| Psilocin and psilocybin are the main active components in hallucinogenic mushrooms. However, their chromatographic quantification has been problematic due in part to the poor chromatographic separation of these compounds, co-elution of interfering compounds, and incomplete extraction of psilocin and psilocybin from mushroom samples. Therefore, a study was undertaken to optimize (1) the extraction of these compounds from mushrooms and (2) the quantification of psilocin and psilocybin by reversed phase high performance liquid chromatography (RP-HPLC) with diode array or mass spectrometry (MS) detection. The effects of the mobile phase on the presence of interfering compounds, the relative separation, and peak shape were examined for psilocybin, psilocin, bufotenine and tryptamine (internal standard). Factors examined included, buffer concentration, buffer type, buffer pH and organic phase type. These parameters all affected chromatographic separations, with the largest effect seen when the buffer type or pH were changed. A short and effective extraction procedure was also developed to quantify these analytes by RP-HPLC with diode array detection and to qualitatively identify them by RP-HPLC with mass spectrometry detection. The estimated detection limit of this method, with a signal-to-noise ratio of three, was 10 ng/mL for diode array detection and 0.1 ng/mL for MS detection of psilocin and psilocybin in methanolic mushroom extracts. Psilocin and psilocybin were stable for a minimum of one year when stored in an acidic methanolic solution that was sparged with high purity nitrogen gas, sealed in an amber vial and stored at -11°C.;Keywords. Forensic Science, psilocybin, psilocin, reverse phase high performance liquid chromatography, liquid chromatography mass spectrometry, hallucinogenic mushrooms, HPLC... |
