3.4. Chemical Composition

For chemical analyses, seeds were milled to wholemeal flour (WMF) by a Hammertec mill (mesh size: 0.8 mm) (Foss, Hilleroed, Denmark).

Moisture (g/100 g) and ash (g/100 g dm) content were determined according to ICC methods no. 110 and 104/1, respectively. Total starch content (g/100 g dm) was analyzed as described by Englyst et al. [46]. The nitrogen content was determined using aVarioMax C/N (Elementar Analysesystemen, Langenselbold, Germany) and converted to protein content (g/100 g dm) using a conversion factor of 6.25 [47]. Fat content (g/100 g dm) was determined by Soxhlet extraction with prior acid hydrolysis (ISO 6491). All macronutrient analyses were performed in triplicate.

To determine the total standard amino acid composition (excluding tryptophan), acid hydrolysis was performed on the lyophilized samples using a hydrogen chloride and dithioglycolic acid solution for 4 h at 150 °C. Sample purification was done on an OASIS HLB cartridge (200 mg, 6 cc), followed by separation and detection by an LC–MS2 instrument (Nexera 8040, Shimadzu, Kyoto, Japan). Separation was done in normal phase mode on an Intrada amino acid column (3.0 μm, 100 × 3.0 mm) with acetonitrile (0.3% acetic acid) as solvent A and 20% acetonitrile-80% 100 mM ammonium formate as solvent B for the mobile phase gradient. The solvent gradient started at 80% solvent A and 20% solvent B. After 4 min, solvent B was gradually increased to 100% until the 14th min, at which it was held for 2 min before being returned to the initial gradient for 9 min. Amino acids were ionized by electrospray ionization (4.5 kV) followed by multiple reaction monitoring (MRM) on a triple quadrupole. All amino acids were measured in positive mode, except for aspartic acid, which was measured in negative mode. Methylvaline, homoarginine, and methyl-D3-methionine were used as internal standards.

To determine the tryptophan, alkaline hydrolysis was performed on lyophilized samples using a lithium hydroxide solution for 4 h at 150 °C. Sample purification was done on an OASIS HLB cartridge (200 mg, 6 cc), followed by separation and detection by an LC-MS2 instrument (Nexera 8040, Shimadzu Corporation, Kyoto, Japan). Separation was done in normal phase mode on an Intrada amino acid column (3.0 μm, 100 × 3.0 mm) with acetonitrile (0.3% acetic acid) as solvent A and 20% acetonitrile-80% 100 mM ammonium formate as solvent B for the mobile phase gradient. The solvent gradient started at 80% solvent A and 20% solvent B. After 4 min, solvent B was gradually increased to 100% until the 14th min, at which it was held for 2 min before being returned to the initial gradient for 9 min. Amino acids were ionized by electrospray ionization (4.5 kV), followed by MRM on a triple quadrupole. Tryptophan was measured in positive mode with methyl-tryptophan as the internal standard.

The fatty acid profile of the quinoa samples was determined as described by Foubert et al. [48]. Quinoa WMF was mixed with diethyl ether to extract the fat. The mixture was subsequently filtered over sodium sulfate, and diethyl ether was evaporated to retain the extracted fat. Four droplets of extracted quinoa fat were dissolved in 9 mL hexane and 1 mL 2 N potassium hydroxide/methanol reagent to produce fatty acid methyl esters (FAMEs). The blend was shaken for 30 s and allowed to settle. The FAMEs in the hexane layer were separated by a Varian 3380 gas chromatograph (Varian Inc., Palo Alto, CA, USA) equipped with a WCOT CP-sil 88 column and a flame ionization detector (FID). The conditions for the gas-chromatographic analysis were: temperature of injector: 250 °C, temperature of detector: 250 °C, flow rate of mobile phase (helium): 1 mL min⁻¹, flow rate of hydrogen: 40 mL min⁻¹, flow rate of air: 120 mL min⁻¹, injection volume: 1 mL, and column oven temperature: 120 °C for 2 min, followed by heating at 5 °C min⁻¹ to 200 °C and holding at that temperature for 20 min.

Analysis of the TAG species was performed as described by Rombaut et al. [49]. Quinoa WMF was mixed with diethyl ether to extract the fat. The mixture was subsequently filtered over sodium sulfate, and diethyl ether was evaporated to retain the extracted fat. The extracted fat was dissolved in a concentration of 5 mg/mL in dichloromethane/acetonitrile (30:70 v/v). Separation of the TAG species was performed on a Thermo Finnigan Surveyor RP-HPLC system (Thermo Electron Corporation, Brussels, Belgium) using a 150 × 3.0 mm Alltima HP C18 HL column with a 3 μm particle diameter (Grace Alltech, Lokeren, Belgium) and connected to an Alltech ELSD 2000 evaporative laser light scattering detector (Grace Alltech, Lokeren, Belgium). The injection volume was 25 µL.

The mineral composition was determined via inductive coupled plasma-optical emission spectrometry (ICP-OES, IRIS Intrepid II XSP, Thermo Scientific, Waltham, MA, USA). Prior to the analysis, 1.0 g of WMF was ashed at 500 °C for 4 h in a muffle furnace. The ash was dissolved in hydrochloric acid during a 2 h reflux. The remaining residue was filtered (Whatman filter no. 5) before analysis.