Determination of amino acids
Amino acids analysis was performed by liquid chromatography prior a pre-column derivatization with a commercial kit (AccQ Tag, Waters Corporation, Italy) according to manufacturer’s instructions and using a Nova-PakTMC18 silica based bonded column (Waters Corporation, Italy). Gradient elution with a flow rate of 1.0 mL min-1 used a mixture of: A = AccQ Tag Eluent; B = 100% acetonitrile (HPLC grade), C = HPLC grade I water (Waters application note for amino acids). Gradient summary: 100:0 A/B to 83:17 A/B from min 0 to 38. Then 60:40 B/C from min 38.01 to 55. The derivatized amino acids were detected by fluorescence (λex = 250 nm, λems = 395 nm; 2475 Multi λ Fluorescence Detector, Waters Corporation, Italy) and quantified by the external standard method using a pure amino acids standard mix.