Determination of N,N-dimethyltryptamine in beverages consumed in religious practices by headspace solid-phase microextraction followed by gas chromatography ion trap mass spectrometry.
ABSTRACT A novel analytical approach combining solid-phase microextraction (SPME)/gas chromatography ion trap mass spectrometry (GC-IT-MS) was developed for the detection and quantification N,N-dimethyltryptamine (DMT), a powerful psychoactive indole alkaloid present in a variety of South American indigenous beverages, such as ayahuasca and vinho da jurema. These particular plant products, often used within a religious context, are increasingly consumed throughout the world following an expansion of religious groups and the availability of plant material over the Internet and high street shops. The method described in the present study included the use of SPME in headspace mode combined GC-IT-MS and included the optimization of the SPME procedure using multivariate techniques. The method was performed with a polydimethylsiloxane/divinylbenzene (PDMS/DVB) fiber in headspace mode (70min at 60°C) which resulted in good precision (RSD<8.6%) and accuracy values (71-109%). Detection and quantification limits obtained for DMT were 0.78 and 9.5mgL(-1), respectively and good linearity (1.56-300mgL(-1), r(2)=0.9975) was also observed. In addition, the proposed method showed good robustness and allowed for the minimization of sample manipulation. Five jurema beverage samples were prepared in the laboratory in order to study the impact of temperature, pH and ethanol on the ability to extract DMT into solution. The developed method was then applied to the analysis of twelve real ayahuasca and vinho da jurema samples, obtained from Brazilian religious groups, which revealed DMT concentration levels between 0.10 and 1.81gL(-1).
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ABSTRACT: Plackett-Burman experimental design was applied for the robustness assessment of GC×GC-qMS (Comprehensive Two-Dimensional Gas Chromatography with Fast Quadrupolar Mass Spectrometric Detection) in quantitative and qualitative analysis of volatiles compounds from chocolate samples isolated by headspace solid-phase microextraction (HS-SPME). The influence of small changes around the nominal level of six factors deemed as important on peak areas (carrier gas flow rate, modulation period, temperature of ionic source, MS photomultiplier power, injector temperature and interface temperature) and of four factors considered as potentially influential on spectral quality (minimum and maximum limits of the scanned mass ranges, ions source temperature and photomultiplier power). The analytes selected for the study were 2,3,5-trimethylpyrazine, 2-octanone, octanal, 2-pentyl-furan, 2,3,5,6-tetramethylpyrazine, and 2-nonanone e nonanal. The factors pointed out as important on the robustness of the system were photomultiplier power for quantitative analysis and lower limit of mass scanning range for qualitative analysis.Talanta 11/2014; 129:303–308. · 3.51 Impact Factor
Determination of N,N-dimethyltryptamine in beverages consumed
in religious practices by headspace solid-phase microextraction followed
by gas chromatography ion trap mass spectrometry
Alain Gaujaca,b,c, Nicola Dempsterb, Sandro Navickiened, Simon D. Brandtb,
Jailson Bittencourt de Andradea,e,n
aUniversidade Federal da Bahia, Campus Universita ´rio de Ondina, 40170-115 Salvador-Ba, Brazil
bLiverpool John Moores University, School of Pharmacy and Biomolecular Sciences, L3 3AF, Liverpool, United Kingdom
cInstituto Federal de Educac -~ ao, Ciˆ encia e Tecnologia de Sergipe, Br 101, Km 96, 49100-000 S~ ao Cristo ´v~ ao-Se, Brazil
dUniversidade Federal de Sergipe, Av. Marechal Rondon, s/n, 49100-000 S~ ao Cristo ´v~ ao-Se, Brazil
eInstituto Nacional de Ciˆ encia e Tecnologia, Centro Interdisciplinar de Energia e Ambiente, Campus Universita ´rio de Ondina, 40170-115 Salvador-Ba, Brazil
a r t i c l e i n f o
Received 13 November 2012
Received in revised form
8 January 2013
Accepted 10 January 2013
Available online 1 February 2013
Vinho da jurema
a b s t r a c t
A novel analytical approach combining solid-phase microextraction (SPME)/gas chromatography ion
trap mass spectrometry (GC-IT-MS) was developed for the detection and quantification N,N-dimethyl-
tryptamine (DMT), a powerful psychoactive indole alkaloid present in a variety of South American
indigenous beverages, such as ayahuasca and vinho da jurema. These particular plant products, often
used within a religious context, are increasingly consumed throughout the world following an
expansion of religious groups and the availability of plant material over the Internet and high street
shops. The method described in the present study included the use of SPME in headspace mode
combined GC-IT-MS and included the optimization of the SPME procedure using multivariate
techniques. The method was performed with a polydimethylsiloxane/divinylbenzene (PDMS/DVB)
fiber in headspace mode (70 min at 60 1C) which resulted in good precision (RSDo8.6%) and accuracy
values (71–109%). Detection and quantification limits obtained for DMT were 0.78 and 9.5 mg L?1,
respectively and good linearity (1.56–300 mg L?1, r2¼0.9975) was also observed. In addition, the
proposed method showed good robustness and allowed for the minimization of sample manipulation.
Five jurema beverage samples were prepared in the laboratory in order to study the impact of
temperature, pH and ethanol on the ability to extract DMT into solution. The developed method was
then applied to the analysis of twelve real ayahuasca and vinho da jurema samples, obtained from
Brazilian religious groups, which revealed DMT concentration levels between 0.10 and 1.81 g L?1.
& 2013 Elsevier B.V. All rights reserved.
Ayahuasca is an indigenous brew produced as a decoction using
the leaves of chacrona (Psychotria viridis) and sections of the stem
of the yage vine (Banisteriopsis caapi) which originates from the
Amazon region. Vinho da jurema, commonly referred to as jurema
wine probably due to its visual similarity with the ordinary red
wine, is also an indigenous brew but prepared with both root and
stem barks of the jurema preta tree (Mimosa tenuiflora) from the arid
Northeast of Brazil . Both are used worldwide by various religious
groups and in neo-shamanic urban rituals. Brazilian legislation
permits the consumption of ayahuasca within a religious context
and may also include pregnant women and children provided
parental consent is given .
Previous studies based on gas chromatography (GC) have been
reported for the determination of DMT in ayahuasca matrices
which employed liquid–liquid extraction (LLE) [3–5] and solid
phase extraction (SPE) procedures . Sample preparation tech-
niques based on LLE can be manually intensive, often involve
large amounts of toxic organic solvents and may be time-
consuming , in addition to the risk of analyte loss. A reliable
alternative approach is the use of solid-phase microextraction
(SPME). SPME is a solvent-free sample preparation technique that
reduces sample preparation requirements and allows both extrac-
tion and concentration to be achieved in a single step .
There are several SPME applications in chemical analysis,
bioanalysis, food and environmental sciences, and a growing
number of publications describing pharmaceutical and medical
studies . The aim of this study was to evaluate the performance
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/talanta
0039-9140/$-see front matter & 2013 Elsevier B.V. All rights reserved.
nCorresponding author at: Universidade Federal da Bahia, Campus Universita ´rio
de Ondina, 40170-115 Salvador-Ba, Brazil. Tel.: þ55 7132836821;
fax: þ55 7132836805.
E-mail address: email@example.com (J.B.de. Andrade).
Talanta 106 (2013) 394–398
of a new solid phase microextraction method for the determina-
tion of DMT content in ayahuasca and vinho da jurema samples,
using multivariate optimization techniques as factorial design
and central composite design to identify the critical points
involved in the SPME extraction procedure. To the best of the
authors’ knowledge, this is the first study based on a SPME
procedure followed by gas chromatography mass spectrometry
(GC–MS) to determine DMT levels present in twelve real aya-
huasca and vinho da jurema samples, and five jurema beverage
samples prepared in the laboratory.
2.1. Standards, reagents and supplies
Methanol was of HPLC grade (Sigma-Aldrich, Gillingham,
Dorset, UK). Analytical grade anhydrous sodium hydroxide was
supplied by Merck (Darmstadt, Germany). Chemicals were used as
received and without further purification. A manual solid phase
microextraction (SPME) holder and fiber, coated with polydi-
methylsiloxane/divinylbenzene (65 mm, PDMS/DVB), was acquired
from Supelco (Bellefonte, PA, USA). Prior to use, the fiber was
conditioned in accordance with the manufacturer’s recommenda-
tions. A magnetic stirrer (Fisher Scientific, Pittsburg, PA, USA) was
used to homogenize beverage samples prior to SPME. N,N-
M. tenuiflora inner barks, using a previously published method
. This standard was used in a previous work to quantify DMT
in the barks of M. tenuiflora, by matrix solid-phase dispersion
followed by GC–MS . A total number of twelve ayahuasca and
jurema beverage samples were obtained from Brazilian religious
2.2. Preparation of DMT standard solutions and beverage samples
A working standard of 100 mg L?1DMT was prepared by
addition of 50 mL DMT standard stock solution (10 g L?1in
methanol) to 5 mL of 0.001 mol L?1NaOH solution (pH 11).
Standard solutions were prepared similarly with spiked stock
solution, leading to concentration standards between 0.78 to
400 mg L?1. These alkaline standard solutions were stored at
4 1C, and were stable for a period of at least 2 weeks. The real
beverage samples were prepared by either 10- or 25-fold dilution
of ayahuasca or vinho da jurema samples using a 0.001 mol L?1
NaOH aqueous solution. The final volume was 5 mL. 50 mL of
methanol was added and the pH was corrected to pH 11, using
small crystals of sodium hydroxide and a pH meter. These real
samples were analyzed immediately after preparation.
2.3. SPME procedure
A 5 mL beverage sample was added to a 9 mL headspace vial
and sealed with a cap containing a PTFE-faced silicone septum.
The vial was placed into an aluminum block to ensure uniform
heating at 60 1C. The sample was stirred (900 rpm) for 10 min to
ensure thermal equilibration. The SPME syringe needle pene-
trated the vial septum, and the PDMS/DVB (65 mm) fiber was then
lowered into the headspace located above the sample solution.
The extraction time was 70 min followed by removal of the SPME
fiber and insertion into the GC injection port for a desorption (and
cleaning) period of 5 min. Statistical procedures were performed
using Statistica 8.0 (StatSoft, Tulsa, USA).
2.4. GC-ion trap-MS system and operating conditions
GC–MS analysis was performed using a Varian 450-GC gas
chromatograph (Walnut Creek, CA, USA) coupled to a Varian 200-
MS ion trap (IT) mass spectrometer. The 1177 injector was
operated in splitless mode for 60 s and heated at 250 1C. A
straight SPME liner (L?o.d.?i.d., 105?2.75?0.75 mm) was
used for sample introduction. The system was operated by the
Saturn GC/MS Workstation, version 6.9. Separation was carried
out on a Supelco SLB-5 ms capillary column (30 m?0.25 mm i.d.,
0.25 mm film thickness) purchased from Supelco (Bellefonte, PA,
USA). Helium (purity 99.995%) was employed as carrier gas at a
constant column flow of 1.0 mL min?1. The GC oven temperature
was programmed from 50 1C (held for 3.5 min) to 280 1C at
20 1C min?1(held for 10 min). The ion trap mass spectrometer
was operated in the electron ionization (EI) mode. Manifold, ion
trap, ion source and transfer line temperatures were maintained
at 80, 220, 300 and 300 1C, respectively. Helium was also used as
damping gas at a flow of 0.8 mL min?1. In the full scan mode the
mass range was varied from m/z 40 to 400 at 0.6 s scan?1. The
identification of DMT in beverages (GC retention time and mass
spectral comparison) was verified with reference material.
For quantification purposes the scan range was restricted to m/z
57–59 which reflected the dominating abundance of the m/z 58
iminium ion in the mass spectrum of DMT.
3. Results and discussion
3.1. Optimization of the SPME method
Initially, a sample of herbal beverage was used to determine
the optimum sample volume (5 mL), desorption time (5 min)
and pH value (pH 11), respectively, which were carried out by
univariate analyses. The PDMS/DVB fiber was well suited for
analyzing volatile and semi-volatile compounds of medium
polarity. In order to select the optimal experimental conditions
for extraction, a multivariate optimization strategy was employed
to assess the influence of the main factors on the SPME procedure.
Several tests were carried out in order to select the factors and
the domain to be considered in the multivariate experimental
approach to maximize the yield of DMT extracted from beverages,
and to obtain a good precision for the method. The factors
included in the 23factorial design (Table 1) were temperature
(T), equilibrium time (tEQU) and extraction time (tEXT), respec-
tively. The relationship between each investigated variable and
impact of possible cross effects on DMT signal response was
determined by using the Pareto graph (Fig. 1). Analysis of the
Pareto graph indicated that, within the studied domain, the
impact of temperature and extraction time was significant. The
temperature was fixed at 60 1C. In order to find the critical factors
of the sample preparation method, a response surface technique
was employed as a central composite design. Although equilibra-
tion time factor did not appear to show any significant influence
in the domain previous studied (15–25 min), it was decided to
include this in the central composite design with a new domain,
ranging between 5 and 15 min. The extraction time was also
Factors and levels for 23factorial design.
FactorLow level (-) Central point (C) High level (þ)
Equilibrium time (tEQU, min) 15
Extraction time (tEXT, min)
A. Gaujac et al. / Talanta 106 (2013) 394–398
evaluated in the central composite design, which considered time
intervals varying from 20 to 80 min (Table 2). The response surface
model generated by the execution of the central composite design
matrix confirmed that the equilibrium time factor had no impact on
the peak area of DMT (Fig. 2). For this factor, it was adopted on the
central point level, 10 min, long enough to ensure that the fiber
reaches thermal equilibrium with the sample medium, at 60 1C,
before exposition on the headspace. The extraction time factor, on
the other hand, had a major influence on the signal response and
the surface (Fig. 2) revealed that chemical equilibrium was reached
after 60 min, in the region where maximum DMT peak area values
were obtained. In order to verify the prediction a set of extraction
time experiments were carried (5–110 min) while maintaining
all other factors unchanged, i.e. pH 11, equilibrium time 10 min,
desorption time 5 min and temperature 60 1C. The results are
presented in Fig. 3. Following this evaluation an extraction time
of 70 min was chosen in order to achieve improved method
robustness. In the all optimization designs, triplicate analyses were
carried out at the central point of the studied domains in order to
provide statistically meaningful figures.
3.2. Method validation
The DMT calibration curve was prepared using twelve con-
centration levels between 1.56 and 300 mg L?1using the solution
standards described on Section 2, and duplicate analysis per
concentration level. The slope and intercept values, together with
their standard deviations (see below), were determined using
regression analyses which yielded a correlation coefficient for
DMT of r2¼0.9975.
To determine the accuracy of the developed method, a recov-
ery study was carried out using beverage samples and the
standard addition method. In this case, a known amount of
isolated DMT was added to the sample at three different con-
centration levels, i.e. 9.5, 50 and 152 mg L?1, respectively. Each
concentration level analyzed in triplicate employing seven sam-
ples and the results were expressed as mean recovery and %RSD.
Under standard addition conditions, consistent and high recovery
values were obtained and mean absolute recovery values for DMT
were found to range between 71 and 109%. This recovery study
indicated that the method was suitable for the determination of
DMT from herbal preparations.
The precision of the method was determined by repeatability
studies and expressed as relative standard deviation (%RSD). The
repeatability (intra-assay precision) was measured by comparing
standard deviation values obtained from recovery percentages
derived from spiked samples (concentration levels 9.5, 50 and
152 mg L?1) that were run on the same day. Each concentration
level was determined seven times followed by RSD calculations.
The RSD values for DMT levels were found to be below 8.3% at all
spiked levels and considered suitable.
3.2.4. Limits of detection and quantification
The limit of detection (LOD) was calculated considering the
standard deviation of the analytical noise (a value seven times the
standard deviation of the blank) and the slope of the regression
line, and it was equal to 0.78 mg L?1. The limit of quantification
(LOQ) was determined as the lowest concentration that provided
a response of ten times the average of the baseline noise, and
were calculated using seven unfortified samples. The LOQ value
for this compound was 9.5 mg L?1.
Fig. 1. Pareto chart used to describe the optimization of SPME variables.
Factors and levels for central composite design.
FactorLow level (-) Central point (C) High level (þ)
Equilibrium time (tEQU, min)
Extraction time (tEXT, min)
Fig. 2. Surface response model.
Fig. 3. Effect of extraction time on DMT peak area.
A. Gaujac et al. / Talanta 106 (2013) 394–398
The robustness of this proposed method was estimated by
testing the reliability of analysis with respect to small but
deliberate variation of optimized method parameters. A three-
factor face-centered design consisting of fifteen experiments was
employed. The extraction time (69.5–70.5 min), temperature (59–
61 1C) and pH of aqueous phase (pH 10.5–11.5) were considered
critical factors. Pareto graph plots (Fig. 4) indicated that the
obtained response remained unaffected by small changes in these
critical method parameters. Statistical analysis revealed that
there was good agreement between experimental and predicted
values. Furthermore, none of the factors studied in these domains
showed significant effect on system efficiency.
3.2.6. Estimation of DMT content in jurema beverages
The analytical procedure described above was also used to
study the influence of several parameters typically involved in
the preparation of jurema products from plant material on DMT
content. This included an assessment of temperature (room
temperature Vs. 100 1C), pH of aqueous phase and the percentage
of ethanol in the aqueous extraction medium. Thus, five jurema
beverages samples were prepared in the laboratory using 5 g of
inner bark of jurema preta (M. tenuiflora) per 100 mL of sample,
employing the same time of extraction. The concentrations of
DMT found in the analyzed samples are summarized in Table 3.
For example, it was found that simply heating the decoction
during preparation did not lead to increased DMT levels, however,
concentrations of DMT did increase when an aqueous acid
medium (pH 1), or a mix of water and ethanol (50:50, v/v), was
employed. All samples were analyzed in triplicate.
Fig. 4. Evaluation of robustness using the Pareto chart.
DMT concentration in jurema beverage samples prepared in the laboratory under
Jurema beverage sample preparation conditionDMT conc. (g L?1)
1 Extraction into water at room temperature
2 Extraction into acidified water (HCl, pH¼1) at room
3 Extraction into water at 100 1C
4 Extraction into acidified water (HCl, pH¼1) at 100 1C
5 Extraction into water:ethanol (50:50, v/v) at room
Fig. 5. Representative SPME GC-ion trap-MS traces obtained from real samples of ayahuasca (A and B) and vinho da jurema (C and D). A and C: full scan mode; B and D:
quantification of DMT (13.05 min) at a reduced scan range between m/z 57–59 which reflected the formation of the m/z 58 iminium ion base peak.
DMT levels in ayahuasca (A) and vinho da jurema
(J) preparations obtained from Brazilian religious
SamplesDMT conc. (g L?1)
A. Gaujac et al. / Talanta 106 (2013) 394–398
3.3. Application of the method
Twelve samples of ayahuasca (A1–A7) and vinho da jurema
samples (J1–J5) were collected from different Brazilian religious
groups and analyzed in triplicate by using the developed SPME/
GC-IT-MS method. All samples were diluted by a factor of 10 or
25, depending on the level of DMT present in the beverages.
Representative chromatograms are shown in Fig. 5. High levels of
DMT were found in both sample types. The DMT concentration in
the vinho da jurema samples ranged from 0.10 to 1.81 g L?1,
whereas ayahuasca products revealed the presence of DMT in the
range of 0.17 to 1.14 g L?1, (Table 4), which was consistent with
previous reports on liquid samples .
The present study provided a new method for the determina-
tion of DMT in ayahuasca and vinho da jurema matrices based on
headspace solid-phase microextraction gas chromatography ion
trap mass spectrometry. The optimization of SPME-related para-
meters were carried out by multivariate techniques and provided
excellent figures of merit. The fact that it was possible to work
with a small sample size and that the extent of sample manip-
ulation was minimized, made the SPME/GC–MS technique parti-
cularly useful. There were considerable variations in DMT levels
detected in ayahuasca and vinho da jurema samples obtained from
Brazilian religious groups.
The authors wish to thank MCT/CNPq (Process no. 620247/
2008-8) and Pronex-FAPESB/CNPq (Process no. 0015/2009) for the
financial support of this study. We are also grateful to Mark Ian
Collins for providing a large part of ayahuasca and vinho da jurema
samples. Prof. Mark Wainwright is thankfully acknowledged for
proof-reading the manuscript.
 A. Gaujac, S. Navickiene, M.I. Collins, S.D. Brandt, J.B. de Andrade, Drug Test.
Anal. 4 (2012) 636–648.
 B.C. Labate, J. Psychoactive Drugs 43 (2011) 27–35.
 J.C. Callaway, J. Psychoactive Drugs 37 (2005) 151–155.
 C. Gambelunghe, K. Aroni, R. Rossi, L. Moretti, M. Bacci, Biomed. Chromatogr.
22 (2008) 1056–1059.
 S. Moura, F.G. Carvalho, C.D.R. de Oliveira, E. Pinto, M. Yonamine, Phytochem.
Lett. 3 (2010) 79–83.
 A.P.S. Pires, C.D.R. de Oliveira, S. Moura, F.A. D¨ orr, W.A.E. Silva, M. Yonamine,
Phytochem. Anal. 20 (2009) 149–153.
 Y. Chen, Z. Guo, X. Wang, C. Qiu, J. Chromatogr. A 1184 (2008) 191–219.
 F. Bonadio, P. Margot, O. Dele ´mont, P. Esseiva, Forensic Sci. Int. 182 (2008)
 B. Bojko, E. Cudjoe, G.A. Go ´mez-Rı ´os, K. Gorynski, R. Jiang, N. Reyes-Garce ´s,
S. Risticevic, E.A.S. Silva, O. Togunde, D. Vuckovic, J. Pawliszyn, Anal. Chim.
Acta 750 (2012) 132–151.
 A. Gaujac, S.T. Martinez, A.A. Gomes, S.J. de Andrade, A.C. Pinto, J.M. David,
S. Navickiene, J.B. de Andrade, Microchem. J. in press, http://dx.doi.org/10.
 A. Gaujac, A. Aquino, S. Navickiene, J.B. de Andrade, J. Chromatogr. B 881–882
A. Gaujac et al. / Talanta 106 (2013) 394–398