Comparison of saponification methods for characterization of the nonsaponifiable fraction of virgin olive oil

Despite the interest of the nonsaponifiable fraction of olive oil (composed by sterols, tocopherols, fatty alcohols, hydrocarbons, phenols, pigments, and volatile compounds) there are no studies in the literature dealing with comparison of methods to isolate this fraction. Four saponification methods (viz. hot and cold saponification, and microwave- and ultrasound-assisted saponification) have been compared by analysis of the resulting extracts using LC-MS in high-resolution mode and data treatment with multivariate statistical tools. Principal component analysis and Venn diagrams were employed to compare qualitatively and/or semiquantitatively the metabolite profiles provided by the different methods. Hot saponification was the approach which provided the highest efficiency of this step in qualitative and quantitative terms. On the other hand, cold and microwave-assisted saponification led to nonsaponifiable extracts with similar composition according to unsupervised analysis. These results revealed that microwave assistance does not promote chemical alteration of the nonsaponifiable fraction. Finally, ultrasound-assisted saponification provided the extract with a more different composition.