Separation of amino acids was achieved by gas chromatography,
carried out in a PerkinElmer Clarus 400
instrument (PerkinElmer, Massachusetts, USA) equipped
with a flame ionisation detector (FID). The injector was
kept at 250 C and the detector at 260 C. Hydrogen was
used as carrier gas. A DB-1 (30 m, 0.25 mm i.d. and
0.15 lm thickness) fused-silica capillary column (J & W
Scientific) was used with the following temperature programme:
1 min hold at 70 C, increase to 170 C at 2.0 C/
min and then to 250 C (5 min hold) at 16 C min-1. The
compounds were identified by their retention times and
chromatographic comparison with authentic standards.
Quantification was based on the internal standard method
using L-norleucine, and the calibration curves were built for
18 amino acids. For asparagine (Asn) and aspartic acid
(Asp), as well as for glutamine (Gln) and glutamic acid
(Glu), the methodology does not allow the distinction
between the amide and carboxylic acid functions. As such,
those amino acids were quantified together as Asx and Glx,
respectively. Also, the methodology used did not allow the
detection of His. The limit of quantification of analysed
amino acids was determined to be ten times the value of the
residual signal peaks.