6420 triple quadrupole

The HPLC method established by Gomez-Caravaca et al. [53 (link)] was used. MRM analyses were performed on 6420 Triple Quadrupole (Agilent Technologies, Santa Clara, CA, USA) equipped with the Agilent HPLC 1200 series autosampler and a binary pump. Phenolic separation was performed on a 100 mm × 3.0 mm Zorbax Poroshell C18 column (Agilent Technologies, Millford, MA, USA) at 25 °C. MS/MS acquisition parameters (MRM mode) used for identification of the target phenolic compounds are provided in Table 2. The phenolic compounds were quantified as ellagic acid equivalents (ellagic acid and ellagic acid hexoside), rutin equivalent (quercetin pentoside and dehydrofisetin glucoside), quercetin equivalent and gallic acid equivalent (gallic acid and protocatechuic aldehyde). The calibration curves were built, from LOQ-500 mg/L, at six concentration levels, plotting peak area versus analyte concentration. LOQ was calculated based on the formula described in the Section 3.2.

The MS detection system consisted of an Agilent Technologies 6420 triple quadrupole with ESI in positive ionization mode for the search and identification of signature peptides from tryptic hydrolysates. The ionization source conditions were set as follows: capillary voltage, 4.0 kV; source temperature, 100°C; desolvation gas (N₂), 350°C; and gas flow 10 L min^-1. The nebulizer pressure was set at 50 PSI to ensure sufficient nebulization for the chromatographic conditions. Collision gas was highly pure nitrogen. Quantitation was performed under MRM mode. For each MRM transition, a dwell time of 200 ms was chosen, and the pause between mass ranges was 5 ms. The Mass Hunter Workstation Quantitative Analysis software was applied to calculate the integration peak area of the MRM transitions of each analyte. The online BLAST search in UniProt (www.uniprot.org) and NCBI (www.ncbi.nhn.nih.gov) were used for evaluating the specificity of the signature peptide selected for bovine lactoferrin. All LC-MS measurements were performed in triplicate.

Amino acid contents were analyzed following the protocol described by Alaiz et al. (46 (link)). Briefly, 40 mg of fine-powder ground seeds were hydrolyzed with 4 mL of 6 N HCl using DL-2-aminobutyric acid as the internal standard. The solutions were sealed in tubes under nitrogen atmosphere and incubated in an oven at 110°C for 24 h. A mix of amino acids was used as standard. After digestion, samples were evaporated using a rotary evaporator, and then, the amino acids were resuspended in 25 mL of 1 M borate buffer. Derivatization of amino acids was performed by incubating for 50 min at 50°C, 300 μL of the sample with 6 μL of diethyl ethoxymethylenemalonate diluted to 3 mL in 1 M borate buffer. After samples filtration through a 0.22 μm cellulose filter, amino acids were analyzed by high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) at Interdepartmental Investigation Service at UAM (SIdI, UAM, Spain). There, the amino acid determination was carried out using HPLC-MS with an Agilent system detector composed of a 1,100 series HPLC coupled to a 6,420 Triple Quadrupole.

The phenolic compounds in the rocket extracts were analyzed using the method proposed by [21 (link)] with some modifications. Agilent 1290 Infinity series was used for performing HPLC analysis. The series was coupled with Triple Quadrupole 6420 purchased from Agilent Technology (Santa Clara, CA, USA). The operation was done in both negative and positive electrospray ionization (ESI) modes. The phenolic compounds were separated on a Synergi Polar–RP C18 analytical column (250 mm × 4.6 mm, 4 µm) from Phenomenex (Chesire, UK), using a mixture of water as solvent A and methanol as solvent B, both with formic acid 0.1%, in gradient elution mode. The following elution program was used for separation: isocratic condition from 0 to 1 min, followed by 20% mobile phase B from 1 to 25 min, 20–85% mobile phase B from 25 to 26 min, then an isocratic condition was used for 85% mobile phase B. Finally, from 26 to 32 min, from 85% to 20% mobile phase B was used. The injection volume of samples was 2 μL, and the flow rate was set at 0.2 mL/min. Dynamic-multiple reaction monitoring mode was used for detecting compounds, and the peak areas were integrated for quantification. The mass spectrometer parameters for the analyzed compounds are reported in Table 1.

HPLC-MS/MS studies were performed using an Agilent 1290 Infinity series and a Triple Quadrupole 6420 from Agilent Technology (Santa Clara, CA) equipped with an electrospray ionization (ESI) source operating in negative and positive ionization modes. All details are given in Supplementary Material.

Spermidine was detected by HPLC/MS according to methods described previously with slight modifications.^{49 (link)} All experiments were carried out on an Agilent 1290 Infinity Series and Triple Quadrupole 6420 from Agilent Technology (Santa Clara, CA, USA). The final data were normalized according to the tissue weight.