**1. Introduction**

Several international organisations, including the European Union through its directives (EC No. 2568/1991 and its amendments) [1] and the International Olive Oil Council (COI/T.15/NC No. 3/ Rev 6) [2], have been at the vanguard in the development of methods and establishing limits for physicochemical parameters of extra virgin olive oil (EVOO) to protect against frauds. The typical approach relies on comparison of the chemical composition with official limits, as it is expected that the presence of adulterants will modify the concentration of these constituents. Nonetheless, this procedure may be inadequate, especially for oils which are classified as 'virgin' but do not conform to official limits of certain constituents due to local climatic or soil peculiarities [3]. Furthermore, these methodologies do not address the problem of geographical traceability and tend to be rather time-consuming with a very low throughput.

During the last decade, nuclear magnetic resonance (NMR) spectroscopy has been shown to be highly effective in the study of oils of vegetable origin [4–8]. In 1999, Vlahov [9] proposed NMR spectroscopy as a new analytical tool to compete with the existing methods for studying olive oil chemistry. Among the vast applications of NMR spectroscopy to the study of EVOO, target analysis of triacylglycerides, fatty acids, unsaturated fatty chains for quantification, seed oil adulteration, and degradation of EVOOs encompass some of the techniques that could employ the use of NMR. Furthermore, NMR spectroscopy could also be extended to the study of minor constituents including phenolic compounds, sterols, and phospholipids for both detection and quantification of markers for geographical origin and cultivar information. The main methods used in NMR include 1H and 13C NMR spectroscopy as reviewed by a number of authors [9–14] together with 31P NMR as employed by Spyros and Dais [15]. Apart from target-based analytical approaches, NMR metabolic fingerprinting [16–18] employs the use of whole NMR spectral data to classify a relevant number of samples according to their origin, harvest, and age. In most cases, fingerprinting analysis is used in conjunction with sophisticated statistical and mathematical procedures.

1H NMR has been much more widely used in the field of olive oil chemistry than 13C NMR. While requiring more concentrated samples than 1H NMR, 13C NMR spectra have a much wider radiofrequency range. Coupled with proton decoupling techniques, this leads to sharp spectra which rarely have overlapping carbon peaks, allowing easy detection of impurities and making the peaks readily interpretable. The main disadvantage in 13C NMR is the long acquisition times which reduces the sample throughput, unlike 1H NMR which takes around 10 min for the entire run to be completed. Preedy and Watson [19] suggest that each type of NMR spectroscopy could be used for a different type of analysis into the composition of olive oil—the 13C technique is useful in characterisation of the genotype of the oil, while the 1H NMR technique is more suited to geographical characterisation of the oils.

The combination of 1H and 13C NMR fingerprinting with multivariate analysis provides a promising approach to studying the profile of olive oils in relation to their geographical origin. The Maltese olive oil industry makes an interesting case, as the industry has only recently been regenerated using an indigenous olive stock. Considering the small state of the market, mislabeled EVOO originating from other countries sold as Maltese EVOO could severely impede the growth of the industry, with severe negative economic repercussions. Recent studies have shown that Maltese EVOOs have a significantly different phenolic composition and mineral composition [20–22]. In this study, a variety of olive oils selected from different areas around the Maltese islands and countries around the Mediterranean were studied. No data is present in the literature regarding the use of 13C and 1H NMR for the authentication of Maltese EVOOs. The aim of this study was to explore the use of 13C NMR and 1H NMR (specifically 1H zg30 and 1H NOESY), in conjunction with chemometrics in order to differentiate the Maltese EVOOs from other EVOOs derived from other countries within the Mediterranean region, thus developing an easy and cost-saving verification method for the origin of EVOOs from the Maltese islands ensuring olive oil chain sustainability.
