ABSTRACT
Saponification probably is the most frequently performed reaction in oil and fat research. Treatment of the sample with a strongly alkaline alcoholic solution converts esters into (dissociated) free acids and free alcohols. In complex samples, such as natural extracts and biomedical samples, part of the sterols might be present as complex conjugates. Upon saponification most of these will also be released, with possibly the exception of sterylglycosides which require acid hydrolysis (39). Because saponification effectively destroys solid matrices, it can also be used as a much faster alternative to liquid extraction because in the solid samples the sterols can be strongly bound to a solid matrix or even captured in matrix particles. To recover the sterols from the saponification mixture after reaction, an extraction with a nonpolar organic solvent has to be performed to obtain the “unsaponifiables” fraction. A wide range of conditions for saponification of fat and fatty food samples has been described in literature. In our laboratory basically three different saponification protocols are used in sterol analysis: (a) strong saponification with a sodium or potassium hydroxide solution in methanol or ethanol; (b) strong saponification with a sodium hydroxide in methanol solution followed by borontrifluoride/methanol esterification; or (c) mild saponification with potassium hydroxide in ethanol. Protocol b, saponification with subsequent methylation of the fatty acids, is only applied if both the sterol and the fatty acid composition of the sample must be characterized. The experimental protocols for these procedures are summarized in Table 6. The details given in this table are the conditions as used in our laboratory. Based on practical experience we are confident in
Procedure Protocol for raw materials or spreads. For samples that contain the analytes of interest at lower levels higher sample weights can be processed.
