Chuo University,School of Science,1-13-27,Kasuga Bunkyo 112-0003 Tokyo,Japan
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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Cholesterol oxidation products; Atherosclerosis; HPLCMS; GLC-MS
Several cholesterol oxidation products might play important roles in the development of atherosclerosis . 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
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).
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|
|Cholesta-3，5-diene-7-one||174||382, 367, 398||1.0|
|Cholesterol-5α,6α-epoxide||402||384, 369, 275||1.20|
Table 1: The principal ions monitored for analysis of cholesterol oxides by HPLCMS with the particle-beam interface.
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.