The analytical standards of 20 amino acids, including aspartic acid (Asp), glutamic acid (Glu), γ-am aminobutyric acid (GAMA), serine (Ser), glutamine (Gln), histidine (His), asparagine (Asn), glycine (Gly), threonine (Thr), arginine (Arg), alanine (Ala), tyrosine (Tyr), valine (Val), methionine (Met), tryptophan (Trp), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), lysine (Lys), and proline (Pro), were obtained from Sigma-Aldrich Co. (St. Louis, Mo, USA) with all purity ≥ 99%. Phenyl isothiocyanate (PITC) (purity ≥ 99%) and triethylamine (purity ≥ 99%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ammonium acetate acetonitrile (HPLC grade) and hexane (HPLC grade) were obtained from Dikma (Beijing, China). Ultra-pure water was prepared using a Milli-Q plus system (Millipore, Boston, Massachusetts, The United States of America).
The individual stock solutions were prepared in 0.1 mol/L HCl solution at the concentration of 10,000 μg/mL. The standard mixture was prepared from the individual stock standard solutions at the concentration of 100 μg/mL. The working solutions of the amino acid were prepared by serial dilutions. The internal standard norleucine was prepared with a concentration of 100 μg/mL. All the solutions were stored at 4 °C in the refrigerator.
The individual stock solutions were prepared in 0.1 mol/L HCl solution at the concentration of 10,000 μg/mL. The standard mixture was prepared from the individual stock standard solutions at the concentration of 100 μg/mL. The working solutions of the amino acid were prepared by serial dilutions. The internal standard norleucine was prepared with a concentration of 100 μg/mL. All the solutions were stored at 4 °C in the refrigerator.