**2.2 NMR analysis**

Aliquots of 40 µL of each VOO were dissolved in 200 µL of deuterated chloroform, shaken in a vortex, and placed in a 2 mm NMR capillary. The 1H-NMR experiments were performed at 300K on a Bruker (Rheinstetten, Germany) Avance 500 (nominal frequency 500.13 MHz) equipped with a 2.5 mm broadband inverse probe. The spectra of the samples used for the study of the geographical origin of VOOs were recorded using a 7.5 µs pulse (90°), an acquisition time of 3.0 s (32k data points) and a total recycling time of 4.0 s, a spectral width of 5500 Hz (11 ppm), 64 scans (+ 4 dummy scans), with no sample rotation. The spectra of the samples used for the VOO stability study were recorded using a 6.7 µs pulse (90°), an acquisition time of 3.5 s (50k data points) and a total recycling time of 7.0 s, a spectral width of 7100 Hz (14 ppm), 32 scans (+ 4 dummy scans), with no sample rotation. Prior to Fourier transformation, the free induction decays (FIDs) were zero-filled to 64k and a 0.3 Hz linebroadening factor was applied. The chemical shifts are expressed in scale (ppm), referenced to the residual signal of chloroform (7.26 ppm) (Hoffman, 2006). The spectra were phase- and baseline-corrected manually, binned with 0.02 ppm-wide buckets, and normalized to total intensity over the region 4.10-4.26 ppm (glycerol signal). TopSpin 1.3 (2005) and Amix-Viewer 3.7.7 (2006) from Bruker BioSpin GMBH (Rheinstetten, Germany)

Quality Assessment of Olive Oil by <sup>1</sup>

H-NMR Fingerprinting 191

*14*

*16*

*15*

*1 2 3*

*4 5*

*6*

*8*

*<sup>7</sup> <sup>10</sup> <sup>9</sup>*

hydrocarbons, fatty acids, pigments and phenolic compounds (Harwood & Aparicio, 2000), are only observed by 1H-NMR when their signals do not overlap with those of the main components and their concentrations are high enough to be detected (R. M. Alonso-Salces et al., 2010a; Rosa M. Alonso-Salces et al., 2010b; D'Imperio et al., 2007; Guillen & Ruiz, 2001; Mannina et al., 2003; Sacchi et al., 1996). Table 1 gathers the common 1H-NMR signals of the major and some minor compounds together with their chemical shifts and their assignments to protons of the different functional groups. Several signals of minor compounds were found in 1H-NMR spectra recorded because they were not overlapped by those of the triglyceryl protons: cycloartenol at 0.318 ppm and 0.543 ppm, -sitosterol at 0.669 ppm, stigmasterol at 0.687 ppm, squalene at 1.662 ppm, sn-1,2 diglyceryl group protons at 3.71 ppm and 5.10 ppm,

*<sup>17</sup> <sup>18</sup> <sup>21</sup>*

x 1 x 8 x 1 x 8

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

The large dataset of VOOs was studied regarding the situations that the antifraud authorities and regulatory bodies face. The PDO "*Riviera Ligure*", some Italian regions and the main countries that produce VOOs were used as examples to prove the potential of the tools to detect the mislabeling of non-PDO oils as PDO VOOs, or the mislabeling of the provenance of VOOs at the regional or national level. With this purpose in mind, several multivariate data analysis techniques, datasets, types of data scaling and cross-validation

After removing 28 extreme samples, the dataset (935 x 342) was analyzed by PCA. The four first principal components, accounting for 63% of total system variability (TSV), showed that samples were distributed in a compact cluster. However, some overlapping sub-clusters due to the harvest year were observed in the score plot of the samples in the space defined by PC2 (13% TSV), PC3 (11% TSV) and PC4 (7% TSV). Taking into account that 70% of the samples were Italian and the rest from countries in the Mediterranean region, seasonal aspects seem to affect all samples in the same way, independently of their geographical origin. Therefore, in the modeling for the authentication of agricultural food products, it is important to include chemical data of several harvests in order to obtain general

and three unknown terpenes at 4.571 ppm, 4.648 ppm and 4.699 ppm.

Fig. 1. 1H-NMR spectra of a VOO (signal numbering, see Table 1).

were evaluated to attain the best classification models for each case study.

classification models that allow for the seasonal variability.

*22*

*23 24 25*

**3.2 Geographical origin of virgin olive oil** 

*26*

*27*

*28*

were used to perform the processing of the spectra. The region of the NMR spectra studied was 0-7 ppm for the geographical origin determination of VOOs, and 0-10 ppm in the VOO stability study. The data tables generated with the spectra of all samples, excluding the eight buckets in the reference region 4.10-4.26 ppm, were submitted to multivariate data analysis.
