2.5. Free amino acid assay
The free amino acid content of the samples were determined as
described by Wang et al. (2010). Different dried samples were
weighed, ground finely, and extracted with 10 mL of distilled water
at 90 C for 20 min. The extract was centrifuged at 4500g and the
supernatant was made up to 10 mL with distilled water and filtered
through a 0.45-lm nylon filter membrane. The SPE-PAKC18
cartridges (Waters Co., Ltd. Milford, MA) were conditioned
with 30 mL of methanol and 10 mL of distilled water. Sample infusion
(1 mL) was loaded onto the conditioned cartridges, and the
retained compounds were eluted with 5.0 mL of 10% ethanol. The
resulted elute was collected, rotary evaporated to dryness and
redissolved in 5 mL distilled water. It was then filtered through a
0.45-lm nylon filter membrane before its pre-column derivatisation
with o-phthalaldehyde (OPA).