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).