This biochemical methods development study (2008) describes a simple gas chromatographic assay for quantifying the composition of ayahuasca’s active alkaloids.
Abstract
“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 β‐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 β‐carbolines found in ayahuasca preparations.
Methodology: The alkaloids were extracted by means of solid phase extraction (C18) 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 (r2 > 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. “
Authors: Ana Paula Salum Pires, Carolina Dizioli Rodrigues De Oliveira, Sidnei Moura, Felipe Augusto Dörr, Wagner Abreu E. Silva & Mauricio Yonamine
Summary
A method was developed and validated for the simultaneous determination of dimethyltryptamine and the main b-carbolines found in ayahuasca preparations. The method uses Banisteriopsis caapi and Psychotria viridis as samples.
Introduction
Ayahuasca is a psychoactive plant tea obtained by infusing the pounded stems of Banisteriopsis caapi in combination with the leaves of Psychotria viridis. These plants contain -carbolines, which inhibit monoamine oxidase, and allow dimethyltryptamine to reach the central nervous system.
In Brazil, ayahuasca has been incorporated in rituals of modern syncretic religious groups, and is currently protected by law. It has also spread outside South America and has attracted the attention of researchers interested in its pharmacological and toxicological aspects.
Peruvian ayahuasca contains dimethyltryptamine, harmine, harmaline and tetrahydroharmine, whereas Brazilian ayahuasca contains 24, 170, 20 and 107 mg respectively of the same four alkaloids. No detailed analytical procedure seems to have been previously reported for the simultaneous quantification of these alkaloids in ayahuasca tea.
In the present work, a method was developed for simultaneous determination of the main active constituents found in ayahuasca.
Experimental
Reagents and chemicals were purchased from Merck, Waters, Sigma and a religious group in Araçoiaba da Serra, So Paulo state, Brazil.
Tetrahydroharmine was synthesized by adding harmaline hydrochloride to a methanolic sodium borohydride solution at 0°C, acidifying the mixture with HCl, alkalising with NaOH, and extracting the residue with dichloromethane. It was then twice recrystallised with ethanol, and its structure was confirmed by 1H NMR.
Dimethyltryptamine was synthesized by adding sodium borohydride, sulfuric acid and formaldehyde to a solution of tryptamine in tetrahydrofuran at 0°C. The product was purified by a silica chromatographic column and ESI-MS and identified by its melting point of 64°C.
Standard solutions of dimethyltryptamine, harmine, harmaline and tetrahydroharmine were prepared in volumetric glassware and stored in the freezer at 20°C for 30 days. Working solutions were prepared by diluting stock solutions.
GC-NPD analyses were performed on a 6890 Agilent gas chromatograph using a HP Ultra-2 fused-silica capillary column and ultra-pure-grade nitrogen as carrier gas at 0.6 mL/min in a constant flow rate mode.
Ayahuasca sample extraction was performed on a C18 cartridge mounted on a vacuum manifold. The analytes were identified based on comparison of the relative retention time with the corresponding values of standards assayed in the same run.
Limit of detection and lower limit of quantification were determined by analyzing a series of water samples containing decreasing amounts of dimethyltryptamine, harmine, harmaline and tetrahydroharmine.
The recovery efficiency of the SPE method was evaluated by analyzing two sets of samples, one with processed samples and the other with unprocessed samples. The unprocessed samples represented 100% recovery.
Intra- and inter-assay precision was determined by analysing solutions of dimethyltryptamine, harmine, harmaline and tetrahydroharmine on three different days.
The accuracy of the entire SPE and GC-NPD method was evaluated by analysing aqueous solutions of dimethyltryptamine, harmine, harmaline and tetrahydroharmine.
Stability tests were performed on ayahuasca samples and spiked water samples after 24 h at room temperature.
Results and Discussion
A method was developed to determine the main alkaloids found in ayahuasca samples. It was based on the procedure described by Yritia et al. (2002) for the determination of -carbolines in plasma samples using SPE and HPLC.
Calibration curves were linear over the specified range (0.02 – 4.0 mg/mL), and linear regression equations and coefficients of correlation were found for dimethyltryptamine, harmine, harmaline and tetrahydroharmine.
The validated method showed good linearity over a broad concentration range (0.02 – 4.0 mg/mL) and good accuracy and precision over the studied concentration range (CV 10%). The solid-phase extraction produced clean extracts with good recovery (better than 68%).
The analytical method was applied to eight different ayahuasca preparations. The results showed that the concentration of alkaloid in ayahuasca varies considerably depending on the method of preparation and the amount and proportion of the source plants.