CN114740110A - Method for detecting purity of distearoyl phosphatidylcholine - Google Patents
Method for detecting purity of distearoyl phosphatidylcholine Download PDFInfo
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- CN114740110A CN114740110A CN202210332662.3A CN202210332662A CN114740110A CN 114740110 A CN114740110 A CN 114740110A CN 202210332662 A CN202210332662 A CN 202210332662A CN 114740110 A CN114740110 A CN 114740110A
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- 238000000034 method Methods 0.000 title claims abstract description 38
- NRJAVPSFFCBXDT-HUESYALOSA-N 1,2-distearoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCCCCCCCCCCC NRJAVPSFFCBXDT-HUESYALOSA-N 0.000 title claims abstract description 26
- 239000012071 phase Substances 0.000 claims abstract description 46
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical group OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000012074 organic phase Substances 0.000 claims abstract description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 23
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000004128 high performance liquid chromatography Methods 0.000 claims abstract description 20
- 239000000741 silica gel Substances 0.000 claims abstract description 14
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 14
- YXMVRBZGTJFMLH-UHFFFAOYSA-N butylsilane Chemical group CCCC[SiH3] YXMVRBZGTJFMLH-UHFFFAOYSA-N 0.000 claims abstract description 8
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 7
- 239000007864 aqueous solution Substances 0.000 claims abstract description 4
- WSGYTJNNHPZFKR-UHFFFAOYSA-N 3-hydroxypropanenitrile Chemical group OCCC#N WSGYTJNNHPZFKR-UHFFFAOYSA-N 0.000 claims abstract description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 81
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 33
- 238000001514 detection method Methods 0.000 claims description 20
- 238000010828 elution Methods 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000008346 aqueous phase Substances 0.000 claims description 6
- 239000002798 polar solvent Substances 0.000 claims description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 4
- 238000010829 isocratic elution Methods 0.000 claims description 4
- 238000003908 quality control method Methods 0.000 claims description 2
- 238000003556 assay Methods 0.000 claims 1
- 238000011084 recovery Methods 0.000 abstract description 5
- 238000011002 quantification Methods 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 17
- 239000000243 solution Substances 0.000 description 14
- HVVJCLFLKMGEIY-UHFFFAOYSA-N 2,3-dioctadecoxypropyl 2-(trimethylazaniumyl)ethyl phosphate Chemical compound CCCCCCCCCCCCCCCCCCOCC(COP([O-])(=O)OCC[N+](C)(C)C)OCCCCCCCCCCCCCCCCCC HVVJCLFLKMGEIY-UHFFFAOYSA-N 0.000 description 13
- 239000000945 filler Substances 0.000 description 11
- 239000007788 liquid Substances 0.000 description 11
- 239000002502 liposome Substances 0.000 description 10
- 239000012488 sample solution Substances 0.000 description 8
- 239000000126 substance Substances 0.000 description 8
- 150000003904 phospholipids Chemical class 0.000 description 6
- 238000004587 chromatography analysis Methods 0.000 description 5
- 239000011550 stock solution Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 239000012535 impurity Substances 0.000 description 4
- YTJSFYQNRXLOIC-UHFFFAOYSA-N octadecylsilane Chemical compound CCCCCCCCCCCCCCCCCC[SiH3] YTJSFYQNRXLOIC-UHFFFAOYSA-N 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000000377 silicon dioxide Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000003643 water by type Substances 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 229940067606 lecithin Drugs 0.000 description 3
- 235000010445 lecithin Nutrition 0.000 description 3
- 239000000787 lecithin Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 2
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 2
- 239000012490 blank solution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000003607 modifier Substances 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 2
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000012085 test solution Substances 0.000 description 2
- 238000004679 31P NMR spectroscopy Methods 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
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- 238000012512 characterization method Methods 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- -1 choline phospholipid Chemical class 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- 230000000214 effect on organisms Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
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- 238000010200 validation analysis Methods 0.000 description 1
Images
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
-
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- 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
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N30/54—Temperature
-
- 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
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention provides a method for detecting the purity of distearoyl phosphatidylcholine, which comprises the following steps: dissolving distearoyl phosphatidylcholine, detecting by high performance liquid chromatography, and adopting a chromatographic column using butylsilane bonded silica gel as a stationary phase; the organic phase of the mobile phase is methanol-acetonitrile, the water phase is trifluoroacetic acid aqueous solution, and the volume ratio of the trifluoroacetic acid to the water is (0.1-0.3): 100, respectively; the method is convenient to operate and good in separation, the purity of the product is effectively controlled by a high-low concentration method, and the method meets standards in the aspects of quantification, linear range, repeatability, recovery rate and the like and has high durability.
Description
Technical Field
The invention relates to the technical field of chemical analysis, in particular to a method for detecting the purity of distearoyl phosphatidylcholine.
Background
Phosphatidylcholine (PC), also known as lecithin or choline phospholipid, is the basic substance of vital activities. It is widely present in the form of a mixture with other phospholipid components such as Phosphatidylethanolamine (PE), Phosphatidic Acid (PA) and inositol Phosphatide (PI), etc., in the cell membranes and egg cells of animals, plants and humans. The liposome (liposome) is an artificial membrane, in which the hydrophilic head of phospholipid molecule is inserted into water, and the hydrophobic tail of liposome is extended into air, and stirred to form double-layer spherical liposome of lipid molecule. The liposome is a directional drug carrier, and belongs to a new dosage form of a targeted drug delivery system. The liposome has little toxic and side effect on organisms, and the lipid bilayer of the liposome has greater similarity and tissue compatibility with a biological membrane and is easy to be absorbed by tissues. Distearoyl phosphatidylcholine (DSPC) is one of important auxiliary materials for preparing liposome, and has wide application in the research aspect of liposome, so a method capable of quickly and accurately detecting distearoyl phosphatidylcholine (DSPC) is urgently needed, and technical support is provided for further research of DSPC and application in the fields of medical treatment, chemical industry, food, medicines, health care products and the like.
In the prior art, a plurality of methods are used for detecting phospholipid, and the main detection methods comprise a chemical method, an enzymatic method, a chromatography method, a nuclear magnetic resonance spectroscopy method and the like. The chemical cost method is relatively low, but the method has low accuracy and is easy to interfere; the precision of the thin layer chromatography is not high; patent document CN 106404683a discloses a technique for detecting phospholipid by an enzymatic method, but has disadvantages such as low accuracy of results, complicated operation, and high requirement for experimental operators. The prior art discloses (arica C, LutzN W, Confort-Gounes S, et al. phospholipidic reagents of milk from differential mammalians defined by 31PNMR: todards specific interest in human health [ J ] Food Chemistry 2012,135(3):1777 and 1783.) analysis of the content of lecithin in milk by nuclear magnetic resonance. However, nmr spectroscopy is not suitable for large-scale popularization because it requires high requirements for instruments and experimental conditions. Patent document CN101387587 discloses a method for detecting lecithin content by colorimetry, but the pretreatment is complicated and the efficiency is low. Patent document CN110007032A discloses a method for detecting phospholipids and the application thereof, which adopts a high performance liquid chromatography evaporation light detection method to detect phospholipids, but the method has weak specificity and cannot separate distearoyl phosphatidylcholine from impurities well, so that the detection requirement for detecting the purity of distearoyl phosphatidylcholine cannot be met well.
Therefore, it is highly desirable to establish a method for detecting the purity of distearoyl phosphatidylcholine, which has high sensitivity, strong specificity, stable method and high analysis efficiency.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for detecting the purity of distearoyl phosphatidylcholine, which has high sensitivity, strong specificity, stable method and high analysis efficiency, and comprises the following technical scheme:
the method dissolves distearoyl phosphatidylcholine, and adopts high performance liquid chromatography to detect;
further, the high performance liquid chromatography adopts a chromatographic column taking butylsilane bonded silica gel as a stationary phase;
further, the organic phase of the mobile phase of the high performance liquid chromatography is methanol-acetonitrile, the aqueous phase is trifluoroacetic acid aqueous solution, and the volume ratio of the trifluoroacetic acid to the water is (0.1-0.3): 100, respectively; for example, the ratio of trifluoroacetic acid to water by volume may be 0.1: 100. 0.125: 100. 0.15: 100. 0.175: 100. 0.2: 100. 0.225: 100. 0.25: 100. 0.275: 100 and 0.3: 100, respectively; preferably, the ratio of the volume of the trifluoroacetic acid to the volume of water is 0.1: 100.
further, the volume ratio of methanol to acetonitrile in the organic phase is 3-5: 1; for example, the volume ratio of methanol to acetonitrile in the organic phase may be 3: 1. 3.5: 1. 4: 1. 4.5: 1 and 5: 1, preferably, the volume ratio of methanol to acetonitrile in the organic phase is 4: 1.
further, the chromatographic column using the butylsilane bonded silica gel as the stationary phase has the following parameters: 4.6X 250mm,5 μm, 300A.
Further, the temperature of the chromatographic column is 38-42 ℃; for example, the column can be 38 ℃, 39 ℃, 40 ℃, 41 ℃ and 42 ℃.
Further, the flow rate of the mobile phase is 0.2-1.5 mL/min, for example, the flow rate of the mobile phase can be 0.2mL/min, 0.3mL/min, 0.4mL/min, 0.5mL/min, 0.6mL/min, 0.7mL/min, 0.8mL/min, 0.9mL/min, 1.0mL/min, 1.1mL/min, 1.2mL/min, 1.3mL/min, 1.4mL/min, and 1.5 mL/min;
preferably, the flow rate of the mobile phase is 0.8-1.0 mL/min; more preferably 1.0 mL/min.
Further, the detector of the high performance liquid chromatography is an electrospray detector.
Further, the sample amount of the high performance liquid chromatography is selected from 1 to 15 μ L, for example, the sample amount of the high performance liquid chromatography can be 1 μ L, 2 μ L, 3 μ L, 4 μ L, 5 μ L, 6 μ L, 7 μ L, 8 μ L, 9 μ L, 10 μ L, 12 μ L, 13 μ L, 14 μ L and 15 μ L, preferably, the sample amount is 2 to 10 μ L, more preferably, the sample amount is 10 μ L.
Further, the high performance liquid chromatography adopts gradient elution;
the gradient elution procedure was:
0min, the volume ratio of the water phase to the organic phase is 90: 10;
and (3) 0-2 min, uniformly changing the volume ratio of the water phase to the organic phase to 10: 90;
2-15 min, the volume ratio of the water phase to the organic phase is changed to 5: 95;
15-18 min, and maintaining the volume ratio of the water phase to the organic phase at 5: 95, performing isocratic elution;
and (3) for 18-20 min, uniformly changing the volume ratio of the water phase to the organic phase to 90: 10,;
20-25 min, and maintaining the volume ratio of the water phase to the organic phase at 90: 10, performing isocratic elution;
further, the dissolution adopts a polar solvent to dissolve the distearoyl phosphatidylcholine, and the polar solvent is selected from the group consisting of: a combination of one or more of methanol, ethanol, isopropanol, acetonitrile and water; preferably, the polar solvent is methanol.
The invention also provides application of the detection method in the quality control of distearoyl phosphatidylcholine.
Compared with the prior art, the invention has at least the following beneficial effects:
the method can quickly, conveniently and quantitatively determine the impurities in the distearoyl phosphatidylcholine (DSPC) so as to effectively control the quality of the distearoyl phosphatidylcholine (DSPC). Therefore, the problems of sample purity detection and impurity control are better solved, and the research of the liposome is helped.
Drawings
FIG. 1 is a liquid chromatogram of a blank solution detected by the method of the invention;
FIG. 2 is a liquid chromatogram of a sample solution detected by a chromatographic column using butylsilane-bonded silica gel as a filler (DAISOPAKSP-300-5-C4-BIO 4.6X 250mm,5 μm,300A) in example 1 of the present invention;
FIG. 3 is a liquid chromatogram of a test sample solution using a column using octadecylsilane chemically bonded silica as a filler (GL Science InertSustain C18, 4.6X 250mm,5 μm) in example 1 of the present invention;
FIG. 4 is a liquid chromatogram of a test sample solution using a column packed with butylsilane-bonded silica gel (Waters Xbridge Protein BEH C44.6X 150mm,3.5 μm,300A) according to example 1 of the present invention;
FIG. 5 is a liquid chromatogram of a sample solution detected by the method of using methanol as a mobile phase in example 2 of the present invention;
FIG. 6 is a quantitative limit chromatogram for detecting a sample by the method of the present invention;
FIG. 7 is a linear relationship using the method of the present invention.
Detailed Description
Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In the present invention, the term "stationary phase" is a phase which will be stationary in the chromatographic separation, resulting in retention of the sample.
In the present invention, the term "mobile phase" refers to a substance that moves forward with a component to be measured during chromatography and is called a mobile phase. And the other phase which is in an equilibrium state with the stationary phase and drives the sample to move forwards.
In the present invention, the term "mobile phase modifier" refers to a reagent added to a mobile phase to adjust the pH, and the addition of the modifier to the mobile phase to adjust the pH suppresses the ionization of solute and adjusts the ionic strength, makes the peak profile of the chromatographic peak more symmetrical, suppresses the tailing of the peak profile, and improves the separation effect.
In the present invention, the term electrospray detector is a general-purpose detector capable of detecting any non-volatile and partially semi-volatile substances, compatible with gradients, and suitable for liquid chromatography quantification, characterization, etc. of compounds without ultraviolet absorption, the principle is that the amount of analyte in a sample is proportional to the signal current, and all non-volatile substances can be detected, regardless of the molecular structure to be detected.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
EXAMPLE 1 optimization of chromatography columns
1.1 chromatographic column using butylsilane bonded silica gel as filler (DAISOPAKSP-300-5-C4-BIO 4.6X 250mm,5 μm,300A)
The HPLC detection method specifically comprises the following steps:
(1) chromatographic conditions are as follows:
the instrument comprises: shimadzu LC-16 high performance liquid chromatograph
A detector: thermo Veo RS CAD detector
A chromatographic column: butylsilane bonded silica gel as filler (DAISOPAKSP-300-5-C4-BIO 4.6X 250mm,5 μm,300A)
Aqueous phase (mobile phase a): 0.1% aqueous trifluoroacetic acid.
Organic phase (mobile phase B): the volume ratio of methanol to acetonitrile is 4: 1, and (2) solution.
Flow rate: 1.0 mL/min.
Column temperature: at 40 ℃.
Sample introduction amount: 10 μ L.
A workstation: labsolution
Gradient elution was performed as follows:
TABLE 1.1 elution procedure
The chromatogram is shown in fig. 2, and it can be seen from fig. 2 that the method of example 1 of the present invention is good in peak shape.
1.2 chromatography column using octadecylsilane chemically bonded silica as filler (GL Science InertSustain C18, 4.6X 250mm,5 μm)
The HPLC detection method specifically comprises the following steps:
(1) chromatographic conditions are as follows:
the instrument comprises the following steps: shimadzu LC-16 high performance liquid chromatograph
A detector: thermo Veo RS CAD detector
A chromatographic column: octadecylsilane chemically bonded silica as filler (GL Science Inertsustain C18, 4.6X 250mm,5 μm)
Aqueous phase (mobile phase a): 0.1% aqueous trifluoroacetic acid.
Organic phase (mobile phase B): and (3) acetonitrile.
Flow rate: 1.0 mL/min.
Column temperature: at 40 ℃.
Sample introduction amount: 10 μ L.
A workstation: labsolution
Gradient elution was performed as follows:
TABLE 1.2 elution procedure
| Time/min | Mobile phase A/%) | Mobile phase B/%) |
| 0 | 90 | 10 |
| 2 | 30 | 70 |
| 15 | 5 | 95 |
| 20 | 5 | 95 |
| 21 | 90 | 10 |
| 25 | 90 | 10 |
The concrete results are shown in FIG. 3, which shows that C takes octadecylsilane chemically bonded silica as filler18The column did not completely separate the impurity peaks and the tailing was severe.
1.3 chromatography column using butylsilane-bonded silica gel as filler (Waters Xbridge Protein BEH C44.6X 150mm,3.5 μm,300A)
The HPLC detection method specifically comprises the following steps:
(1) chromatographic conditions are as follows:
the instrument comprises: shimadzu LC-16 high performance liquid chromatograph
A detector: thermo Veo RS CAD detector
A chromatographic column: butylsilane bonded silica gel as filler (Waters Xbridge Protein BEH C44.6X 150mm,3.5 μm,300A)
Aqueous phase (mobile phase a): 0.1% aqueous trifluoroacetic acid.
Organic phase (mobile phase B): methanol.
Flow rate: 1.0 mL/min.
Column temperature: at 40 ℃.
Sample introduction amount: 10 μ L.
A workstation: labsolution
Gradient elution was performed as follows:
TABLE 1.3 elution procedure
| Time/min | Mobile phase A/%) | Mobile phase B/%) |
| 0 | 90 | 10 |
| 2 | 10 | 90 |
| 15 | 5 | 95 |
| 18 | 5 | 95 |
| 20 | 90 | 10 |
| 25 | 90 | 10 |
FIG. 4 shows that the column using butylsilane-bonded silica gel as a filler (Waters Xbridge Protein BEH C44.6X 150mm,3.5 μm,300A) has a poorer resolution than that of FIG. 2.
The above results indicate that the peak pattern is good in a column packed with butylsilane-bonded silica gel (DAISOPAKSP-300-5-C4-BIO 4.6X 250mm,5 μm, 300A).
EXAMPLE 2 optimization of the Mobile phase
2.1, the mobile phase is organic phase methanol
The HPLC detection method specifically comprises the following steps:
(1) chromatographic conditions are as follows:
the instrument comprises the following steps: shimadzu LC-16 high performance liquid chromatograph
A detector: thermo Veo RS CAD detector
A chromatographic column: butylsilane bonded silica gel as filler (DAISOPAKSP-300-5-C4-BIO 4.6X 250mm,5 μm,300A)
Aqueous phase (mobile phase a): 0.1% aqueous trifluoroacetic acid.
Organic phase (mobile phase B): methanol.
Flow rate: 1.0 mL/min.
Column temperature: at 40 ℃.
Sample introduction amount: 10 μ L.
A workstation: labsolution
Gradient elution was performed as follows:
TABLE 2.1 elution procedure
As shown in fig. 5, when methanol was used as the organic phase, the main peak appeared later, and acetonitrile was added to the B phase, as shown in fig. 5 and fig. 2, the volume ratio of methanol to acetonitrile in the B phase was 4: 1 solution, the main peak-off time is about 15min, and the peak-off time is proper.
The above results demonstrate that the volume ratio of methanol to acetonitrile in phase B is 4: 1 solution, the time to peak is appropriate.
Example 3 method validation
3.1 solution preparation:
blank solution: methanol, Merck, HPLC grade.
Preparation of a control stock solution: precisely weighing 20mg distearoyl phosphatidylcholine (DSPC), placing in a 10mL volumetric flask, adding methanol for dissolving, adding methanol for constant volume till the volume is scaled, and shaking up to obtain 2mg/mL stock solution. Precisely weighing 24mg distearoyl phosphatidylcholine (DSPC), placing in a 10mL volumetric flask, adding methanol for dissolving, adding methanol for constant volume to scale, and shaking up to obtain 2.4mg/mL stock solution.
Linear control solution: accurately transferring 2mg/mL control stock solutions of 2.5mL, 1mL, 0.5mL and 0.25mL to 5mL volumetric flasks respectively, adding methanol to a constant volume to a scale, and shaking up to obtain 1mg/mL, 0.4mg/mL, 0.2mg/mL and 0.1 mg/mL. Accurately transferring 2.5mL to 5mL volumetric flasks of 2.4mg/mL reference stock solution, adding methanol to a constant volume to a scale, and shaking up to obtain 1.2 mg/mL. Accurately transferring 1mL, 0.5mL, 0.25mL, 0.125mL to 5mL volumetric flasks of 0.4mg/mL control solution, adding methanol to constant volume to scale, and shaking up to obtain 0.08mg/mL, 0.04mg/mL, 0.02mg/mL, and 0.01 mg/mL. Accurately transferring 1mL, 0.5mL, 0.25mL and 0.125mL to 5mL volumetric flasks of 0.04mg/mL reference substance solution respectively, adding methanol to a constant volume to scale, and shaking up to obtain 0.008mg/mL, 0.004mg/mL, 0.002mg/mL and 0.001 mg/mL.
Preparation of 100% test solution: weighing a distearoyl phosphatidylcholine (DSPC) sample 20mg to 10ml in a volumetric flask, adding methanol to completely dissolve the sample, fixing the volume to a scale, and mixing uniformly to obtain the distearoyl phosphatidylcholine (DSPC).
1% test solution: measuring the 100% test sample solution 1ml to 100ml volumetric flask, adding methanol to dilute to constant volume to scale, and taking the volume as 1% test sample solution.
Limit of quantitation (LOQ) solution: the above 0.001mg/L is a quantitative Limit (LOQ) solution.
3.2 creation of Standard Curve
The linear solution control samples of the above concentrations were subjected to linear regression under the above chromatographic conditions with the concentration X (. mu.g) as abscissa and the corresponding peak area Y as ordinate, and the results and linear equation are shown in FIG. 7 and Table 3, showing that the linear relationship is good.
TABLE 3 Distearylphosphatidylcholine (DSPC) linearity results
| Concentration, mg/mL | Corresponding percentage of | Area of main peak |
| 0.001007 | 0.05％ | 86636 |
| 0.002014 | 0.1％ | 114550 |
| 0.004028 | 0.2％ | 202345 |
| 0.008056 | 0.4％ | 372229 |
| 0.01007 | 0.5％ | 429961 |
| 0.02014 | 1％ | 871948 |
| 0.04028 | 2％ | 1641912 |
| 0.08056 | 4％ | 3123552 |
| 0.1007 | 5％ | 3509985 |
| 0.2014 | 10％ | 6117524 |
| 0.4028 | 20％ | 11175927 |
| 1.007 | 50％ | 16937366 |
| 1.234 | 60％ | 20240129 |
| 2.014 | 100％ | 21910964 |
| 2.468 | 120％ | 24606102 |
(2)3.3 sample detection
Precisely weighing 100% sample solution and 10 μ L of 1% sample solution in 3.1, injecting into a liquid chromatograph, recording 30 min chromatogram under the chromatographic conditions of 1.1 in example 1, and calculating the purity of the sample according to high and low concentration method.
(3)3.4 detection of recovery:
and (2) precisely measuring 10 mu L of distearoyl phosphatidylcholine in the table 4, injecting into a liquid chromatograph, detecting the recovery rate of the distearoyl phosphatidylcholine under different concentrations by adopting the chromatographic conditions in the step (1), wherein the detection result is shown in the table 4, and as can be seen from the table, the recovery rate and RSD of the method both meet the requirements.
TABLE 4 Distearylphosphatidylcholine (DSPC) recovery results
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A method for detecting the purity of distearoyl phosphatidylcholine, which is characterized by comprising the following steps: dissolving distearoyl phosphatidylcholine, and detecting by high performance liquid chromatography;
the high performance liquid chromatography adopts a chromatographic column taking butylsilane bonded silica gel as a stationary phase;
the organic phase of the mobile phase of the high performance liquid chromatography is methanol-acetonitrile, the water phase is trifluoroacetic acid aqueous solution, and the volume ratio of the trifluoroacetic acid to the water is (0.1-0.3): 100.
2. the detection method according to claim 1, wherein the volume ratio of methanol to acetonitrile in the organic phase is 3 to 5: 1.
3. the detection method according to claim 1, wherein the aqueous phase is an aqueous solution of trifluoroacetic acid, and the volume ratio of trifluoroacetic acid to water is 0.1: 100.
4. the detection method according to claim 1, wherein the chromatographic column in which the butylsilane-bonded silica gel is a stationary phase has parameters of: 4.6X 250mm,5 μm, 300A.
5. The detection method according to claim 1, wherein the temperature of the chromatographic column is 38 to 42 ℃;
the flow rate of the mobile phase is 0.2-1.5 mL/min, preferably 0.8-1.0 mL/min; more preferably 1.0 mL/min.
6. The detection method according to claim 1, wherein the detector of the high performance liquid chromatography is an electrospray detector.
7. The detection method according to claim 1, wherein the amount of the sample to be subjected to the high performance liquid chromatography is selected from 1 to 15. mu.L, preferably 2 to 10. mu.L, and more preferably 10. mu.L.
8. The detection method according to any one of claims 1 to 7, wherein the high performance liquid chromatography employs gradient elution;
the gradient elution procedure was:
0min, the volume ratio of the water phase to the organic phase is 90: 10;
and (3) 0-2 min, uniformly changing the volume ratio of the water phase to the organic phase to 10: 90;
2-15 min, the volume ratio of the water phase to the organic phase is changed to 5: 95;
15-18 min, and maintaining the volume ratio of the water phase to the organic phase at 5: 95, performing isocratic elution;
and (3) for 18-20 min, uniformly changing the volume ratio of the water phase to the organic phase to 90: 10,;
20-25 min, and maintaining the volume ratio of the water phase to the organic phase at 90: 10, isocratic elution is carried out.
9. The detection method of claim 1, wherein the dissolving employs a polar solvent to dissolve distearoylphosphatidylcholine, the polar solvent being selected from the group consisting of: a combination of one or more of methanol, ethanol, isopropanol, acetonitrile and water; preferably, the polar solvent is methanol.
10. Use of the assay of any one of claims 1 to 9 for the quality control of distearoylphosphatidylcholine.
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