alexa Determination of Cholesterol and its Oxides | Open Access Journals
ISSN : 2153-2435
Pharmaceutica Analytica Acta
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Determination of Cholesterol and its Oxides

Hideharu Shintani*

Chuo University,School of Science,1-13-27,Kasuga Bunkyo 112-0003 Tokyo,Japan

*Corresponding Author:
Hideharu Shintani
Chuo University, School of Science
1-13-27, Kasuga Bunkyo 112-0003 Tokyo, Japan
Tel: +81425922336
E-mail: [email protected]

Received date: June 09, 2013; Accepted date: June 15, 2013; Published date: June 19, 2013

Citation:Shintani H (2013) Determination of Cholesterol and its Oxides. Pharm Anal Acta S1:003. doi: 10.4172/2153-2435.S1-003

Copyright: © 2013 Shintani H. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Keywords

Cholesterol oxidation products; Atherosclerosis; HPLCMS; GLC-MS

Introduction

Several cholesterol oxidation products might play important roles in the development of atherosclerosis [1]. This concept is fostered by mounting interest in the role of oxidatively modified lipoproteins and cholesterol in atherogenesis. Cholesterol oxides are widely encountered in foods and biological tissues. Here the analysis of plasma cholesterol oxidation products by high-performance liquid chromatography-mass spectrometry (HPLC-MS) [2,3] is described

Protocol

1. Bllod is drawn into 10-mL Vacutainer tubes containing EDTA (15% w/v, 0.10 mL).

2. Lipids are extracted from plasma (1.0-mL) in 12-mL borosilicate screw cap tubes by addition of chloroform-methanol (2:1, v/v 6.0 mL) containing BHT (0.01% w/v).

3. Internal standard stock solution (5α-cholestane in toluene, 1.0 mg mL-1, 100 μL) is added to each sample and the tubes are sealed under argon and mixed in a rotary drum for 30 min at room temperature.

4. After centrifugation at 3,000 rpm for 15 min, the organic phase is isolated and the aqueous phase is re-extracted with chloroform-methanol (3.0 mL).

5. The pooled organic phases are evaporated to dryness under nitrogen and the residue, dissolved in ethanol or hexaneisopropanol (1:1, v/v), is analysed by HPLC-MS. (HPLCMS analysis was performed by means of a Hewlett-Packard HPLC 1090A chromatograph, a particle beam interface, and a Hewlett-Packard 5988A mass spectrometer. Compounds were separated on an Altex-Ultrasphere-Si 250 column with 96:4 (v/v) hexane-isopropanol as mobile phase at a flow rate of 0.4 mL min-1).

Results

The principal ions monitored for analysis of cholesterol oxides are listed in Table 1.

Compound name Major ions Minorions Detection limit (ng)
Cholesterol 386, 275 301, 368, 353 0.70
Cholestanetriol 402, 384 369, 303,331 1.50
Cholesterol-5β,6β-epoxide 402 384, 369, 275 1.20
Cholesterol-7-hydroperoxide 400 367, 382, 287 0.75
7-Ketocholesterol 400 367, 382, 287 0.75
5α-Cholestane 372 - -
7a-Hydroxycholesterol 384 402, 369 1.0
Cholesta-3,5-diene-7-one 174 382, 367, 398 1.0
Cholesterol-5α,6α-epoxide 402 384, 369, 275 1.20
25-Hydroxycholesterol 402 400 1.0

Table 1: The principal ions monitored for analysis of cholesterol oxides by HPLCMS with the particle-beam interface.

Conclusion

Together with HPLC-MS, capillary gas chromatography-mass spectrometry (GC-MS) can also be used for the analysis of cholesterol oxides after derivatization, usually to the trimethylsilyl ethers.

References

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