Method M2

SPE/GC-FID method to detect both free and esterified sterols

Virgin olive oils are one of the major components of the Mediterranean diet. For that reason, they can be rather expensive products and fraud is frequent. For instance, EVOO is often diluted with other olive oils of inferior quality or with cheaper vegetable oils, such as seeds oils. 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 of oils. The methods that are available (COI/T.20/Doc. Nº 26, Reg. (CEE) 2568/1991, Annex XIX) are suitable to determine the total composition of sterols, not depending on being in the free or in the esterified form. In different vegetable oils, sterols can be differently distributed between these two forms.

For these reasons, a novel analytical method for the determination of both 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) of olive oils and seed oils has been proposed. The methodology, which is focused not only on the detection of free and esterified sterols but also on some characteristic triterpenic alcohols, could be utilized as a screening tool to detect adulteration by seed oils.

The procedure is quite simple, rapid and does not require expensive and/or complex instruments. It consists of direct derivatization of free sterols in oil samples that are converted into silyl derivatives. In such a way, their polarity became the same of esterified sterols. The oil sample is therefore fractioned for the removal of triacylglycerols and other matrix interferences by solid-phase extraction and the fraction containing free and esterified sterols is analysed by capillary GC with on column injection. The method has the potential to detect illegal blends of olive oil and other vegetable oils based on the results expressed as percentages or absolute concentration in free and esterified forms. It must be stressed however that the method herein proposed is not intended to replace total sterols method (COI/T.20/Doc. Nº 26, Reg. (CEE) 2568/1991, Annex XIX) since they provide different types of information. The two methods could be complementary and used together to obtain a higher degree of information regarding the nature of the oil sample and therefore to reinforce analytical methods available for the prevention of fraud.

 

The analytical procedure has been in-house validated with satisfactory intra-laboratory precision with RSD_r ranging between 3-6 %. Then, the first part of an international method validation study has been carried out. For this purpose, ten European laboratories, who received two different olive oil samples, took part the initial “pre-trial” study. The participants received a Standard Operating Procedure (SOP) as well as instructions for the preparation and storage of the samples. The inter-laboratory precision was worse than the one observed during in-house validation. However, many of the causes of these anomalous results have been identified and corrective measures taken for the trial proper. Valuable feedback has been also received from participants on the SOP with the addition of reporting requirements for the trial proper to better assess whether the laboratories reporting results are correctly following the SOP.
 

The pre-trial has successfully identified causes of aberrant results and allowed several improvements, hoping that method performance data in trial proper, that is due to start in Autumn 2020, will be satisfactory.

 

For the University of Udine: Paolo Lucci