Rapid and robust bioanalytical assay for the quantification of circulating tryptophan-derived indole-3 metabolites in human plasma

Abstract

Tryptophan-derived indole-3 metabolites are increasingly recognized for their role as central mediators in health and disease by regulating systemic metabolism and immunity. Hence, there is a growing interest in methods that enable robust and accurate quantification of this family of metabolites. Therefore, we developed a new bioanalytical assay, using FDA and EMA guidelines, to quantify these metabolites in human plasma using stable isotope labeled internal standards and liquid chromatography coupled to a triple quadrupole mass spectrometric detector. After protein precipitation with acetonitrile and separation on a short reversed-phase column in gradient mode, compounds were quantified in positive selected reaction monitoring mode of an electrospray source. Responses were linear for indole-3-propionic acid (5–2000 ng/ml) and DL-indole-3-lactic acid (10–4000 ng/ml), whereas a quadratic least-squares regression model was used for indole-3-acetic acid (10–4000 ng/ml) and indole-3-carboxaldehyde (2.5–1000 ng/ml). Precisions in human plasma, within- and between-run, were within 2.4–12.1%, accuracies ranged from 89.1 to 107.7%. Indoles were all stable under all investigated conditions, including repeated freeze-thaw cycles. Significant extraction losses or matrix effects were not observed. Overall, the method provides a relatively fast and simple bioanalytical assay suitable for quantification of indoles in clinical setting.

The authors' abstract, as published at the source. Journal of Chromatography B, 2026 · DOI ↗