The TRP metabolites were measured by a previously described HPLC method [22 (link)]. Briefly, analytes were extracted from samples and calibrators/controls using Waters Oasis MCX extraction cartridges (Waters, Milford, MS). The eluent was then evaporated to dryness and reconstituted with 0.1 M PBS for injection into the HPLC system. Analyses were carried out on a Waters 2695 chromatograph with a 250 mm × 4 mm Supersphere 60 RP-select B, C8 column (Merck, Darmstadt, Germany) connected to a Waters 2487 dual-λ UV detector and a 2475 fluorescence detector. TRP (λex: 300 nm; λem: 350 nm) and 5-HIAA (λex: 300 nm; λem: 340 nm) were measured by fluorescence detection; KYN (365 nm), KYNA (330 nm), and 3-HK (365 nm) were measured by UV detection. Data were processed using EMPOWER software (Waters). The concentrations were established through comparison of peak heights of the single analytes with the peak heights of the respective calibration curves, always including the internal standard into the calculation. The method has been validated showing good absolute recovery and precisions (e.g. for 3-HK: recovery of 85,8%, intra-day precision of 3.9%, and inter-day precision of 7.5%).
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