Tryptophan-derived metabolites, a group of neurotransmitters essential for various brain functions, play key roles in regulating mood, movement, sleep, and cognition. However, the comprehensive characterisation of tryptophan-melatonin pathway metabolites is challenging due to factors such as their structural diversity, chemical complexity, low concentrations, and instability of these metabolites. In this study, we developed and validated an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) methodology with electrospray ionisation for the simultaneous separation and quantification of tryptophan metabolites in human plasma. The analytical calibration ranges in plasma were 0.50-200 ng/mL for serotonin, 0.01-5 ng/mL for N-acetylserotonin, 0.01-20 ng/mL for tryptamine, 0.01-20 ng/mL for 6-sulfatoxymelatonin, 0.01-20 ng/mL for 6-hydroxymelatonin, 0.01-100 ng/mL for melatonin, and 0.10-20 ng/mL for N-acetyltryptamine, with correlation coefficients ranging from 0.954 for N-acetyltryptamine to 0.997 for tryptamine. The intraday and interday precision remained consistently below 15 % for all analytes. Most analytes met the accuracy criteria, except for N-acetyltryptamine at the lowest quality control level (0.2 ng/mL), where the intraday and interday accuracy were 22.4 % and 17.4 %, respectively. In conclusion, this novel method allows for rapid identification of tryptophan-melatonin pathway intermediates in less than ten minutes, including seven distinct melatonin-related analytes. This suggests that it may find use in everyday clinical and scientific endeavours.

Department of Chemistry BMC Analytical Chemistry and Neurochemistry Uppsala University SE 75124 Uppsala Sweden

Laboratory of Growth Regulators Faculty of Science Palacký University and Institute of Experimental Botany of the Czech Academy of Sciences Šlechtitelů 27 783 71 Olomouc Czech Republic

Laboratory of Growth Regulators Faculty of Science Palacký University and Institute of Experimental Botany of the Czech Academy of Sciences Šlechtitelů 27 783 71 Olomouc Czech Republic; Department of Neurology Faculty of Medicine and Dentistry Palacký University 779 00 Olomouc Czech Republic

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