APPLICATION NOTE

E184VPG-001

Separation of Cholesterol and its oxidation products

Abstract

Cholesterol is an important membrane lipid providing structural strength for the cell as well as

regulating membrane fluidity. As one of the main constituents of lipid rafts, it is known to influence

processes like signal transduction and membrane trafficking [1]. By partial depletion of cholesterol

from membranes and subsequently substituting it with oxycholesterols, these functions can be

studied more closely.

With this method, we are able to link cholesterol and oxycholesterol content in cell membranes to

certain observations that are made in cell culture when for example varying oxycholesterol content

in culture medium or challenging cells with stress conditions.

Keywords

• Natural products

• Sterols

• Oxysterols

• Cholesterol

• Cholesterol derivatives

Compound information

Classification Compound name

Sterol Cholesterol

Oxysterols

22(S)-hydroxycholesterol

25-hydroxycholesterol

7-beta-hydroxycholesterol

beta-epoxy-cholesterol

alpha-epoxy-cholesterol

Chromatographic conditions

Column

Particle Size, Length × inner diameter

Order number

VDSpher® PUR 100 C18-H

3 µm, 150 × 2.0 mm

N1520E184VPG

Separation mode descriptions analytical, reverse phase

Mobile Phase A: 0.1 % Formic acid,

B: Acetonitrile/Methanol (50:50 v/v)

Elution conditions Isocratic

0-25 min: 95% B

Flow rate 0.2 ml/min

Injection 10 µl

Column temperature 12°C

HPLC system Shimadzu Prominence HPLC unit

Detector ABSciex API 2000 mass spectrometer with APCI

ion source; selected ion monitoring at

m/z = 367, 369 and 385 amu

Sample and sample preparation Sample: Lipid extract from cell membranes

Preparation: cell membranes are solubilized by

detergent (e.g. Triton X) and extracted with

chloroform/methanol [2].

c = 500 ng/ml cholesterol, 50 ng/ml cholesterol

oxidation products

Chromatograms

Origin

Anna Becker, Prof. Dr. Rainer Buchholz

Friedrich-Alexander-Universität Erlangen Nürnberg

Technische Fakultät (Faculty of Engineering)

Department Chemie- und Bioingenieurwesen (Department Chemical and Biological Engineering)

Lehrstuhl für Bioverfahrenstechnik (Institute of Bioprocess Engineering)

References

“Differential regulation of cell death in head and neck cell carcinoma through alteration of

cholesterol levels in lipid rafts microdomains”

Clara Bionda, Anna Athias, Delphine Poncet, Gersende Alphonse, Amel Guezguez, Philippe Gambert,

Claire Rodriguez-Lafrasse, Dominique Ardail

Biochem. Pharmacol. 2008, 75(3) 761–772.

“A rapid method of total lipid extraction and purification”

E. G. Bligh, W. J. Dwyer

Can. J. Biochem. Physiol. 1959, 37(8), 911-917.

22(S

)-O

H

25-O

H

Chol

7b-O

H

b-E

P

a-E

P

0.00E+00

5.00E+03

1.00E+04

1.50E+04

2.00E+04

2.50E+04

3.00E+04

3.50E+04

4.00E+04

4.50E+04

0 5 10 15 20 25

Inte

nsity (

cps)

Time (min)

m/z 367 amu Intensity (cps) m/z 369 amu Intensity (cps) m/z 385 amu Intensity (cps)

VDSpher PUR C18-H 150 x 2.0 mm, 3 µm A: 0.1% formic acid, B: ACN/MeOH (50:50)Isocratic 95% B at 0.2 mL/min and 12°C for 25 min APCI-MS: Q1 MI at m/z 367.4, 369.4 and 385.4 amu

Sample: 50 ng/ml COPs, 500 ng/ml CholVinj = 10 µl

Year of application: 2016

© 2016 VDS optilab Chromatographietechnik GmbH

VDS optilab does not warrant the correctness of the application.

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