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Thin-Layer Chromatography Method for the Detection of N,N-

Dimethyltryptamine in Seized Street Samples

Article  in  JPC - Journal of Planar Chromatography - Modern TLC · November 2014

DOI: 10.1556/JPC.27.2014.6.13

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Thin-Layer Chromatography Method for the Detection of
N,N-Dimethyltryptamine in Seized Street Samples
Boris Duffau*, Sonia Rojas, Sebastian Jofre, Marcelo Kogan, Iván Triviño, and Paula Fuentes

Key Words
High-performance thin-layer chromatography
Dimethyltryptamine
Hallucinogens
Uncertainty

1 Introduction
Dimethyltryptamine (DMT) is an endogenous hallucinogen
with traditional use as a ceremony in the orally active prepara-
tion of Ayahuasca, a hallucinogenic beverage used by indige-
nous communities in the Amazon. Although the religious use of
Ayahuasca has been studied extensively, very little is known
about the recreational use of DMT [1]. Recently, many people
have shown prominent attention in traditional indigenous prac-
tices and popular medicine, involving the ingestion of natural
psychotropic drugs [2]. The consumption of Ayahuasca is
increasing worldwide due to the expansion of syncretic religions
founded in the north of Brazil in the first half of the twentieth
century, such as Santo Daime. This product contains N,N-
dimethyltryptamine which needs coadministration of naturally
occurring monoamine oxidase inhibitors, for example, β-carbo-
line derivatives, in order to induce its psychoactive effects in
humans, so this substance is required by users of DMT to be
effective orally [3]. Dimethyltryptamine dose-dependently ele-
vated blood pressure, heart rate, pupil diameter, and rectal tem-
perature, in addition to elevating blood concentrations of b-
endorphin, corticotrophins, cortisol, and prolactin. Growth hor-
mone blood levels rose equally in response to all doses of DMT,
even though melatonin levels are unaffected [4]. While previous
studies of DMT use had examined Ayahuasca use exclusively
[5], several studies had demonstrated the ubiquity of smoking as
the most prevalent route of administration among recreational
DMT users. A wide spread of analytical methods had been
developed in order to establish the composition of Ayahuasca,
but there is not much information available about the analysis of
DMT in street samples. Due to this reason, we implemented a
fast and reliable method for the determination of DMT by
HPTLC that offers many advantages such as rapidness, low
cost, accuracy, and precision. The use of validated methods is
significant for an analytical laboratory to show its qualification
and capabilities [6]. In this scenario, we developed and validat-
ed a rapid and simple method for the detection and quantitation
of DMT in illicit powder samples. Besides validation parame-
ters, the uncertainty of the method was also evaluated [7].
2 Experimental
2.1 Chemicals and Reagents
N,N-Dimethyltryptamine was generously provided by Drug
Enforcement Administration, Washington DC. Methanol and
conc. ammonia were of analytical grade, purchased from Merck,
Darmstadt, Germany.
2.2 Chromatography
The analysis was performed on 10 × 10 cm precoated silica gel
F254 plates (Merck, Darmstadt, Germany), previously activated
at 80°C for 20 min. One microliter of standards and samples was
applied in bands of 4 mm with an ATS 4 automatic TLC sampler,
using a spray band technique; the first application x axis was
10 mm and y axis was 8.0 mm, and the distance between the
tracks was 4.2 mm. Plates were developed with an automatic
developing chamber ADC-2 without saturation to a distance of
70 mm, with methanol–ammonia 100:1.5 as the mobile phase
(10 mL, freshly prepared) and drying time of 2.0 min; the spots
were scanned with a TLC Scanner 4 densitometer by absorbance
at 220 nm. Spectra of each peak were recorded in the range of
190–400 nm on all detected peaks mode; slit dimension, 4.00 ×
0.30 mm; scanning speed, 20 nm s−1; data resolution, 100 µm
step−1; reference spectrum, x = 10.0 m and, y = 5.0 mm. All
processes were controlled with the software winCATS Planar
Chromatography Manager, version 1.4.7 (CAMAG, Muttenz,
Switzerland).

3 Results and Discussion

B. Duffau, S. Rojas, S. Jofre, P. Fuentes, and I. Triviño, Drug Analysis Section,
Institute of Public Health of Chile, Chile; and M. Kogan, Faculty of Chemical and
Pharmaceutical Sciences, University of Chile, Chile. The proposed method was validated according to parameters of
E-mail: bduffau@gmail.com the International Conference on Harmonization (ICH) guide-

Journal of Planar Chromatography 27 (2014) 6, 477–479

0933-4173/$ 20.00 © Akadémiai Kiadó, Budapest
DOI: 10.1556/JPC.27.2014.6.13
477
Short Communications
Figure 1
Densitogram of N,N-DMT at 220 nm (RF 0.50).
lines [8]. The mobile phase resulted in a symmetrical and
resolved peak at RF 0.50 ± 0.01 (Figure 1). The specificity of the
method was evaluated by analyzing the standard and real sam-
ples. The band for DMT was confirmed by comparing the RF
value as well as the ultraviolet (UV) spectra of the band from the
standards and samples. The peak purity of DMT was measured
by comparing the spectra acquired at the peak start (S), peak
apex (M), and peak end (E) of each band. It was found that r(S,
M) = 0.9991 and r (M, E) = 0.9961. The peak purity of >0.99
indicated the specificity of the method; there was no interfer-
ence from any impurities in the separation and determination of
the DMT peak. Linearity was established by least squares linear
regression analysis of the calibration curve. The constructed cal-
ibration plot was linear over the concentration ranges of 11.50 to
34.25 μg per band. Peak heights of DMT were plotted against
their respective concentrations, and linear regression analysis
was achieved on the resulting plots. Equations for the calibration
plots of DMT were Y = 1.790x − 6.346 by peak height.
Correlation coefficient was 0.99531, and relative standard devi-
ation (RSD) was 4.36% (Figure 2). The analysis of variance
showed that p-value was less than 0.05; there was a statistically
significant relationship between height and added amount of
DMT at the 95.0% confidence level. The Durbin–Watson (DW)
statistical tests (1.39856; p = 0.0324) revealed that there was a
strong correlation at the 95.0% confidence level. Limit of detec-
tion (LOD) and limit of quantification (LOQ) were calculated
by the formulas LOD = 3.3xSa/b and LOQ = 10xSa/b, where Sa
is standard deviation of the intercept and b is the slope of the cal-
ibration curve. In this case, LOD was 3.28 µg per band and LOQ
was 9.94 µg per band. The precision (repeatability) of the
method was examined by analyzing six independent replicates
of one sample, injected in duplicate in the same plate by the
same analyst on the same day. Intermediate precision was inves-
tigated by analyzing the same sample in triplicate on three dif-
ferent days, at three concentration levels by the same analyst.
Accuracy was determined on the basis of standard addition per-
formed at three levels, each in triplicate; for this purpose, a
known amount of standard powder of DMT was added and then
analyzed by the proposed method. Results from repeatability
478
Figure 2
Calibration plot of N,N-DMT at 220 nm.
Table 1
Summary of validation parameters.
Parameter Results
RF 0.50 ± 0.1
Linearity 11.50 to 34.25 μg band−1,
Y = 1.790x − 6.346
Correlation coefficient 0.99531
LOD and LOQ 3.28 μg band−1 and 9.94 μg band−1
Precision (repeatability and
intermediate precision) 4.89% and 3.85%
Accuracy (% Recovery) 117.9%
and intermediate precision were expressed as coefficient of vari-
ation (CV%) or RSD. The low values showed that the method is
precise (% RSD less than 5% for both parameters). Robustness
was determined by changing the distance of migration of the
plate (70 mm to 80 mm) and then evaluating the RSD of the cal-
ibration curve. Low values of % RSD (3.54%–4.67%) were
obtained after introducing this change in migration distance.
A brief summary of validation results is shown in Table 1. No
other peaks were found, and no significant changes in peak
shape and linearity were found for standards solutions after stor-
age at room temperature (20 ± 5°C) for 24 h and after storage at
2–8°C for 24 h. Uncertainty of the method was evaluated with
results of validation parameters, i.e., standard deviation (SD) of
linearity, precision, and accuracy. The expanded uncertainty was
calculated with the formula:
U = 2 × √SDcal2 + SDR2 + SDr2+ SDRec2
where U is the expanded uncertainty (α = 0.05; K = 2), SDcal is
the standard deviation of calibration, SDR is the standard devia-
tion of repeatability, SDr is the standard deviation of intermedi-
ate precision, and SDRec is the standard deviation of recovery
results. The expanded uncertainty was 5.13 µg.
Journal of Planar Chromatography 27 (2014) 6

With the validated method, we analyzed one real sample of an
unknown yellow powder; the obtained results revealed the pres-
ence of DMT, and its concentration was 92.5 µg/100 µg.
4 Conclusion
The HPTLC method developed for the quantitation of DMT was
found to be fast, simple, accurate, reproducible, and sensitive
and is appropriate for the analysis of illicit samples of DMT. The
proposed method for the quantification of DMT is the first vali-
dated HPTLC method to the best of our knowledge, with many
advantages such as short time, low sample preparation, small
cost, and it can be easily implemented in forensic laboratories in
order to establish the profile of illicit seized samples of DMT.
This is the first time that we found DMT in street samples seized
from Chilean police; this fact, besides the rise of confiscations
of substances such as mescaline, phenyl ethylamine derivatives
(i.e., 25 C NBOMe), could lead us to suspect that hallucinogenic
compounds are becoming popular among people who consume
drugs of abuse. In this perspective, analytical methods are a
powerful tool in order to test the presence or absence of illegal
drugs; this objective can be easily reached by using HPTLC.
Acknowledgments
The authors want to thank to the Drug Enforcement Administ-
ration DEA for providing the reference material for this study,
Journal of Planar Chromatography 27 (2014) 6
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Short Communications
and also, we want to thank the Public Health Institute of Chile
for making this research available and pharmacist Lorena
Delgado Rivera for her collaboration in our work.
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Ms received: June 17, 2014
Accepted: August 27, 2014
479