Ayahuasca Lyophilization (Freeze-drying) Protocol with Pre- and Post-procedure Alkaloids Quantification

This methodological paper (2021) describes a standardized protocol for freeze-drying ayahuasca and compares its alkaloid composition before and after the procedure.


Introduction: Ayahuasca is a psychoactive brew from the decoction of different Amazonian plants, traditionally used in several cultures, religions, and rituals. Scientific studies with ayahuasca are rapidly increasing due to its subjective effects and therapeutic potential. Although ayahuasca is traditionally used in its liquid presentation, lyophilized (freeze-dried) ayahuasca is often used in scientific experimentation settings. However, there is no standard process or guideline to freeze-dry ayahuasca nor comparison of the chemical profile between the liquid and freeze-dried presentations.

Methods/Results: Therefore, we describe a reproducible five-day protocol for ayahuasca lyophilization with alkaloids quantification by liquid chromatography coupled to tandem mass spectrometry of both the liquid and the final freeze-dried ayahuasca. By the end of the protocol, approximately 295 g of freeze-dried extract with similar alkaloids concentration were obtained from two liters of ayahuasca (dry matter: 14.75 %). The final extract was stored for three years inside a vacuum desiccator (approximately 6°C) with its texture quality preserved.

Discussion: Further studies should address the impact of different storage conditions and the lyophilization on the alkaloids’ quantity of the freeze-dried ayahuasca, especially the use of heat in regards to the β-carbolines.”

Authors: Dimitri Daldegan-Bueno, Vanessa Manchim Favaro, Luís Fernando Tófoli, Alessandra Sussulini, Fernando Cassas & Maria Gabriela Menezes Oliveira


Psychedelics may be effective treatments for smoking-related disease.


In 2015, 19.8% of people worldwide smoked, accounting for 11.5% of deaths. Although rates are decreasing in developed countries, they are increasing in middle- and low-income countries, including Brazil, where the prevalence of smoking is still a major public health problem. Despite the high prevalence of smoking in Brazil and globally, most smokers intend to quit. However, current interventions for smoking cessation seldom exceed a success rate of 30%.

Over the last decade, a growing body of evidence has emerged suggesting that psychedelics could be potential tools in the treatment of addiction. Psilocybin has been shown to successfully reduce or stop tobacco smoking in many people. Psychedelics may have an anti-addiction potential through several mechanisms, including downregulation of 5-HT2A receptors, increase in BDNF in frontolimbic regions and neuroplasticity, and subjective aspects of the experience. These effects may also account for at least partially the therapeutic potential of psychedelics in addiction.

Ayahuasca is a psychedelic brew made from the Banisteriopsis caapi vine and Psychotria viridis leaves. Pre-clinical studies have shown that ayahuasca reduces smoking and alcohol dependence by reducing withdrawal symptoms and decreasing the amount of substance used.

We used a mixed-model design with multivariate quantitative and qualitative thematic analysis to explore factors predicting smoking cessation in ayahuasca users.


An anonymous survey was conducted using the LimeSurvey platform to investigate the factors associated with smoking cessation or reduction after ayahuasca use. Participants had to give their informed consent by checking ‘I agree’ at the end of the page to join the survey.

Demographic, smoking and ayahuasca‑related characteristics

We used both closed and open questions to collect data on demographics, smoking characteristics, overall ayahuasca experience, and variables related to smoking cessation or reduction.

Questionnaire on Smoking Urges—Brief (QSU‑B)

A ten-item questionnaire was used to assess craving for smoking with a two-factor structure. Participants were asked to identify a single experience with ayahuasca that was the most important for their smoking cessation/reduction.

Fagerström Test for Cigarette Dependence (FTCD)

A six-item single-factor questionnaire was completed retrospectively by cigarette smokers 6 months before the occurrence of the index ayahuasca experience.

Mystical Experience Questionnaire (MEQ30)

A questionnaire derived from the State of Consciousness Questionnaire (SOCQ) is used to assess the occurrence of a mystical-type experience. It has four factors: mystical, positive mood, transcendence of time/space and ineffability.

Qualitative assessment

Qualitative data from two mandatory open questions were submitted to thematic analysis using an exploratory, inductive and semantic approach. Themes were identified based on the relevance of the expressed content and the frequency of responses to each theme.

Statistical analysis

Participants were grouped according to self-attributed smoking status: cessation (smoking abstinence since the index ayahuasca experience) or reduction/relapse (reduction in smoking since the experience, or temporary cessation/reduction in smoking after the experience). Descriptive statistics were performed and three models were developed to identify variables associated with smoking user status.


A total of 504 individuals completed the survey, of which 54 were excluded for not meeting study criteria and 9 were excluded for inconsistent responses. Of the 441 participants, 305 reported quitting smoking, 81 reported reducing smoking and 55 reported relapsing.

Demographics and smoking

The overall sample consisted of women and men with a mean age of 34.19 years, with a postgraduate education level (49.7%), currently employed (71.6%), without children (61.5%) and residents of the Southeast part of Brazil (53.5%).

Respondents started smoking when 16.29 years old, smoked more than ten cigarettes a day, for at least 10 years, and had one or more attempts to quit smoking.

Ayahuasca use and index experience

Ayahuasca use data and statistics are presented in Table 2. Most respondents used ayahuasca for the first time at 27.94 ( 9.24) years of age, drank ayahuasca fortnightly or more frequently, did not attend the ayahuasca ritual with the intention of quitting smoking, and needed more than one experience to quit/reduce smoking.

Smoking outcomes, mystical experience and multivariate model

The cessation group had higher FTCD scores 6 months before the experience, and the interaction between groups and time was significant. The cessation group also had a higher QSU score 6 months before the experience.

Multivariate logistic regression results show that age, being in a relationship, the frequency of ayahuasca consumption, a longer period since the occurrence of the experience and the mystical experience score are predictors of tobacco cessation. The positive mood subscale of the MEQ30 score is a risk factor.

Qualitative thematic analysis

The participants’ reports included eight themes, including characteristics of the index experiences with ayahuasca that led to smoking cessation/reduction and the process of quitting.

Acquired awareness of the harmful effects of smoking on the body, increased self-love and self-care, and triggered smoking cessation/reduction.

In the ayahuasca experience, participants reported physical/psychological cleansings, spiritual experiences, contact with spiritual entities, non-specific or inexplicable experiences, and changes in lifestyle. Some participants reported that the experience is ‘inexplicable’ or that they could not describe it (ineffable). Participants reported a decrease in the desire to smoke and a repulsion to cigarettes after ayahuasca rituals, as well as immediate or gradual smoking cessation. Some participants reported struggling with craving and anxiety, while others described a natural process.

Some reports describe a decrease in the desire to smoke after ayahuasca experiences, but many participants start smoking again when not participating in rituals.


We collected a self-selected sample of people who either quit or reduced smoking after one or more ayahuasca experiences and attributed this to their smoking cessation/reduction. The cessation group had a worse smoking pattern before the index ayahuasca experience.

To assess the factors associated with smoking cessation, a multivariate logistic regression was conducted including sociodemographic, smoking and ayahuasca-related characteristics. The mystical experience subscale score was a significant predictor of smoking cessation. Psychedelics such as ayahuasca are known to induce mystical experiences, which may facilitate meaningful personal insights and lasting behaviour changes. They may also alleviate psychological symptoms such as anxiety and depression, which are often associated with substance misuse and addiction. The psychological effects of psychedelics, such as insights and awe, are increasingly being studied as an important factor for the therapeutic potential of psychedelics. These effects can be associated with positive outcomes, such as the reduction of depressive symptoms.

A systematic review of qualitative studies on the use of psychedelic treatments for mental disorders found that participants had insights that resulted in improved self-awareness, an expanded emotional spectrum, and transcendental experiences. Additionally, participants reported a reduction in craving and increased motivation to quit smoking after the experience. In our sample of ritualistic ayahuasca users, the mystical experience was a protective factor against tobacco smoking, whereas a study by Johnson et al. (2017b) found no statistical significance in respect of mystical experience scores and tobacco consumption after a psychedelic experience.

We found that the positive mood subscale of the MEQ30 was a risk factor for tobacco smoking, although there is evidence that psychedelics may induce better mood outcomes. A possible explanation could be that difficult/challenging experiences motivated by inner unresolved issues could lead to greater motivation to make behavioural changes. This is especially true for ayahuasca drinkers who often undergo difficult experiences that combine psychological suffering and physical distress.

The frequency of ayahuasca intake was the strongest protective factor for smoking cessation. Moreover, people in the cessation group drunk more ayahuasca in their lifetime and needed more ayahuasca rituals to stop smoking when compared to the reduction/relapse group. We found that ayahuasca intake was associated with positive changes in drug consumption and health habits, including improved diet and physical exercise.

This research does not show the prevalence of non-smokers within an ayahuasca sample, and the self-selected nature of our sample means that it is naturally biassed.

We defined our groups based on self-reports of tobacco abstinence, which is a major limitation since self-reports can be biased towards a socially desirable response. Furthermore, several participants had their experience within a formal religious setting, which can influence their smoking habits through social pressure to stop smoking. We did not correct for multiple comparisons, and did not assess other forms of tobacco consumption, such as traditional snuffs (‘rapé’), which may influence smoking behaviours. Furthermore, we did not include a control group.


We found that the mystical experience and frequency of ayahuasca intake were protective factors for smoking cessation, whereas positive mood during a psychedelic experience was a risk factor for smoking. Most participants described other positive changes related to ayahuasca consumption, including reducing drug use and increasing healthy habits.


Authors associated with this publication with profiles on Blossom

Luís Tófoli
Luís Tófoli is a professor at UNICAMP and one of the organizers of ICARO, his work is mostly done in Brazil and focused on ayahuasca.

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