Optimization and Validation of the Weber Color Test for the Presence of Psilocyn

In forensic laboratory settings, the analysis of hallucinogenic mushrooms has historically been a tedious process. The two prominent psychoactive compounds present in mushrooms, psilocyn and psilocybin, are difficult to distinguish due to their sensitivity and propensity to decompose. Thin-layer chromatography is commonly used to differentiate between the two compounds, although it is time-consuming and results can be affected by many external factors. The Weber Color Test was originally proposed by the Weber State University Laboratory of Criminalistics as a preliminary test that is used to differentiate between psilocyn and psilocybin in the analysis of hallucinogenic mushrooms. It requires two drops of the Weber reagent (a 0.1% solution of Fast Blue B in deionized water) and one drop of hydrochloric acid be applied to the sample, with the color change after each step being observed. This color test indicates the presence of psilocyn with a red to blue color change, while a negative result is produced for psilocybin. In this work, iterations of the original Weber Color Test were considered to develop the optimal procedure for performing the test efficiently while still producing clear and consistent results. Two different reagents (a 0.1% Fast Blue B solution and a 0.1% Fast Blue BB solution) were tested, as Fast Blue B is a known carcinogen. Four different sample preparations were also tested. The optimized procedure was then validated by considering the following requirements and acceptance criteria as set forth by the United Nations Office on Drugs and Crime: specificity (selectivity), precision (repeatability), precision (reproducibility), limit of detection, and robustness. The validation was performed using 21 hallucinogenic mushroom samples and 2 other plant material samples obtained from the Illinois State Police, as well as a variety of chemical standards including hallucinogens, cannabinoids, and theobromine. Optimizing the Weber Color Test would ensure that time and sample are used efficiently, while also considering the analyst’s safety. By then validating the Weber Color Test for use in forensic casework, the analysis of hallucinogenic mushrooms would become simpler and faster.