Amino Acid Analysis of Food Samples

Sample preparations for analysis of total amino acids were performed as described by Mæhre et al. [11 ]. For the raw material samples, approximately 200 mg of fish and shrimp samples and approximately 50 mg of flour and dulse samples, were dissolved in 0.7 mL distilled H₂O and 0.5 mL 20 mM norleucine (internal standard). For the protein extract samples, 500 µL extract was mixed with 50 µL 20 mM norleucine. Subsequently, for all samples, concentrated hydrochloric acid (HCl, 12 M) was added, to a final concentration of 6 M. The sample mixtures were flushed with nitrogen gas for 15 s in order to minimize oxidation, before hydrolysis at 110 °C for 24 h according to Moore and Stein [12 ]. Following hydrolysis, 100 µL aliquots of the hydrolysates were evaporated under nitrogen gas until complete dryness and re-dissolved to a suitable concentration in lithium citrate buffer at pH 2.2. All amino acids were analyzed chromatographically using an ion exchange column followed by ninhydrin post column derivatization on a Biochrom 30 amino acid analyzer (Biochrom Co., Cambridge, UK). Amino acid residues were identified using the A9906 physiological amino acids standard (Sigma Chemical Co., St. Louis, MO, USA) as described previously [13 (link)]. Protein content was calculated as the sum of individual amino acid residues (the molecular weight of each amino acid after subtraction of the molecular weight of H₂O).