Analysis of minor constituents of lipidsis essential as they are used as a reference for edible oil regulation and forthe 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. Nonpolarsolvents 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 complextask, because these compounds occur both as free alcohols and as variousconjugates, and therefore may be easily extractable or tightly bound to the foodmatrix (246).
Typically, the analysis includes saponification,separation of sterols, e.g. bythin-layerchromatography (TLC) or solid-phaseextraction (SPE), formation of sterol derivatives, and their analysis by gaschromatography- flame ionizationdetector (GC-FID) (247-249).Free sterols can be determined directlyby analysis of a lipid extract, while the total amount of sterols (free plusesterified) is determined similarly after saponification of the sample (250).
Direct saponification may be applied to dryand liquid samples. For saponification, hydroxide solution is added to thesample which is already dissolved in an organic solvent. Saponificationtemperatures vary from 30?Cto 85?C and time from 8 min to 60 min, and thefinal 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 thesaponification reaction (251).Sterolsare challenging analytes for mass spectrometry, since their low polarity makesthem difficult to ionize by both electrospray ionization and matrix-assistedlaser desorption ionization, typically requiring derivatization steps toovercome their low ionization efficiencies (252).
Recently, a new method fordetermination some PS by GC coupled with electron impact ionization mode-tandemmass spectrometry without derivatization in general food was developed (253).Modernchromatographic techniques can use various specific enrichment and purificationprocedures. Chromatographic methods are commonly used during sample preparationto purify and concentrate phytosterols from other lipids. Normal-phase (NP),reversed phase (RP), and argentation chromatography are used in sample clean-upprocedures. PS are most commonly purified from the unsaponifiable matter ortotal lipid extracts with silicic acid and alumina.
RP materials, e.g., Lipidex5000 and octadecyl-bonded silica, may also be used. In analytical andpurification works, TLC (Figure 12) and SPE are the two most commonly usedtechniques (254). TLC is a conventional preparative method inanalysis of PS, but this method has some drawbacks.
Therefore, SPE as a simpleand inexpensive chromatographic method can be used instead of TLC in most cases(255).