Optimal Conditions for the Determination of Tryptophan and Its Metabolites Using Various Chromatographic Techniques: A New Perspective

Background. Tryptophan (Trp) is an essential amino acid whose metabolism is distributed among the kynurenine, serotonin and microbiota-derived indole pathways. Their products differ markedly in polarity, stability, abundance and ionization, which makes simultaneous chromatographic determination difficult. Aim. The aim of this review is to critically compare chromatographic strategies for the determination of Trp and its metabolites and to formulate an updated analytical framework for method selection and validation. Material and methods. PubMed-indexed literature was reviewed with emphasis on recent analytical papers, systematic reviews and studies describing HPLC, UHPLC, LC-MS, LC-MS/MS and high-resolution MS workflows. Polish contributions were specifically considered, including work from Medical University of Lublin groups. Results. The literature demonstrates a transition from conventional HPLC-UV/fluorescence toward targeted LC-MS/MS and high-resolution metabolomics. Recent methods improve multiplexing, sensitivity and matrix tolerance, but methodological heterogeneity remains substantial. The newest direction is chromatographic optimization based on chemical diversity rather than the assumption that a single C18 phase is universally optimal. Conclusions. An optimal method should be selected according to the intended analyte panel, biological matrix and quantitative purpose. Biphenyl, mixed-mode or carefully optimized reversed-phase systems, isotope-labelled internal standards, matrix-matched calibration and explicit validation of recovery, matrix effects and stability provide a strong contemporary framework.