1260 hplc

Amino acid analysis was carried out using a modified version of the method previously described by Özcan et al. [41 (link)]. A total of 8 mL of 6 mol/L HCl was added to 100 mg of dried and ground sample, and the mixture was hydrolyzed for 24 h at 110 °C. After hydrolysis, the volume was adjusted to 10 mL using Milli-Q water, and an aliquot of 2 μL was injected into the LC-MS system: an Agilent 1260 HPLC with a Phenomenex column (C18 (2) 250 μm × 4.6 μm × 3 μm), coupled to an Agilent 6120 Quadrupole in the SIM-positive mode (Agilent Inc., Santa Clara, CA, USA). The composition of mobile phase A was 3% MeOH, 0.2% formic acid and 0.01% acetic acid (HAc), and mobile phase B was 50% MeOH with 0.2% formic acid and 0.1% HAc. The initial gradient, held for the first 8 min, contained 94% A and 6% B, which was gradually changed until it reached 80% A and 20% B after 20 min. This gradient was held for a run time of 27 min, then gradually altered until it reached 94% A and 6% B at a run time of 28 min, and then held again for a total run time of 40 min. Twenty-four amino acids diluted in 0.2 mol/L HAc in the concentration range of 1–20 mg/L were used to derive the standard curve. Due to the use of acidic hydrolysis, tryptophan could not be quantified. LOD of the method is 0.025 µmol/mL.