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.