Food Lipids: Chemistry, Nutrition, and Biotechnology

sample preparation is usually employed. Samples can be extracted directly, with little
or no preparation; after drying and powdering; after homogenization of the fresh
materials; or after freeze-drying or fresh-freezing, followed by powdering, sonication,
or homogenization. Extraction procedures vary. The analyst may simply mix the
prepared material with the extraction solvent (the most frequently used solvents include
mixtures of chloroform and methanol, or dichloromethane and acetone) for a
short time (0.5–1 hour) and separate the organic solvent phase from the aqueous
phases and debris by centrifugation. Another common procedure is extraction from
a homogenized material by means of a refluxing solvent in a Soxhlet apparatus for
18 hours or with a boiling solvent for 1 hour. To obtain a total lipid extraction,
saponification under basic (i.e., in 10% KOH in 95% ethanol) or acidic conditions
is usually conducted prior to the organic solvent extraction. Most of the glycosylated
sterols and some esterified sterols cannot be easily extracted into organic solvents
without the hydrolysis step.
Many oxysterols contain functional groups (e.g., epoxides and ketones) that
may be sensitive to high concentration of acids or bases. Epoxides may undergo
nucleophilic attack by strong bases (e.g., NaOH and KOH), followed by ring opening.
Moreover, treatment with strong acids can result in ring opening to form to
alcohols, alkenes, and ketones. The hydrolysis of cholesterol epoxides under mildly
acidic conditions has been studied [56]. Modified procedures are available for the
isolation of steroidal epoxides from tissues and cultured cells by saponification and/
or extraction [57–59]. Ketones are known to form enolates under the influence of
strong bases, which may then form condensation products of higher molecular weight
[60]. To circumvent these potential problems, procedures using different extraction
techniques are sometimes preferred to a saponification followed by extraction. Mild
methods for the removal of the ester function without ketone enolization include
extraction by means of sodium or potassium carbonate in heated aqueous solutions
of methanol or ethanol. The addition of tetrahydrofuran to these mixtures has been
found to significantly increase the solubility of the more polar oxysterols [61].
There are not many studies on the efficiency of various extraction methods.
Most extraction procedures were designed to compare the extraction of lipids from
cells or tissues of a single source and have been applied subsequently to plants and
animals of various types. The errors in the quantitative analysis of sterols, which are
probably introduced in the extraction steps, could be eliminated by using in situ
labeling of key sterols. Sterols labeled with deuterium and 14 C have been used to
monitor the extraction recovery in human plasma oxysterol analysis.
2. Isolation of Sterols
Conventional column chromatography, with ordinary phase (silica gel or alumina
oxide), reversed phase, and argentation stationary phase, is still the most important
method for the isolation and purification of sterols, especially if the total lipid extraction
is complex and high in weight (>200 mg) [62]. Chromatographic methods
with an organic phase involve the binding of a substrate to the surface of a stationary
polar phase through hydrogen bonding and dipole–dipole interaction. A solvent gradient
with increasing polarity is used to elute the substrate from the stationary phase.
The order of substrate movement will be alkyl > ketone > hindered alcohol > unhindered
alcohol. The elution profile is routinely monitored by GC or TLC. Reversed
phase column chromatography involves the use of lipophilic dextran (Sephadex LH-
Copyright 2002 by Marcel Dekker, Inc. All Rights Reserved.