CN115932098A - Method for separating cholesterol, epi-cholesterol and isocholesterol by HPLC method - Google Patents
Method for separating cholesterol, epi-cholesterol and isocholesterol by HPLC method Download PDFInfo
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- CN115932098A CN115932098A CN202211607266.3A CN202211607266A CN115932098A CN 115932098 A CN115932098 A CN 115932098A CN 202211607266 A CN202211607266 A CN 202211607266A CN 115932098 A CN115932098 A CN 115932098A
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- cholesterol
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- isocholesterol
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- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 title claims abstract description 94
- 235000012000 cholesterol Nutrition 0.000 title claims abstract description 47
- HVYWMOMLDIMFJA-VEIPTCAHSA-N (3r,8s,9s,10r,13r,14s,17r)-10,13-dimethyl-17-[(2r)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1h-cyclopenta[a]phenanthren-3-ol Chemical compound C1C=C2C[C@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-VEIPTCAHSA-N 0.000 title claims abstract description 32
- HVYWMOMLDIMFJA-UHFFFAOYSA-N 3-cholesterol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)CCCC(C)C)C1(C)CC2 HVYWMOMLDIMFJA-UHFFFAOYSA-N 0.000 title claims abstract description 32
- 238000000034 method Methods 0.000 title claims abstract description 22
- 238000004128 high performance liquid chromatography Methods 0.000 title claims abstract description 15
- 238000004458 analytical method Methods 0.000 claims abstract description 7
- 238000003556 assay Methods 0.000 claims abstract description 7
- 239000007788 liquid Substances 0.000 claims abstract description 7
- QEVHRUUCFGRFIF-MDEJGZGSSA-N reserpine Chemical compound O([C@H]1[C@@H]([C@H]([C@H]2C[C@@H]3C4=C(C5=CC=C(OC)C=C5N4)CCN3C[C@H]2C1)C(=O)OC)OC)C(=O)C1=CC(OC)=C(OC)C(OC)=C1 QEVHRUUCFGRFIF-MDEJGZGSSA-N 0.000 claims abstract description 7
- 238000000926 separation method Methods 0.000 claims abstract description 6
- 238000004587 chromatography analysis Methods 0.000 claims abstract description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 16
- 239000012071 phase Substances 0.000 claims description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 9
- 239000012074 organic phase Substances 0.000 claims description 8
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 6
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 3
- 239000000126 substance Substances 0.000 abstract description 6
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 abstract description 2
- 239000000945 filler Substances 0.000 abstract description 2
- 239000000741 silica gel Substances 0.000 abstract description 2
- 229910002027 silica gel Inorganic materials 0.000 abstract description 2
- 239000002502 liposome Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 150000003904 phospholipids Chemical class 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 229910004298 SiO 2 Inorganic materials 0.000 description 4
- 239000012085 test solution Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 3
- UKJFVOWPUXSBOM-UHFFFAOYSA-N hexane;oxolane Chemical compound C1CCOC1.CCCCCC UKJFVOWPUXSBOM-UHFFFAOYSA-N 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000011259 mixed solution Substances 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 238000005034 decoration Methods 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000035699 permeability Effects 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000003908 quality control method Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 125000002252 acyl group Chemical group 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
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- 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
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- Steroid Compounds (AREA)
Abstract
The invention belongs to the technical field of analytical chemistry, and provides a method for separating cholesterol, epi-cholesterol and isocholesterol by using an HPLC method. The present invention provides an assay solution comprising cholesterol, epi-cholesterol, and iso-cholesterol; and injecting the obtained analysis solution into a liquid chromatograph, carrying out chromatographic analysis, and recording a chromatogram. The invention adopts silica gel as a filling agent (Elite Hypersil SiO) 2 5um4.0mm x 150mm), can effectively separate cholesterol, epi-cholesterol and isocholesterol; the invention solves the separation of cholesterol, epi-cholesterol and iso-cholesterol, thereby ensuring the quality of cholesterol products to be controllable; the method of the invention can simply, rapidly and accurately analyze cholesterol related substances.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a method for separating cholesterol, epicholesterol and isocholesterol by using an HPLC method.
Background
Cholesterol is an important adjuvant in the process of liposome pharmaceutical preparation. Phospholipids are the basis of the material constituting the bilayer and, in theory, liposomes can be formed in the presence of phospholipids. However, the long acyl chains of phospholipids have very strong mobility, like aquatic weeds in ocean waves, and are constantly in a state of rocking. While this fluidity gives liposomes good flexibility and deformability, it also allows very high membrane permeability. This means that the exchange of substances between the inside and outside of the liposome becomes very easy. When liposomes are used to entrap drugs, this excessive permeability will allow the drug to easily "escape" from the internal aqueous phase, resulting in a reduced drug loading. It is a skill of people to learn from nature to add a small amount of cholesterol to phospholipids to regulate membrane fluidity. The biomembrane contains a large amount of cholesterol, and in addition to certain physiological functions, the cholesterol plays a role in the structural function of the biomembrane more importantly. If the phospholipid molecules forming the bilayer are arranged in order to form a fence, cholesterol is like a rope binding the fence, so that the combination between the phospholipid molecules is tighter and firmer, and the liposome is more stable. The fact proves that the drug loading and the stability of the liposome can be greatly improved by adding 30-50% of cholesterol when the liposome is prepared.
The molecular structure of cholesterol is as follows:
in the course of synthesis, other cholesterol impurities (related substances) need to be strictly controlled (isocholesterol and epicholesterol), and its structural formula is
The quality control is required for extracting relevant substances existing in cholesterol, namely, in a bulk drug or a preparation. Therefore, the separation of cholesterol and related substances is realized, which has practical significance in the aspects of cholesterol extraction and quality control of preparation process.
Disclosure of Invention
The invention aims to provide a method for separating cholesterol, epi-cholesterol and isocholesterol by using an HPLC method, which realizes the separation of cholesterol and related substances thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for separating cholesterol, epi-cholesterol and isocholesterol by using an HPLC method, which comprises the following steps:
preparing an assay solution comprising cholesterol, epicholesterol, and isocholesterol;
injecting the obtained analysis solution into a liquid chromatograph, carrying out chromatographic analysis, and recording a chromatogram;
wherein the mobile phase comprises n-hexane and an organic phase comprising dichloromethane or tetrahydrofuran; the flow rate of the mobile phase is 0.8-1.2 ml/min, the detection wavelength is 205-209 nm, and the column temperature is 20-40 ℃.
Optionally, the volume ratio of the n-hexane to the organic phase is 99: 1-90: 10.
Optionally, the concentration of cholesterol, epi-cholesterol and iso-cholesterol in the assay solution is independently 0.8-2.0 mg/ml.
Optionally, the sample amount is 5-40 μ L.
Optionally, the use of Elite Hypersil SiO 2 And, 5um4.0mm 150mm separation column.
Optionally, the volume ratio of the n-hexane to the organic phase is 97: 3.
The invention adopts silica gel as a filling agent (Elite Hypersil SiO) 2 5um4.0mm × 150mm), can effectively separate cholesterol, epi-cholesterol and isocholesterol;
the invention solves the separation of cholesterol, epi-cholesterol and isocholesterol, thereby ensuring the controllable quality of the cholesterol raw material.
Drawings
FIG. 1 is an HPLC overlay of cholesterol, epi-cholesterol, and iso-cholesterol in example 1.
FIG. 2 is a nuclear magnetic diagram of cholesterol isolated in example 1.
FIG. 3 is a nuclear magnetic map of the isolated epi-cholesterol in example 1.
FIG. 4 is a nuclear magnetic map of the isolated cholesterol in example 1.
Detailed Description
The invention provides a method for separating cholesterol, epi-cholesterol and isocholesterol by using an HPLC method, which comprises the following steps:
preparing an assay solution comprising cholesterol, epi-cholesterol and iso-cholesterol;
injecting the obtained analysis solution into a liquid chromatograph, carrying out chromatographic analysis, and recording a chromatogram;
wherein the mobile phase comprises n-hexane and an organic phase comprising dichloromethane or tetrahydrofuran; the flow rate of the mobile phase is 0.8-1.2 ml/min, preferably 0.9-1.1 ml/min, more preferably 1.0ml/min; the detection wavelength is 205-209 nm, preferably 206-208 nm, more preferably 207nm; the column temperature is 20 to 40 ℃, preferably 25 to 35 ℃, more preferably 30 to 32 ℃.
In the invention, the volume ratio of the n-hexane to the organic phase is 99: 1-90: 10, preferably 97: 3-98.
In the present invention, the concentration of cholesterol, epi-cholesterol and iso-cholesterol in the assay solution is independently 0.8 to 2.0mg/ml, preferably 1 to 1.8mg/ml, more preferably 1.2 to 1.6mg/ml.
In the present invention, the amount of the sample is 5 to 40. Mu.L, preferably 10 to 30. Mu.L, and more preferably 20 to 25. Mu.L.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
(1) Apparatus and conditions
A high performance liquid chromatograph, a Saimer fly UlltiMate 3000 high performance liquid chromatography system and a workstation; chromatographic column Elite Hypersil SiO 2 ，5um 4.0mm*150mm；
The mobile phase is normal hexane-tetrahydrofuran (97: 3), the flow rate is 1.0mL/min, the detection wavelength is 207nm, the column temperature is 30 ℃, and the injection volume is 20 mu L.
(2) Experimental procedure
Taking 80mg of cholesterol, 80mg of epicholesterol and 80mg of isocholesterol, adding the mobile phase for dissolving, fixing the volume to 50ml, and uniformly mixing to obtain a mixed solution containing 1.6mg/ml of cholesterol, epicholesterol and isocholesterol, which is used as a test solution.
Taking the sample solution, performing high performance liquid chromatography analysis according to the above conditions, and recording chromatogram. The results are shown in FIG. 1, in which 19 minutes is epicholesterol, 25.5 minutes is cholesterol, and 29.5 minutes is isocholesterol; under the condition, the main peak of cholesterol can be completely separated from the epi-cholesterol and the iso-cholesterol.
Example 2
(1) Apparatus and conditions
A high performance liquid chromatograph, a Saimer fly UlltiMate 3000 high performance liquid chromatography system and a workstation; chromatographic column Estrit Hypersil SiO 2 ，5um4.0mm*150mm；
The mobile phase is normal hexane-tetrahydrofuran (97: 3), the flow rate is 1.0mL/min, the detection wavelength is 207nm, the column temperature is 25 ℃, and the injection volume is 20 mu L.
(2) Experimental procedure
Taking 80mg of cholesterol, 80mg of epicholesterol and 80mg of isocholesterol, adding the mobile phase for dissolving, fixing the volume to 50ml, and uniformly mixing to obtain a mixed solution containing 1.6mg/ml of cholesterol, epicholesterol and isocholesterol, which is used as a test solution.
Taking the test solution, performing high performance liquid chromatography analysis according to the conditions, and recording the chromatogram. Under the condition, the main peak of cholesterol can be completely separated from the epi-cholesterol and the iso-cholesterol.
Example 3
(1) Instruments and conditions
A high performance liquid chromatograph, a Saimeri Fei UlltiMate 3000 high performance liquid chromatography system and a workstation; chromatographic column Estrit Hypersil SiO 2 ，5um4.0mm*150mm；
The mobile phase is normal hexane-tetrahydrofuran (97: 3), the flow rate is 1.0mL/min, the detection wavelength is 207nm, the column temperature is 35 ℃, and the injection volume is 20 mu L.
(2) And the experimental procedure
Taking 80mg of cholesterol, 80mg of epicholesterol and 80mg of isocholesterol, adding the mobile phase for dissolving, fixing the volume to 50ml, and mixing uniformly to obtain a mixed solution containing 1.6mg/ml of cholesterol, epicholesterol and isocholesterol, which is used as a test solution.
Taking the sample solution, performing high performance liquid chromatography analysis according to the above conditions, and recording chromatogram. Under the condition, the main peak of cholesterol can be completely separated from the epi-cholesterol and the iso-cholesterol.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (6)
1. A method for separating cholesterol, epi-cholesterol and iso-cholesterol by HPLC, comprising the steps of:
preparing an assay solution comprising cholesterol, epicholesterol, and isocholesterol;
injecting the obtained analysis solution into a liquid chromatograph, carrying out chromatographic analysis, and recording a chromatogram;
wherein the mobile phase comprises n-hexane and an organic phase comprising dichloromethane or tetrahydrofuran; the flow rate of the mobile phase is 0.8-1.2 ml/min, the detection wavelength is 205-209 nm, and the column temperature is 20-40 ℃.
2. The method of claim 1, wherein the volume ratio of n-hexane to organic phase is 99: 1 to 90: 10.
3. The method according to claim 1 or 2, wherein the concentration of cholesterol, epi-cholesterol and iso-cholesterol in the assay solution is independently 0.8-2.0 mg/ml.
4. The method of claim 3, wherein the sample size is 5 to 40 μ L.
5. The method as claimed in claim 1, 2 or 4, wherein Hypersil SiO is used 2 5um4.0mm 150mm separation column.
6. The method of claim 5, wherein the volume ratio of n-hexane to organic phase is 97: 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211607266.3A CN115932098A (en) | 2022-12-14 | 2022-12-14 | Method for separating cholesterol, epi-cholesterol and isocholesterol by HPLC method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211607266.3A CN115932098A (en) | 2022-12-14 | 2022-12-14 | Method for separating cholesterol, epi-cholesterol and isocholesterol by HPLC method |
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| CN202211607266.3A Pending CN115932098A (en) | 2022-12-14 | 2022-12-14 | Method for separating cholesterol, epi-cholesterol and isocholesterol by HPLC method |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5503988A (en) * | 1992-06-10 | 1996-04-02 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing a cholesterol-reduced substance |
| US20110282587A1 (en) * | 2010-05-17 | 2011-11-17 | Emory University | Computer readable storage mediums, methods and systems for normalizing chemical profiles in biological or medical samples detected by mass spectrometry |
| WO2022173531A1 (en) * | 2021-02-10 | 2022-08-18 | Oncorus, Inc. | Compounds, compositions, and methods of using thereof |
-
2022
- 2022-12-14 CN CN202211607266.3A patent/CN115932098A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5503988A (en) * | 1992-06-10 | 1996-04-02 | Kyowa Hakko Kogyo Co., Ltd. | Process for producing a cholesterol-reduced substance |
| US20110282587A1 (en) * | 2010-05-17 | 2011-11-17 | Emory University | Computer readable storage mediums, methods and systems for normalizing chemical profiles in biological or medical samples detected by mass spectrometry |
| WO2022173531A1 (en) * | 2021-02-10 | 2022-08-18 | Oncorus, Inc. | Compounds, compositions, and methods of using thereof |
Non-Patent Citations (2)
| Title |
|---|
| ASHOK K. BATTA ET AL: "Chromatographic separation of putative precursors of cholestanol", STEROIDS, 31 December 1988 (1988-12-31) * |
| 刘贵银 等: "精制橄榄油中甾醇的分离与测定", 食品科技, 31 December 2018 (2018-12-31) * |
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