Kinetex 5μm evo c18 100a

Samples were prepared by extracting camelina cake and diets with ethanol using a ratio of 1:10 (w/v). Antioxidant activity was determined as 2.2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity according to methodology described by Brand-Williams et al. (1995) [32 (link)]. After 100 min of reaction, the reaction mixture was transferred to 96-well transparent plates and absorbance read at 517 nm. DPPH* (scavenging activity %) was calculated as (1 − Abs_sample/Abs_control) × 100, where Abs_control is ethanol instead of the sample. The total phenolic content (TPC), flavonoids and tannins were extracted and assayed in all samples as previously described by Russo and Reggiani (2018) [33 (link)]. Tocopherol isomers were analyzed in High-Performance Liquid Chromatography (HPLC) by direct injection of oil [34 (link)]. Samples were loaded into a Kinetex 5μm EVO C18 100A (Phenomenex) column (100 × 4.6 mm) and eluted with 0.8 mL/min MeOH 93%. Tocopherols were quantitated at 290 nm by the Borwin software system.

The HPLC analyses were performed using Shimadzu LC20 Performance HPLC chromatograph (Shimadzu, Kyoto, Japan) equipped with UV and fluorescence detector. For simultaneous chromatographic separation of all tested compounds, Phenomenex Kinetex 5 μm EVO C18 100 A 150 × 3 mm with a guard column was used. An isocratic elution, at a flow rate of 0.5 mL/min, was performed with mobile phase consisting of 0.1 M acetic acid, pH 4.5 (adjusted with NaOH), and methanol 97 + 3. All analytes were eluted within 8.5 min.
Excitation and emission wavelengths of fluorescence detector were set for individual compounds: 275/333 nm for 5-OH-TRP from 0 to 3.1 min and 280/334 nm for 5-HT and TRP from 3.1 min. KYN was detected by UV detection with wavelength set to 369 nm. Additionally, in cases of TRP concentrations higher than the range of fluorescence detection, UV detection was used with wavelength set to 300 nm.