Application of the optimized and validated LC-MS method for simultaneous quantification of tryptophan metabolites in culture medium from cancer cells.

Kynurenine pathway is the main route of tryptophan degradation generating a number of immunoregulatory compounds. Some conditions like oxidative stress, inflammatory factors might enhance tryptophan degradation. Process is active in several cells including fibroblasts, cancer cells, and immune cells, therefore it is intensively studied in context of cancer microenvironment. The validated and standardized methodology for kynurenine quantification is crucial for reliable comparison of results obtained in different studies. This paper concerns an approach for simultaneous quantification of four major tryptophan metabolites of the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in cell culture supernatants by liquid chromatography coupled with single quadrupole mass spectrometer. During development of the novel method, the principal component analysis was used to select the best mobile phase and to ensure the optimal conditions for simultaneous quantification of metabolites. The analysis involves simple protein precipitation with acidified methanol and 3-nitrotyrosine as an internal standard. The obtained limits of detection and quantification in cell culture medium were in the range of 3.31-10.80 nmol/L and 9.60-19.50 nmol/L, respectively. At the validation step, other method parameters (linearity, precision, accuracy, recovery, matrix effects) were also evaluated and satisfactory results were obtained for all target compounds. The method was applied to study tryptophan metabolites by determination of kynurenines in cell culture medium from two different human cancer cell lines (MDA-MD-231 and SK-OV-3) in context of exposure to glycation products.