4.7.2. HPLC–UV–MS Analyses

After alkaline hydrolysis, the chemical content of each flaxseed meal extract, together with camelina meal extracts, was analyzed by HPLC–PDA/UV–ESI–MS/MS technique. The LC–PDA/UV ESI–MS system was composed by a Surveyor LC pump, a Surveyor autosampler, coupled with a Surveyor PDA detector, and a LCQ Advantage ion trap mass spectrometer (ThermoFinnigan, San Jose, CA, USA) equipped with Xcalibur 3.1 software.

Analyses were performed using a 4.6 × 250 mm, 4 µm, Synergi Fusion-RP column (Phenomenex, Bologna, Italy). The eluent was a mixture of methanol (solvent A) and a 0.1% v/v aqueous solution of acetic acid (solvent B). For flaxseed meal extract analysis, a linear gradient of increasing 5 to 35% A was developed within 30 min, while for camelina meal extract, a linear gradient of increasing 5 to 60% A was used within 55 min. The column was successively washed with methanol an equilibrated with 5% A for 10 min.

Elutions were performed at a flow rate of 0.8 mL/min with a splitting system of 2:8 to MS detector (160 µL/min) and PDA detector (640 µL/min), respectively [53]. The volume of the injected methanol solutions was 20 µL. Analyses were performed with an ESI interface in the negative ion mode. The ionization conditions were optimized and the parameters used were as follows: capillary temperature, 270 ◦C; capillary voltage, −16.0 V; tube lens offset, −5 V; sheath gas flow rate, 60.00 arbitrary units; auxiliary gas flow rate, 3.00 arbitrary units; spray voltage, 4.50 kV; scan range of *m/z* 150–1500. N<sup>2</sup> was used as the sheath and auxiliary gas. PDA data were recorded within the 200–600 nm range, with the UV preferential channel as the detection wavelength of 280 nm.

For quantitative analyses of glucosinolates and flavonoids in camelina seed cake extracts, calibration curves were constructed by using glucoraphanin (concentration range 0.06–0.5 mg/mL) and rutin (concentration range 0.01–0.25 mg/mL), as external standards, respectively. Standard methanol solutions at different concentrations were prepared by serial dilution from stock solution (1 mg/mL), then analyzed by triplicate injections, and finally used with respect to the area obtained from the integration of the MS base peak [M − H]<sup>−</sup> of each standard to generate a calibration curve. The relations between variables were analyzed by using a linear simple correlation (*R* <sup>2</sup> = 0.9829 for rutin and 0.9837 for glucoraphanin). The phenol amounts were obtained by using a Microsoft® Office Excel (Redmond, WA, USA) and finally expressed as mg/g of dried cakes.

#### *4.8. Free Radical-Scavenging Assay*

The free radical-scavenging activity was evaluated by the DPPH (1,1-diphenyl-2 picryl-hydrazil radical) and ABTS free radical assay according to the method described by Brand-Williams et al. [54] and spectrophotometrically estimating the solution discoloration at 515 and 734 nm, respectively. The concentration required to obtain a 50% antioxidant effect (EC50) was evaluated as the concentration of extract (mg of defatted seed meal × mL−<sup>1</sup> of extraction solvent) causing the 50% inhibition of the initial color production. A series of dilutions in 80% methanol was prepared for each extract and standard. An Infinite M200 PRO microplate reader was used for spectrophotometric assays. Trolox and butylated hydroxyanisole (BHA) were used as the reference standards. Measurements were replicated three times for each sample.

## *4.9. Statistical Analyses*

All the agronomic and phytochemical variables were subjected to the analysis of variance (ANOVA) using the statistical software CO-STAT Cohort, 2002 (CoHort Software, Monterey, CA, USA). A factorial design with variety (V) and cultivation site (S) as main treatments was used for flaxseed deriving data. Means were separated on the basis of least significance difference (LSD) post-hoc test only when the ANOVA *F*-test per treatment was significant at ≤ 0.05 probability level. For the camelina data, a Student's *t*-test analysis was performed in order to estimate the effect of a cultivation site.
