HIGH QUALITY BERGAMOT OIL FROM CEPHALLONIA (GREECE): CHEMICAL ANALYSIS USING ENANTIOMERIC GAS CHROMATOGRAPHY-MASS SPECTROΜΕΤRY Melliou Eleni, Magiatis Prokopios, Skaltsounis Alexios-Leandros Department of Pharmacognosy and Natural Products Chemistry, Faculty of Pharmacy, University of Athens, Panepistimiopolis Zografou, Τhe essential oil contained in the rind of the beramot fruit (Citrus aurantium subsp. bergamia, syn. Citrus bergamia) is a high value product with many applications in perfume industry, beverage industry and cosmetology. Up today the highest quality bergamot oil is produced in the Ionian coast of Calabria (Italy). It is noteworthy that although the Ionian coasts are considered as the best region in the world for the cultivation of bergamot, in Greece the bergamot tree had never been cultivated for the production of essential oil. The herein studied bergamot trees were cultivated in Cephallonia island (region Vlachata) in a total area of m 2. The variety of bergamot found in Cephallonia is botanically distinct from the Calabrian bergamot as well as from other bergamot varieties found in Greece. Although the morphology of the fruit is different, the composition of the obtained oil clearly shows a strong similarity with the oil obtained from Calabrian bergamots. The mature fruit is yellow with diameter cm, weight gr and peel thickness approx. 2 cm. These characters make the fruit very different from other common varieties of bergamot and could be considered as important advantages for its use for the production of essential oil or for the preparation of sweets. Plant material: The plant material (leaves and fruits) were collected in January The rind of the fruit (300 g) was separated manually and the essential oil was obtained either by cold pressing or by hydrodistillation for 3 hours in a Clevenger apparatus. The total yield calculated on wet weight of fresh rinds was 0.8%. The leaves (4 Kg) were submitted to hydrodistillation for 3 hours using a semi-industrial distiller affording 0.55% of yellow oil. GC-MS analysis with enantiomeric column Methodology The obtained bergamot oils were analyzed using gas chromatography-mass spectrometry first with a HP-5MS column and then with an enantiomeric column b-Dex sm. The GC-MS analyses were carried out using a Hewlett Packard GC-MS system operating on EI mode (equipped with a HP 5MS 30m x 0.25mm, 0.25 μm film thickness capillary column). He (1 ml/min) was used as carrier gas. The initial temperature of the column was 60 o C and then it was heated to 280 o C at a rate of of 3 o C /min. GC-MS analyses were also performed on a Finnigan Trace spectrometer in both the EI and chemical ionization (CI) modes at a flow rate of 1.0 ml/min, using CH 4 as the CI ionization reagent. Enantiomeric GC-MS analysis was performed using a b-Dex sm column on a Finnigan Trace spectrometer. The initial temperature of the column was 60 o C and then it was heated to 230 o C at a rate of of 4 o C /min, He (1 ml/min) The identification of the compounds in the HP 5MS column was based on comparison of their retention indices (RI), obtained using n-alkanes (C9-C25), and on comparison of their EI-mass spectra with the NIST/NBS, Wiley library spectra and literature. Additionally, the identity of all compounds was performed by comparison of the expected molecular weights with the results obtained from the CI spectra. The enantiomeric identification of the constituents was based on the co-injection with commercial or previously isolated standards as well as comparison with the corresponding mass spectra. Results It was found that the main constituent of the cold press essential oil of the fruit rind was (-)- linalyl acetate with optical purity >99.9% which is responsible for the characteristic odour of the bergamot fruit. Other important constituents were (-)-linalool, (+)-limonene and γ-terpinene. It should be noted that the main difference between Cephallonian bergamot oil and Calabrian or Turkish bergamot oil is the 10-fold reduced concentration of β-pinene/sabinene and the relatively increased concentration of γ-terpinene. One important index for the characterization of the high quality bergamot oil is the ratio linalool/linalyl acetate which must be 0.3. The performed analysis showed that the value of this specific index in the last stage of the fruit maturity was Another characteristic index is the sum of linalool+linalyl acetate which was found to be >56%. In the bergamot oil of Calabria (Italy) which is considered as the best bergamot oil in the world the corresponding index is reported to be <50%. Concerning the essential oil of the leaves it should be noted that it could be used as an alternative cheap source of bergamot-like oil. It is characterized by high linalool (34.6%) and linalyl acetate (29.8%) content and low concentration of limonene (0.65%). Additionally neryl acetate (4.9%) and geranyl acetate (9.4%) are highly increased. It is also important to note that the quality of the fruit oil obtained by hydrodistillation is lower due to the decomposition of linalyl acetate and the isomerization of linalool. Comparison between the same sample treated by cold press or distillation reveals that the percentage of linalyl acetate falls from 41.5% to only 10.7% in the distilled oil. Additionally, the thermal treatment for the distillation procedure leads to partial isomerization of linalool which can be confirmed by the presence of (+)-linalool The high quality of the Cephallonian bergamot oil confirmed the observation that the best region in the world for the cultivation of bergamot tree is the Ionian coasts. The present work offers important supporting data for the exploitation of Cephallonian bergamot as a new agroindustrial product in Greece. Bergamot oil cold press Bergamot oil distilled (-)-linalyl acetate (+)-limonene γ-terpinene (-)-linalool (-)-limonene (-)-linalool (+)-linalool Region of cultivation of Citrus bergamia Citrus bergamia from Cephallonia Common bergamot (left) and bergamot from Cephallonia (right) Enantiomeric analysis of cold press oil of bergamot fruit from Cephallonia Composition of distilled essential oil from bergamot leaves from Cephallonia