A method for clinical potency determination of psilocybin and psilocin in hallucinogenic mushroom species Psilocybe cubensis was developed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Five strains of dried, intact mushrooms were obtained and analyzed: Blue Meanie, Creeper, B-Plus, Texas Yellow, and Thai Cubensis. An extraction protocol was developed; this included an evaluation of sample milling technique, extraction solvents, and recovery/stability. Reversed phase chromatography on fused-core particle phases was developed for the determination of the two analytes using internal standard calibration with deuterated isotopologues of each analyte. The separation takes less than 5 min. Matrix effects were investigated by comparing signal response of calibration samples in neat solution and several mushroom matrices; no significant matrix effects were observed. The limit of detection for psilocybin was 1.5 ng/mL (1.5 pg on-column; 300 ng/g mushroom) and for psilocin was 0.15 ng/mL (0.15 pg on-column; 30 ng/g mushroom) using a Shimadzu LCMS-8050 triple quadrupole mass spectrometer. Assessment of the accuracy and precision of the method indicated percent error and RSD were <6 % at all concentration levels. Three whole, intact mushrooms from each strain were analyzed individually to obtain average content differences both between strains and between mushrooms of the same strain. From most to least potent, the study found that the average total psilocybin and psilocin concentrations for the Creeper, Blue Meanie, B+, Texas Yellow, and Thai Cubensis strains were 1.36, 1.221, 1.134, 1.103, and 0.879 % (w/w), respectively. A subset of these mushrooms was also tested in a separate non-affiliated laboratory, and the results were comparable between the two laboratories. Results from the secondary laboratory showed improved precision when multiple mushrooms were homogenized together, prior to extraction.
