Simultaneous Analysis of Tryptophan and Its Metabolites in Human Plasma Using Liquid Chromatography–Electrospray Ionization Tandem Mass Spectrometry

Abstract

A simplified method for analyzing tryptophan (Trp) and its metabolites in human plasma was developed using liquid chromatography–electrospray ionization tandem mass spectrometry. Trp and its metabolites have various chemical properties but have no common functional group for derivatization. Using a reversed-phase pentafluorophenyl (PFP) column for liquid chromatography separation, Trp and its 15 metabolites (3-hydroxyanthranilic acid, 3-hydroxy-kynurenine, 3-indoleacetic acid, 5-hydroxyindole-3-acetic acid, 5-hydroxy-L-tryptophane, anthranilic acid, indole-3-lactic acid, kynurenine, kynurenic acid, melatonin, nicotinic acid, picolinic acid, quinolinic acid, serotonin, and xanthurenic acid were successfully separated within 15 min without derivatization. Neopterin which is known as a biomarker for inflammation and is often evaluated with Trp metabolites in several reports could also be simultaneously analyzed. With this method, Trp and its metabolites were detected with good sensitivity and selectivity without derivatization and solid-phase extraction. The method was validated in this study, showing that the relative standard deviation of 14 Trp metabolites was <15%, and accuracy was within 100% ± 20%, with the exception of nicotinic acid. The quantification range was optimized to 0.0150–100 µM covering the concentration of Trp metabolites in human plasma. Overall, 12 Trp metabolites in healthy human plasma were quantified with good precision and accuracy.