Chromatographic Separation of Phenolic Compounds from Extra Virgin Olive Oil: Development and Validation of a New Method Based on a Biphenyl HPLC Column

Three different high performance liquid chromatography columns were accessed for phenolic compounds (PC) separation in the hydrophilic fraction of extra virgin olive oil (EVOO). Two fully porous C(18) bonded silica phases and one partially porous biphenyl column were used. Biphenyl column allowed for an increase of more than 30% in peak capacity (n(c)), higher selectivity (α) (1.045), and improved retention (k), with a reduction of 22.1% in the retention time. The higher resolution (R(s)) was obtained by using the biphenyl column, with a fair separation of oleuropein aglycone isomers (OAI) and a good identification of caffeic acid (CA). Tyrosol (T), hydroxytyrosol (HT), and dihydroxyphenyl glycol (DHPG) were also well separated and identified. Moreover, the method using a biphenyl column was fully validated according to the requirements for new methods. For all parameters, the method applying the biphenyl column proved to be a reliable, accurate, and robust tool for separation, identification, and quantification of the main PCs in EVOOs.