Validation of an analytical method for the determination of the main ayahuasca active compounds and application to real ayahuasca samples from Brazil

doi:10.1016/j.jchromb.2019.06.014

Journal of Chromatography B

Volume 1124, 15 August 2019, Pages 197-203

https://doi.org/10.1016/j.jchromb.2019.06.014 Get rights and content

Highlights

•
Ayahuasca is a psychotropic Amazonian brew that has therapeutic properties.
•
DMT, THH, HME and HML are the main active compounds present in ayahuasca.
•
An analytical method was developed to simultaneously quantify these compounds.
•
The LC-MS/MS method was validated and applied to real ayahuasca samples.
•
This is a valuable tool for studies of alkaloid effects in biological responses.

Abstract

Ayahuasca is a brew prepared from the water decoction of two Amazonian plants, which is legally used for religious, cultural or therapeutic activities. The potential use of ayahuasca as a natural or phytotherapeutic drug is directly linked to the action of its active compounds and their connection with the therapeutic efficacy of the beverage. In this context, the aim of the present study was to establish a selective, sensitive and reproducible analytical method for the quantification of the main active ayahuasca compounds. Thirty-eight samples from the state of São Paulo, Brazil, were analyzed and the simultaneous quantifications of N,N-dimethyltryptamine (DMT), tetrahydroharmine (THH), harmine (HME) and harmaline (HML) were performed. This study enabled the development of a fast validated analytical method with minimal matrix interference and high reproducibility for the tracing of active ayahuasca compound concentrations for the first time. This method is important as an auxiliary tool for the study of active compound effects in biological responses using different multi-omic platforms.

Introduction

Ayahuasca is a psychotropic Amazonian beverage formulated from the water decoction of Banisteriopsis caapi vines and Psychotria viridis leaves. This drink is legally used in Brazil, as well as some European countries and the USA [1], for religious, cultural and therapeutic purposes [[2], [3], [4]]. Its effects can be described as displaying potential efficacy within a model that involves biomedical, psychological, anthropological and theological elements [5,6]. It is believed that the psychoactive effects of ayahuasca may exhibit the potential for the treatment of mental disorders due to the effects of β-carboline-derived alkaloids present in vines and N,N-dimethyltryptamine (DMT) present in leaves [7,8]. Because of its effects on the nervous system, as well as the presence of alkaloids, ayahuasca is classified as psychoactive and has also been described as psychedelic or hallucinogenic [9].

Psychoactive substances, both natural and synthetic, were a significant object of study especially between 1950 and 1960, as they display therapeutic potential for the treatment of alcohol addiction, as well as other drugs of abuse [10,11]. Preliminary studies, for example, have demonstrated a positive effect of the action of substances such as LSD (lysergic acid diethylamide) in the treatment of alcohol addiction [11]. In the 1970s, however, LSD was considered a dangerous substance due to its recreational use and was prohibited [1,12]. Only in the 1990s did studies regarding this and other substances resume, boosted by success in treating anxiety and depression [11], such as, for example, the use of psilocybin in the treatment of anxiety and anxiety disorders in patients presenting potentially terminal diseases, like cancer [[13], [14], [15]].

A growing number of studies indicate the potential increase of ayahuasca in general addiction treatments, namely against alcohol, tobacco, and illicit drugs, such as cocaine and heroin [[15], [16], [17], [18]], as well as in the treatment of depression and anxiety [19,20]. According to the Brazilian National Health Surveillance Agency (ANVISA), herbal drugs are obtained exclusively using active raw plant materials [21], and should be characterized concerning their efficacy and usage risks, as well as their quality reproducibility and constancy. Products comprising isolated active substances of any origin or their associations with plant extracts are not considered phytotherapeutic medicines [22].

In view of ANVISA recommendations and regulations, the establishment of reliable analytical methods for the quantification of active compounds present in ayahuasca beverages is essential for its application as a possible psycho-phytotherapeutic. In this context, the main active ayahuasca compounds, DMT, tetrahydroharmine (THH), harmine (HME) and harmaline (HML) were quantified by ultra-high performance liquid chromatography (UHPLC) coupled to tandem mass spectrometry (MS/MS) operating in selected reaction monitoring (SRM) mode. SRM is a targeted mass spectrometry method, performed on triple quadrupole-like instruments, used to increase selectivity and sensitivity in the analysis of specific compounds, especially in complex matrices, such as ayahuasca. In the present study, a considerable number of samples from the state of São Paulo, Brazil, which are used in traditional União do Vegetal (one of the Brazilian ayahuasca religions) rituals, were investigated for the first time by a rapid and validated analytical method for the quantification of ayahuasca active compounds.

Section snippets

Material and methods

A general analytical workflow is presented in Suppl. Fig. 1.

Analytical method optimization

Initially, procedures were performed to optimize the chromatographic conditions in order to guarantee better alkaloid and IS separation efficiency, as well as the establishment of short threshold times for elution using a C18 chromatographic column (BEH). The separation conditions were assessed both in the isocratic and gradient elution mode using different mobile phase compositions. Acetonitrile:water and methanol:water, with the addition (or not) of formic acid 0.1% (v/v) were assessed as

Conclusions

In the present study, the LC-MS/MS technique allowed for the development, optimization and validation of an efficient and fast analytical method capable of simultaneously quantifying the four main active ayahuasca compounds with minimum sample preparation. Reliable quantification results for these active compounds in 38 samples from the state of São Paulo were obtained, and demonstrated the significant heterogeneity of this type of extract, which makes ayahuasca tea standardization for future

Acknowledgements

Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP, Brazil (grant number 2018/01525-3) and INCT of Bioanalytics (FAPESP 2014/50867-3 and CNPq, Brazil 465389/2014-7 grant numbers) are acknowledged for financial support. Prof. Dr. Luiz Fernando Milanez is acknowledged for helping in the collection of the samples, as well as all UDV members who donated the ayahuasca samples.

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L’ayahuasca, ou yagé, est une boisson hallucinogène consommée principalement lors de rituels chamaniques à des fins thérapeutiques et divinatoires. Traditionnellement, ce breuvage est préparé à partir de lianes de Banisteriopsis caapi, sources de β-carbolines (harmine, harmaline et tétrahydroharmine) mélangées à différentes feuilles provenant de plantes telles que Psychotria viridis qui contiennent de la N,N-diméthyltryptamine (DMT). Suite aux recherches d’un historien parisien, trois flacons contenant du « yagé » ont été retrouvés au domicile d’un pharmacien français ayant vécu au début du xx^e siècle. Le laboratoire de toxicologie de l’institut de médecine légale de Strasbourg a été chargé d’analyser le contenu des bouteilles afin de déterminer la présence d’alcaloïdes de l’ayahuasca et de déterminer l’état de conservation des breuvages. Les échantillons ont fait l’objet d’une recherche d’éthanol par HS-GC/FID, de substances volatiles par HS-GC/MS et des principaux alcaloïdes psychoactifs par LC-HRMS et LC-MS/MS après dilutions appropriées dans l’eau. La recherche d’éthanol se révèle positive pour tous les liquides avec des concentrations entre 67,3° et 81,1°. La recherche d’alcaloïdes a révélé la présence d’harmine dans les trois échantillons avec des concentrations comprises entre 0,13 et 1,5 g/L, d’harmaline et de tétrahydroharmine seulement dans deux échantillons avec des concentrations comprises entre 53 et 80 mg/L pour le premier composé et entre 49 et 142 mg/L pour le deuxième. La DMT ainsi que les tryptamines 5-OH-DMT et 5-MeO-DMT n’ont été trouvées dans aucun échantillon. Ces analyses montrent une probable absence totale de plantes contenant des tryptamines et une composition complexe de β-carbolines due à des processus de dégradation et d’interconversion.
Ayahuasca, or yagé, is a hallucinogenic drink mainly consumed during shamanic rituals for therapeutic and divinatory purposes. Traditionally, this drink is prepared from Banisteriopsis caapi vines, sources of β-carbolines (harmine, harmaline and tetrahydroharmine) mixed with various leaves from plants such as Psychotria viridis that contain N,N-dimethyltryptamine (DMT). Following the investigations of a Parisian historian, three bottles containing “yagé” were found in the home of a French pharmacist who lived at the beginning of the 20th century. The laboratory of toxicology of the Institute of Legal Medicine of Strasbourg was asked to analyse the contents of the bottles in order to determine the presence of ayahuasca alkaloids and, after the dosage, to determine the state of conservation of the beverages. The samples were analysed for ethanol by HS-GC/FID, for solvents by HS-GC/MS and for the main psychoactive substances by LC-HRMS and LC-MS/MS after appropriate dilutions in water. The ethanol test was positive for all solutions with an alcohol content between 67.3° and 81.1°. The alkaloid test revealed the presence of harmine in all three samples with concentrations between 0.13 and 1.5 g/L, harmaline and tetrahydroharmine only in two samples with concentrations between 53 and 80 mg/L for the first compound and between 49 and 142 mg/L for the second. DMT as well as 5-OH-DMT and 5-MeO-DMT were not found in any of the three samples. These analyses suggest a probable total absence of plants containing tryptamines and a complex composition of β-carbolines due to degradation and interconversion processes.
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After being informed of the study design and objectives, the institution head signed a consent form. The ayahuasca was lyophilised (freeze-dried), and its alkaloids were quantified using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in the Institute of Chemistry of the University of Campinas (UNICAMP) [40]. The results are as follow (mean ± standard deviation of three analysis): DMT = 250 ± 4 mg/L, tetrahydroharmine = 629 ± 27 mg/L, harmine = 966 ± 6 mg/L and harmaline = 52 ± 2 mg/L. Details on the lyophilisation and analysis process are published elsewhere [41].
Considering the long-lasting effects of ayahuasca on the brain and emotional processing, the objective of this study was to evaluate the behavioural and neurobiological effects of repeated ayahuasca administration in an animal model of exploratory behaviour related to novel-environment anxiety. Male Wistar rats received water, 120, 240, 480 or 3600 mg/kg of resuspended freeze-dried ayahuasca by gavage once a day for 30 days; there was also a non-manipulated homecage group. One hour after the last administration, animals were placed individually in the open field for 20 min. We analysed the weight gain, the behavioural response through a stochastic analysis, and c-Fos immunoreactive levels in the hippocampus, amygdala, pre-frontal and barrel field cortex. Ayahuasca at 120 mg/kg increased ambulation, and at 3600 mg/kg decreased vertical exploration and reduced weight gain. Aya3600 had higher c-Fos expression in regions of the hippocampus and infralimbic cortex than homecage, water or aya120 groups. Water-receiving animals had less c-Fos expression in the anterior basolateral amygdala than others groups. Our results show different behavioural effects of ayahuasca: a stimulant-like effect in small doses, and decreased activity in extreme high-dose, probably due to adverse effects. Higher activation of areas involved in emotional processing and the serotonergic pathway adds to the neurobiological literature on repeated/chronic ingestion of ayahuasca. Our data do not support an anxiolytic effect of repeated ayahuasca related to exploring new anxiogenic-environment but suggest that low ayahuasca doses should be further studied. The absence of severe impairment and behavioural syntax alteration reinforce ayahuasca safety.
A rapid analytical strategy for the determination of ayahuasca alkaloids in non-ritualistic approaches by UHPLC-MS/MS
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A number of studies in the literature consisting of different experimental procedures and analytical methods have been proposed for the determination of the major constituents of ayahuasca in brewed preparations. Generally, these studies describe the use of high-performance liquid chromatography coupled to diode array and/or mass spectrometry detection systems (MS) [5,13,14], although other techniques can also be applied such as gas chromatography coupled to nitrogen-phosphorus detector and/or MS [15–17]. The indicated concentrations of constituents ranged from 62 to 3190 mg/L for DMT, 210 to 11,900 mg/L for THH, 295 to 16,000 mg/L for harmine and 28–1720 mg/L for harmaline [5,13,16,17].
Ayahuasca is a beverage composed by a mixture of herbs which contain the compound N,N-dimethyltriptamine (DMT) and the β-carbolines. Although its use is legalized in Brazil only for religious and spiritual ceremonies, there is a growing black market specialized in the distribution of these compounds in form of herbal material through internet and mail. The purpose of this work was the development of an ultra-high-performance liquid chromatography-tandem mass spectrometry method for the determination of ayahuasca alkaloids and its application in seized ayahuasca products.
An aliquot of seized products was weighted and diluted with methanol. An aliquot of this solution was added with internal standard (DMT-d₆), followed by injection in the analytical system.
The limit of quantitation was 10 ng/mL for DMT and 25 ng/mL for harmine, harmaline and tetrahydroharmine. The concentration ranges used were 10–100 ng/mL for DMT, harmine and harmaline and all analytes presented a coefficient of determination (r²) ≥ 0,99. Analysis of four seized samples presented concentrations of DMT ranging between 31.5 and 46.5 mg/g. Presence of β-carbolines was not detected in the products. The variability of DMT concentrations can be correlated with the potential intoxications described in the literature.
This work successfully established a determination method for ayahuasca alkaloids in herbal material. In addition, the workflow proved to be simple, rapid and useful to estimate the concentration of psychoactive compounds in seized materials, leading to further investigation of ayahuasca ritualistic or recreational exposure.
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¹: Both authors contributed equally to this work.

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Validation of an analytical method for the determination of the main ayahuasca active compounds and application to real ayahuasca samples from Brazil

Highlights

Abstract

Introduction

Section snippets

Material and methods

Analytical method optimization

Conclusions

Acknowledgements

Int. J. Drug Policy

Psychiatry Res.

Int. J. Antimicrob. Agents

J. Chromatogr. A

Food Chem.

Food Chem.

Talanta

Biochemistry and pharmacology of tryptamines and β-carbolines A minireview

J. Psychoactive Drugs

Ayahuasca, Ritual and Religion in Brazil

Res. n. 5

Human pharmacology of ayahuasca: subjective and cardiovascular effects, monoamine metabolite excretion, and pharmacokinetics

J. Pharmacol. Exp. Ther.

The therapeutic potentials of ayahuasca: possible effects against various diseases of civilization

Front. Pharmacol.

Hitting highs at rock bottom: LSD treatment for alcoholism, 1950–1970

Social History of Medicine

Therapeutic mechanisms of classic hallucinogens in the treatment of addictions: from indirect evidence to testable hypotheses

Drug Test. Anal.

Shyness as a risk factor for adolescent substance use

J. Sch. Health

Antidepressive, anxiolytic, and antiaddictive effects of ayahuasca, psilocybin and lysergic acid diethylamide (LSD): a systematic review of clinical trials published in the last 25 years

Ther. Adv. Psychopharmacol.

Psilocybin produces substantial and sustained decreases in depression and anxiety in patients with life-threatening cancer: a randomized double-blind trial

J. Psychopharmacol.

Rapid and sustained symptom reduction following psilocybin treatment for anxiety and depression in patients with life-threatening cancer: a randomized controlled trial

J. Psychopharmacol.