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ABSTRACT:A method was developed and validated for the simultaneous determination of dimethyltryptamine and β-carfbolines alkaloids (harmine, harmaline and tetrahydroharmine) in ayahuasca preparations. The alkaloids were extracted by menas of solid phase extraction (C18) and detected by gas Introduction -Ayahuasca is obtained by infusing the pounded stems of Banisteriopsis caapi in combination with the leaves of Psychotria viridis. P. viridis is rich in the psychedelic indole N,N-dimethyltryptamine, whereas B. caapi contains substantial amounts of b-carboline alkaloids, mainly harmine, harmaline and tetrahydroharmine, which are monoamine-oxidase inhibitors. Because of differences in composition in ayahuasca preparations, a method to measure their main active constituents is needed. Objective -To develop a gas chromatographic method for the simultaneous determination of dimethyltryptamine and the main b-carbolines found in ayahuasca preparations. Methodology -The alkaloids were extracted by means of solid phase extraction (C 18 ) and detected by gas chromatography with nitrogen/phosphorous detector. Results -The lower limit of quantification (LLOQ) was 0.02 mg/mL for all analytes. The calibration curves were linear over a concentration range of 0.02-4.0 mg/mL (r 2 > 0.99). The method was also precise (RSD < 10%). Conclusion -A simple gas chromatographic method to determine the main alkaloids found in ayahuasca was developed and validated. The method can be useful to estimate administered doses in animals and humans for further pharmacological and toxicological investigations of ayahuasca.