# The Potential of Apulian Olive Biodiversity: The Case of Oliva Rossa Virgin Olive Oil
## 2 Materials and Methods
### VOO Analysis  
The determination of fatty acid composition was carried out after sample transesterification with KOH 2N in methanol [31] by a GC (Agilent 7890A gas chromatograph, Agilent Technologies, Santa Clara, CA, USA) equipped with an FID detector (set at 220 \({}^{\circ}\)C) and an SP2340 capillary column of 60 m \(\times\) 0.25 mm (i.d.) \(\times\) 0.2 mm film thickness (Supelco Park, Bellefonte, PA, USA). The identification of each fatty acid was carried out by comparing the retention time with that of the corresponding standard methyl ester (Sigma-Aldrich, St. Louis, MO, USA). The amount of single fatty acids was expressed as area % with respect to the total area. Basic quality analyses of the VOOs (free fatty acids, FFA; peroxide value, PV; K\({}_{232}\), K\({}_{270}\)) were carried out according to the official regulations [31]. VOO hydrophilic antioxidants were extracted as previously described for drupes with slight modifications. Basically, about 1 g of oil was mixed with 5 mL of methanol/water mixture and 2 mL of hexane. Then the extraction protocol, the Folin assay and the quantitative determination were done the same as previously reported (see Section 2.2). Pigments (chlorophylls and carotenoids) were measured spectrophotometrically (Agilent Cary 60 spectrophotometer, Agilent Technologies, Santa Clara, CA, USA), as reported in [32; 33], respectively. Tocopherols were determined by RP-UHPLC-FLD (ThermoScientific, Waltham, MA, USA), as reported in previous work [28]. For the analysis of single phenolic compounds, phenolic extracts were obtained according to the procedure already described for the Folin assay with minor modifications. In detail, 5 g of sample were used with 2 mL of methanol/watermixture and 2 mL of hexane. Then, 250 uL of a 100 mg L\({}^{-1}\) solution of gallic acid was added as internal standard for quantitation. The separation was obtained by RP-UHPLC-DAD (ThermoScientific, Waltham, MA, USA), as described in [34]. The identification was carried out by comparison of the retention times with those of pure standards and, if not available, with the literature data. Quantification was carried out on the signal recorded at 280 nm and the results were expresses as mg of gallic acid equivalent (GAE) per kg of oil. The phenolic profile was reported only for the harvest season 2017/2018.  
The analysis of volatile compounds was carried out by means of HS-SPME-GC-MS, as reported in a previous work [35]. Briefly, for the extraction of volatiles, about 1 g of sample was sealed in a 20 mL vial after the addition of 100 uL of a 60 mg kg\({}^{-1}\) solution of 1-octanol in purified olive oil as internal standard for quantification. Then, it was first conditioned at 40 \({}^{\circ}\)C for 2 min and, thereafter, the SPME fiber (50/30 um DVB/CAR/PDMS; Supelco, Bellefonte, PA, USA) was exposed in the vial headspace for 20 min. The desorption was carried out directly in the GC-MS (Agilent 6850 series gas chromatograph coupled to a mass spectrometer Agilent 5975 series; Agilent Technologies, Santa Clara, CA, USA) injector at 250 \({}^{\circ}\)C for 2 min. The stationary phase was an HP-Innovax polar column. Volatile compounds were identified by comparison of their mass spectra with those in the NIST library. Only those with a match quality above 70% were considered. Results were expressed as mg of 1-octanol equivalents (OE) per kg of oil. The volatile profile was reported only for the harvest season 2017/2018.