The authors developed and validated a simple, low‑cost SPE–LC method with UV diode‑array detection for quantifying N,N‑dimethyltryptamine, tryptamine and β‑carbolines in ayahuasca, showing good linearity (r>0.9902), excellent repeatability (RSD<0.8%) and an LOD of 0.12 mg/L. Applied to 20 samples from a Fortaleza religious group's brew, the method found target compound concentrations ranging widely from 0.3 to 36.7 g/L.
Ayahuasca is a potent hallucinogenic beverage prepared from Banisteriopsis caapi in combination with other psychoactive plants. N,N-dimethyltryptamine, tryptamine, harmine, harmaline, harmalol, and tetrahydroharmine were quantified in ayahuasca samples using a simple and low-cost method based on SPE and LC with UV diode-array detection. The experimental variables that affect the SPE method, such as type of solid phase and nature of solvent, were optimized. The method showed good linearity (r > 0.9902) and repeatability (RSD < 0.8%) for alkaloid compounds, with an LOD of 0.12 mg/L. The proposed method was used to analyze 20 samples from an ayahuasca cooking process from a religious group located in the municipality of Fortaleza, state of Ceará, Brazil. The results showed that concentrations of the target compounds ranged from 0.3 to 36.7 g/L for these samples.
Ayahuasca is a traditional hallucinogenic beverage typically prepared as a decoction of Banisteriopsis caapi (providing reversible monoamine oxidase inhibitors: harmine, harmaline, tetrahydroharmine) and Psychotria viridis (leaves containing N,N-dimethyltryptamine, DMT). The reversible MAO inhibitors in B. caapi render orally ingested DMT active. In Brazil, consumption of ayahuasca is permitted in a religious context under specific national regulations, which also highlight the need for multidisciplinary research including chemical characterisation and analytical methods for tryptamines and β-carbolines. Such characterisation is important for monitoring use, informing clinical or forensic investigations, and understanding risks from combinations with other psychoactive substances. Santos and colleagues set out to develop and validate a simple, low-cost analytical method to quantify major alkaloids in ayahuasca—DMT, tryptamine, harmine, harmaline, harmalol, and tetrahydroharmine—using solid-phase extraction (SPE) on silica cartridges coupled with liquid chromatography and UV diode-array detection (LC-UV/DAD). The study applied the method to 20 samples taken throughout a two-stage ayahuasca cooking process from a religious group in Fortaleza, Ceará, Brazil, and included structural characterisation of tetrahydroharmine isolated during the work.
Papers in Blossom that reference this study
Rodriguez, L., Lopez, A., Moyna, G. et al. · ACS Omega (2022)
The investigators obtained reference standards for tryptamine, harmalol, harmine and harmaline from commercial sources; DMT and tetrahydroharmine were prepared according to cited methods. Stock standard solutions were prepared in methanol (2 mg/mL) and working standards were prepared by dilution. The extracted text contains inconsistent concentration units reported for working standards and calibration ranges; the Method Validation section reports calibration over 0.001–3.0 mg/L. Ayahuasca samples were produced by members of a religious group from freshly harvested B. caapi stems and P. viridis leaves. The cooking process lasted 20 h in the first step, during which hourly samples produced 16 fractions. These 16 fractions were subdivided into five sets, recombined and subjected to a second 4 h cooking step that produced four further hourly ‘‘apuro’’ fractions (samples 17–20). A total of 20 aqueous samples were bottled and sent for analysis; samples 1–16 were processed directly, while the viscous apuro fractions (17–20) were diluted 1:100 prior to SPE to avoid cartridge clogging. Solid-phase extraction used silica cartridges preconditioned with methanol and 1 mol/L HCl. A 20 mL aliquot of ayahuasca (adjusted to pH 8 with 0.01 mol/L NaOH) was percolated at ~0.5 mL/min, dried, then eluted with acidified methanol (pH 3). The eluent was concentrated under nitrogen to 1 mL and 20 μL injections were analysed by HPLC-UV/DAD. SPE here refers to concentrating and cleaning analytes on a solid sorbent prior to analysis. Chromatography employed a reversed-phase Zorbax Eclipse Plus C8 column (150 × 4.6 mm, 5 μm) with a binary gradient of 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B) at 1.5 mL/min. The gradient started at 5% B for 2 min, ramped linearly to 50% B at 30 min, and returned to initial conditions over 10 min. Spectral data were collected across 190–800 nm. The authors report characteristic UV absorption maxima used for identification: 278 nm for DMT and tryptamine, 246 nm for harmine, 320 nm for tetrahydroharmine, and 372 nm for harmalol and harmaline. Tetrahydroharmine isolated in the study was characterised by melting point (199.3 ± 0.2°C), GC–MS (major peak at 18.600 min, molecular ion m/z 216, base peak m/z 201, 89% similarity to library spectrum) and 1H/13C NMR (recorded in CDCl3 on a 400 MHz instrument), supporting its identity. Method optimisation compared different sorbents and solvents and found silica and methanol produced cleaner chromatograms. The pH of extraction was adjusted because indole alkaloids are basic; washing the sorbent with methanol and acidic aqueous solution decreased ion suppression. Acidified methanol provided average recoveries of 71.2–82.5% in optimisation experiments and was selected as the eluent. Validation steps included assessment of linearity, recovery, repeatability (intra-assay precision) and intermediate (between-day) precision, and determination of limits of detection (LOD) and quantification (LOQ). The extracted text reports specific calibration regression equations and correlation coefficients for each compound and provides recovery and precision summaries (see Results).
Linearity of calibration curves was reported across 0.001–3.0 mg/L, with correlation coefficients (r) ≥ 0.9902 for all analytes: harmine r = 0.9905, harmaline r = 0.9904, tetrahydroharmine r = 0.9902, harmalol r = 0.9929, tryptamine r = 0.9943, and DMT r = 0.9919. Regression equations for peak area versus concentration were provided for each compound. Recovery experiments used fortified aqueous samples at 1, 5, 10, 50, 100 and 250 μg/mL with five replicates per level. For most alkaloids, recoveries ranged from 71.7% to 107.4% with RSDs between 1.1% and 9.8%. Harmalol showed lower recoveries (45.0–58.4%) at the 1 and 5 μg/mL fortification levels with RSDs of 2.3–5.5% at those levels. Repeatability (intra-assay) assessments at 100 μg/mL gave RSDs for retention times lower than 0.8%. Intermediate precision over three alternate days (n = 5 per day) reported RSD < 0.3% for all compounds studied, indicating good precision. Limits of detection (LOD) and quantification (LOQ) were calculated using signal-to-noise ratios of 3 and 10, respectively. Reported LODs ranged from 6.8 to 18.8 μg/mL (compound-specific values: tryptamine 6.8 μg/mL, DMT 18.8 μg/mL, harmalol 13.8 μg/mL, harmine 11.6 μg/mL, harmaline 6.8 μg/mL, tetrahydroharmine 17.5 μg/mL). LOQs ranged from 20.6 to 57.1 μg/mL. Application to the 20 ayahuasca samples from the cooking process showed substantial concentrations of the target alkaloids. Reported concentrations for harmine, harmaline, tetrahydroharmine, harmalol and DMT ranged from 0.3 to 36.7 g/L across samples. The four ‘‘apuro’’ fractions (samples 17–20) exhibited increased concentrations, which the authors attribute to recombining fractions 1–16 into the apuro and thereby concentrating DMT and β-carbolines. Tryptamine was not detected in the analysed samples; the authors suggest this absence may reflect complete bioconversion of tryptamine to DMT in the plant material. Structural characterisation of the isolated tetrahydroharmine was consistent with literature values: melting point approximately 199°C, GC–MS spectrum showing molecular ion at m/z 216 and a base peak at m/z 201, and corroborative 1H and 13C NMR data.
Santos and colleagues conclude that the SPE on silica coupled with HPLC-UV/DAD provides acceptable accuracy, precision and selectivity for determination of major DMT and β-carboline alkaloids in ayahuasca prepared by the studied religious group. The authors interpret the marked increases in alkaloid concentrations in the apuro fractions as a consequence of combining earlier cooking fractions, which concentrates active constituents. The study situates its method as a simple, low-cost option suitable for chemical characterisation of ritual ayahuasca and for use in contexts where documentation of alkaloid content is valuable, such as forensic investigation of adverse events, monitoring of ceremonial preparations, or research on pharmacology and toxicology. The absence of detectable tryptamine is discussed as plausibly reflecting plant bioconversion to DMT rather than analytical failure. The authors acknowledge method limitations implicitly through the validation data: harmalol showed lower recovery at low fortification levels, and LOD/LOQ values are compound-dependent. The extracted text contains some inconsistent reporting of concentration units for standards and calibration ranges, which the paper does not explicitly reconcile in the available extraction. The investigators emphasise that analytical characterisation is important given growing use of ayahuasca in religious, recreational and clinical research contexts, and that reliable measurement methods can inform safety assessments and multidisciplinary studies.
The authors state that the proposed SPE–HPLC-UV/DAD method demonstrates acceptable accuracy, precision and selectivity for measuring DMT and major β-carbolines in ayahuasca samples from the described cooking process. They present the method as suitable for routine analysis of these alkaloids in ritual ayahuasca preparations.
A yahuasca is a beverage that is mostly prepared using a decoction of two plants: the leaves of chacrona (Psychotria viridis), which contain the psychoactive/ hallucinogenic N,N-dimethyltryptamine (DMT); and sections of the stem of the jagube vine (Banisteriopsis caapi), which provide three major monoamine oxidase (MAO) inhibitors (i.e., harmine, harmaline, and tetrahydroharmine). The combination of the leaves of P. viridis (containing DMT) and the reversible MAO inhibitors renders the DMT orally active. Brazilian legislation, based on a constitutional right to freedom of religion, permits the consumption of ayahuasca within a religious context. Norms concerning the use of ayahuasca in Brazil for religious purposes were published by the Brazilian National Council on Drug Policies in January 2010; they prohibit the marketing of ayahuasca, its therapeutic use, ayahuasca tourism, and its use with illicit drugs. Under this resolution, consumption is permitted in a religious context. The same document also emphasized the need for more multidisciplinary areas of research of ayahuasca. Studies involving the chemical characterization of these plants, together with the development of analytical techniques for the measurement of tryptamines and β-carbolines in plant matrixes and in ritual beverages are essential, given the current expansion in their use for religious, recreational, and clinical research purposes. The need for an in-depth approach toward analytical characterization becomes obvious in cases of untoward effects or even fatal intoxications that can, e.g., arise from ill-informed combinations of plant products with other psychoactive substances. The present work reports a simple method for the determination of major alkaloid components, namely DMT, tryptamine, harmine, harmaline, harmalol, and tetrahydroharmine, in ayahuasca beverage samples collected from a religious group located in the municipality of Fortaleza, state of Ceará, Brazil, using SPE on silica cartridges and LC with UV diode-array detection (DAD).
Tryptamine, harmalol, harmine, and harmaline were purchased from Sigma-Aldrich (St. Louis, MO). DMT was prepared as previously described. Tetrahydroharmine was prepared according to the method described by Begum. The HPLC grade solvents acetonitrile and methanol were purchased from Tedia (Fairfield, OH). Research grade Florisil (80-100 mesh) was supplied from Sigma-Aldrich (Büchs, Switzerland), and silica-gel 60 (70-230 mesh) was from Merck (Darmstadt, Germany). C 18 -bonded silica (50 μm) SPE cartridges (6 mL, 500 mg) were obtained from Phenomenex (Torrance, CA).
Ayahuasca samples were prepared from specimens of B. caapi stems and P. viridis leaves by members of a religious group located in the municipality of Fortaleza, state of Ceará, Brazil. At the start of the cooking process, plant materials and water were at room temperature. Aqueous decoctions of each plant were prepared from freshly harvested material. The cooking process for obtaining ayahuasca tea takes 20 h. At each cooking hour a sample is collected and stored. This is the first step in the cooking process, which produces a total of 16 samples (1-16 samples, Figure). The 16 samples that have been stored are subdivided into five sets (1-3, 4-6, 7-9, 10-13, and 14-16 samples), from which a sample is taken from each of the five sets. These five samples are combined and are subjected to a new cooking process which takes 4 h. This is the second step of the cooking process that produces another four samples, which are also obtained at each hour of this second step of the cooking process (17-20 samples, Figure). These four samples are called apuro or ayahuasca tea. A total of 20 aqueous dark brown liquid samples were separately bottled and shipped to the Department of Chemistry of the Federal University of Sergipe. Before analysis, ayahuasca samples were taken out of the refrigerator and maintained for 30 min to reach room temperature. Samples 1-16 were analyzed directly by the SPE method, whereas samples 17-20 were initially diluted (1:100) due to their viscous characteristics that could clog the filter of the SPE cartridges.
UV-Vis molecular absorption spectrometry.-A Cary 100 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA) was used for measurements (in triplicate, at 326 nm) of five harmine standard solutions at concentrations in the range of 0.01-0.1 mmol/L to generate an analytical curve (6). The percentage of tetrahydroharmine was determined using a solution concentration of 0.1 mmol/L. The measurements were performed at a wavelength of 296 nm. NMR spectroscopy.-Proton NMR ( 1 H NMR) spectra were recorded at 400 MHz, using a CDCl 3 solution. Chemical shifts were referenced to the residual solvent peak or to tetramethylsilane as an external reference. The data were reported in terms of the chemical shift (δ, in parts per million), multiplicity, coupling constant (J, in hertz), and integrated intensity. 13 C NMR spectra were recorded at 100 MHz (using a CDCl 3 solution). The chemical shifts were referred to the CDCl 3 solvent peak. The multiplicity of a particular signal was indicated as s (singlet), d (doublet), qdd (quartet of doublet of doublets), dd (doublet of doublets), ddd (doublet of doublet of doublets) and dddd (doublet of doublet of doublet of doublets). The 1 H NMR and 13 C NMR spectra were measured using a Bruker Spectrospin Avance DPX-400 spectrometer (Fällanden, Switzerland). GC-MS.-GC/MS analyses were performed with a Shimadzu GC-2010 Plus gas chromatograph (Shimadzu, Kyoto, Japan) coupled to a Shimadzu TQ8040 mass spectrometer. Samples were injected into a split/splitless injector using an autosampler. A Supelco SBL-5ms capillary column (30 m × 0.25 mm id, 0.25 μm film thickness) was used. The mass spectrometer was operated in electron ionization (EI) mode at 70 eV. The computer that controlled the system also contained an EI-MS library. The mass spectrometer was calibrated with perfluorotributylamine. Helium (99.999%), at a flow rate of 1.2 mL/min, was used as the carrier gas. The injector temperature was 250°C. The oven temperature program was as follows: initial temperature of 80°C for 1 min, followed by a ramp to 280°C at 10°C/min and a hold at 280°C for 5 min. The mass spectrometer was operated in SCAN mode, and the temperatures of the transfer line and ionization source were set at 280 and 250°C, respectively. The total run time was 26 min.
Individual stock standard solutions of the indole alkaloids (harmine, harmaline, tetrahydroharmine, harmalol, tryptamine, and DMT) were prepared by dissolving the reference standard in methanol to obtain a concentration of 2 mg/mL. The stock solutions were stored at -18°C. Mixed working standard solutions were prepared at various concentrations by diluting the stock solutions in methanol as required. This solution was prepared monthly to obtain the analytical curves (0.001, 0.005, 0.01, 0.025, 0.05, 0.1, 0.15, 0.02, 0.025 and 0.30 mg/mL) and the optimum experimental conditions.
A silica-based cartridge was preconditioned with 4 mL methanol, followed by 4 mL aqueous hydrochloric acid (1 mol/L). The cartridge was placed on top of a vacuum block. An analytical aliquot of 20 mL ayahuasca sample (pH 8, with 0.01 mol/L NaOH solution) was transferred to the cartridge at a flow rate of around 0.5 mL/min, and the solid phase was allowed to dry for 5 min. The alkaloids were eluted with 3.0 mL acidified methanol (pH 3). The eluent was collected into a round-bottom flask and concentrated using a gentle stream of nitrogen to a volume of 1 mL. A 20 μL aliquot was analyzed by HPLC-UV/DAD.
The analysis of the alkaloid components of ayahuasca in the SPE extracts was carried out using an HPLC system (Shimadzu) equipped with a binary solvent pump (LC-20AT), a DGU-20A3 degasser, a Sil-20A autosampler with injection volume set at 20 μL, and an SPD-M20A UV/DAD. Data acquisition and processing were performed with LCsolution software (LabSolutions Series WorkStation v. 2.0). The chromatographic separation was performed on a reversed-phase Zorbax 5 μ Eclipse Plus C 8 analytical column (150 × 4.6 mm id, 5 μm particle size), protected by a guard cartridge (4.6 × 12.5 mm, 5 μm), both obtained from Agilent Technologies (Palo Alto, CA). The elution was carried out with a binary gradient composed of 0.1% formic acid in water (A) and 0.1% formic acid in methanol (B), delivered at ambient temperature with a flow rate of 1.5 mL/min. The initial mobile phase composition was 5% B held constant for 2 min, followed by a linear gradient to 50% B at 30 min, and then a return to the initial conditions in 10 min. Spectral data for all peaks were collected in the range of 190-800 nm. The identification of compounds present in the ayahuasca samples was achieved by comparing the characteristics of the DAD spectra and the retention times with those of the standard compounds. The injection volume was 20 μL.
The best conditions for analysis of the investigated alkaloids were provided by gradient elution using 0.1% formic acid in methanol-0.1% formic acid in water at an initial proportion of 5 + 95 (v/v) maintained for 2 min, and then ramped to 50 + 50 (v/v) at 30 min. Alkaloid components are compounds with high molar absorptivity in the UV-Vis region. They display their highest UV absorption maxima at 278 nm for DMT and tryptamine, at 246 nm for harmine, at 320 nm for tetrahydroharmine, and at 372 for harmalol and harmaline. For this reason, HPLC with DAD could be one of the methods used in their determination.
Melting point.-The melting point of tetrahydroharmine recrystallized was 199.3 ± 0.2°C (n = 3), which was compatible with the literature value (199°C; 5). GC-MS analysis.-An aliquot of the synthesized compound was analyzed by GC-MS in full scan mode and showed a prominent peak at 18.600 min. To confirm the identity of the compound, the spectrum of the peak was compared with the spectra available in the Wiley EI-MS library. There was 89% similarity between the measured spectra and the library spectra, and with a molecular ion peak at m/z 216 and a base peak at m/z 201. These and other peaks in the tetrahydroharmine spectrum were similar to the spectrum provided in the Wiley Registry of Mass Spectral Data. NMR
To select the optimal experimental conditions for extraction, an optimization strategy was used to assess the influence of the main factors on the SPE procedure. Silica and methanol were chosen over the other solid phase and solvent, respectively, for producing the cleanest chromatographic profiles with lower baselines. Indole alkaloids are basic compounds, and the pH of the solution was expected to have effect on their recovery. Therefore, before the elution step, the adsorbent was washed with 4 mL methanol and 4 mL aqueous HCl (1 mol/L). The washing of the adsorbent with this solvent composition was found to decrease ion suppression and, thus, improve the detectability of the compounds. The compounds were dissolved in aqueous solution at a concentration of 100 μg/mL, the pH was adjusted to 8, and the sorbent was eluted with 4 mL acidified methanol. Average recoveries of the analytes of 71.2-82.5% were achieved with acidified methanol. Therefore, acidified methanol was chosen as the eluent.
Linearity.-Calibration curves were linear over the specified range (0.001-3.0 mg/L). The linear regression equations and coefficients of correlation were as follows: harmine (y = 7303x + 58996, r = 0.9905); harmaline (y = 42766x + 298629, r = 0.9904); tetrahydroharmine (y = 41180x + 462149, r = 0.9902); harmalol (y = 19228x + 71563, r = 0.9929); tryptamine (y = 15839x + 96013, r = 0.9943); and DMT (y = 9753x -169.9, r = 0.9919), where y and x = the peak area and the corresponding calibration concentration, respectively. Recovery.-Aqueous samples were fortified at 1, 5, 10, 50, 100, and 250 μg/mL. Five replicates spiked at each fortification level were assayed. The recovery obtained for alkaloids ranged from 71.7 to 107.4%, and the obtained RSDs ranged from 1.1 to 9.8%, with the exception of harmalol, for which the recovery ranged from 45.0 to 58.4%, with RSDs ranging from 2.3 to 5.5% for 1 and 5 μg/mL. Repeatability.-The repeatability (intra-assay precision) was measured by comparing the SD of the recovery percentages of spiked aqueous samples at one concentration level (100 μg/mL) run the same day. The samples were injected five times using an autoinjector, and the RSD values obtained for the retention times were lower than 0.8%. The intermediate precision (between-day precision) was determined by analyzing spiked aqueous samples at one concentration level (100 μg/mL) for 3 alternate days. Replicate (n = 5) samples were run, and the RSD values were calculated for the alkaloid compounds. The method was found to be precise (RSD < 0.3%) for all compounds studied. LOD and LOQ.-LOD and LOQ were calculated at an S/N of 3 and 10, respectively. LODs ranged from 6.8 to 18.8 μg/mL (7.5, 18.8, 13.8, 11.6, 6.8, and 17.5 μg/mL for tryptamine, DMT, harmalol, harmine, harmaline, and tetrahydroharmine, respectively). The LOQ values ranged from 20.6 to 57.1 μg/mL.
Twenty samples were collected from an ayahuasca preparation process from a religious group of the municipality of Fortaleza, Brazil, and analyzed by the developed SPE method combined with HPLC-UV/DAD. The harmine, harmaline, tetrahydroharmine, harmalol, and DMT concentrations in the samples ranged from 0.3 to 36.7 g/L (Figure). The increase observed for fractions 17-20 can be explained by the addition of all fractions (1-16) to form the apuro (ayahuasca tea) fractions, which contain significant concentrations of DMT and β-carbolines. It is suggested that the absence of tryptamine alkaloid is related to the complete bioconversion to DMT in plants.
The results demonstrate that the accuracy, precision, and selectivity of the proposed method are acceptable for the determination of these alkaloids in ayahuasca samples from an ayahuasca cooking preparation process from a religious group of the municipality of Fortaleza, Brazil.
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