Lichrocart rp 18 column

The products were purified using a semi preparative HPLC. Separation of oxidation products was carried out using LiChroCART RP-18 column (Merck, 25 × 3 × 5). The products were detected at 280 nm using UV/Vis detector. The two solvents were used to separate the oxidation products: solvent A composed from water and TFA at 0.03 v/v), and solvent B composed from 80% acetonitrile and 20% solvent A using 5 mL/min as a flow rate. An amount of 1 mL of sample (10 mg/mL) was used as an injection volume. The used gradient was linear from 5% to 30% of solvent B for 15 min, from 30% to 50% of solvent B for 15 min and from 50% to 75% of solvent B for 15 min.

In order to compare the phenolic composition, extracts from all samples, maize flour and corresponding broa, were analysed in a Thermo Fisher Scientific Surveyor HPLC system, equipped with a DAD (Waltham, MA, USA). The analytical conditions are described in Appendix A.
Extracts of the SF and IF of Verdeal de Aperrela sample, as well as the commercial wheat and rye flours, were analysed on an Alliance 2695 separation module HPLC system (Waters, Dublin, Ireland) coupled to a 2996 Photodiode Array Detector and a Micromass^® Quattro Micro triple quadrupole (TQ) (Waters, Dublin, Ireland). The analytical conditions are described in Appendix B. MS/MS experiments were performed in order to identify the major phenolic compounds. Additionally, when standards were commercially available, MS/MS conditions were optimised (Table S1) and extracts were analysed in multiple reaction monitoring (MRM) mode in order to increase selectivity and sensitivity.
In both types of equipment, the injection volume was 20 µL, and the chromatographic separation procedure was carried out using a Lichrocart^® RP-18 column (250 × 4 mm, 5 µm) and a Manu-cart^® RP-18 pre-column (Merck, Darmstadt, Germany) in a thermostated oven at 35 °C.

LC-MS system (LTQ-MS) was used to assess the mass spectra at atmospheric pressure ionization interface operating in Atmospheric Pressure Chemical Ionization (APCI) positive mode using LiChroCART RP-18 column (Merck, 25 × 0.4 × 5 µm). The spray voltage was used at 6.0 kV. The temperatures of the APCI vaporizer and of the heated capillary were used at 400 °C and 225 °C, respectively. The flow rates of auxiliary gas, sweep gas and sheath gas were determined at to 5, 5 and 48 (units/min), respectively. A voltage of 50 V was used in tube lens, 1 kV was used as capillary voltage and the values of front lens and split lens were −6.75 V and 70 V, respectively. To optimize all parameters, the FA solution at 0.1 mg/mL was infused using two solvents (solvent A was composed from water and TFA (0.03%)/solvent B was composed from 80% acetonitrile and 20% solvent) with a flow rate of 5 µL × min⁻¹. The full scan was measured from 50 m/z to 1000 m/z.