UHPLC for Kynurenine Pathway Metabolites

Serum KYNA, KYN and TRP levels were determined by the ultra-high pressure liquid chromatography (UHPLC) method (Waters Acquity UHPLC system; Waters C18 analytical column), as described before [40 (link)]. Briefly, the mobile phase containing 20 mM sodium acetate, 3 mM zinc acetate and 7% acetonitrile was run with the flow rate of 0.1 mL/min. Quantification of TRP and its metabolites was performed by a UV variable wavelength detector (KYN at 365 nm; TRP at 250 nm) and by a fluorescence detector (KYNA-344 nm excitation and 398 nm emission). 3-OH-KYN levels were determined fluorometrically with an electrochemical detector (potential of working electrode: + 0.20 V; Coulochem III, ESA), as described before [28 (link)]. The HPLC column (HR-80; 3µm; C18 reverse-phase column; ESA) was perfused at 0.6 mL/min using a mobile phase consisting of 2% acetonitrile, 0.9% triethylamine, 0.59% phosphoric acid, 0.27 mM sodium EDTA and 8.9 mM heptane sulfonic acid.