Usefulness of HS-SPME-GC-MS quantification of volatile compounds for quality control of virgin olive oil

1 Introduction Extra Virgin Olive Oil (EVOO) is considered as the highest quality product among edible oils mainly thanks to its pleasant taste and smell and the high content of bioactive phenolic compounds. In this context, economic frauds regarding false claim of geographical origin [1] and commercial category [2] cannot be fully avoid to date. Moreover, virgin olive oil classification is based, among other, on its sensory assessment carried out by a panel of trained experts, according to International Olive Council (IOC) trade standards and European legislation [3]. Over the last years, the possibility of using reliable chemical data related to the composition of the volatile fraction of virgin olive oils, has been recognized as more and more crucial. The use of HS-SPME-GC-MS analysis for quantification of volatile organic compounds (VOCs) has gained great attention in the last years and quantification of 29 VOCs by this technique has been validated [4]. In this study, carried out over many years of work, we aimed at developing chemometric approaches for the quality control of virgin olive oil. First we optimized and validated the HS-SPME-GC-MS quantification of virgin olive oil VOCs by using several internal standards. Then, we used the validated method for analyzing more than 1000 oil samples; the obtained set of data has been statistically treated for proposing reliable and robust approaches to support the panel test in virgin olive oil classification and to authenticate the geographical origin of virgin olive oils from the main worldwide producing countries. 2 Experimental A refined olive oil free from VOCs was used for preparing internal and external standard solution, used for both method validation and samples analysis. More than 1000 virgin olive oil samples from different geographical origin (Italy, Spain, Greece, Tunisia, Portugal) and category (EVOO, VOO, LVOO) were collected in the Carapelli laboratory. Method optimization and validation was carried out using a Trace CG-MS Thermo Fisher Scientific, equipped with a ZB-FFAP capillary column (Zebron) 30 m × 0.25 mm ID, 0.25 μm DF. Method was then applied to virgin olive oils using a 6890N GC system equipped with a MS detector, model 5975 by Agilent, equipped with a HP-Innowax capillary column 50 m × 0.2 mm ID, 0.4 μm DF. In both cases, analysis of VOCs was carried out weighting 4.3 g of sample and 0.1 g of internal standard solution into a 20 ml screw cap vial fitted with a PTFE/silicone septa. A SPME fiber 50/30 µm DVB/CAR/PDMS was exposed for 20 min in the vial headspace under orbital shaking at 400 rpm after equilibration for 5 minutes at 45°C, then the adsorbed VOCs were desorbed in the injection port of the GC system. Mass detector worked in scan mode within the range of 30-350 Th, 1500 Th/s at ionization energy of 70 eV. Each VOC was quantified using a calibration curve in which the area ratio (ratio between areas of that VOC and the selected ISTD) was plotted versus the amount ratio. Suitable statistical tools were employed for data analysis, aiming at building the chemometric approaches above mentioned: Principal Component Analysis (PCA), Linear Discriminant Analysis (LDA), Analysis of Variance (ANOVA) and t-test 3 Results and Conclusion In the first step of the work [5], quantification of 73 VOCs was optimized and validated using 11 internal standard and selecting the more suitable one for each of the quantified VOCs. This approach allowed overcoming several issue previously reported as critical when HS-SPME-GC-MS is used for quantification purpose [6], resulting in a good linearity in ranges of calibration wider than those previously reported in studies performed only using a single internal standard. The validated method was then applied for quantifying the 73 VOCs in virgin olive oils selected for building chemometric approaches aimed to: • Supporting the panel test in virgin olive oil classification (1223 samples) • Authentication of the geographical origin of virgin olive oils from the main worldwide producing countries (1217 samples). The main results can be summarized as follow: Four chemometric approaches for supporting the panel test in virgin olive oil classification have been proposed and externally validated with a set of independent samples. The PCA-LDA model gave the best results, when the classification was compared with the results of the panel test (Table 1). The t-test-DSV model allowed strongly simplifying the analytical work, in that it gave a very good predictive performance only using quantitative data of 10 VOCs. During development of the proposed approaches, it also emerged that, from the qualitative point of view, octane, heptanal, pent-1-en-3-ol, Z-3-hexenal, nonanal and 4-ethylphenol must be considered the VOCs more suitable for discriminating different classes of virgin olive oils during their classification. Three chemometric approaches for authentication of the geographical origin of virgin olive oils from the main worldwide producing countries have been proposed. The best results (Table 2) were obtained by the ANOVA-LDA model, only using 25 VOCs, selected using ANOVA. For some origins, the proposed model showed a prediction capability higher than 97% In conclusion, quantitative data obtained by HS-SPME-GC-MS using several internal standard are suitable for building reliable and robust chemometric approaches for virgin olive oil quality control, thus allowing protecting consumers and producers from incorrect claiming of geographic origin and commercial classification