qNMR: An applicable method for the determination of dimethyltryptamine in ayahuasca, a psychoactive plant preparation

Article abstract of DOI:10.1016/j.phytol.2009.12.004

Ayahuasca is an Amazonian plant beverage obtained by infusing the pounded stems of Banisteriopsis caapi in combination with the leaves of Psychotria viridis. P. viridis contains the psychedelic indole N,N-dimethyltryptamine (DMT). This association has a wide range of use in religious rituals around the world. In the present work, an easy, fast and non-destructive method by Nuclear Magnetic Resonance of proton (¹H NMR) for quantification of DMT in ayahuasca samples was developed and validated. 2,5-Dimethoxybenzaldehyde (DMBO) was used as internal standard (IS). For this purpose, the area ratios produced by protons of DMT (N(CH₃)₂) at 2.70 ppm, singlet, (6H) and for DMBO (Ar(OCH₃)₂) at 3.80 and 3.89 ppm, doublet, (6H) were used for quantification. The lower limit of quantification (LLOQ) was 12.5 μg/mL and a good intra-assay precision was also obtained (relative standard deviation < 5.1%). The present ¹H NMR method is not time consuming and can be readily applied to monitor this tryptamine in plant preparations. We believe that qNMR can be used for identification and quantification of many plant-based products and metabolites with important advantages, while comparing with other analytical techniques.

Full text of DOI:10.1016/j.phytol.2009.12.004

Phytochemistry Letters 3 (2010) 79–83
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
qNMR: An applicable method for the determination of dimethyltryptamine
in ayahuasca, a psychoactive plant preparation
Sidnei Moura *, Felipe Garcia Carvalho, Carolina Dizioli Rodrigues de Oliveira, Ernani Pinto,
Mauricio Yonamine
Department of Clinical and Toxicological Analysis, Faculty of Pharmaceutical Sciences, University of S a˜ o Paulo, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history:
Ayahuasca is an Amazonian plant beverage obtained by infusing the pounded stems of Banisteriopsis
caapi in combination with the leaves of Psychotria viridis. P. viridis contains the psychedelic indole N,N-
dimethyltryptamine (DMT). This association has a wide range of use in religious rituals around the
world. In the present work, an easy, fast and non-destructive method by Nuclear Magnetic Resonance of
Received 9 November 2009
Received in revised form 28 December 2009
Accepted 29 December 2009
Available online 6 January 2010
1
proton ( H NMR) for quantification of DMT in ayahuasca samples was developed and validated. 2,5-
Dimethoxybenzaldehyde (DMBO) was used as internal standard (IS). For this purpose, the area ratios
Keywords:
produced by protons of DMT (N(CH
3.89 ppm, doublet, (6H) were used for quantification. The lower limit of quantification (LLOQ) was
2.5 g/mL and a good intra-assay precision was also obtained (relative standard deviation < 5.1%). The
3 2 3 2
) ) at 2.70 ppm, singlet, (6H) and for DMBO (Ar(OCH ) ) at 3.80 and
N,N-Dimethyltryptamine (DMT)
1
H NMR quantification
1
m
Ayahuasca
1
present H NMR method is not time consuming and can be readily applied to monitor this tryptamine in
plant preparations. We believe that qNMR can be used for identification and quantification of many
plant-based products and metabolites with important advantages, while comparing with other
analytical techniques.
Psychotria viridis (Rubiaceae)
ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
1
. Introduction
alkaloids is the basis of the psychotropic action of ayahuasca
(McKenna, 2004).
Recently, epidemiologic data have alerted scientists around the
In Brazil, other species also contains this tryptamine in their
world about the consumption of a new drink in Europe and the
USA, the ‘‘Santo Daime’s’’ tea. This is also known as Hoasca,
Ayahuasca, Daime, Yaj e´ , Natema and Vegetal. It is a hallucinogenic
drink and it is prepared using two different plants: the stems from
Banisteriopsis caapi and the leaves from Psycotria viridis (Callaway
et al., 1999). It was originally prepared for magic-religious
ceremonies in the Amazon basin (Riba et al., 2003).
P. viridis contains a potent psychedelic agent, N,N-dimethyl-
tryptamine (DMT), an indole alkaloid, which is structurally similar
to the monoamine neurotransmitter serotonin and acts on
receptors 5-HT2A/2C (O’Brien, 1996). Nevertheless, the tea has
leaves, such as Psychotria cartagenensis and Diplopterys cabrerana
(McKenna et al., 1984). In the United States, appreciable
concentrations of this compound are found in extracts of Phalaris
(Phalaris arundinacea, Phalaris tuberosa and Phalaris aquatica).
There are many other plant sources of this active compound in the
region of South America. It can be extracted from seeds of
Anadenanthera peregina or bark of stems of Virola spp. Mimosa
hostilis, Lespedeza bicolor, Diplopterys cabrerana (syn. Banisteriopsis
rusbyana) and Psychotria viridis (Thompson et al., 1987).
In the last years, the use of ayahuasca has spread outside South
America and some religious groups have established on United
States and some European countries (Halpern, 2004). A relative
small number of analytical procedures have been reported for
evaluation of this compound, which make use of liquid (Callaway
et al., 1996) or gas (Gambelunghe et al., 2008; Pires et al., 2009)
chromatographic methods and capillary electrophoresis (Huhn
et al., 2005).
another active compound, the b-carbolines, which is represented
by harmine, harmaline and tetrahydroharmine. They have capacity
to inhibit MAO-A, enabling DMT to reach its site of action in the
central nervous system. The synergistic interaction of these
The aim of this study was the implementation of an alternative
method to detect and quantify DMT by Nuclear Magnetic
Resonance (NMR) with some advantages in relation of chro-
matographic methods generally used for the determination of
*
Corresponding author at: Department of Clinical and Toxicological Analysis,
Faculty of Pharmaceutical Sciences, University of S a˜ o Paulo, 558 B13B, Prof. Lineu
Prestes Avenue, 05508-900, S a˜ o Paulo, SP, Brazil. Tel.: +55 11 30912192;
fax: +55 11 30911555.
1
E-mail address: smoura@usp.br (S. Moura).
plant metabolites. Therefore, a procedure by H NMR was
1
874-3900/$ – see front matter ß 2010 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.phytol.2009.12.004

8
0
S. Moura et al. / Phytochemistry Letters 3 (2010) 79–83
developed and validated. This method can be useful to estimate
administered doses in animals and humans for further pharmaco-
logical and toxicological investigations of ayahuasca. Like demon-
strated in this paper, it is possible to use the qNMR concept for the
analyses of plant-based products with reproducibility.
2
. Results and discussion
The application of qNMR concept for natural products was
described (Pauli, 2001). This technique can offer some major
advantages in comparison with chromatographic methods. Some
information about chemical structure can be obtained at the same
time while quantitative measurement is performed, while only in
chromatographic hyphenated techniques (such as GC–MS or LC–
MS) this feature can be provided. This is particularly true for the
1
measurement of H signals, especially when dealing with analogues.
Additionally, a further advantage of quantitative procedures for
NMR is that there is no necessity for clean-up and derivatization
steps. The non-destructive nature of this technique make possible
that samples can be kept for future analyses and/or confirmations if
necessary. However, sometimes the higher quantification limit
might be a problem and it depends on each case. A complete review
about qNMR comparing with other analytical methods was recently
written (Holmes et al., 2006).
1
Fig. 1. H NMR spectra of a water sample containing N,N-dimethyltryptamine and
internal standard (2,4-dimethoxybenzaldehyde). Signals relative to protons of DMT
and DMBO used for quantification are indicated, beyond its chemical shift and
signal integration.
constituents of ayahuasca was found in the analysis. This
concentration is in accordance with the result obtained by the
gas chromatographic method previously published by our research
group (Pires et al., 2009) for similar samples. McKenna (2004) also
determined concentrations of about 500
P. viridis preparation depending on its origin.
1
In this study, dimethyltryptamine was quantified by H NMR
after a treatment with a simple liquid–liquid extraction. The
extraction conditions evaluated for the ayahuasca tea indicated
that hexane was better in relation to chloroform because the
second one carried out endogenous compounds that interfered
with the analyses. Using hexane, the method was not affected by
the constituents of the extract. This could be proved by the
application of the method in a real ayahuasca sample.
mg/mL of this molecule in
1
Quantitative analyses by the H NMR technique need some
precautions aside from the necessity to adequate the choice of
specific signals in the spectrum and NMR parameters. The delay for
total relaxation of spins must be observed to be within 0.7% of the
1
Different compounds can be directly compared by H NMR
through integration ratios of their signals. In DMT case we have
chosen the highest signal level that was representative of six
1 1
maximum that occurs after a time 5T , where T is the spin-lattice
relaxation time for a given nucleus. In this procedure, with pulse of
308, was used 10.0 s, which is considered enough to completely
relax all analyzed protons (Saito et al., 2004). Also, signal-to-noise
ratio (S/N) effect has a significant influence in qNMR methods can
lead to systematic mistakes. This effect, which is influenced by
variations of pulse flip angle, number of scans and the line
broadening (lb) can affect strongly the precision of the measure-
ment. The S/N of at least 150 is required for the target uncertainty
of <1% (Malz and Jancke, 2005).
3 2
hydrogen’s (N(CH ) ), with chemical shift (d) of 2.70 ppm. This
signal, that represent six hydrogens, show a good intensity which
lead to increase the sensitivity of the method. The signals used for
quantification of both analyte and internal standard do not suffer
any interference of possible residual proton of organic solvent
(CHCl
3
at 7.26 ppm).
Dimethoxybenzaldehyde (DMBO) was chosen as internal
standard (IS), which was used for this purpose because its
spectrum contains signals that do not overlap with the signals
of analyzed psychoactive compound. Besides, it presents other
advantages: low cost, easy availability, it is soluble in the same
solvent of the extraction method and it is not volatile. The internal
standard is also stable in the standardized conditions and did not
react with other components of the ayahuasca. This was confirmed
for the proportional responses between integration values of
hydrogen of aldehyde (1H) and dimethoxy (6H) present in DMBO
in the analysis of the real sample. Thus, both methoxy signals with
The data processing and integration of the spectra are the
potentially most subjective aspect of NMR quantification. Burton
d
of 3.80 and 3.89 ppm were used for quantification. In Fig. 1, it is
possible to see the characteristic spectra obtained with the
practical use of this method to the analysis of a water sample
spiked with dimethyltryptamine and internal standard. In order to
take better quality to representative figures showed here, the
1
spectra were processed with MestRec 4.7.4.0.
Considering that the original goal was to implement a new and
easy method that could be employed to analyze DMT in
preparations of P. viridis, we selected some ayahuasca samples
1
for analyses. In Fig. 2, it can be observed the H NMR obtained for
ayahuasca tea sample in which internal standard was added. In this
specific case, were used 3 mL of tea and 500
application of the method, a concentration of 400
dimethyltryptamine was obtained. No interference of other
m
g of IS. With the
1
Fig. 2. H NMR spectra of an ayahuasca tea sample submitted to the method where
m
g/mL of N,N-
internal standard was added. Signals referents of protons of DMT and internal
standard used for quantification are indicated.

S. Moura et al. / Phytochemistry Letters 3 (2010) 79–83
81
and colleagues, after comparison between operators for manual
integration, reported an error less than 1% (Burton et al., 2005). The
errors in case of automatic integration should not be higher that
preparation samples. In addition, with the optimization of
instrumental parameters, qNMR may be adapted for the analysis
of other constituents in plant-based products.
1
%. However, with the evolution of software, equipment and
adequate spectral processing such as zero-filling, FID weighting
and baseline correction, the NMR signal integration cannot be
considered as a principal source of systematic error.
3. Materials and methods
3.1. Reagents
The parameters of validation of quantitative NMR analyses for
this compound in ayahuasca using internal standard showed good
results. The obtained data are an indicative of robustness and
reproducibility of the method in instrumental optimized condi-
tions. The calibration curve was linear over the specified range
The internal standard, 2,5-dimethoxybenzaldehyde (DMBO),
and the analytical solvent used for extraction (hexane) were
purchased from Sigma–Aldrich Chem. Co. (Milwaukee, WI, USA).
The deuterated solvent (chloroform, CDCl ) was purchased by
3
(
25–1000 mg/L). The linear regression equation and coefficient of
Cambridge Isotope Laboratories (Andover, MA, EUA). N,N-Dimeth-
2
correlation were: Y = 8.4713X ꢀ 0.1019, r = 0.999, where Y and X
yltryptamine is not founded in commercial forms. For this reason,
it was obtained from synthesis. The purity of dimethyltryptamine
obtained after the synthetic reaction was >99% with regard to
represents the relationship between the peak signal ratio
(
compound/internal standard) and the corresponding calibration
1
13
concentrations, respectively. In Fig. 3, it is possible to examine the
response for three different concentrations of DMT (25, 50 and
analysis (NMR H, C and HPLC–ESI-MS). The purity of DMBO was
1
>99.9% in accordance with Sigma–Aldrich report.
1
00 mg/L) used for calibration curve are showed.
The intra-assay precision varied slightly indicating that the
3.2. Synthesis of N,N-dimethyltryptamine
reproducibility is acceptable over the studied concentration range
(
(
RSD < 5.1%). Good sensitivity was also obtained for the analyses
LOD and LLOQ = 12.5 mg/L). During the experiments to obtain
The synthesis of dimethyltryptamine was performed according
to a modified procedure based on the selective dimethylation
method (Giumanini et al., 1980), Scheme 1. Sodium borohydride
(57 mg, 1.5 mmol) was slowly added to a stirred solution of
tryptamine (160 mg, 1.0 mmol) in tetrahydrofuran at 0 8C. After-
wards, sulfuric acid (2 M, 5.0 mL) and an aqueous solution of
formaldehyde (40% v/v, 2.0 mL) were also added. This solution was
diluted with water and alkalinized with NaOH pellets until
reaching pH 14. The obtained product was extracted with diethyl
ether (3ꢁ 20 mL). The organic layer was dried with magnesium
sulfate, filtered and evaporated under vacuum. The product was
purified by means of a silica chromatographic column (eluted with
n-hexane/ethyl acetate 80:20) and recrystallization with n-
hexane/ethyl acetate (80:20). White crystals with a melting point
LOD and LLOQ, it was verified that lower concentrations than
2.5 mg/L, no signal was detected, so we considered this value as
1
being LOD and LLOQ, since the RSD at this concentration was better
than 20% (Armbrsuter and Pry, 2008). The recovery was 70% in
media for the three concentrations evaluated. The variation of 5%
was indicative of reproducibility of the optimized method for
sample extraction.
The concentration of psychoactive compound in tea can vary
considerably among plants and, consequently, the ayahuasca
prepared with them (McKenna et al., 1984). Therefore, depending
on every one of these factors, different ayahuasca preparations can
produce changeable intensity in psychotropic response.
In conclusion, the results described in this work demonstrate
that it is possible to analyze N,N-dimethyltryptamine, the
of 64 8C were obtained. The structure was confirmed on the basis of
1
the H NMR (300 MHz, CDCl , ppm):
3
3 2
d 2.70 (6H, s, N(CH ) ); 3.25
psychoactive compound presented in the preparation of P. viridis
2 2 3 2
(2H, t, J = 7.22, CH CH N(CH ) ); 3.27 (2H, t, J = 6.80,
1
(ayahuasca tea) by H NMR. The main advantages in comparison
2 2 3 2
CH CH N(CH ) ); 6.96 (1H, s, C 55 C–H); 7.2–7.5 (4H, m, Ph); and
+
+
with chromatographic methods can be summarized: (i) to be very
fast (the NMR measurement can be performed in less than 30 s for
sample), do not need extra-time for column equilibration; (ii) do
not need extra-reaction (derivatization); (iii) the sample is not
destroyed; (iv) give information about chemical structure in the
same analyses; (v) have good validation parameters; and (vi) easy
sample preparation because its good specificity. This technique can
be an efficient tool to monitor the presence of this drug in plant
LC–ESI-MS (m/z) data: 189 [M+H] ; 145 [MꢀN(CH ) +H] ; 117
3
2
+
[Mꢀ(CH ) N(CH ) +H] .
2
2
3 2
3.3. Preparation of standard solutions
Working solutions of DMT at a concentration of 1.0 mg/mL were
prepared in chloroform with volumetric glassware. The internal
standard solutions were prepared at a concentration of 1.0 mg/mL
in hexane with volumetric glassware. Stock solutions were stored
at 4 8C when it is not in use.
3.4. Instrumentation
1
Proton NMR experiments were carried out using Bruker
(
Karls-ruhe, Germany) DPX 300 spectrometer operating at 300.13.
The instrument was equipped with a multinuclear 5 mm probe
1
Fig. 3. Three typical H NMR used for the determination of linearity with different
concentration of dimethyltryptamine: (A) 25
00 g/mL.
mg/mL; (B) 50 mg/mL; and (C)
1
m
Scheme 1. Synthesis of N,N-dimethyltryptamine.

8
2
S. Moura et al. / Phytochemistry Letters 3 (2010) 79–83
operating in a temperature of 298 K. The chemical shifts were
reported in ppm with tetramethylsilane (TMS) add in solvent
3.8. Linearity
(
CDCl
parameters optimized for qNMR experiments were as follows:
single pulse with 308, preacquisition delay of 5 s, acquisition time
.4 s, relaxation delay 10 s (in order to ensure that all protons were
totally relaxed), window 10 ppm, 32 k data points after16 scans
AQ = 5.30 s), yielding
3
) as internal standard (0.0 ppm). The typical acquisition
The study of linearity was performed by the analyses of water
samples in triplicate submitted to the method spiked with the
following concentrations for DMT: 25, 50, 100, 200, 500 and
1000 mg/L.
m
3
(
a
digital resolution of ꢂ0.3 Hz. Data
3.9. Recovery
1
processing was performed offline with Top Spin (Bruker , Karls-
ruhe, Germany, 2006) program package. Digital filter was used and
each FID was multiplied by an exponential window function (lb
The efficiency of the liquid–liquid extraction method was
evaluated through the recovery studies that were performed by
preparing two sets of samples of each concentration. One of them
(set A), consisting of three concentrations of dimethyltryptamine
(75, 300 and 750 mg/L) was extracted using the method described
in Section 3.6 (processed). The analyses were performed in six
replicates for each concentration. The other one (set B) also
consisted on six replicates of each concentration (75, 300 and
750 mg/L). However, the extract was spiked with standard
solutions of DMT before drying under nitrogen stream (unpro-
cessed). To both sets (A and B), the internal standard was added
prior to the extraction of the matrix. The absolute recovery was
evaluated through the comparison of the mean response of
extracted samples spiked before the extraction (processed) and the
response of the extracted blank matrix to which analytes had been
added at the same concentration just before the drying step
0
.3 Hz) before Fourier transformation. Phase correction and base
line were done manually using two expanded regions at both ends
after performing a standard PH correction of the full spectrum.
Automatic integration for selected signals was adopted through-
out.
The DMT content of the solution was determined after
integration of well separated signals of N(CH
3
) at 2.70 ppm
singlet, 3H) and for 2,5-dimethoxybenzaldehyde Ph(OCH at
.80 and 3.89 ppm, (doublet, 6H). This choice was made taking into
(
3
3 2
)
consideration some important aspects: its chemical shift, the
absence or negligible (that does not affect the validation
parameters) overlapping, and their intensity, that lead to lower
limit of detection. The area ratios produced by protons of DMT and
internal standard were used for quantification.
(
unprocessed). The unprocessed response represented 100%
3.5. Plant material
recovery.
P. viridis used in ayahuasca extract was collected by some
3.10. Intra-assay precision
specimens cultivated at the Santo Ant oˆ nio farm in Ara c¸ oiaba da
0
00
0
00
Serra, S a˜ o Paulo state, Brazil (23831 48 S 47837 36 W). The
species were identified by Dr. Sigrid Luiza Jung-Menda c¸ olli from
the Agronomic Institute of Campinas (Department of Agriculture
and Supply, Government of S a˜ o Paulo) where exsiccates are
deposited under the code 48679. The leaves were collected in
January 2007, from the apical, intermediate and basal sections of
a bush of 6.5 feet high, cultivated under solar radiation. The
collected materials were blended to obtain a homogeneous
sample.
Precision defined as the relative standard deviation (RSD) was
determined by intra-assay. They were carried-out by analyzing
water samples spiked with DMT in the concentration of 75, 300
and 750 mg/L in six replicates.
3.11. Specificity
The specificity of the method was tested by analyzing eight
samples of different ayahuasca preparations, which was obtained
from a religious group settled in the city of Ara c¸ oiaba da Serra,
S a˜ o Paulo state, Brazil. The tea was prepared by the religious
group, boiling the leaves of Psychotria viridis together with the
stems of Banisteriopsis caapi. The chemical shifts of signals of
dimethyltryptamine and dimethoxybenzaldehyde were selected
taking into consideration the intensity of signals and to avoid
3.6. Sample extraction
Initially, an aliquot of ayahuasca sample was centrifuged at
1
0,000 ꢁ g for 5 min to separate the solid phase of the tea from the
1
aqueous phase. The latter was filtered by filter Millex HV (PVDF)
.45 m and only after this process the sample went to the stage of
0
m
the possibility of overlapping: DMT (
3.25 (CH CH N(CH ), 3.27 (CH CH N(CH
O), 3.89 (CH O), 7.1–7.3 (Ph), 10.4
d
ppm) 2.70 (N(CH
3 2
) ),
extraction. During the extraction, the following solutions were
added in a flask with 20 mL of capacity: 3 mL of the liquid phase of
filtered tea, 0.5 mL of solution of sodium hydroxide 5N, 0.5 mg of
2
2
3
)
2
2
2
3 2
)
), 6.80 (C 55 C–H),
7.2–7.5 (Ph); DMBO 3.80 (CH
(COH).
3
3
solid NaCl, 500
was shaken in an oscillating table for 20 min. The organic phase
was separated and 0.5 g of anhydrous MgSO was added. This
mg DMBO and 5 mL of hexane. Then the mixture
Acknowledgements
4
suspension was filtrated and, subsequently, the solvent was
evaporated under vacuum. The residue was reconstituted with
The authors thank FAPESP, CAPES and CNPq for financial
support. Thanks to Dr. Sigrid Luiza Jung-Menda c¸ olli for plant
identification and to Dra. Maria Ines Almeida Gon c¸ alves for NMR
analyses and support.
0
3
.6 mL of deuterated chloroform (CDCl ), placed in NMR 5 mm
Norell XR-55 tubes (Norell, Landisville, NJ, USA) in order to be read
in the NMR equipment.
3.7. Limit of detection (LOD) and lower limit of quantification (LLOQ)
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