Analysis because these compounds occur both as free

Analysis of minor constituents of lipids
is essential as they are used as a reference for edible oil regulation and for
the analytical assessment of oil quality, its origin, extraction method,
refining procedures used, and possible adulteration of the oils (244).
Most common methods for the extraction of lipids also extract phytosterols. Nonpolar
solvents such as hexane (commonly used to extract most types of vegetable oils)
quantitatively extract free phytosterols and phytosteryl fatty-acid esters (5, 245).

Analysis of PS in foods is a complex
task, because these compounds occur both as free alcohols and as various
conjugates, and therefore may be easily extractable or tightly bound to the food
matrix (246). Typically, the analysis includes saponification,
separation of sterols, e.g. by
thin-layer
chromatography (TLC) or solid-phase
extraction (SPE), formation of sterol derivatives, and their analysis by gas
chromatography- flame ionization
detector (GC-FID) (247-249).

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Free sterols can be determined directly
by analysis of a lipid extract, while the total amount of sterols (free plus
esterified) is determined similarly after saponification of the sample (250). Direct saponification may be applied to dry
and liquid samples. For saponification, hydroxide solution is added to the
sample which is already dissolved in an organic solvent. Saponification
temperatures vary from 30?C
to 85?C and time from 8 min to 60 min, and the
final concentration of potassium hydroxide is 0.33 to 0.5 M in ethanol (246). In addition, ultrasonic frequency can be used to provide energy for the
saponification reaction (251).

Sterols
are challenging analytes for mass spectrometry, since their low polarity makes
them difficult to ionize by both electrospray ionization and matrix-assisted
laser desorption ionization, typically requiring derivatization steps to
overcome their low ionization efficiencies (252). Recently, a new method for
determination some PS by GC coupled with electron impact ionization mode-tandem
mass spectrometry without derivatization in general food was developed (253).

Modern
chromatographic techniques can use various specific enrichment and purification
procedures. Chromatographic methods are commonly used during sample preparation
to purify and concentrate phytosterols from other lipids. Normal-phase (NP),
reversed phase (RP), and argentation chromatography are used in sample clean-up
procedures. PS are most commonly purified from the unsaponifiable matter or
total lipid extracts with silicic acid and alumina. RP materials, e.g., Lipidex
5000 and octadecyl-bonded silica, may also be used. In analytical and
purification works, TLC (Figure 12) and SPE are the two most commonly used
techniques (254). TLC is a conventional preparative method in
analysis of PS, but this method has some drawbacks. Therefore, SPE as a simple
and inexpensive chromatographic method can be used instead of TLC in most cases
(255).