Simultaneous determination of serotonin and its precursor tryptophan in small volumes of human serum using HPLC-ECD

Serotonin (5-HT) is not only a key neurotransmitter in the brain but also plays an important role in peripheral tissues e.g. in the gastrointestinal (GI) tract. The multitude of processes in which 5-HT is involved explains why recent studies have linked altered 5-HT levels to Long Covid (LC) and fatigue. LC is a post-viral syndrome which can occur after a SARS-CoV-2 infection with various symptoms, amongst others including fatigue. We present an optimized bioanalytical HPLC method with electrochemical detection (ECD) for the simultaneous detection of 5-HT and its precursor tryptophan (W) in small volumes of human serum. We validated the method with a calibration curve better representing concentrations expected in human serum; with the possibility to concurrently measure W and low 5-HT concentrations. Sample comparability and standardization were improved through an adapted sample preparation, reducing transfer errors and increasing recovery of the internal standard (IS). The simple preparation involved only two steps: protein precipitation and sample dilution. After a brief series of validation measurements of the optimized method, it was applied to human serum from an observational study cohort. • Easy-to-use method for detecting 5-HT and W in human serum without the need for expensive buffer ingredients or pre-filter units. • Enables measurement of triplicates with less than 100 µL serum.