This study presents the development and validation of a high-performance liquid chromatography method with diode array detection (HPLC-DAD) for determining the content of tryptamines in methanolic extracts of dried fruiting bodies of mushrooms belonging to species of the Psilocybe genus. The objective is to initiate the assessment of tryptamine content in fungi marketed as Psilocybes in Colombia, using a standard chromatographic method that can be replicated. The separation was conducted in reverse phase using a Synergi 4 μm Hydro-RP C18 column (150 × 4.6 mm), eluted in a gradient mode with water-trifluoroacetic acid (100:0.1 v/v) and acetonitrile-trifluoroacetic acid (100:0.1 v/v) as the mobile phase. The gradient started at 5 % B at 0 min, reaching 30 % B at 15 min, with a flow rate of 0.20-0.22 mL/min, a column temperature of 35 ± 2 °C, and an injection volume of 10 μL. After establishing chromatographic conditions, the method was validated in terms of selectivity, linearity, system suitability, precision, robustness, and limits of detection and quantification, following the guidelines recommended by the International Conference on Harmonization (ICH) and the U.S. Food and Drug Administration (FDA). Once the mentioned parameters were evaluated, it was concluded that the method is suitable for the analysis of tryptamines in mushrooms. These conditions enabled the comparison of tryptamine content in 19 fungal samples from the Antioquia region (near Medellín city, Colombia), revealing a total content ranging from 0.01 % to 0.73 % of tryptamines in psilocin equivalents per fungal biomass.
