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Anal Chem. 2019 Apr 16;91(8):5207-5216. doi: 10.1021/acs.analchem.8b05884. Epub 2019 Mar 25.

Ultrahigh-Performance Liquid Chromatography Tandem Mass Spectrometry with Electrospray Ionization Quantification of Tryptophan Metabolites and Markers of Gut Health in Serum and Plasma-Application to Clinical and Epidemiology Cohorts.

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UK Dementia Research Institute, Burlington Danes Building , Imperial College London , Hammersmith Hospital, London W12 0NN , United Kingdom.
MRC-NIHR National Phenome Centre, IRDB Building , Imperial College London , Hammersmith Hospital, London W12 0NN , United Kingdom.
Division of Integrative Systems and Digestive Medicine, Department of Surgery and Cancer , Imperial College London , Sir Alexander Fleming Building, South Kensington Campus , London SW7 2AZ , United Kingdom.
Waters Corporation , Milford , Massachusetts 01757 , United States.
St. Marks Hospital and Academic Institute , Watford Road , Middlesex, London HA1 3UJ , United Kingdom.
Australian National Phenome Centre , Murdoch University , Harry Perkins Building , Perth , Western Australia 6150 , Australia.


A targeted ultrahigh-performance liquid chromatography tandem mass spectrometry with electrospray ionization (UHPLC-ESI-MS/MS) method has been developed for the quantification of tryptophan and its downstream metabolites from the kynurenine and serotonin pathways. The assay coverage also includes markers of gut health and inflammation, including citrulline and neopterin. The method was designed in 96-well plate format for application in multiday, multiplate clinical and epidemiology population studies. A chromatographic cycle time of 7 min enables the analysis of two 96-well plates in 24 h. To protect chromatographic column lifespan, samples underwent a two-step extraction, using solvent protein precipitation followed by delipidation via solid-phase extraction (SPE). Analytical validation reported accuracy of each analyte <20% for the lowest limit of quantification and <15% for all other quality control (QC) levels. The analytical precision for each analyte was 2.1-12.9%. To test the applicability of the method to multiplate and multiday preparations, a serum pool underwent periodic repeat analysis during a run consisting of 18 plates. The % CV (coefficient of variation) values obtained for each analyte were <15%. Additional biological testing applied the assay to samples collected from healthy control participants and two groups diagnosed with inflammatory bowel disease (IBD) (one group treated with the anti-inflammatory 5-aminosalicylic acid (5-ASA) and one group untreated), with results showing significant differences in the concentrations of picolinic acid, kynurenine, and xanthurenic acid. The short analysis time and 96-well plate format of the assay makes it suitable for high-throughput targeted UHPLC-ESI-MS/MS metabolomic analysis in large-scale clinical and epidemiological population studies.

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