In samples. They concluded that transesterification isIn samples. They concluded that transesterification is

In order to increase the
sensitivity and effectiveness of the analytical method, the purification step
should remove interfering compounds from the sample. The first step in the purification
procedure of the POP is transesterification or saponification of the oil/fat
samples. Cold saponification is preferable because during hot saponification there
is a possibility of labile sterol oxidation products (SOP) being lost and
artefacts being formed. Transesterification is less time-consuming (60 min),
easy to perform and no artefacts are formed during this reaction. Saponification
has also practical drawbacks; the emulsion formed during saponificationmay
reduce extraction of the unsaponifiable materials such as SOP because of the
difficulty in separation of the evolved emulsions (150). Azadmard-damirchi and Dutta (150) compared the transesterification and cold saponification of
rapeseed oil samples with a single step SPE method. The results obtained showed
that recovery of the POP fraction was significantly higher (33%, p < 0.05) with transesterification compared with cold saponification of rapeseed oil samples. They concluded that transesterification is much more efficient than cold saponification in purifying and enriching the POP fraction before quantification (150). The second step consisted of the isolation and enrichment of the phytosterol oxides from the unsaponifiable matter (135). Preparative TLC is a rather simple method, but owing to long exposure of the sample to air during workup, it has considerable drawbacks. Different column chromatographic and preparative HPLC methods are also used, but these methods are quite tedious and time-consuming. A simple and quick alternative is the SPE method (137). This operation is generally carried out by SPE with silica gel or aminopropyl as stationary phases (135). Reports are abundant on separation and enrichment of SOP after cold saponification or transesterification of oil samples by single or two-fold SPE using hexane, diethyl ether and finally elution with acetone (155). In two-fold SPE, losses of POP can occur during loading of the POP fraction collected from the first SPE to the second SPE and a proportion of the POP can also be retained in first SPE sorbent (150). It has been reported that the single-step SPE method can be used to complete separation of SOP from both cold saponified and transesterified oils from the unoxidized sterols and other lipid components with high recoveries (88–96%) (150). Figure 9 shows work-up procedure of a single step SPE method to separate and enrich POP for further analysis by GC and GC–MS. The 1 g silica SPE could be used for high amounts, up to 1 g of different oil samples and samples rich in PS. The 1 g silica SPE could be used for high amounts, up to 1 g of different oil samples and samples rich in PS (150).