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.
Papers cited by this study that are also in Blossom
Domínguez-Clavé, E., Soler, J., Elices, M. et al. · Brain Research Bulletin (2016)
Favaro, V. M., Yonamine, M., Soares, J. C. K. et al. · PLOS ONE (2015)
McKenna, D., Riba, J. · Current Topics in Behavioral Neurosciences (2016)
Palhano-Fontes, F., Barreto, D., Onias, H. et al. · Psychological Medicine (2018)
Ayahuasca is a traditional Amazonian psychoactive brew most commonly prepared from Banisteriopsis caapi (a source of β-carbolines such as harmine, harmaline and tetrahydroharmine) and Psychotria viridis (the main source of N,N-dimethyltryptamine, DMT). The β-carbolines act as reversible monoamine oxidase A inhibitors and thereby enable oral activity of DMT. Previous research interest has expanded because of ayahuasca's subjective effects and potential therapeutic applications. Although ritual use is of a liquid decoction, many experimental studies use a freeze-dried (lyophilised) form to improve stability, enable encapsulation or precise dosing, and facilitate administration in animal models; nevertheless, there is no widely accepted, reproducible protocol for lyophilising ayahuasca nor a systematic comparison of alkaloid content before and after the process. Daldegan-Bueno and colleagues set out to fill this gap by describing a reproducible five-day protocol for ayahuasca lyophilisation, and by quantifying the principal alkaloids (DMT, THH, harmine (HME) and harmaline (HML)) in the liquid brew and in the resulting freeze-dried material using UHPLC-MS/MS. The paper also outlines practical pre- and post-procedure considerations that may influence product quality and presents simple storage guidance based on the authors' experience.
Overall approach: The investigators developed and executed a five-day lyophilisation protocol on a donated ayahuasca batch, and analysed alkaloid concentrations in the original liquid and the resuspended freeze-dried product using an established UHPLC-MS/MS method. Material source and handling: The ayahuasca batch was donated by the Barquinha religious institution in Ji-Paraná, Rondônia (Brazil), prepared from plants cultivated by the church at the end of 2017. The liquid brew was stored at approximately 6 °C prior to processing; the church provided consent for research use. Lyophilisation protocol: Pre-treatment and freezing (day 1) began with volume reduction using a rotary evaporator (FISATOM) operating at 80 rpm and an average temperature of 70 °C; this reduced 2,000 mL to approximately 1,000 mL in about two hours. Concentrated liquid (250 mL per tray) was frozen overnight in four AISI 304 stainless steel trays using a standard freezer at about -25 °C. Primary drying (day 2 onward) employed a LIOBRAS L101 lyophiliser with an acrylic chamber and inox stand; the chamber was cooled to about -55 °C, vacuum applied (<500 µHg), and foaming stabilised within 30 minutes. The authors report a primary drying period of 48–72 hours and used 72 hours to ensure complete dryness. They did not perform a secondary drying step. Final handling (day 4 or 5) involved careful venting, mechanically pestling the brittle cake into powder, homogenising to account for sedimentation layers, and pooling any slightly moist residues into subsequent batches to avoid waste. Storage: The homogenised freeze-dried material was portioned into small plastic containers (~25 g) and stored inside a vacuum desiccator with silica gel at a constant temperature of approximately 6 °C. Analytical methods: Alkaloids were quantified by UHPLC-MS/MS using a Quattro Micro API mass spectrometer with electrospray ionisation in positive mode coupled to an Acquity UPLC system with a BEH C18 column (50 mm × 2.1 mm, 1.7 μm). Diphenhydramine hydrochloride served as an internal standard. Samples and standards were injected at 1 µL and analysed by selected reaction monitoring (SRM) of protonated molecular ions. The freeze-dried sample was resuspended in water at the same proportion as the original liquid for direct comparison. The reported alkaloid concentrations are mean values from three analyses (n = 3).
Yield and dry matter: Applying the described procedure to two litres of ayahuasca produced approximately 295 g of freeze-dried extract, corresponding to 14.75% dry matter of the original infusion. The rotary evaporator step halved the volume (2,000 mL to 1,000 mL) in roughly two hours under the conditions reported. Alkaloid quantification: The authors report that qualitative comparisons indicated similar alkaloid quantities between the liquid and the resuspended freeze-dried presentations. For the dried extract, mean equivalent concentrations were reported as: DMT 37.5 mg/g; THH 94.44 mg/g; harmine (HME) 149.55 mg/g; harmaline (HML) 7.80 mg/g. The paper indicates detailed concentrations for both sample types are presented in a table within the text (values above are the reported means per gram of dried material). Stability and use: Within the storage conditions described (vacuum desiccator with silica gel at ~6 °C), the freeze-dried ayahuasca retained its dry texture for three years. The final product was used in animal experiments and, according to the authors, produced behavioural and neurobiological changes compared with water-receiving controls. The investigators did not, however, report alkaloid analyses performed after the three-year storage period.
The authors interpret their findings as demonstrating a practicable, reproducible five-day protocol to produce freeze-dried ayahuasca suitable for experimental use, with the main alkaloids qualitatively preserved relative to the liquid form. They emphasise that lyophilisation is time- and energy-intensive and that key stages—pre-treatment (volume reduction), freezing, primary drying, and storage—can affect final product quality. On pre-treatment, the researchers argue that reducing volume with a rotary evaporator optimises primary drying time and resources but may pose risks: heating could alter β-carboline concentrations. They tested temperatures from 50 °C to 90 °C and selected 70 °C as a pragmatic compromise between speed and thermal exposure, while noting that traditional ayahuasca decoction already involves prolonged heating above 100 °C. Nevertheless, the impact of the evaporative heating step on alkaloid composition remains unclear. With respect to freezing, the paper notes that freezing is not part of traditional preparation but that prior work found no alkaloid changes after multiple freeze–thaw cycles at -20 °C; their use of a -25 °C standard freezer produced satisfactory frozen material for primary drying. The authors caution, however, that freezing is complex (cooling rate, annealing, freeze temperature/time) and that the effects of these parameters on ayahuasca quality have not been investigated. Primary drying was set at 48–72 hours; the team chose 72 hours to avoid incomplete drying and the need to repeat the process. They did not perform secondary drying, reasoning that secondary drying typically involves elevated temperatures for extended periods and could therefore affect temperature-sensitive β-carbolines. Regarding storage, the authors place importance on conditions: prior studies have reported reductions in harmaline and THH over 12 months in refrigerated liquid samples, and the present freeze-dried batch maintained dryness for three years under vacuum dessicator storage, although no post-storage alkaloid assays were conducted. The final dry matter percentage (14.75%) aligns with values reported from other institutional ayahuasca batches (approximately 15–16%). Limitations and recommendations the authors acknowledge include the lack of systematic investigation of freezing parameters, omission of secondary drying and the unknown impact of heat during volume reduction and potential secondary drying on β-carbolines, and the absence of alkaloid analysis after long-term storage. They recommend further studies to quantify how different lyophilisation phases and storage conditions affect chemical profiles, with particular attention to heat-sensitive β-carbolines.
Ayahuasca is an ancient psychedelic concoction of different Amazonian plants, and is an important facet of many practices, including therapeutic, of South American native people. Ayahuasca's most common preparation consists of the decoction of the Banisteriopsis caapi vine and the Psychotria viridis leaves). The vine possesses several β-carboline alkaloids, from which the most studied are harmine (HME), harmaline (HML), and tetrahydroharmine (THH). The main compound of the leaves is the N,N-dimethyltryptamine (DMT), a tryptamine with no psychoactive effect if orally ingested given the deamination process performed by the monoamine oxidases (MAO) present in the liver and the gut. Therefore, the psychoactive effect of ayahuasca occurs mainly due to the synergy between the βcarbolines, which act as competitive inhibitors of the MAO-A enzyme, and DMT. There has been a growing scientific interest in ayahuasca due to its subjective effects and therapeutic potential. Although ayahuasca is traditionally consumed as a brew, its freeze-dried form is frequently used in research settings both with human populations and pre-clinical animal models. Lyophilization, or freeze-drying, is a process to improve stability and long-term storage of products by removing the water through sublimation. It generally consists of three phases that can be adjusted toward optimization: (1) freezing, (2) primary drying, and (3) secondary drying. First, most of the water is frozen to provide the condition for the next, low-temperature primary drying phase. On the primary drying, the phase that consumes the highest amount of time and energy, the water is removed by sublimation in a lowtemperature vacuum chamber. Then, in the secondary drying, the water is removed by letting the freeze-dried product in heated shelves with higher temperatures than the environment, usually around 40 °C, for three to six hours.. Apart from the better dosage control and better longterm storage condition due to reduced water and volume, the freeze-dried presentation may offer specific benefits in the context of ayahuasca research. When using some animal models (e.g., rodents), different concentrations of freeze-dried ayahuasca resuspended in water can be administered directly into the stomach (i.e., gavage) with a manageable and constant volume. It also enables using extremely high doses, e.g., up to 50 times higher than the ones used in most of the ritual contexts, that would otherwise be impossible to achieve with the concentration of the majority of the traditional, liquid ayahuasca. In the context of human research, it is possible to encapsulate the freeze-dried ayahuasca, and thus, facilitate the placebo strategy by hiding its organoleptic properties (e.g., color, taste, and smell) from volunteers. Despite being used in its non-traditional freeze-dried presentation in many research settings, to the best of our knowledge, there is neither a study comparing the liquid and freeze-dried ayahuasca (e.g., pharmacokinetics, alkaloids) nor a guideline or protocol to perform the lyophilization. Nevertheless, data on the chemical profile of liquid and freeze-dried ayahuasca is critical to allow a better comparison between studies that use different ayahuasca presentations and, therefore, a standardized and replicable method of lyophilizing ayahuasca makes a timely and topical contribution to the field. On this basis, the objective of this work is to describe a replicable protocol for ayahuasca lyophilization, provide an alkaloids quantification before and after the procedure, and discuss key factors that may influence the final product in light of the available literature.
The lyophilization was conducted in the LIOBRAS L101 lyophilizer using its AISI 304 tray and the acrylic chamber. The concentration of the alkaloids DMT, THH, HME, and HML was determined by UHPLC-MS/MS using a previously validated method. The instrumentation consisted of a mass spectrometer Quattro Micro API series (Waters Corp., Milford, MA, USA), with electrospray ionization (ESI) operating in the positive ion mode and a triple quadrupole mass analyzer, coupled to an Acquity UPLC system equipped with BEH C18 column (50 mm × 2.1 mm, 1.7 μm) using gradient elution with a mobile phase composed of water and methanol. Diphenhydramine hydrochloride was used as an internal standard. Samples and standards were analyzed using a 1 µL injection volume, and MS/MS analyses were performed using selected reaction monitoring (SRM) of the protonated molecular ions for the analytes and internal standard. Both the original liquid and the final freeze-dried ayahuasca were analyzed; the freeze-dried sample was resuspended in water with the original liquid proportion for the analysis.
The ayahuasca used in this work was donated by the religious institution Barquinha located in Ji-Paraná, Rondônia (Brazil), which produces ayahuasca from plants (Banisteriopsis caapi and Psychotria viridis) cultivated in their church to be used as a sacrament in their rituals, following the current national norms (Norma Federal 2010). The batch was prepared at the end of 2017, and the lyophilization was conducted in early 2018, from February to March. The liquid ayahuasca was always stored in a cold (approximately 6°C) environment. The head of the church was informed of the research procedures and objectives, including the lyophilization process, and signed a consent form for the ayahuasca use.
First, we used a rotary evaporator (FISATOM) at 80 rpm and an average temperature of 70 °C to decrease the ayahuasca volume (Figure)). Then, we froze 250 mL of the concentrated ayahuasca directly in each of the four AISI 304 lyophilizer trays. The trays hold up to 500 mL each, but we recommend leaving an extra space given the frozen ayahuasca may expand due to foam formation. We froze our batch by letting it overnight in a standard freezer (approximately -25 °C).
Once the ayahuasca is frozen, it is necessary to turn on the lyophilizer with the acrylic chamber attached and wait until it reaches the temperature of -55°C (approximately 20 minutes). Then, the four trays with the frozen ayahuasca should be placed on the inox stand inside the acrylic chamber, and the vacuum turned on. In the beginning, foaming will occur, which should stabilize in approximately 30 minutes (Figure). After the vacuum stabilization (<500µHg), the entire process may last from 48 to 72 hours. Parts of this process are also explained in the machine's user guide and may vary according to the model used.
By the end of the primary drying, turn off the vacuum and open the air drain carefully and slowly to avoid spreading the dried ayahuasca with the air influx. The completely freeze-dried ayahuasca can be identified by an easy-to-crack and crumble texture (Figure). However, it may be difficult to visually distinguish whether the primary drying has ended, as the content may be dry in the upper part but contain moisture in the bottom. If the drying is incomplete, the freezing phase and primary drying should be repeated. Once removed from the chamber, quickly pestle the freeze-dried ayahuasca to obtain its powder. When pestling the final material, a minor part may retain humidity -to ensure the optimization of resources, we diluted it in the next ayahuasca batch to be lyophilized. When the lyophilization of all ayahuasca available is finished, the resulted freeze-dried material should be homogenized. It is important to homogenize the final powder due to ayahuasca sedimentation; different concentrations can be noted by the different colors of layers (Figure).
After homogenized, we kept the final freeze-dried ayahuasca in small plastic containers (25 g each) inside a vacuum desiccator with silica gel at a constant temperature of approximately 6 °C, following the standard procedure with desiccators (Figure).
We safely halved the ayahuasca volume in approximately two hours (i.e., 2,000 mL to 1,000 mL) using the rotary evaporator. By the end of the protocol, two liters of ayahuasca yielded approximately 295 g of lyophilized extract, corresponding to 14.75 % of the dry matter of the original infusion. The quantities of alkaloids between the liquid and the freezedried presentations were similar when compared qualitatively, and the final freeze-dried ayahuasca was used in experiments with animal models that induced both behavioral and neurobiological alterations compared to water-receiving controls. The concentrations of DMT, HME, HML, THH in both samples can be found in Table. The equivalent mean concentrations of alkaloids for the dried extract were: DMT: 37.5 mg/g; THH: 94.44 mg/g; HME: 149.55 mg/g, and HML: 7.80 mg/g. Within the presented storage conditions, our freeze-dried ayahuasca lasted with its dryness preserved for three years.
Freeze drying is a time-and energy-intensive process with key stages that may impact the final product. To design an optimum freeze-drying process, the experimenter must be aware of the characteristics of the product to be lyophilized and the different phases involved in the process. Such properties include elements of the pre-procedure (e.g., volume reduction, freezing), primary drying, and post-treatment storage conditions. In the first phase (i.e., pre-treatment and freezing), reducing the volume of the ayahuasca will significantly optimize time and resources; this process is usually done by a rotary evaporator. There is no ideal concentration of ayahuasca to start the second phase (i.e., primary dryin), and this should be achieved accounting for the original ayahuasca concentration. Excessive volume reduction may result in unwanted decantation, causing material loss or unnecessary use of solvents. On the contrary, a low volume reduction will imply lower efficiency in the primary dry phase. We tested reducing the volume with temperatures varying from 50°C to 90°C and determined 70°C to be a satisfactory mean temperature considering the time and thermal increase of the liquid. It is hypothesized that the βcarbolines are transformed through a chemical reduction pathway by the high temperatures of decoction during ayahuasca preparation; also, a study reported changes in β-carboline concentrations when left for six days at a temperature of 37 °C. Overall, suggesting the βcarbolines may be sensible to temperature variations and that reducing the volume through heating may change the chemical profile of ayahuasca. However, the preparation of ayahuasca involves long decoctions periods with temperatures above 100 °C; therefore, any possible chemical modification caused by a heat up to 100 °C should not (theoretically) be different from what occurs naturally in the traditional ayahuasca preparation. Nevertheless, while the impact of the pre-process volume reduction in the alkaloids remains unclear, this volume reduction phase aims to optimize the subsequent primary dry and, therefore, configures a skippable, non-essential process. The next important phase is to freeze the ayahuasca. There is no freezing in the process of making ayahuasca, and freezing is not a common practice within the traditional uses. However, a recent study found no significant modification in the alkaloids quantities after three freeze-thaw cycles at -20 °C, suggesting that freezing does not affect the concentration of the alkaloids. Using a standard freezer with approximately the same temperature (-25 °C), we achieved a satisfactory freeze quality for the primary dry phase. Nevertheless, freezing is a complex stage of lyophilization that can determine the quality of the final product, and yet, there is no investigation on the techniques to freeze ayahuasca and the impact of different conditions (e.g., cooling rate, freeze temperature/time, annealing) on the final product. Ayahuasca alkaloids quantification by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Mean value ± standard deviation of three analyzes (n = 3). Abbreviations: N,N-dimethyltryptamine (DMT), tetrahydroharmine (THH), harmine (HME), and harmaline (HML) concentrations by milligrams in a liter. To finalize the primary drying phase, we established a period from 48 to 72 hours as satisfactory to achieve a good quality dry ayahuasca. However, we used 72 hours as an optimal time to avoid the possibility of incomplete dryness and, consequently, the necessity to repeat the primary drying procedure. This may be further optimized considering specificities of ayahuasca, the lyophilizer (e.g., ideal temperature and chamber pressure), and the freezing phase. Moreover, we did not conduct the secondary drying step. Since it involves temperatures higher than the environment through several days, and that the β-carbolines may be sensitive to temperature, it is reasonable to expect, as discussed before, that the secondary drying might influence the final chemical profile of ayahuasca. Another crucial phase is the ayahuasca storage condition. Regarding liquid ayahuasca, some studies investigated the stability of alkaloids when stored over different time-frames. The most comprehensive study did not find an influence of the material used (plastic versus glass containers) to store liquid ayahuasca but found significant decreases of harmaline and tetrahydroharmine quantities over 12 months when stored in a fridge. To the best of our knowledge, there is no study investigating the stability of stored freeze-dried ayahuasca. Nevertheless, studies in humans published through the period of ten years found relevant alterations in biological outcomes using the same freeze-dried ayahuasca batch, showing that the lyophilized ayahuasca remain active for long periods. Our freeze-dried sample was stored in a vacuum desiccator inside a fridge and lasted with its dryness preserved for three years; however we did not analyze the alkaloids afterward. Finally, our final dry matter percentage (14.75 %) was similar to others found on lyophilized ayahuasca batches from the União do Vegetal (UDV) and Santo Daime churches in Brazil (16 % and 15.7 %, respectively). In conclusion, we demonstrated a five day protocol to lyophilize ayahuasca resulting in a product with adequate quality for experimentation use and preserved main alkaloids when qualitatively compared to the liquid form. We developed a first reproducible guideline and presented key aspects of pre-and post-lyophilization process that should be investigated. Further studies are required to address the impact of different storage conditions and the impact of the difference phases of lyophilization on the chemical profile of ayahuasca, especially the use of heat (e.g., for volume reduction or in the secondary dry) in regards to the β-carbolines.
Create a free account to open full-text PDFs.
Riba, J., Anderer, P., Jané, F. et al. · Neuropsychobiology (2004)
Riba, J., Mcilhenny, E. H., Valle, M. et al. · Drug Testing and Analysis (2012)
Riba, J., Urbano, G., Morte, A. et al. · Psychopharmacology (2001)