A Novel Selective and Sensitive HPLC-ESI-Tandem MS/MS Method for Indole Structure-Retaining Metabolites of Tryptophan: Application in Beverages

The essential amino acid tryptophan yields a plethora of secondary metabolites with key roles in plants and animals. Its fate in different living organisms is crucial for their own health, and metabolic profiling is a valuable tool for investigating it. Among the various metabolites, those retaining the indole structure were examined for qualitative and quantitative profiling. Liquid chromatography coupled with a tandem mass spectrometry detector with an electrospray ionization source (HPLC-ESI-MS/MS), acquiring in multiple reaction monitoring (MRM) mode, was used to develop a selective and sensitive method for the simultaneous analysis of tryptophan and 10 indole structure-retaining metabolites of it. Satisfactory values were obtained for linearity (R2 ≥ 0.99 for all compounds except two), sensitivity (LOD, within 6–31 ng/mL, and LOQ, within 17–94 ng/mL, where minimum and maximum values were relative to serotonin and 5-methoxytryptamin, respectively), reproducibility (interday and intraday precision and accuracy), and effect of the matrix (recovery and matrix effect). The method was then successfully applied to the analysis of different types of beverage, such as herbal products, like Eschscholzia californica and a sleep herbal tea marketed with added melatonin (consumed to reduce anxiety and improve sleep quality), and fermented beverages, like beer and kefir. High amounts of tryptophan (from 77 ng/mL in kefir to 26,974 ng/g in the sleep herbal tea) followed by lower contents of serotonin (from 29 ng/mL in kefir to 2207 ng/g in the sleep herbal tea), were found in all samples along with the serotonin pathway-related compounds 5-hydroxytryptophan and tryptamine. Melatonin was detected in the plant matrix Eschscholzia c. for the first time to our knowledge (446 ng/g) and in the fermented beverages (96 ng/mL in beer and 39 ng/mL in kefir), regardless of their vegetable or animal origin, along with the melatonin route metabolites 5-methoxytryptamine and tryptophan ethyl ester. The amount of melatonin in the sleep herbal tea (556,464 ng/g) was in strong agreement with the declared content. Suggested applications include the search for biomarkers in phytochemical characterization, mechanistic studies of tryptophan’s chemistry, valorization of foods, beverages, and tryptophan-rich agro-food by-products and waste for nutraceutical and pharmacological purposes.