High performance liquid chromatography (HPLC)
Quantification of metabolites tryptophan (TRP), KYN, KYNA, and PLP was
performed by HPLC-fluorescence detection.
Sample preparation : Within 5 min of resection from the brain, the
tissue samples were kept in 1.0 ml of 0.1 M perchloric acid and stored
at -80°C. The frozen tissues were thawed on ice, weighed and placed in
2ml cryo-tubes. The samples were homogenised in ten volume of
methanol/water (85:15, v/v), centrifuged for 15 min at 10,000 rpm at
0°C. The supernatant was removed and stored at -80°C until the time of
chromatographic analysis.
For HPLC-fluorescence detection, reverse phase isocratic HPLC was
performed (Shimadzu Prominence). Separation of the metabolites was
achieved with a C18 column (Phenomenex Luna; 250 mm length, 4.6 mm
internal diameter, 100 Å pose size and 5 µm particle size). The column
temperature was maintained at 37ºC. The mobile phase, pumped at a flow
rate of 1ml/min, was consisted of 50 mM acetic acid, 100 mM zinc acetate
containing 3% acetonitrile (pH 4.7). Ten microliter sample volume was
injected into the system through an autosampler. The detection system
was a fluorescence detector (RF 20A, Shimadzu, Japan) with dual
wavelength simultaneous monitoring capability. Excitation, emission
wavelength spectra and retention time for TRP, KYN, KYNA, and PLP were
297nm/348nm, 365nm/480nm, 344nm/404nm and 300nm/400nm (Xiao et al.,
2008; Cabo et al., 2014).