Because ofsome drawbacks of TLC, a novel SPE method has been developed to separate4-desmethyl-, 4-monomethyl-, and 4,4-dimethyl sterols in vegetable oils, whichis shown in Figure 13 (170). After separation of different sterolfractions by TLC or SPE, they are analysed by GC to obtain their compositionand also quantity. Figure 14 shows separation of trimethylsilyl etherderivatives of the purified 4,4?-dimethyl sterols of hazelnut oil and virginolive oil by GC.Todetermine the free and esterifiedsterol concentrations of different edible oils, free and esterified sterolsseperated by SPE, and then saponified, and quantified as trimethylsilyl etherderivatives using GC. Determination of individual values for free andesterified ?-sitosterol,campesterol, stigmasterol, brassicasterol, ?5-avenasterol,sitostanol, campestanol, and cholesterol was possible (38).ComparableGC methods are recommended for total plant sterols, where several distinctcomponents can be determined (e.g. campesterol, stigmasterol, sitosterol, ?5-avenasterol)(250).

Steryl classes (freeforms and conjugates) and structural groups are separated by adsorptionchromatography on the basis of their polarity (256). GC-FID chromatogram of sterols in refinedrapeseed oil is shown in Figure 15. HPLC techniques are less often used toseparate free PS than GC, but some applications exist. One of the reasons forlimited use of HPLC may be that there is not such a general and sensitive HPLCdetector for PS as the flame ionization detector in GC and also its poor separationquality.

However, with new separation techniques and detection possibilities,attempts have been made to apply HPLC to the analysis of free phytosterols infoods (246).A highly sensitive method for quantification of PSbased on HPLC has been developed by derivatization with the benzoylchromophore. Introduction of the chromophore, benzoyl group, to PS viaderivatization greatly improved the UV response at 254 nm. Quantification of PSwas effectively performed by HPLC analysis with methyl benzoate as the internalstandard after derivatization (258).