
| mPEG-DMG-2K test sample | 10.0 | 0 | 2.0 |
| Mixing sample | 10.0 | 0.5 | 2.0 |
Example 3.3: linearity test
Different standard solutions were prepared in the indicated amounts of 25-175% and tested as follows.
Linear solution S-1 (25%): taking 0.5mL of the solution 1, placing the solution into a 10mL volumetric flask, fixing the volume by using a diluent solution, mixing and shaking uniformly to be measured.
Linear solution S-2 (50%): 1.0mL of the solution 1 is taken and placed in a 10mL volumetric flask, the volume is fixed by using the diluent solution, and the mixture is uniformly mixed and shaken for measurement.
Linear solution S-3 (75%): 1.5mL of the solution 1 is taken and placed in a 10mL volumetric flask, the volume is fixed by using the diluent solution, and the mixture is uniformly mixed and shaken for measurement.
Linear solution S-4 (100%): 2.0mL of the solution 1 is taken and placed in a 10mL volumetric flask, the volume is fixed by using the diluent solution, and the mixture is uniformly mixed and shaken for measurement.
Linear solution S-5 (125%): 2.5mL of the solution 1 is taken and placed in a 10mL volumetric flask, the volume is fixed by using the diluent solution, and the mixture is uniformly mixed and shaken for measurement.
Linear solution S-6 (150%): 3.0mL of the solution 1 is taken and placed in a 10mL volumetric flask, the volume is fixed by using the diluent solution, and the mixture is uniformly mixed and shaken for measurement.
Linear solution S-7 (175%): 3.5mL of the solution 1 is taken and placed in a 10mL volumetric flask, the volume is fixed by using the diluent solution, and the mixture is uniformly mixed and shaken for measurement.
The linear ranges of the different concentrations and peak areas are shown in table 3 and the linear curves are shown in fig. 5. Linear equation: y=0.0756x+2.1911, r 2 =0.9960 (. Gtoreq.0.990), indicating that mPEG-DMG-2K is linear in the range of 50-350 μg/mL.
TABLE 3 Linear test results
Example 3.4: reproducibility test
Transferring 2.0mL of the solution 2, placing the solution in a 10.0mL volumetric flask, adding a diluent to fix the volume, shaking uniformly, preparing 6 parts in parallel, carrying out sample injection test, and observing the relative standard deviation of the results. The results are shown in Table 4, and the RSD of the sample injection test is 0.58% and less than 5.0%, which indicates good reproducibility.
TABLE 4 repeatability test results
Example 3.5: intermediate precision test
Different experimenters remove 2.0mL of the solution 2 on different days, place the solution in a 10.0mL volumetric flask, add diluent to fix the volume and shake the solution uniformly, prepare 6 parts in parallel, sample injection test, and investigate the relative standard deviation of the results. The results are shown in Table 5, the RSD of the sample injection test of different experimenters and different days are respectively 0.58% and 2.52%, the RSD of the test result of 12 test sample solutions of the two experimenters is 1.78%, and the RSD is less than 5.0%, which shows that the intermediate precision is good.
TABLE 5 results of intermediate precision test
Example 3.6: accuracy test
The accuracy solutions were prepared as in Table 6, and were all sized to 10.0mL and tested. Wherein A0 is a sample solution without a reference substance solution, and A1, A2 and A3 are solutions prepared by the reference substance solutions with different concentrations and the sample solution. A0, A1, A2 and A3 were each formulated in parallel in 3 parts. The recovery rate is calculated by an external standard method, the recovery rate level is 80% -120%, and the relative standard deviation of the standard addition recovery rate result is not more than 5.0%.
TABLE 6 accuracy solution formulation table
| Sample numbering | A0 | A1 | A2 | A3 |
| Solution 1 (mL) | 0 | 0.4 | 0.5 | 0.6 |
| Solution 2 (mL) | 2.0 | 2.0 | 2.0 | 2.0 |
| Reference concentration (μg/mL) | 0 | 40 | 50 | 60 |
| Sample concentration (μg/mL) | 200 | 200 | 200 | 200 |
The results are shown in Table 7, with recovery rates between 98.06% and 99.91% and RSD of 0.63% and less than 5.0%, indicating good process accuracy.
TABLE 7 accuracy test results
Example 3.7: solution stability test
Recovery was determined after 24 hours and 68 hours of standing the proprietary solution prepared in example 3.2. If the recovery rate is in the range of 80% -120%, the solution is stable in the period of time, otherwise, the solution is unstable, and the preparation and sample introduction time is required to be regulated according to the stability result. The results are shown in Table 8, with recovery ranging from 80% to 120% and solution stable over 68 hours.
TABLE 8 stability of solutions results
Example 4: method durability
The conditions of HPLC-CAD test are changed, the solution in test example 3.2 is tested, and the content of the measured result of the test sample solution is 90% -110.0%.
Example 4.1: mobile phase investigation
(1) Mobile phase B was changed to acetonitrile based on the chromatographic conditions of example 2.
Test results: cannot be eluted.
(2) Based on the chromatographic conditions of example 2, mobile phase A was changed to a 0.6g/L aqueous ammonium acetate solution and mobile phase B was changed to a methanol solution.
Test results: the peak type is not good, the separation effect is poor, impurities cannot be separated, and the content of the sample cannot be accurately measured.
(3) Mobile phase B was changed to 0.4g/L methanolic ammonium acetate based on the chromatographic conditions of example 2.
Test results: the blank solution has no interference, the sample content is 98.51%, the standard adding recovery rate is 100.82%, and the system applicability, the specificity and the sample measurement result all accord with the verification standard.
(4) Mobile phase B was changed to 0.5g/L methanolic ammonium acetate based on the chromatographic conditions of example 2.
Test results: the blank solution has no interference, the sample content is 100.78%, the standard adding recovery rate is 104.28%, and the system applicability, the specificity and the sample measurement result all accord with the verification standard.
(5) Durability inspection results: the method has good durability between 0.4-0.6g/L of ammonium acetate.
Example 4.2: inspection by detector
The detector was changed to a light scattering detector based on the chromatographic conditions of example 2.
Test results: the sensitivity is low.
Example 4.3: flow rate investigation
(1) The flow rate was changed to 0.8mL/min based on the chromatographic conditions of example 2.
Test results: the blank solution has no interference, the sample content is 98.69%, the standard adding recovery rate is 94.70%, and the system applicability, the specificity and the sample measurement result all accord with the verification standard.
(2) The flow rate was changed to 1.2mL/min based on the chromatographic conditions of example 2.
Test results: the blank solution has no interference, the sample content is 99.74%, the standard adding recovery rate is 96.16%, and the system applicability, the specificity and the sample measurement result all accord with the verification standard.
(3) Durability inspection results: the method has good durability between 0.8-1.2 mL/min.
Example 4.4: investigation of elution pattern
(1) Non-gradient elution: only mobile phase B was used.
Test results: the peak type is not good, the separation effect is poor, impurities cannot be separated from main peaks and impurities, and the content of the sample cannot be accurately and reliably measured, as shown in fig. 6.
(2) Gradient elution comparative example:
0-20min, mobile phase B:50% gradient to 80%, mobile phase a: the balance;
20-30min, mobile phase B:80%, mobile phase a: the balance;
30-31min, mobile phase B:80% gradient to 50%, mobile phase a: the balance;
40min: stopping.
Test results: the separation effect is poor, impurities and main peaks cannot be separated, and the content of a sample cannot be accurately and reliably measured, as shown in fig. 7.
(3) Gradient elution comparative example:
0-20min, mobile phase B:60% gradient to 90%, mobile phase a: the balance;
20-30min, mobile phase B:90%, mobile phase a: the balance;
30-31min, mobile phase B:90% gradient to 60%, mobile phase a: the balance;
40min: stopping.
Test results: the separation effect is poor, impurities and main peaks cannot be separated, and the content of a sample cannot be accurately and reliably measured.
(4) Gradient elution procedure:
0-10min, mobile phase B:50% gradient to 70%, mobile phase a: the balance;
10-20min, mobile phase B:70% gradient to 100%, mobile phase a: the balance;
20-30min, mobile phase B:100%;
30-31min, mobile phase B: from 100% gradient to 50%, mobile phase a: the balance;
stopping for 40 min;
test results: the impurities and the main peaks can be well separated, and the content of the sample can be accurately and reliably measured.
(5) Gradient elution procedure:
0-20min, mobile phase B:60% gradient to 100%, mobile phase a: the balance;
20-30min, mobile phase B:100%;
30-31min, mobile phase B: from 100% gradient to 60%, mobile phase a: the balance;
and stopping the operation after 40 min.
Test results: the impurities and the main peaks can be well separated, and the content of the sample can be accurately and reliably measured.
Example 4.5: column temperature investigation
(1) Based on the chromatographic conditions of example 2, the column temperature was changed to 35 ℃.
Test results: the blank solution has no interference, the sample content is 99.65%, the standard adding recovery rate is 95.88%, and the system applicability, the specificity and the sample measurement result all accord with the verification standard.
(2) Based on the chromatographic conditions of example 2, the column temperature was changed to 45 ℃.
Test results: the blank solution has no interference, the sample content is 99.01%, the standard adding recovery rate is 97.24%, and the system applicability, the specificity and the sample measurement result all accord with the verification standard.
(3) Durability inspection results: the method has good durability at column temperature of 35-45deg.C.
The foregoing description is only illustrative of the present invention and is not intended to limit the scope of the invention, and all equivalent structures or equivalent processes or direct or indirect application in other related arts are included in the scope of the present invention.
It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains.
Claims (16)
1. The method for detecting the content of the PEGylated lipid is characterized in that a reference substance solution and a test substance solution are respectively diluted and then detected by adopting a high performance liquid chromatography, wherein a detector of the high performance liquid chromatography is an electrospray detector, and a chromatographic column of the high performance liquid chromatography takes octadecylsilane chemically bonded silica as a stationary phase;
the elution mode of the detection method is gradient elution;
the structural formula of the polyethylene glycol lipid isWherein B is 1 、B 2 Each independently is a bond or C 1-3 An alkylene group; l (L) 1 、L 2 Each independently is a bond, -O-, -C (=o) O-, or-OC (=o) -; r is R 1 、R 2 Each independently is C 4-20 An alkyl group; l (L) d Is- (CH) 2 ) i -、-(CH 2 ) i OC(＝O)(CH 2 ) i -、-(CH 2 ) i OC(＝O)NH(CH 2 ) i -、-(CH 2 ) i NHC(＝O)(CH 2 ) i -、-(CH 2 ) i NHC(＝O)O(CH 2 ) i -any one of the following, wherein i is an integer from 1 to 4; n is the polymerization degree of polyethylene glycol chain and is selected from the integer of 4-250.
2. The method for detecting the content of the PEGylated lipid according to claim 1, wherein R is 1 、R 2 Each independently is C 10-20 An alkyl group; more preferably each independently is tridecyl, tetradecyl, hexadecyl or heptadecyl; further preferably R 1 、R 2 Are the same alkyl groups.
3. The method for detecting the content of the PEGylated lipid according to claim 1, wherein L d is-CH 2 -、-CH 2 OC(＝O)CH 2 CH 2 -、-CH 2 CH 2 NHC(＝O)CH 2 CH 2 -、-CH 2 CH 2 NHC(＝O)OCH 2 CH 2 -、-CH 2 OC(＝O)NHCH 2 CH 2 CH 2 -any one, preferably-CH 2 -、-CH 2 OC(＝O)CH 2 CH 2 -or-CH 2 OC(＝O)NHCH 2 CH 2 CH 2 -。
4. The method for detecting the content of the PEGylated lipid according to claim 2, wherein the structure of the PEGylated lipid is selected from any one of the following:
preferably selected from any one of the following:
more preferably
5. The method for detecting the content of the PEGylated lipid according to claim 4, wherein the molecular weight of the PEGylated lipid is 200-20000 Da, preferably 1000-3000 Da, more preferably 1400-2600 Da, and most preferably 2000Da.
6. The method for detecting the content of the PEGylated lipid according to claim 1, wherein the pore size of the stationary phase is selected from the group consisting ofPreferably +.>More preferably +.>
7. The method for detecting the content of pegylated lipids according to claim 1, characterized in that the particle size of the stationary phase is chosen from 2-15 μm, preferably 3-10 μm, most preferably 5 μm.
8. The method for detecting the content of the PEGylated lipid according to claim 1, wherein the mobile phase of the high performance liquid chromatography comprises a mobile phase A and a mobile phase B; wherein the mobile phase A is ultrapure water, and the mobile phase B is ammonium acetate methanol solution; preferably, the ammonium acetate concentration in the mobile phase B is 0.4-0.6g/L, more preferably 0.4g/L, 0.5g/L or 0.6g/L, most preferably 0.6g/L.
9. The method for detecting the content of the PEGylated lipid according to claim 8, wherein the flow rate of the mobile phase is 0.5-1.5 mL/min, preferably 0.8-1.2mL/min, more preferably 1.0mL/min.
10. The method for detecting the content of the PEGylated lipid according to claim 8, wherein the gradient elution is performed as follows:
0-T 1 min, mobile phase B: gradient of 50-80% to 100%, mobile phase A: the balance;
T 1 -T 2 min, mobile phase B:100%;
T 2 -T 3 min, mobile phase B: changing from 100% gradient to 50-80%, mobile phase A: the balance;
T 4 min, stopping;
wherein T is 1 、T 2 、T 3 、T 4 An integer selected from 1 to 60, and wherein any one of the time intervals is composed of 1 to 2 sub-intervals;
preferably the gradient elution procedure is selected from any one of the following:
procedure (1):
0-20min, mobile phase B:50% gradient to 100%, mobile phase a: the balance;
20-30min, mobile phase B:100%;
30-31min, mobile phase B: from 100% gradient to 50%, mobile phase a: the balance;
stopping for 40 min;
procedure (2):
0-20min, mobile phase B:60% gradient to 100%, mobile phase a: the balance;
20-30min, mobile phase B:100%;
30-31min, mobile phase B: from 100% gradient to 60%, mobile phase a: the balance;
stopping for 40 min;
procedure (3):
0-10min, mobile phase B:50% gradient to 70%, mobile phase a: the balance;
10-20min, mobile phase B:70% gradient to 100%, mobile phase a: the balance;
20-30min, mobile phase B:100%;
30-31min, mobile phase B: from 100% gradient to 50%, mobile phase a: the balance;
stopping for 40 min;
more preferably procedure (3).
11. The method for detecting the content of the PEGylated lipid according to claim 1, wherein the control solution is prepared by the following method: taking 0.02-0.2 g of polyethylene glycol lipid reference substance, precisely weighing, precisely adding 10-100 mL of diluent, dissolving and quantitatively diluting to prepare a reference substance solution with the concentration of the reference substance of 1 mg/mL.
12. The method for detecting the content of the PEGylated lipid according to claim 11, wherein the sample concentration is 200. Mu.g/mL after the dilution of the control solution.
13. The method for detecting the content of the PEGylated lipid according to claim 1, wherein the sample solution is prepared by the following method: taking 0.02-0.2 g of the PEGylated lipid sample, precisely weighing, precisely adding 10-100 mL of diluent, dissolving and quantitatively diluting to prepare a sample solution with the concentration of the sample of 1 mg/mL.
14. The method for detecting the content of the PEGylated lipid according to claim 13, wherein the sample solution is diluted to have a sample concentration of 100-300 μg/mL, preferably 140-260 μg/mL, more preferably 200 μg/mL.
15. The method for detecting the content of the PEGylated lipid according to claim 1, wherein the sample injection amount of the chromatographic test is 6-10. Mu.L, preferably 8. Mu.L.
16. The method for detecting the content of the PEGylated lipid according to claim 1, wherein the column temperature of the chromatographic test is 30-50 ℃, preferably 35-45 ℃, more preferably 40 ℃.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310795704.1A CN117491546A (en) | 2023-06-30 | 2023-06-30 | Detection method for content of PEGylated lipid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310795704.1A CN117491546A (en) | 2023-06-30 | 2023-06-30 | Detection method for content of PEGylated lipid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117491546A true CN117491546A (en) | 2024-02-02 |
Family
ID=89673192
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310795704.1A Pending CN117491546A (en) | 2023-06-30 | 2023-06-30 | Detection method for content of PEGylated lipid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117491546A (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107951861A (en) * | 2016-10-17 | 2018-04-24 | 南京绿叶制药有限公司 | A kind of lipid nano particle membrane material feed composition |
| CN113960182A (en) * | 2020-07-21 | 2022-01-21 | 苏州艾博生物科技有限公司 | Method for detecting lipid component in lipid nanosphere |
| CN114507342A (en) * | 2022-03-18 | 2022-05-17 | 江苏东南纳米材料有限公司 | Preparation method of 1, 2-dimyristoyl-RAC-glycerol-3-methoxypolyethylene glycol and intermediate thereof |
| CN116024237A (en) * | 2023-01-05 | 2023-04-28 | 中国人民解放军军事科学院军事医学研究院 | mRNA vaccine for preventing heat of rift grains and preparation method thereof |
| CN116134020A (en) * | 2020-07-17 | 2023-05-16 | 赛诺菲巴斯德有限公司 | Lipid compounds comprising at least one terminal group of formula-NH-CX-A or-NH-CX-NH-A, compositions containing them and uses thereof |
-
2023
- 2023-06-30 CN CN202310795704.1A patent/CN117491546A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107951861A (en) * | 2016-10-17 | 2018-04-24 | 南京绿叶制药有限公司 | A kind of lipid nano particle membrane material feed composition |
| CN116134020A (en) * | 2020-07-17 | 2023-05-16 | 赛诺菲巴斯德有限公司 | Lipid compounds comprising at least one terminal group of formula-NH-CX-A or-NH-CX-NH-A, compositions containing them and uses thereof |
| CN113960182A (en) * | 2020-07-21 | 2022-01-21 | 苏州艾博生物科技有限公司 | Method for detecting lipid component in lipid nanosphere |
| CN114507342A (en) * | 2022-03-18 | 2022-05-17 | 江苏东南纳米材料有限公司 | Preparation method of 1, 2-dimyristoyl-RAC-glycerol-3-methoxypolyethylene glycol and intermediate thereof |
| CN116024237A (en) * | 2023-01-05 | 2023-04-28 | 中国人民解放军军事科学院军事医学研究院 | mRNA vaccine for preventing heat of rift grains and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 徐洋: "聚乙二醇-脂质衍生物修饰对脂质体稳定性的影响", 药学学报, vol. 46, no. 10, 31 December 2011 (2011-12-31), pages 1178 - 1186 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113125625B (en) | Method for detecting genotoxic impurities in propiofuravir fumarate | |
| CN112129871B (en) | Method for detecting contents of DOPE and M5 phospholipids in composite phospholipid liposome | |
| CN112630365B (en) | Method for determining content of dimyristoyl phosphatidylcholine by high performance liquid chromatography | |
| CN117491546A (en) | Detection method for content of PEGylated lipid | |
| CN104897841A (en) | Method of measuring content of p-Sulfonamide-phenylhydrazine hybrochloride in celecoxib capsules by means of efficient liquid chromatography | |
| CN116297978A (en) | HPLC method for separating and measuring posaconazole Z 3 And methods for impurity and solvent thereof | |
| CN110606846A (en) | Tofacitinib citrate impurity and analysis method and application thereof | |
| CN114689737B (en) | Analysis method of S-o-chlorophenylglycine methyl tartrate related substances | |
| CN115166080A (en) | Method for detecting impurity A and impurity B in ifosfamide bulk drug | |
| CN111220720B (en) | Purity detection method for trypsin and zymogen thereof | |
| CN111426760B (en) | Method for determining genotoxic impurities in doxofylline raw material medicine | |
| CN114137120A (en) | Method for detecting related substances in rapamycin drug stent | |
| CN110045034B (en) | Method for determining content of erucyl phosphatidylcholine by high performance liquid chromatography | |
| CN106483205A (en) | A kind of method that employing high performance liquid chromatography detects pharmaceutic adjuvant carmine content | |
| CN115436525B (en) | LLTS-M3 and detection method and application of related substances thereof | |
| CN112034058B (en) | Method for detecting isomer impurities in vincamine | |
| CN109765316A (en) | A method of detecting right etiracetam from drug | |
| CN114280191B (en) | Method for detecting related substances in double-cysteine and preparation thereof | |
| CN112394112B (en) | Method for detecting content of hydroxychloroquine oxynitride impurities in hydroxychloroquine sulfate | |
| CN117990817A (en) | Method for measuring impurity content in pharmaceutical intermediate acyl chloride iminodibenzyl by utilizing HPLC | |
| CN115902043B (en) | Method for measuring impurity content in 8-bromooctanoic acid sample | |
| CN115078572B (en) | Method for determining folic acid impurity by ultra-high performance liquid chromatography | |
| CN114280190B (en) | Kit for detecting related substances of double cysteines | |
| CN117517547A (en) | Method for measuring content of sodium ascorbate in calcium supplement | |
| CN116359396A (en) | Method for detecting lipid component and content in lipid nanospheres |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |