2.3.2. Fatty Acids, Tocopherols, and Organic Acids

Fatty acids were analysed in a DANI GC 1000 (DANI instruments, Contone, Switzerland) equipped with a split/splitless injector and a flame ionization detector (FID) at 260 ◦C. The fatty acids obtained by Soxhlet extraction were methylated with methanol:sulphuric acid:toluene 2:1:1 (v:v:v) during at least 12 h in a bath at 50 ◦C and 160 rpm. Then, deionised water was added to obtain phase separation, and the fatty acid methyl esters (FAME) were recovered with diethyl ether. The upper phase was dehydrated and filtered through 0.2 μm nylon filters for injection. Chromatographic separation was performed on a Zebron-Kame column (30 m × 0.25 mm i.d. × 0.20 μm film thickness, Phenomenex, Torrance, CA, USA). The oven temperature program was as follows: the initial temperature of the column was 100 ◦C, held for 2 min, then a 10 ◦C/min ramp to 140 ◦C, 3 ◦C/min ramp to 190 ◦C, 30 ◦C/min ramp to 260 ◦C, held for 2 min [18]. The carrier gas (hydrogen) flow rate was 1.1 mL/min, measured at 100 ◦C. Split injection (1:50) was carried out at 250 ◦C. The identification was made by chromatographic comparison of the retention times of the sample FAME peaks with those of commercial standards (standard 47885-U). The results were processed using the Clarity 4.0.1.7 Software (DataApex, Podohradska, Czech Republic) and given as relative percentage of each fatty acid.

Tocopherols were analysed in a high-performance liquid chromatography (HPLC) system (Knauer, Smartline system 1000, Berlin, Germany) coupled to a fluorescence detector (FP-2020, Jasco, Easton, MD, USA) programmed for excitation at 290 nm and emission at 330 nm, following an previously described analytical procedure [19]. The samples (500 mg) were spiked with a BHT solution (10 mg/mL) and tocol (internal standard, 50 μg/mL) and homogenized with methanol (4 mL) by shaking in vortex (1 min) and then with hexane (4 mL). After that, a saturated NaCl aqueous solution (2 mL) was added, the mixture was homogenized, centrifuged (5 min, 4000× *g*) and the clear upper layer was collected. The extraction was repeated twice with hexane. The obtained extracts were dried under a nitrogen stream, redissolved in 2 mL of *n*-hexane, dehydrated, and filtered through 0.22 μm disposable syringe filters for injection. Chromatographic separation was performed in normal phase on a Polyamide II column (5 μm particle size, 250 × 4.6 mm; YMC, Kyoto, Japan). Elution was performed with a mixture of *n*-hexane and ethyl acetate (70:30, v/v). The detected compounds were identified by chromatographic comparisons with authentic standards (α, β, γ, and δ isoforms) and quantified using the internal standard method. The results were processed using the Clarity 4.0.1.7 Software and given as μg per 100 g of plant material.

Organic acids were analysed in a ultra-fast liquid chromatography system (Shimadzu 20 A series UFLC, Shimadzu Corporation, Kyoto, Japan) coupled to a photodiode array detector (UFLC-PDA), as previously described [20]. The samples (1 g) were stirred with meta-phosphoric acid (25 mL) for 45 min and filtered, first through Whatman No. 4 paper and then through 0.2 μm nylon filters. Chromatographic separation was achieved in reverse phase on a C18 column (5 μm particle size, 250 × 4.6 mm; Phenomenex, Torrance, CA, USA). The elution was performed with sulphuric acid (3.6 mM). PDA detection was carried out at 215 and 245 nm (for ascorbic acid). The detected compounds were identified and quantified by chromatographic comparison of the peak area with calibration curves obtained from commercial standards (oxalic, quinic, malic, ascorbic, citric, and fumaric acids). The results were processed using the LabSolutions Multi LC-PDA software and expressed in mg per 100 g of plant material.
