Targeted LC-MS/MS for Tryptophan Metabolites

An analytical protocol based on targeted LC-MS/MS was developed to measure tryptophan metabolites. The latter (with tryptophan) includes 5-hydroxytryptophan, serotonin, kynurenin, 3-hydroxy kynurenin, 3-hydroxy anthranilic acid, xantherunic acid, kynurenic acid, quinaldic acid, 8-hydroxy quinaldic acid, anthranilic acid, indol-3-acetic acid, tryptamine, indole-3-lactic acid, 5-hydroxy indole-3-acetic acid, quinolinic acid, picolinic acid, indole-3-carboxaldehyde, and indol-3-propionic acid. Serum or cecal content (50 µL) was mixed with 50 µL pure acetonitrile (for protein precipitation) containing deuterated compounds at 50 μ µM as an internal standard (CDN isotopes, Pointe-Claire, QC, Canada). The supernatant (50 µL) was then added to deionized water (600 µL). Ten microliters of this mixture were injected onto an UPLC-MS/MS system (Acquity TQ-XS Detector, Waters, Milford, MA) equipped with a C18-XB column (1.7 µm-100Å-150 × 2.1 mm from Phenomenex®, Torrance, USA). The ions of each analyzed compound were detected in positive ion mode using multiple reaction monitoring. The Masslinks software (Waters) was used for data acquisition and processing.