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Forensic Science International: Genetics Supplement Series 1 (2008) 600–602

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Research Article
Forensic detection of marijuana trace
Thitika Kitpipit a,1, Nathinee Panvisavas a,b,*,
Nuntavan Bunyapraphatsara c
a
Forensic Science Graduate Programme, Faculty of Science, Mahidol University,
Rama 6 Road, Bangkok 10400, Thailand
b
Department of Plant Science, Faculty of Science, Mahidol University, Thailand
c
Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, Thailand
Received 28 August 2007; received in revised form 23 October 2007; accepted 7 November 2007

Abstract
In this study, we compared the use of both chemical and biological tests for precise screening. Marijuana leaves had been treated in simulated
conditions according to the way they are consumed; leaves materials were boiled in water for 5 min to 8 h, dried in hot-air oven, air-dried in shade
and sunlight, and burned to black and white ashes. The THC band was detected in the TLC fingerprints of all samples, except the white ash extract.
In contrast, the 197-bp mitochondrial trnL-F fragment was amplified in two samples, i.e., the DNA extracted from fresh marijuana leaves that were
boiled for 5 min and some of the dried marijuana sample. The results suggested that TLC was a robust method for the detection of THC in
marijuana. However, DNA analysis seems to be limited when DNA from heat-treated materials were analyzed.
# 2008 Elsevier Ireland Ltd. All rights reserved.

Keywords: Forensic; Marijuana; Trace; TLC; DNA

1. Introduction
Chemical examination is routinely used to detect the presence
of the hallucinogenic substance in alleged materials called
‘‘tetrahydrocannabinol’’ (THC), which is specific to Cannabis
sativa. Although marijuana DNA markers have been developed
from regions such as trnL-F [1] and the THCA synthase gene [2],
DNA analysis is not widely used in the Thai forensic community.
In this study, we compared the use of chemical and biological
techniques to detect marijuana materials, which had been treated
in various simulated conditions according to the way they are
consumed.
2. Materials and methods
Marijuana materials were obtained from the Office of the
Narcotics Control Board (ONCB), Thailand. Marijuana leaves
* Corresponding author at: Forensic Science Graduate Programme, Faculty of
Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand.
Tel.: +66 2 201 5213; fax: +66 2 354 7096.
E-mail address: scnpv@mahidol.ac.th (N. Panvisavas).
1
Present address: Forensic Science Graduate Programme, Faculty of
Science, Prince of Songkla University, Songkhla 90110, Thailand.
1875-1768/$ – see front matter # 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.fsigss.2007.11.013
were boiled in water for 5 min to 8 h, dried in hot-air oven, air-
dried in shade and sunlight, and burned to black and white
ashes. Cannabinoids were extracted from the treated materials
and separated by TLC in hexane:dioxane:methanol (7:2:1),
petroleum ether:diethyl ether (8:2), hexane:diethyl ether (8:2),
or hexane:dioxane (9:1). DNA was extracted for PCR analysis
using the trnL-F Cannabis-specific primer pair described by
Ref. [1].
3. Results
The 197-bp DNA fragment was amplified only in C. sativa,
hence demonstrating the specificity of the trnL-F primers
(Fig. 1). When testing the DNA of the treated marijuana
materials, the 197-bp trnL-F fragment was detected in only two
samples, one is the fresh marijuana leaves boiled for 5 min and
a dried-marijuana sample (Fig. 2).
Comparison of four TLC solvent systems for cannabinoid
separation showed that Rf value of the eight bands separated in
hexane:dioxane:methanol (7:2:1) were in the range of 0.2–0.6,
suggesting the best separation resolution (Fig. 3). TLC
fingerprint of the treated marijuana samples showed the major
bands of D9-THC, CBN, and CBD in all samples, except the
white ashes (Fig. 4).
 T. Kitpipit et al. / Forensic Science International: Genetics Supplement Series 1 (2008) 600–602 601

Fig. 1. A 2% (w/v) agarose gel depicting the 197-bp PCR product amplified from DNA extracted from Cannabis sativa L. (lane 2), but not from other plants (lanes 3–9).

Fig. 2. A 2% (w/v) agarose gel depicting the 197-bp DNA fragment amplified from the trnL-F region of fresh marijuana leaves that were boiled for 5 min (lane 2), and
air-dried marijuana (lane 28).

Fig. 3. Comparison of cannabinoid separation in four different TLC solvent systems on GF254 TLC plates: (a) hexane:dioxane:methanol (7:2:1); (b) petroleum
ether:diethyl ether (8:2); (c) hexane:diethyl ether (8:2); (d) hexane:dioxane (9:1). The hexane:dioxane:methanol (7:2:1) system (a) gave the best resolution.

Fig. 4. TLC fingerprint of treated marijuana samples. The THC bands and similar TLC fingerprint pattern were present in all treated marijuana samples, except in
lane 24 which marijuana was burned into white ash, and all showed.
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4. Conclusions amount of DNA template recovered from different sample

types.
The study demonstrated that TLC fingerprinting, being
simple and rapid, is a robust method for the detection of THC Conflict of interest
in treated marijuana materials when compared to DNA
analysis. It can detect the controlled substances in all None.
treated marijuana samples, except the white ash extract. In
contrast, DNA analysis is limited when DNA from heat- References
treated materials were analyzed. However, chemical refer-
[1] A. Linacre, J. Thorpe, Detection and identification of cannabis by DNA,
ence standards (controlled substances) are required for the
Forensic Sci. Int. 91 (1998) 71–76.
TLC analysis of unknown samples. It is also suggested that [2] M. Kojoma, H. Seki, S. Yoshida, T. Muranaka, DNA polymorphisms in the
DNA recovery from the other types of samples generated tetrahydrocannabinolic acid (THCA) synthase gene in ‘‘drug-type’’ and
should be further optimized as there are factors affecting the ‘‘fiber-type’’ Cannabis sativa L., Forensic Sci. Int. 159 (2005) 132–140.