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

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.