Stable isotope dilution ultra-high performance liquid chromatography–tandem mass spectrometry quantitative profiling of tryptophan-related neuroactive substances in human serum and cerebrospinal fluid

•Profiling of L-tryptophan (L-TRP) and its related compounds in human bodily fluids.•Simultaneous determination of endogenous L-TRP and its 16 metabolites by LC–MS/MS.•Isotope dilution mass spectrometry—17 isotope labelled internal standards used.

Many compounds related to L-tryptophan (L-TRP) have interesting biological or pharmacological activity, and their abnormal neurotransmission seems to be linked to a wide range of neurodegenerative and psychiatric diseases. A high-throughput method based on ultra-high performance liquid chromatography connected to electrospray tandem mass spectrometry (UHPLC–ESI-MS/MS) was developed for the quantitative analysis of L-TRP and 16 of its metabolites in human serum and cerebrospinal fluid (CSF), representing both major and minor routes of L-TRP catabolism. The combination of a fast LC gradient with selective tandem mass spectrometry enabled accurate analysis of almost 100 samples in 24 h. The standard isotope dilution method was used for quantitative determination. The method’s lower limits of quantification for serum and cerebrospinal fluid ranged from 0.05 to 15 nmol/L and 0.3 to 45 nmol/L, respectively. Analytical recoveries ranged from 10.4 to 218.1% for serum and 22.1 to 370.0% for CSF. The method’s accuracy ranged from 82.4 to 128.5% for serum matrix and 90.7 to 127.7% for CSF matrix. All intra- and inter-day coefficients of variation were below 15%. These results demonstrate that the new method is capable of quantifying endogenous serum and CSF levels of a heterogeneous group of compounds spanning a wide range of concentrations. The method was used to determine the physiological levels of target analytes in serum and CSF samples from 18 individuals, demonstrating its reliability and potential usefulness in large-scale epidemiological studies.