Triethylamine

Serum leucine levels were measured using HPLC (Perkin Elmer, USA). To prepare the sample solution, 200 μL of amino acid standards and serum samples were added into individual 1.5-mL microcentrifuge tubes. Fifty μL of norleucine (1 g/L diluted in 10 mol/L; Sangon Biotech, China) was added to each tube as internal standard. Then, 100 μL of acetonitrile triethylamine solution (triethylamine: acetonitrile (v/v)=500: 8.6, Sangon Biotech, China) and 100 μL of phenyl isothiocyanate acetonitrile solution (isothiocyanate: acetonitrile (v/v)=250: 1, Sangon Biotech, China) were added to each tube, mixed well, and kept aside for 1 h at room temperature. Following that, 400 μL of hexane was added into each tube, mixed well, and kept aside for 10 min at room temperature to form 2 phases. The lower phase, which contained amino acids, was filtered and transferred into a fresh tube using a 0.45-μm syringe filter. Then, 200 μL of phenylthiocarbamoyl amino acid was diluted in 800 μL of sterile water as sample solution. A Venusil AA chromatographic column was used (4.6×250 mm and 5 μm; Agela Technologies, China), and column temperature was kept at 40°C. Ten μL of each sample solution was used for the measurement of amino acid concentration, and the wavelength was 254 nm.