Cholesteryl acetate detection

The hydroperoxides obtained on thermal oxidation of cholesteryl acetate (191e) can be selectively separated by SPE and elution with a polar solvent. After reduction to the corresponding alcohols by NaBH4 and further derivatization to the trimethylsilyl ether, the products can be subjected to GLC with ion-trap MS detection. It can be thus demonstrated with the aid of standards that under the oxidation conditions (160 °C for 90 min) only the 7-position is attacked, leading to the la- and 7/3-hydroperoxy derivatives, while the plausible 4-position remains unscathed . Treatment of erythrocite ghosts with t-BuOOH causes a manyfold content increase of 5-hydroxyeicosatetraenoic acid (5-HETE), 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and 5-oxoeicosatetraenoic acid (5-oxo-ETE) residues of phospholipids. These acids can be separated by HPLC, identified and quantitized by tandem MS . ... 

Cholesteryl acetate was used as a model to illustrate the application of tailored detection to the assignment of spectra of complex molecules. The noise-decoupled spectrum contains several regions where signals almost overlap, so that off-resonance decoupling, aimed at ascertaining the multiplicity of individual signals as a result of coupling, can cause much confusion. The tailored... 

In the present experiment cholesterol is dissolved in acetic acid and allowed to react with acetic anhydride to form the ester, cholesteryl acetate. The reaction does not take place rapidly and consequently does not go to completion under the conditions of this experiment. Thus, when the reaction is over, both unreacted cholesterol and the product, cholesteryl acetate, are present. Separating these by fractional crystallization would be extremely difficult but because they differ in polarity (the hydroxyl group of the cholesterol is the more strongly adsorbed on alumina), they are easily separated by column chromatography. Both molecules are colorless and hence cannot be detected visually. Each fraction should be sampled for thin-layer chromatography. In that way not only the presence but also the purity of each fraction can be assessed. It is also possible to put a drop of each fraction on a watch glass and evaporate it to see if the fraction contains product. Solid will also appear on the tip of the column while a compound is being eluted. 

Fig. 11.6.1. HPLC separation of cholesterol and cholesteryl ester standards. Chromatographic conditions column, Supelcosil LC-18 (250x4.6 mm I.D.) mobile phase, acetonitrile-methanol-chloroform (1 1 1, v/v/v) flow rate, 1.0 ml/min temperature, ambient detection, differential refractometer. Peaks 1, cholesterol, 2, acetate 3, propionate 4, butyrate 5, nonanoate 6, decanoate 7, arachidonate 8, laurate 9, linoleate 10, oleate 11, elaidate 12, palmitate 13, stearate. The average mass of lipid chromatographed was 20-40 ng. Reproduced from Perkins et al. (1981), with...

Figure 15.1 Separation of yolk-saline medium (see text) following extraction in chloroform-methanol (2 1). Lipids were deveioped in petroleum ether-diethyl ether-acetic acid (80 20 1) and detected by spraying with PMA. Lane 1 contains neutral lipid standard mixture 18-4A, which consists of cholesterol (c), oleic acid (o), triolein (t), methyl oleate (m), and cholesteryl oleate (co). Lane 2 shows presence of triacylglycerols and free sterols that are the predominant neutral lipids in the yolk-saline medium. Lane 3 contains saline alone that is neutral lipid negative.

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