Session: Session 3

Quantification of tryptophan metabolites in complex biological matrices

Tryptophan is an amino acid known to improve sleep and help reduce depression. This essential amino acid is metabolized in the gut and in the brain into kynurenine and then into kynurenic acid or quinolinic acid. While the first metabolite promotes neuroprotection, the second one exerts neurotoxic effects. Thus, their monitoring provides valuable information even though their identification / quantification in complex matrices is still challenging, especially at low concentration.

In this work, tryptophan metabolism was studied in the brain and plasma of anorexic mice. We developed an MRM-based method to detect its metabolites at low concentrations. First, a heavy internal standard was added to samples; then these metabolites were enriched thanks to a protein precipitation step. Thereafter, the Optimizer software was used to determine parameters for MRM analysis, especially the definition of transitions and associated collision energies. These developments allowed to achieve quantification ranges of 12 fmol/µL to 50 pmol/µL in plasma and of 6 fmol/mg to 0.8 nmol/mg in mouse cerebral tissue (hypothalamus, hippocampus, amygdala). Despite the complexity of the matrix, we managed to reach very low limits of quantification through the use of a triple quadrupole coupled to an UHPLC system equipped with a 15 cm Polaris 3 column.