Periodic Reporting for period 1 - MACOOIL (Multidimensional Approaches for Comprehensive Olive Oil Authentication)
Période du rapport: 2022-10-01 au 2024-09-30
Virgin olive oil (VOO) is probably the most representative and iconic component of the Mediterranean diet. No industrial processes or chemicals are added during production, thus providing an extra value reflected in its price higher than most edible oils. However, it is one of the most counterfeited commodities worldwide according to the EU. The EU is the largest producer of olive oil globally, accounting for 69% of the world production and Spain is the main producer. Increasing competitiveness, the expansion of markets in non-producing countries, and the lack of validated methods, has favoured fraud. The assessment of VOO quality, safety, and authenticity is of utmost importance from economic and health perspectives. Current gaps in EU analytical methods in olive oil (OO) include quantifying selected blends of OOs with other vegetable oils (seed oils) and the specific detection of OOs markers in VOO to identify fraud. Hence, it is vital to identify and improve analytical solutions that can detect both standard and emerging frauds and meet all the information required by the international market.
MACOOIL aims to propose novel cutting-edge solutions based on hyphenated techniques such as mass spectrometry, and multidimensional chromatography -previously unexplored in the field-to provide adequate solutions to decipher novel food fraud practices. In particular, MACOOIL will provide novel approaches to extend the knowledge on chemical markers of adulteration of VOO with refined olive oils.
MACOOIL aims to propose novel cutting-edge solutions based on hyphenated techniques such as mass spectrometry, and multidimensional chromatography -previously unexplored in the field-to provide adequate solutions to decipher novel food fraud practices. In particular, MACOOIL will provide novel approaches to extend the knowledge on chemical markers of adulteration of VOO with refined olive oils.
To ensure the quality and authenticity of olive oil there are several analytical methods established in European Union regulations and IOC standards, including the analysis of sterols and triterpenic alcohols. These compounds constitute the unsaponifiable fraction, which represents 2% of the total weight of the oil. However, despite being a minority fraction, knowing its content and composition is of great importance, not only to verify the authenticity of olive oil as well as their potential for discrimination of geographical oil origin or varietal differentiation.
Most of the methods described in the literature for the determination of sterols and triterpenic alcohols are based on gas chromatography analysis coupled with a flame ionization detector (GC-FID). This is the analytical technique proposed also by the official EU method, after a previous process of saponification, separation, and derivatization. However, the lack of selectivity of this detection system and the complexity of the matrix analyzed, make GC-FID methods not always reliable and robust enough to analyze sterols in oil samples.
Another important consideration is that, in vegetable oils, sterols can be found in free form or esterified with fatty acids. The distribution of fatty acids available for esterification varies greatly between oils. However, the official method and most of the applications are based on the saponification process. Therefore, only the total sterol content can be determined, regardless of whether the sterols were originally in free or esterified form. This represents a significant loss of information that could be particularly useful for distinguishing oils with similar sterol profiles, characterization purposes or identifying adulterations. Only a few studies have attempted to cover the simultaneous analysis of free and esterified sterols, using solid phase extraction to achieve the separation of both fractions and employing GC-FID or GC-MS.
For these reasons, the MACOOIL project has focused on developing methods for the analysis of triterpenic alcohols and sterols, both in their free and esterified form, trying to overcome some of the drawbacks found in the published methods for oil analysis and using liquid chromatography as an alternative analytical method with advanced ionization sources and mass spectrometry.
Most of the methods described in the literature for the determination of sterols and triterpenic alcohols are based on gas chromatography analysis coupled with a flame ionization detector (GC-FID). This is the analytical technique proposed also by the official EU method, after a previous process of saponification, separation, and derivatization. However, the lack of selectivity of this detection system and the complexity of the matrix analyzed, make GC-FID methods not always reliable and robust enough to analyze sterols in oil samples.
Another important consideration is that, in vegetable oils, sterols can be found in free form or esterified with fatty acids. The distribution of fatty acids available for esterification varies greatly between oils. However, the official method and most of the applications are based on the saponification process. Therefore, only the total sterol content can be determined, regardless of whether the sterols were originally in free or esterified form. This represents a significant loss of information that could be particularly useful for distinguishing oils with similar sterol profiles, characterization purposes or identifying adulterations. Only a few studies have attempted to cover the simultaneous analysis of free and esterified sterols, using solid phase extraction to achieve the separation of both fractions and employing GC-FID or GC-MS.
For these reasons, the MACOOIL project has focused on developing methods for the analysis of triterpenic alcohols and sterols, both in their free and esterified form, trying to overcome some of the drawbacks found in the published methods for oil analysis and using liquid chromatography as an alternative analytical method with advanced ionization sources and mass spectrometry.
Under MACOIL project different approaches have been developed to go beyond the state of the art. For instance the use of mass spectrometry offers unique features that map well against the challenges described above, either those direct methods based on atmospheric pressure ionization or those occurring under vacuum environment. Notably, the combination of state-of-art high-resolution MS instrumentation with novel ionization methods based on low temperature plasma sources such as DBDI further extends the analyte coverage and ionization efficiency of the standard ionization methods towards non polar species, which, in the particular case of VOOs are highly relevant. On the other hand, together with the fatty acid composition, the total amount of sterols and their profile is one of the most informative determinations to assess the nature of a fat. However, methods for this purpose are based on saponification and these approaches do not allow the physical separation of the enormous diversity of sterols conjugates and their oxidized forms coming out from industrial processing methods that may alter the original nature of the fat. These forms might be highly informative to understand possible changes that may assist the detection of fraudulent practices that are not feasible with current approaches. For this one of the purpose of the project has been to develop an easy sample preparation method where the two fractions, sterols and their conjugates are considered for analysis.