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Ayahuasca is an ancient psychoactive ethnobotanical medicine tea used by indigenous S. American tribes as a religious sacrament. It is made from the vine of B. caapi and the leaves of P. viridis and represents an ancient yet 'novel' short and long-term serotonin agonist. Here we present preclinical pharmacognosy data as an early step towards phase I and II clinical trials of ayahuasca in psychiatric medicine. The purpose of this study was: 1) to compare the alkaloid constituents in samples of ayahuasca decoctions made in S. and N. America that have been used in healing ceremonies, and 2) to investigate the effects of various methods of decoction and storage on active alkaloid constituents. HPLC and direct injection/liquid chromatography-electrospray ionization-tandem mass spectrometry procedure were used for the simultaneous quantitation of ayahuasca alkaloids. The indolamine dimethyltryptamine varied more than a log fold among ayahuasca brews used in several ceremonies. Monoamine oxidase inhibiting beta-carbolines varied widely in decoctions made of the two plants together versus the decoction of each plant separately. Freezing ayahuasca samples at -80o C degrades active alkaloids, while refrigeration and room temperature storage do not. DMT concentrations vary greatly among ayahuasca teas that have been used in ceremonies. Analytical chemistry methods are now in place to conduct phase I and II clinical studies of ayahuasca. Dose-escalation clinical studies of ayahuasca are needed to evaluate both the safety and efficacy of ayahuasca as a novel 5-HT agonist with potential for the treatment of several psychiatric disorders.