IN-HOUSE VALIDATION OF AN SPE-GC-FID METHOD FOR THE DETECTION OF FREE AND ESTERIFIED HYDROXYLATED MINOR COMPOUNDS IN VIRGIN OLIVE OILS

INTRODUCTION Virgin olive oils suffer from adulterations as observed in the EC reports related to food fraud [1]. In this context, the analytical evaluation of the composition of sterols is a well-established tool for assessing the purity of olive oils, as it depends on the botanical origin. The available official methods are suitable to determine the total composition of sterols, not depending on being in the free or in the esterified form [2, 3]. However, it should be highlighted that, in different vegetable oils, sterols can be differently distributed between these two forms. Information related to the esterified fraction of minor compounds (hydroxylated minor compounds, HMCs) in oil is inevitably lost when applying official procedures involving a saponification step. A method for the determination of the free and esterified minor components (waxes, alkyl esters, free fatty alcohols, free and esterified sterols, free and esterified triterpenic alcohols, sterenes, and free and esterified tocopherols) in olive and seed oils has been developed and in-house validated in this experimental work. The proposed offline SPE-GC-FID methodology, that takes inspiration from previous research works [4, 5], has been applied to pure olive and sunflower oil samples and tested for its ability to reveal extra virgin olive oil (EVOO) adulteration with small percentages (up to 10%, w/w) of refined sunflower oil (RSO). RESULTS A simplified method based on offline SPE-GC-FID for analysis of free and esterified HMCs in olive and seed oils has been developed and in-house validated. This method is accessible to most analytical laboratories and replace toxic solvents usually employed in sterol analysis while reducing at minimum the amounts of reagents needed. The procedure allows the conversion of free minor compounds into silyl derivatives, in such a way, their polarity become the same of esterified minor components. Oil is then fractioned by silica solid-phase extraction and the fraction with free and esterified minor compounds is analyzed by capillary GC with on column injection. The method has been optimized and then in-house validated using three different oils [EVOO, refined pomace oil (PO), and RSO] samples. The method has been applied to pure EVOO and RSO samples (see Figure). As an example, results showed three times higher level of esterified minor compounds in RSO compared to EVOO, thus confirming that the esterified fraction could represent the most diagnostic one for detecting the fraudulent addition of RSO to EVOO. Additionally, the ability of the method to quantify free and esterified HMCs has been investigated by analyzing EVOO samples added with small amounts of RSO.