A method for separating cholesterol, epicholesterol and isocholesterol by HPLC

Abstract

The present invention belongs to the technical field of analytical chemistry, and provides a method for separating cholesterol, epicholesterol and isocholesterol by HPLC. The present invention configures an analytical solution containing cholesterol, epicholesterol and isocholesterol; injects the obtained analytical solution into a liquid chromatograph, performs chromatographic analysis, and records a chromatogram. The present invention uses silica gel as a filler (HypersilSiO 2 , 5um4.0mm*150mm), can effectively separate cholesterol, epicholesterol and isocholesterol; the present invention solves the separation of cholesterol, epicholesterol and isocholesterol, thereby ensuring the controllable quality of cholesterol products; the method of the present invention can simply, quickly and accurately analyze cholesterol-related substances.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, and in particular to a method for separating cholesterol, epicholesterol and isocholesterol by using HPLC. Background Art

[0002] Cholesterol is an important auxiliary material in the process of liposome drug preparation. Phospholipids are the material basis of the bilayer. In theory, liposomes can be formed as long as phospholipids exist. However, the long acyl chain of phospholipids has very strong fluidity, just like water grass in the waves, always in a swaying state. Although this fluidity gives liposomes good flexibility and deformability, it also makes the membrane permeability very high. This means that the exchange of substances inside and outside the liposome becomes very easy. When liposomes are used to encapsulate drugs, this excessive permeability will make the drugs easily "escape" from the inner water phase, and the drug loading will decrease. Adding a small amount of cholesterol to phospholipids to regulate the fluidity of the membrane is a skill that people have learned from nature. There is a large amount of cholesterol in the biological membrane. In addition to assuming certain physiological functions, it is more important to participate in the structural function of the biological membrane. If the neatly arranged phospholipid molecules that constitute the bilayer are described as fences, cholesterol is like the ropes that bind the fences, making the binding between phospholipid molecules tighter and firmer, making the liposomes more stable. It has been proven that adding 30-50% cholesterol when preparing liposomes can greatly increase the drug loading capacity and stability of liposomes.

[0003] The molecular structure of cholesterol is as follows:

[0004]

[0005] In the process of synthesis, there are other cholesterol impurities (related substances) that need to be strictly controlled (isocholesterol, epicholesterol), and its structural formula is:

[0006]

[0007] For the related substances present in the extracted cholesterol, quality control is required in both the raw material and the preparation. Therefore, separation of cholesterol and its related substances is of practical significance in the quality control of cholesterol extraction and preparation process. Summary of the invention

[0008] The object of the present invention is to provide a method for separating cholesterol, epicholesterol and isocholesterol by HPLC, so as to achieve the separation of cholesterol and its related substances.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides a method for separating cholesterol, epicholesterol and isocholesterol by HPLC, comprising the following steps:

[0011] preparing an analytical solution comprising cholesterol, epicholesterol and isocholesterol;

[0012] injecting the obtained analytical solution into a liquid chromatograph, performing chromatographic analysis, and recording a chromatogram;

[0013] The mobile phase comprises n-hexane and an organic phase, and the organic phase comprises 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° C.

[0014] Optionally, the volume ratio of n-hexane to the organic phase is 99:1 to 90:10.

[0015] Optionally, the concentrations of cholesterol, epicholesterol and isocholesterol in the analytical solution are independently 0.8-2.0 mg / ml.

[0016] Optionally, the injection volume is 5 to 40 μL.

[0017] Optionally, use Elite Hypersil SiO2, 5um 4.0mm*150mm separation column.

[0018] Optionally, the volume ratio of n-hexane to the organic phase is 97:3.

[0019] The present invention uses silica gel as a filler (Hypersil SiO2, 5um4.0mm*150mm), which can effectively separate cholesterol, epicholesterol and isocholesterol;

[0020] The invention solves the separation of cholesterol, epicholesterol and isocholesterol, thereby ensuring the controllable quality of cholesterol raw materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is an HPLC overlay of cholesterol, epicholesterol and isocholesterol in Example 1.

[0022] Figure 2 This is the NMR image of the cholesterol separated in Example 1.

[0023] Figure 3 This is the NMR image of the epicholesterol separated in Example 1.

[0024] Figure 4 This is the NMR image of isocholide separated in Example 1. DETAILED DESCRIPTION

[0025] The present invention provides a method for separating cholesterol, epicholesterol and isocholesterol by HPLC, comprising the following steps:

[0026] preparing an analytical solution comprising cholesterol, epicholesterol and isocholesterol;

[0027] injecting the obtained analytical solution into a liquid chromatograph, performing chromatographic analysis, and recording a chromatogram;

[0028] Wherein, the mobile phase comprises n-hexane and an organic phase, and the organic phase comprises dichloromethane or tetrahydrofuran; the flow rate of the mobile phase is 0.8-1.2 ml / min, preferably 0.9-1.1 ml / min, and more preferably 1.0 ml / min; the detection wavelength is 205-209 nm, preferably 206-208 nm, and more preferably 207 nm; the column temperature is 20-40°C, preferably 25-35°C, and more preferably 30-32°C.

[0029] In the present invention, the volume ratio of n-hexane to the organic phase is 99:1 to 90:10, preferably 97:3 to 98:2.

[0030] In the present invention, the concentrations of cholesterol, epicholesterol and isocholesterol in the analysis solution are independently 0.8 to 2.0 mg / ml, preferably 1 to 1.8 mg / ml, and more preferably 1.2 to 1.6 mg / ml.

[0031] In the present invention, the injection volume is 5 to 40 μL, preferably 10 to 30 μL, and more preferably 20 to 25 μL.

[0032] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] Example 1

[0034] (1) Instruments and conditions

[0035] High performance liquid chromatography: Thermo Fisher UltiMate 3000 high performance liquid chromatography system and workstation; Chromatographic column: Elite Hypersil SiO2, 5um 4.0mm*150mm;

[0036] Mobile phase: n-hexane-tetrahydrofuran (97:3), flow rate: 1.0 mL / min, detection wavelength: 207 nm, column temperature: 30°C, injection volume: 20 μL.

[0037] (2) Experimental procedures

[0038] Take 80 mg of cholesterol, 80 mg of epicholesterol and 80 mg of isocholesterol, add mobile phase to dissolve and make up to 50 ml, mix well to obtain a mixed solution containing 1.6 mg / ml of cholesterol, epicholesterol and isocholesterol as the test solution.

[0039] Take the test solution and perform HPLC analysis under the above conditions and record the chromatogram. Figure 1 In the figure, epi-cholesterol is at 19 minutes, cholesterol is at 25.5 minutes, and iso-cholesterol is at 29.5 minutes. It can be seen that under this condition, the main peak of cholesterol can be completely separated from epi-cholesterol and iso-cholesterol.

[0040] Example 2

[0041] (1) Instruments and conditions

[0042] High performance liquid chromatography: Thermo Fisher UltiMate 3000 high performance liquid chromatography system and workstation; Chromatographic column: Elite Hypersil SiO2, 5um4.0mm*150mm;

[0043] Mobile phase: n-hexane-tetrahydrofuran (97:3), flow rate: 1.0 mL / min, detection wavelength: 207 nm, column temperature: 25°C, injection volume: 20 μL.

[0044] (2) Experimental procedures

[0045] Take 80 mg of cholesterol, 80 mg of epicholesterol and 80 mg of isocholesterol, add mobile phase to dissolve and make up to 50 ml, mix well to obtain a mixed solution containing 1.6 mg / ml of cholesterol, epicholesterol and isocholesterol as the test solution.

[0046] Take the test solution and perform HPLC analysis under the above conditions and record the chromatogram. Under this condition, the main peak of cholesterol can be completely separated from epicholesterol and isocholesterol.

[0047] Example 3

[0048] (1) Instruments and conditions

[0049] High performance liquid chromatography: Thermo Fisher UltiMate 3000 high performance liquid chromatography system and workstation; Chromatographic column: Elite Hypersil SiO2, 5um4.0mm*150mm;

[0050] Mobile phase: n-hexane-tetrahydrofuran (97:3), flow rate: 1.0 mL / min, detection wavelength: 207 nm, column temperature: 35°C, injection volume: 20 μL.

[0051] (2) Experimental steps

[0052] Take 80 mg of cholesterol, 80 mg of epicholesterol and 80 mg of isocholesterol, add mobile phase to dissolve and make up to 50 ml, mix well to obtain a mixed solution containing 1.6 mg / ml of cholesterol, epicholesterol and isocholesterol as the test solution.

[0053] Take the test solution and perform HPLC analysis under the above conditions and record the chromatogram. Under this condition, the main peak of cholesterol can be completely separated from epicholesterol and isocholesterol.

[0054] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for separating cholesterol, epicholesterol and isocholesterol by HPLC, characterized in that: The following steps are included: preparing an analytical solution comprising cholesterol, epicholesterol and isocholesterol; injecting the obtained analytical solution into a liquid chromatograph to perform chromatographic analysis and record a chromatogram; The mobile phase is n-hexane and 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 °C; The volume ratio of n-hexane to tetrahydrofuran is 99:1-90:10; Elite Hypersil SiO2, 5um 4.0mm*150mm separation column was used.

2. The method according to claim 1, characterized in that The concentrations of cholesterol, epicholesterol and isocholesterol in the analytical solution are independently 0.8-2.0 mg / ml.

3. The method according to claim 2, characterized in that The injection volume is 5~40μL.

4. The method according to claim 3, characterized in that: The volume ratio of n-hexane to tetrahydrofuran is 97:3.

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