Quantifying Free Amino Acids in Fish Tissues

FAA analysis of fish tissues was performed according to Kwasek et al. [27 (link)]. Muscle samples of three fish from each tank were combined and homogenized together with 0.1 mol/L HCl in 1:9 (w/v) and spun at 12,000× g (4 °C, 15 min). Supernatants were collected, filtered (Milipore, 10 kDa cutoff at 15,000× g, 4 °C, 30 min), and later diluted with 0.1 mol/L HCl (1:19 v/v) containing norvaline and sarcosine (40 μmol/L) as internal standards. Blanks (0.1 mol/L HCl + 40 μmol/L norvaline and sarcosine) and external standards (Sigma acid/neutral and basic AA) were prepared along with the sample preparation. The same concentration of glutamine in 0.1 mol/L HCl as an external standard was prepared and added to the basic AA standard. Free amino acids were quantified using Shimadzu Prominence Nexera—i LC-2040C Plus (Shimadzu, Japan) according to the Shimadzu protocol No. L529 with modifications. Free amino acid concentrations (expressed as μmol/kg wet body weight) were calculated in LabSolutions software version 5.92 (Shimadzu, Japan) using internal and external standards.