Development and validation of an LC-MS method based on derivatization for quantification of kynurenine pathway...

Simultaneous determination of a broad panel of kynurenine pathway metabolites remains analytically challenging due to their diverse polarity, poor chromatographic retention, and wide concentration range in biological matrices. In this work, a liquid chromatography-mass spectrometry method, which includes a derivatization step to form ester derivatives of tryptophan metabolites, was developed to measure serum levels of kynurenine, kynurenic acid, 3-hydroxykynurenine, picolinic acid, nicotinic acid, quinolinic acid, and xanthurenic acid. Sample preparation procedure was thoroughly optimized. As part of method development, different strategies for sample purification were tested. Ultimately, serum samples after derivatization were subjected to clean-up using solid-phase extraction. This approach enables the separation of isomeric pairs (picolinic and nicotinic acids) and improved the detection of quinolinic acid. Data were normalized to a single internal standard (3-nitro-l-tyrosine), which undergoes derivatization in the same manner as the target analytes. The method demonstrated excellent linearity (R² > 0.99) over wide concentration ranges, typically from low nanomolar to several tens of micromolar levels. Low limits of quantification were determined in a surrogate matrix: 0.35 µM for picolinic acid, 0.11 µM for 3-hydroxykynurenine, and <80 nM for the other metabolites.Inter- and intraday accuracy ranged between 90-110%, with recoveries >70% for all analytes. The method was validated and successfully applied to the quantification of kynurenine pathway metabolites in serum from healthy donors and patients with gastric or pancreatic cancer.